The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistryamp Cell Biology
journa l h o me page wwwelsev ier com locate b ioce l
eview
odal signalling in embryogenesis and tumourigenesis
aniela F Quail Gabrielle M Siegers Michael Jewer Lynne-Marie Postovit lowast
epartment of Anatomy and Cell Biology University of Western Ontario and Robarts Research Institute London ON Canada
r t i c l e i n f o
rticle historyeceived 28 September 2012eceived in revised form9 December 2012ccepted 24 December 2012vailable online xxx
eywordsodalancermbryogenesisetastasisorphogenesis
tem cells
a b s t r a c t
With few exceptions most cells in adult organisms have lost the expression of stem cell-associatedproteins and are instead characterized by tissue-specific gene expression and function This cell fate spec-ification is dictated spatially and temporally during embryogenesis It has become increasingly apparentthat the elegant and complicated process of cell specification is ldquoundonerdquo in cancer This may be becausecancer cells respond to their microenvironment and mutations by acquiring a more permissive plasticepigenome or because cancer cells arise from mutated stem cells Regardless these advanced cancer cellsmust use stem cell-associated proteins to sustain their phenotype One such protein is Nodal an embry-onic morphogen belonging to the transforming growth factor- (TGF-) superfamily First described inearly developmental models Nodal orchestrates embryogenesis by regulating a myriad of processesincluding mesendoderm induction leftndashright asymmetry and embryo implantation Nodal is relativelyrestricted to embryonic and reproductive cell types and is thus absent from most normal adult tissuesHowever recent studies focusing on a variety of malignancies have demonstrated that Nodal expressionre-emerges during cancer progression Moreover in almost every cancer studied thus far the acquisition
of Nodal expression is associated with increased tumourigenesis invasion and metastasis As the list ofcancers that express Nodal grows it is essential that the scientific and medical communities fully under-stand how this morphogen is regulated in both normal and neoplastic conditions Herein we reviewthe literature relating to normal and pathological Nodal signalling In particular we emphasize the rolethat this secreted protein plays during morphogenic events and how it signals to support stem cellmaintenance and tumour progression
f Medicine amp Dentistry University of Western Ontario Medical Sciences Building
886 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
41 Nodal signalling in the placenta 89142 Nodal signalling in the mammary gland 89243 Nodal signalling in the cycling endometrium 89344 Nodal expression in human adult stem cells 89345 Nodal mutations in human disease 893
5 Nodal signalling in cancer 89351 Melanoma 89352 Brain cancers 89453 Prostate and testicular cancers 89454 Endometrial cancer 89455 Ovarian cancer 89456 Hepatocarcinoma 89457 Pancreatic cancer 89458 Breast cancer 894
6 Regulation of Nodal expression 89561 Silencing of Nodal expression in development 89562 Re-expression of Nodal in cancer 895
7 Conclusions 895Conflict of interest statement 896Acknowledgements 896References 896
1
ttbacmccmesGT
FdswdbegictiN
Introduction
Embryonic development is characterized by the spatial andemporal differentiation of stem cells leading to the genera-ion of tissue diversity This process of differentiation is dictatedy microenvironmental mediators such as morphogen gradientsnd oxygen availability (Quail et al 2012a) The instructiveues of the embryonic programme culminate in an organismade of tissues and cell types that maintain homeostatic balance
oncomitant with exquisite structurendashfunction relationships Inancer the embryonic programme is disrupted resulting in theanifestation of stem cell-like characteristics (Fig 1) (Hendrix
t al 2007) Alternatively some cancers may arise from residenttem cell populations (Chen et al 2012 Driessens et al 2012ilbertson and Graham 2012 Visvader and Lindeman 2006)his stem-cell-like nature of cancer is correlated with metastatic
ig 1 Nodal signalling is a developmental phenomenon that is ldquore-awakenedrdquouring cancer progression tumour progression is characterized by a loss of tis-ue structure and by the acquisition of a more stem cell-like phenotype In manyays this process represents an ldquoundoingrdquo of the differentiation that occurs duringevelopment and mimics certain aspects of induced pluripotency The mechanismy which cancer cells aberrantly acquire the expression of Nodal likely involvespigenetic alterations facilitated by microenvironmental mediators such as oxy-en availability Normal embryonic stem cells maintain a balance of activators andnhibitors of self-renewal in order to facilitate differentiation in response to specificues Here we highlight Nodal and its inhibitor Lefty In contrast cancer cells hijackhese elegant signalling pathways in a manner that favours uncontrolled growthn the absence of normal differentiation In this example the cancer cells expressodal in the absence of Lefty
progression resistance to therapy and a poor clinical progno-sis Hence understanding and targeting molecules that sustainstem cell-like phenotypes could be of tremendous therapeuticvalue
A number of proteins have been shown to regulate both normaland neoplastic stem cell populations One example is Nodal Nodalis a member of the transforming growth factor- (TGF-) super-family which includes TGF-s activins growthdifferentiationfactors (GDFs) and bone morphogenic proteins (BMPs) (Quailet al 2012a Schier 2009) Originally discovered in mouse Nodalhomologues have been characterized in humans Xenopus (frogs)and zebrafish amongst other model organisms (Schier 2009)As is the case for mouse Nodal there is currently only oneannotated isoform for human Nodal mRNA in the National Cen-tre for Biotechnology Information (NCBI) Entrez Gene database(httpwwwncbinlmnihgov80sitesentrez) corresponding toone Nodal ligand Human Nodal is found on chromosome 10 andconsists of three protein-coding exon regions that are translatedinto a 347 amino acid pro-protein Nodal is further processed intoa mature form (amino acids 238ndash347) with a C-terminus thatmight be important for interactions with other proteins such asitrsquos co-receptor Cripto (Schier 2009) Unlike mammals zebrafishhave three Nodal orthologues (cyclops squint and southpaw)whilst frogs have six ldquoXenopus Nodal-relatedrdquo members (Xnr1-6)although only 5 of these (Xnr124ndash6) have canonical Nodal func-tions (Schier 2009) Regardless of the species in chordates Nodalproteins are essential for the induction of mesoderm and endo-derm and leftndashright patterning during embryogenesis (Hendrixet al 2007 Schier 2009 Shen 2007)
Nodal is relatively restricted to embryonic and reproductive celltypes and is not detectable in most normal adult tissues (Hendrixet al 2007 Hooijkaas et al 2011) However recent studies havedemonstrated that Nodal expression re-emerges during cancerprogression (Lawrence et al 2011 Lee et al 2010 Lonardo et al2011 Strizzi et al 2012 Topczewska et al 2006) Moreoverin almost every cancer studied thus far the acquisition of Nodalexpression is associated with increased tumourigenesis invasionand metastasis (Table 1) (De et al 2012 Lawrence et al 2011Lee et al 2010 Lonardo et al 2011 Papageorgiou et al 2009
Quail et al 2012a 2012b in press Strizzi et al 2012 Topczewskaet al 2006) As more cancers expressing Nodal are added to thelist it is becoming increasingly important that we understandthe mechanisms by which this morphogen is regulated Here we
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 887
Table 1The pro- and anti-tumourigenic roles of Nodal in different types of cancer
Phenotype Pro-tumourigenic Anti-tumourigenic
Cancer types References Cancer types References
Invasion and migration Breast cancer glioma melanoma prostate cancerpancreatic cancer
Quail et al (2011 in press) Lee et al(2010) Postovit et al (2007b)Topczewska et al (2006) Lawrenceet al (2011) and Lonardo et al (2011)
Angiogenesis andvascular phenotypes
Melanoma breast cancer GBM Hardy et al (2010) McAllister et al(2010) Quail et al (2012c) and Huenget al (2011)
Proliferation andapoptosis
GBM endometrial cancer hepatocellular carcinomabreast cancer
De et al (2012) Papageorgiou et al(2009) Cavallari et al (2012) andQuail et al (2012b)
Ovarian cancerbreast cancerchoriocarcinoma
Xu et al (2004)Zhong et al (2009)Munir et al (2004)Fu and Peng (2011)
Stem cell phenotypes Self-renewal of GBM anchorage-independent growthof prostate cancer pancreatic spheroids and CSCsplasticity in breast cancer
De et al (2012) Lawrence et al (2011)Lonardo et al (2011) and Meyer et al(2009)
EMT Breast cancer choriocarcinoma Quail et al (in press)
Metastasis andtumourigenesisin vivo
Melanoma GBM prostate cancer hepatocellularcarcinoma pancreatic cancer breast cancer
Postovit et al (2007b) Topczewskaet al (2006) Lee et al (2010) Adkinset al (2003) Cavallari et al (2012)Lonardo et al (2011) and Quail et al(2012b in press)
Patient correlations Invasion and advanced stage in melanoma and breast Topczewska et al (2006) Hooijkaaset aet a(20
ra
2
2
cpc4wbIc(wtasNffiF2naaadA
namp
cancer MVD in breast cancer and glioma tumourgrade in GBM endometrial cancer and breast cancerlymph node status in breast cancer
eview the function and regulation of Nodal signalling in normalnd pathological conditions
Nodal signalling
1 Canonical SMAD-associated signalling
Like most TGF- family members Nodal is secreted from theell as a precursor (Fig 2) In order to form an active ligand therecursor protein must dimerize and the pro-domain must beleaved by substilisin-like pro-protein convertases such as Pace-
and Furin This event can occur in the extracellular space orithin the cell (Schier 2009) The active homodimer signals by
inding to activin-like kinase receptors type I (ALK47) and typeI (ActRIIB) Upon activating this receptor complex (ALK receptoromplex) mothers against decapentaplegicdrosophila homolog 2SMAD2) and possibly SMAD3 is phosphorylated and interactsith SMAD4 before translocating to the nucleus (Schier 2003) In
he nucleus transcription factors such as forkhead box HI (FOXH1)nd Mixer work with the activated SMADs to increase the expres-ion of Nodal responsive genes such as Goosecoid (Gsc) and Leftyodal also induces its own expression thereby creating a positive
eedback loop (Schier 2009 Topczewska et al 2006) The speci-city of Nodal is established by the epidermal growth factor-criptoRL1 cryptic (EGF-CFC) family co-receptor Cripto-1 (Strizzi et al008 Topczewska et al 2006) Cripto-1 has an N-terminal sig-al peptide an EGF-like domain that directly interacts with Nodal
conserved cysteine-rich (CFC) domain that interacts with ALK4nd a hydrophobic C-terminal glycosyl-phosphatidylinositol (GPI)nchor (Minchiotti 2005) The adjacent positioning of the EGF-likeomain and the CFC domain helps bring Nodal into proximity withLK4 to facilitate enhanced binding (Minchiotti 2005)
Endocytosis is an important early step in Nodal-mediated sig-
al transduction (Blanchet et al 2008a 2008b) Specifically Nodalnd its co-receptor Cripto enter cells via a lipid-raft-associatedechanism (Blanchet et al 2008a) This NodalndashCripto com-
lex is subsequently sorted to the limiting membrane of early
l (2011) Lee et al (2010) Quaill (2012a) Papageorgiou et al09) and Strizzi et al (2012)
endosomes marked by the small GTPases Rab4 and Rab5 (Blanchetet al 2008b) These early endosomes also contain the ALK4receptor complex and the SMAD Anchor for Receptor Activation(SARA) a scaffold protein that couples the ALK receptors to effec-tor SMADs (Blanchet et al 2008b) Hence endosomes may act assignalling platforms where Nodal-activated receptors can phos-phorylate SMAD23
Nodal signalling is inhibited spatially and temporally dur-ing development by inhibitors such as Lefty A Lefty B andCerberus (Constam 2009b Schier 2009) These inhibitors aretranscribed in response to Nodal signalling and act as a nega-tive feedback mechanism to control Nodal localization and actionin the developing embryo (Schier 2003) Lefty in particular isalso regulated by alternate SMAD pathways canonical Wnt andOct34 and is up-regulated with differentiation (Tabibzadeh andHemmati-Brivanlou 2006) Lefty inhibits Nodal signalling throughinteractions with Nodal andor Cripto-1 that prevent activation ofthe ALK receptor complex (Schier 2003) Notably Nodal is uniqueamongst TGF- ligands in its requirement for the co-receptorCripto to signal effectively This dependence is also responsible forthe ability of Lefty proteins to inhibit Nodal in near exclusivityIndeed other TGF- members such as Activin signal in a Cripto-independent fashion and thus are not susceptible to inhibition byLefty (Cheng et al 2004) Interestingly whilst both Nodal and Leftyare expressed in most embryonic and adult stem cell contexts (asdescribed below) Lefty negative feedback is not present in cancercells (Hendrix et al 2007 Postovit et al 2008 Quail et al 2012a)
22 Non-canonical (non-SMAD) Nodal signalling
Non-SMAD pathway activation by Nodal is poorly understoodOne important exception is the requirement for p38 activationduring Nodal-induced anterior visceral endoderm (AVE) speci-
fication (Clements et al 2011) Activated p38 signalling in theAVE in turn amplifies Nodal signalling during anteriorndashposterior(AP) axis formation through phosphorylation of the SMAD2 linkerregion (which is receptive to phosphorylation by mitogen activated
888 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 2 The Nodal signalling pathway and mechanisms of regulation Nodal signals through activation of its receptor complex (ALK47 ActRIIB Cripto) and subsequentphosphorylation of SMAD23 SMAD23 forms a complex with SMAD4 and translocates to the nucleus to regulate target genes including Nodal (for autoregulation) andits endongenous inhibitor Lefty (for negative feedback) As embryogenesis proceeds Nodal signalling eventually activates differentiation programmes before it becomestranscriptionally silenced Nodal initiates differentiation via association of SMAD23 with TRIM33 and subsequent activation of poised chromatin in promoter regions ofgenes associated with mesendoderm specification Later Nodal is silenced possibly through polycomb-mediated trimethylation of histone H3 at Lysine 27 (H3K27me3) Thism ates thm amonga
pSsmtoIprpcNt
awptAcotNwoir
piNnaNe
ethylation signature is reversible as re-activated Nodal-SMAD23 signalling facilitethylations and re-activate transcription of Nodal This may be one mechanism (
berrant contexts
rotein kinase (MAPK) proteins) and subsequent enhancement ofMAD2 activity (Clements et al 2011) In keratinocytes of thekin SMAD and Extracellular Regulated Kinase (ERK) signalling areutually required for TGF--induced epithelial-to-mesenchymal-
ransition (EMT) as ERK substrates mediate nuclear translocationf SMADs and alter target gene expression (Davies et al 2005)n cancer non-SMAD pathway activation by other TGF--familyroteins has been characterized For instance the TGF- type 1eceptor is capable of activating MAPK signalling through phos-horylation of ShcA and subsequent interaction with the GRB2SOSomplex (Lee et al 2007) Given the similarities between TGF- andodal these studies lend insight into putative non-SMAD Nodal
argets during disease progressionWe recently determined that Nodal promotes cellular invasion
nd EMT-like phenomena via activation of the ERK12 MAPK path-ay in breast cancer and choriocarcinoma cells (Quail et al inress) Specifically we found that Nodal induces ERK12 activa-ion and that this ability of Nodal to activate ERK12 is mediated byLK47 activity Our results indicate there may be multiple points ofrosstalk between the SMAD23 and ERK12 pathways at the levelf receptor activation intracellular signalling and gene transcrip-ion (Fig 3) Importantly Nodal-induced ERK activation mediatesodal-induced EMT and invasion such that inhibition of this path-ay with U0126 (a MEK12 inhibitor) abrogates the effects of Nodal
n these phenotypes (Quail et al in press) Together these find-ngs indicate that putative Nodal-targeted therapies would likelyequire interference of both SMAD and ERK pathways
As described below Nodal plays paradoxical roles in humanluripotent stem cells maintaining pluripotency whilst initiat-
ng mesendoderm induction Such disparate functions suggest thatodal likely regulates chromatin accessibility by altering epige-
etic phenomena such as DNA or histone methylation Indeed
recent study by Massagueacute and colleagues (2011) showed thatodal signalling is capable of inducing the expression of differ-ntiation signals in human embryonic stem cells by activating
e recruitment of the demethylase Jmjd3 to the Nodal locus to lift polycomb-inducedst others such as hypoxia-induced HIF signalling) of how Nodal is re-activated in
ldquopoisedrdquo chromatin (Xi et al 2011) Poised chromatin markedby polycomb-mediated trimethylation of histone H3 at lysine 9(H3K9me3) and acetylation of lysine 18 (H3K18ac) ensures trans-criptional repression of embryonic genes however these genesare responsive to acute activation by developmental signals inthe environment Chromatin assumes a poised state in responseto pluripotency factors such as Oct4 and Nanog to ensure thetimely activation of differentiation cues during development In thisstudy NodalActivin signalling through phosphorylated SMAD23caused recruitment of TRIM33 to H3K9me3-K18ac at the promo-ters of genes associated with mesendoderm specification (such asNodal targets MixI1 and Gsc) Association of SMAD23 and TRIM33in these regions lifted methylations to activate poised chromatinrendering promoter regions accessible to SMAD23ndashSMAD4 trans-criptional complexes (Fig 2) This study is the first to propose amechanism of how Nodal mediates widespread differentiation cuesduring embryogenesis and lends insight into how other mediatorsof embryonic differentiation may work in a similar fashion
3 Nodal signalling in developmental models
31 Role of Nodal in mouse development
The first gradient that Nodal establishes during development isthe proximalndashdistal (PD) axis which later rotates to become the APaxis Nodal is activated through the developing epiblast by conver-tase enzymes that are secreted from the extraembryonic ectoderm(Beck et al 2002 Schier 2009) These convertases including Pace4and Furin cleave the pro-domain of Nodal to activate its signallingin the proximal epiblast adjacent to the extraembryonic ectoderm(Beck et al 2002 Schier 2003) Here Nodal is maintained and
concentrated either through autoregulation andor by activatingBMP in the extraembryonic ectoderm which signals back to theadjacent epiblast to induce Wnt (Vincent et al 2003) The Nodalgene contains a proximal epiblast enhancer (PEE) upstream of
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 889
Fig 3 Putative crosstalk between ERK and SMAD pathways during Nodal signalling and regulation of E-cadherin trafficking Studies from our laboratory and others suggestthere may be multiple points of crosstalk between the SMAD23 and ERK12 pathways We have recently shown that recombinant Nodal induces phosphorylation of ERK12This may be indirect through receptor phosphorylation of an upstream mediator of the ERK cascade (such as ShcA) or direct via receptor phosphorylation of ERK12 We havealso shown that MEK inhibition leads to a decrease in Nodal-induced phosphorylation of SMAD2 This may be due to altered phosphorylation of the SMAD23 linker regionby ERK MAPKs We have shown that MEK inhibition reduces transcription of Nodal-induced EMT transcription factors ERK may alter SMAD-mediated gene expression byregulating translocation of SMAD proteins to the nucleus or by interfering with gene transcription We also found that MEK inhibition rescued Nodal-induced mislocalizationo n thatp
ttbiivastedpShC
itmpiseNa(
left
f E-Cadherin In accordance with these observations previous reports have showlasma membrane after internalization during EMT
he transcription start site that is Wnt-responsive Activation ofhis enhancer by Wnt concentrates Nodal to the proximal epi-last (Fig 4A) (Brennan et al 2001) Activated Nodal signalling
n the proximal epiblast subsequently induces gene expression ofts endogenous inhibitors Lefty1 and Cerberus (CER1) in the distalisceral endoderm (DVE) to establish a Nodal PD gradient Lefty1nd CER1 subsequently restrict Nodal expression to the proximalide of the embryo where the primitive streak will form to ini-iate gastrulation The PD axis graded by Nodal expression willventually rotate to become the AP axis whereby the DVE will beisplaced to yield the AVE and the proximal epiblast will moveosteriorly (Fig 4B) (Collignon et al 1996 Constam 2009a 2009btrizzi et al 2008 Zhou et al 1993) It is not entirely understoodow this rotation is regulated however it has been shown to beripto-dependent (Ding et al 1998)
Following AP axis formation gastrulation occurs as the prim-tive streak elongates from the posterior to the anterior side ofhe embryo This process is orchestrated by ingression of proxi-
al posterior cells to form mesendoderm cells which elongate therimitive streak towards the anterior side of the embryo Nodal
s expressed in the primitive endoderm overlying the primitivetreak and is thought to co-ordinate its directional migration andlongation (Conlon et al 1994) Once gastrulation is completeodal expression becomes restricted to the periphery of the nodet the anterior end of the primitive streak hence the name ldquoNodalrdquoSchier 2003)
Following gastrulation the node is the central initiator of
eftndashright (LR) asymmetry (Schier 2003 Smith 1995 Takaokat al 2006 Zhou et al 1993) TGF--family members secretedrom the ventral node including Nodal and growth differen-iation factor 1 (GDF1) define the left side of the embryo
inhibition of the ERK pathway is required for relocalization of E-Cadherin to the
through interactions with mouse Cripto family member Cryp-tic co-receptors in the lateral plate mesoderm (Constam 2009a)Meanwhile a leftward flow induced by active cilia prevents Nodalfrom signalling on the right side of the embryo in conjunc-tion with the right side-specific expression of CER1 and Lefty1(Fig 4C) (Constam 2009a) Of note this is how Nodalrsquos endogenousinhibitor Lefty received its name It has been shown that murineembryos lacking node-specific Nodal expression are not capableof establishing LR asymmetry and exhibit asymmetric patterningdefects including random positioning of the stomach and abnormalbranching of lung lobes (Brennan et al 2002) Furthermore thesestudies have shown that short-range Nodal signalling may also playa role in establishing a midline between the left and right sides ofthe embryo (Brennan et al 2002)
Following axis patterning of the embryo somitogenesis occurswhereby cells condense into transient epithelial structures byundergoing mesenchymal-to-epithelial transition At the begin-ning of this process Nodal is restricted to mesoderm cells on the leftside of the embryo (Collignon et al 1996) Interestingly as Nodal isa critical regulator of gastrulation (one of the first major EMT eventsto occur during development) somitogenesis is accompanied byan abrupt silencing of Nodal signalling by 8 days postcoitum (dpc)(Collignon et al 1996) Until recently it was not completely under-stood how Nodal silencing during development occurred Howevernew investigations suggest that Nodal is silenced by polycomb-mediated methylation of histones in a reversible manner (Dahleet al 2010 Xi et al 2011) (Fig 2)
Several studies using genetically modified mice have been per-formed to assess the role of different aspects of the Nodal signallingpathway during development Murine Nodal null mutants areembryonic lethal as they are unable to initiate the primitive streak
890 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 4 Nodal morphogens pattern the murine embryo to establish multiple criticalaxes (A) Nodal is activated in the epiblast by convertase enzymes (ex Pace4 andFurin) secreted from the extraembryonic ectoderm (ExEct) Convertases cleave andactivate Nodal in the proximal epiblast where Nodal is maintained by autoregula-tion andor by activation of a BMPWnt signalling loop The Nodal gene containsa proximal epiblast enhancer (PEE) upstream of the transcription start site thatis Wnt-responsive activation of the PEE by Wnt concentrates Nodal to the prox-imal epiblast (B) Nodal subsequently induces gene expression of its endogenousinhibitors Lefty1 and CER1 in the distal visceral endoderm (DVE) to establish aproximal-distal (PD) gradient The PD axis graded by Nodal expression eventuallyrotates to become the anterior-posterior (AP) axis whereby the DVE is displacedto yield the anterior visceral endoderm (AVE) and the proximal epiblast movesposteriorly (C) During gastrulation Nodal is expressed in the primitive endodermoverlying the primitive streak and then becomes restricted to the node to initiateleftndashright (LR) asymmetry GDF1 and Nodal from the node define the left side of theedf
aat(amaoot
Fig 5 Comparison of Nodal expression patterns in early versus late gastrulationin mouse frog zebrafish and sea urchin development In mice Nodal is expressedin the node during early gastrulation and in the left lateral plate mesoderm dur-ing specification of LR asymmetry In the frog Xnr proteins are critical initiators ofgastrulation in the dorsal lip and become restricted to the lateral plate mesodermlater during neurulation In zebrafish Cyclops is expressed in the hypoblast of theembryonic shield and Squint is expressed in the dorsal forerunner cells during earlygastrulation During late gastrulation Southpaw induces the expression of Cyclopsin the left lateral plate mesoderm and the expression of Nodal inhibitors on theright side of the embryo (eg Lefty) In sea urchins Nodal is first expressed in the pre-sumptive ectoderm prior to gastrulation where it helps to establish the oralndashaboral
mbryo through interactions with Cryptic co-receptors in the lateral plate meso-erm Meanwhile a cilia leftward flow and LeftyCER1 inhibitors prevent Nodalrom signalling on the right side of the embryo
nd therefore do not gastrulate Cells within the epiblast proliferatend degenerate in these embryos rather than ingressing throughhe primitive streak and therefore gastrulation cannot occurConlon et al 1994 Zhou et al 1993) Nodal hypomorphs havelso been developed via insertion of a neomycin cassette Theseutants are viable however phenotypic analyses of these animals
re as of yet incomplete Given that the Nodal null mutant is embry-nic lethal attempts have been made to knock out other membersf the Nodal signalling pathway in murine embryos In most caseshese embryos recapitulate the effects observed in Nodal mutant
axis and is abruptly downregulated once gastrulation begins Following gastrula-tion Nodal is asymmetrically expressed on the right to restrict the development ofthe rudiment to the left side
embryos For example homozygous ActR1Bminusminus ActRIIBminusminus andSMAD2minusminus mice are embryonic lethal due to impaired embryonicpatterning and a subsequent inability to gastrulate (Brennan et al2001 Goumans and Mummery 2000 Nomura and Li 1998) Het-erozygote mutants are less consistent ActR1Bminus+ heterozygotes areviable and grossly normal whilst SMAD2minus+ heterozygotes exhibitgastrulation defects and usually die Interestingly some SMAD2minus+
embryos do survive however they have severe craniofacial defectswhich is an indication of deregulated neural crest delaminationfrom the dorsal neural tube (Thiery et al 2009)
32 Role of Nodal signalling in the development of non-murineorganisms
In addition to mice and humans Nodal is expressed in all othervertebrates as well as some invertebrates (including sea urchinsascidians and snails) where it appears to predominantly regulatechirality (Duboc and Lepage 2008 Grande and Patel 2009a 2009bSchier 2009) Generally whilst Nodal specifies mesendoderm invertebrates its expression is limited to ectoderm in invertebrateorganisms In all species Nodal proteins are expressed asymmetri-cally throughout the embryo where they play an important role inspecifying the left and right sides (Duboc et al 2005 Mohapatraet al 2009)
There are three Nodal-related proteins in zebrafish calledSquint Cyclops and Southpaw (Feldman et al 1998 Long et al2003 Schier 2005) During gastrulation Cyclops is expressed inthe hypoblast of the embryonic shield and Squint is expressed inthe dorsal forerunner cells of the developing zebrafish (Fig 5) Bothof these Nodal-related proteins are important for specificationof endodermal and mesodermal structures during gastrulation(Dougan et al 2003) Single mutant embryos for either Squint orCyclops are generally normal with only mild defects whilst doublemutant embryos for both Squint and Cyclops lack all endodermand most mesoderm tissue (Dougan et al 2003) This suggeststhat although Squint and Cyclops have distinct roles under normal
circumstances their capacity for redundancy ensures that propergastrulation occurs Following gastrulation Southpaw induces theexpression of Cyclops in the left lateral plate mesoderm and theexpression of Nodal inhibitors on the right side of the embryo
f Bioc
(2odeod
itisr(2futeterocrcuiTb(Xg
aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
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92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
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4
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4
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4
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so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
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5
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5
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5
Nia
94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
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DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
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A
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B
B
B
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B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
dkins HB Bianco C Schiffer SG Rayhorn P Zafari M Cheung AE et al Antibodyblockade of the Cripto CFC domain suppresses tumor cell growth in vivo Journalof Clinical Investigation 2003112575ndash87
l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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H
I
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L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
886 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
41 Nodal signalling in the placenta 89142 Nodal signalling in the mammary gland 89243 Nodal signalling in the cycling endometrium 89344 Nodal expression in human adult stem cells 89345 Nodal mutations in human disease 893
5 Nodal signalling in cancer 89351 Melanoma 89352 Brain cancers 89453 Prostate and testicular cancers 89454 Endometrial cancer 89455 Ovarian cancer 89456 Hepatocarcinoma 89457 Pancreatic cancer 89458 Breast cancer 894
6 Regulation of Nodal expression 89561 Silencing of Nodal expression in development 89562 Re-expression of Nodal in cancer 895
7 Conclusions 895Conflict of interest statement 896Acknowledgements 896References 896
1
ttbacmccmesGT
FdswdbegictiN
Introduction
Embryonic development is characterized by the spatial andemporal differentiation of stem cells leading to the genera-ion of tissue diversity This process of differentiation is dictatedy microenvironmental mediators such as morphogen gradientsnd oxygen availability (Quail et al 2012a) The instructiveues of the embryonic programme culminate in an organismade of tissues and cell types that maintain homeostatic balance
oncomitant with exquisite structurendashfunction relationships Inancer the embryonic programme is disrupted resulting in theanifestation of stem cell-like characteristics (Fig 1) (Hendrix
t al 2007) Alternatively some cancers may arise from residenttem cell populations (Chen et al 2012 Driessens et al 2012ilbertson and Graham 2012 Visvader and Lindeman 2006)his stem-cell-like nature of cancer is correlated with metastatic
ig 1 Nodal signalling is a developmental phenomenon that is ldquore-awakenedrdquouring cancer progression tumour progression is characterized by a loss of tis-ue structure and by the acquisition of a more stem cell-like phenotype In manyays this process represents an ldquoundoingrdquo of the differentiation that occurs duringevelopment and mimics certain aspects of induced pluripotency The mechanismy which cancer cells aberrantly acquire the expression of Nodal likely involvespigenetic alterations facilitated by microenvironmental mediators such as oxy-en availability Normal embryonic stem cells maintain a balance of activators andnhibitors of self-renewal in order to facilitate differentiation in response to specificues Here we highlight Nodal and its inhibitor Lefty In contrast cancer cells hijackhese elegant signalling pathways in a manner that favours uncontrolled growthn the absence of normal differentiation In this example the cancer cells expressodal in the absence of Lefty
progression resistance to therapy and a poor clinical progno-sis Hence understanding and targeting molecules that sustainstem cell-like phenotypes could be of tremendous therapeuticvalue
A number of proteins have been shown to regulate both normaland neoplastic stem cell populations One example is Nodal Nodalis a member of the transforming growth factor- (TGF-) super-family which includes TGF-s activins growthdifferentiationfactors (GDFs) and bone morphogenic proteins (BMPs) (Quailet al 2012a Schier 2009) Originally discovered in mouse Nodalhomologues have been characterized in humans Xenopus (frogs)and zebrafish amongst other model organisms (Schier 2009)As is the case for mouse Nodal there is currently only oneannotated isoform for human Nodal mRNA in the National Cen-tre for Biotechnology Information (NCBI) Entrez Gene database(httpwwwncbinlmnihgov80sitesentrez) corresponding toone Nodal ligand Human Nodal is found on chromosome 10 andconsists of three protein-coding exon regions that are translatedinto a 347 amino acid pro-protein Nodal is further processed intoa mature form (amino acids 238ndash347) with a C-terminus thatmight be important for interactions with other proteins such asitrsquos co-receptor Cripto (Schier 2009) Unlike mammals zebrafishhave three Nodal orthologues (cyclops squint and southpaw)whilst frogs have six ldquoXenopus Nodal-relatedrdquo members (Xnr1-6)although only 5 of these (Xnr124ndash6) have canonical Nodal func-tions (Schier 2009) Regardless of the species in chordates Nodalproteins are essential for the induction of mesoderm and endo-derm and leftndashright patterning during embryogenesis (Hendrixet al 2007 Schier 2009 Shen 2007)
Nodal is relatively restricted to embryonic and reproductive celltypes and is not detectable in most normal adult tissues (Hendrixet al 2007 Hooijkaas et al 2011) However recent studies havedemonstrated that Nodal expression re-emerges during cancerprogression (Lawrence et al 2011 Lee et al 2010 Lonardo et al2011 Strizzi et al 2012 Topczewska et al 2006) Moreoverin almost every cancer studied thus far the acquisition of Nodalexpression is associated with increased tumourigenesis invasionand metastasis (Table 1) (De et al 2012 Lawrence et al 2011Lee et al 2010 Lonardo et al 2011 Papageorgiou et al 2009
Quail et al 2012a 2012b in press Strizzi et al 2012 Topczewskaet al 2006) As more cancers expressing Nodal are added to thelist it is becoming increasingly important that we understandthe mechanisms by which this morphogen is regulated Here we
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 887
Table 1The pro- and anti-tumourigenic roles of Nodal in different types of cancer
Phenotype Pro-tumourigenic Anti-tumourigenic
Cancer types References Cancer types References
Invasion and migration Breast cancer glioma melanoma prostate cancerpancreatic cancer
Quail et al (2011 in press) Lee et al(2010) Postovit et al (2007b)Topczewska et al (2006) Lawrenceet al (2011) and Lonardo et al (2011)
Angiogenesis andvascular phenotypes
Melanoma breast cancer GBM Hardy et al (2010) McAllister et al(2010) Quail et al (2012c) and Huenget al (2011)
Proliferation andapoptosis
GBM endometrial cancer hepatocellular carcinomabreast cancer
De et al (2012) Papageorgiou et al(2009) Cavallari et al (2012) andQuail et al (2012b)
Ovarian cancerbreast cancerchoriocarcinoma
Xu et al (2004)Zhong et al (2009)Munir et al (2004)Fu and Peng (2011)
Stem cell phenotypes Self-renewal of GBM anchorage-independent growthof prostate cancer pancreatic spheroids and CSCsplasticity in breast cancer
De et al (2012) Lawrence et al (2011)Lonardo et al (2011) and Meyer et al(2009)
EMT Breast cancer choriocarcinoma Quail et al (in press)
Metastasis andtumourigenesisin vivo
Melanoma GBM prostate cancer hepatocellularcarcinoma pancreatic cancer breast cancer
Postovit et al (2007b) Topczewskaet al (2006) Lee et al (2010) Adkinset al (2003) Cavallari et al (2012)Lonardo et al (2011) and Quail et al(2012b in press)
Patient correlations Invasion and advanced stage in melanoma and breast Topczewska et al (2006) Hooijkaaset aet a(20
ra
2
2
cpc4wbIc(wtasNffiF2naaadA
namp
cancer MVD in breast cancer and glioma tumourgrade in GBM endometrial cancer and breast cancerlymph node status in breast cancer
eview the function and regulation of Nodal signalling in normalnd pathological conditions
Nodal signalling
1 Canonical SMAD-associated signalling
Like most TGF- family members Nodal is secreted from theell as a precursor (Fig 2) In order to form an active ligand therecursor protein must dimerize and the pro-domain must beleaved by substilisin-like pro-protein convertases such as Pace-
and Furin This event can occur in the extracellular space orithin the cell (Schier 2009) The active homodimer signals by
inding to activin-like kinase receptors type I (ALK47) and typeI (ActRIIB) Upon activating this receptor complex (ALK receptoromplex) mothers against decapentaplegicdrosophila homolog 2SMAD2) and possibly SMAD3 is phosphorylated and interactsith SMAD4 before translocating to the nucleus (Schier 2003) In
he nucleus transcription factors such as forkhead box HI (FOXH1)nd Mixer work with the activated SMADs to increase the expres-ion of Nodal responsive genes such as Goosecoid (Gsc) and Leftyodal also induces its own expression thereby creating a positive
eedback loop (Schier 2009 Topczewska et al 2006) The speci-city of Nodal is established by the epidermal growth factor-criptoRL1 cryptic (EGF-CFC) family co-receptor Cripto-1 (Strizzi et al008 Topczewska et al 2006) Cripto-1 has an N-terminal sig-al peptide an EGF-like domain that directly interacts with Nodal
conserved cysteine-rich (CFC) domain that interacts with ALK4nd a hydrophobic C-terminal glycosyl-phosphatidylinositol (GPI)nchor (Minchiotti 2005) The adjacent positioning of the EGF-likeomain and the CFC domain helps bring Nodal into proximity withLK4 to facilitate enhanced binding (Minchiotti 2005)
Endocytosis is an important early step in Nodal-mediated sig-
al transduction (Blanchet et al 2008a 2008b) Specifically Nodalnd its co-receptor Cripto enter cells via a lipid-raft-associatedechanism (Blanchet et al 2008a) This NodalndashCripto com-
lex is subsequently sorted to the limiting membrane of early
l (2011) Lee et al (2010) Quaill (2012a) Papageorgiou et al09) and Strizzi et al (2012)
endosomes marked by the small GTPases Rab4 and Rab5 (Blanchetet al 2008b) These early endosomes also contain the ALK4receptor complex and the SMAD Anchor for Receptor Activation(SARA) a scaffold protein that couples the ALK receptors to effec-tor SMADs (Blanchet et al 2008b) Hence endosomes may act assignalling platforms where Nodal-activated receptors can phos-phorylate SMAD23
Nodal signalling is inhibited spatially and temporally dur-ing development by inhibitors such as Lefty A Lefty B andCerberus (Constam 2009b Schier 2009) These inhibitors aretranscribed in response to Nodal signalling and act as a nega-tive feedback mechanism to control Nodal localization and actionin the developing embryo (Schier 2003) Lefty in particular isalso regulated by alternate SMAD pathways canonical Wnt andOct34 and is up-regulated with differentiation (Tabibzadeh andHemmati-Brivanlou 2006) Lefty inhibits Nodal signalling throughinteractions with Nodal andor Cripto-1 that prevent activation ofthe ALK receptor complex (Schier 2003) Notably Nodal is uniqueamongst TGF- ligands in its requirement for the co-receptorCripto to signal effectively This dependence is also responsible forthe ability of Lefty proteins to inhibit Nodal in near exclusivityIndeed other TGF- members such as Activin signal in a Cripto-independent fashion and thus are not susceptible to inhibition byLefty (Cheng et al 2004) Interestingly whilst both Nodal and Leftyare expressed in most embryonic and adult stem cell contexts (asdescribed below) Lefty negative feedback is not present in cancercells (Hendrix et al 2007 Postovit et al 2008 Quail et al 2012a)
22 Non-canonical (non-SMAD) Nodal signalling
Non-SMAD pathway activation by Nodal is poorly understoodOne important exception is the requirement for p38 activationduring Nodal-induced anterior visceral endoderm (AVE) speci-
fication (Clements et al 2011) Activated p38 signalling in theAVE in turn amplifies Nodal signalling during anteriorndashposterior(AP) axis formation through phosphorylation of the SMAD2 linkerregion (which is receptive to phosphorylation by mitogen activated
888 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 2 The Nodal signalling pathway and mechanisms of regulation Nodal signals through activation of its receptor complex (ALK47 ActRIIB Cripto) and subsequentphosphorylation of SMAD23 SMAD23 forms a complex with SMAD4 and translocates to the nucleus to regulate target genes including Nodal (for autoregulation) andits endongenous inhibitor Lefty (for negative feedback) As embryogenesis proceeds Nodal signalling eventually activates differentiation programmes before it becomestranscriptionally silenced Nodal initiates differentiation via association of SMAD23 with TRIM33 and subsequent activation of poised chromatin in promoter regions ofgenes associated with mesendoderm specification Later Nodal is silenced possibly through polycomb-mediated trimethylation of histone H3 at Lysine 27 (H3K27me3) Thism ates thm amonga
pSsmtoIprpcNt
awptAcotNwoir
piNnaNe
ethylation signature is reversible as re-activated Nodal-SMAD23 signalling facilitethylations and re-activate transcription of Nodal This may be one mechanism (
berrant contexts
rotein kinase (MAPK) proteins) and subsequent enhancement ofMAD2 activity (Clements et al 2011) In keratinocytes of thekin SMAD and Extracellular Regulated Kinase (ERK) signalling areutually required for TGF--induced epithelial-to-mesenchymal-
ransition (EMT) as ERK substrates mediate nuclear translocationf SMADs and alter target gene expression (Davies et al 2005)n cancer non-SMAD pathway activation by other TGF--familyroteins has been characterized For instance the TGF- type 1eceptor is capable of activating MAPK signalling through phos-horylation of ShcA and subsequent interaction with the GRB2SOSomplex (Lee et al 2007) Given the similarities between TGF- andodal these studies lend insight into putative non-SMAD Nodal
argets during disease progressionWe recently determined that Nodal promotes cellular invasion
nd EMT-like phenomena via activation of the ERK12 MAPK path-ay in breast cancer and choriocarcinoma cells (Quail et al inress) Specifically we found that Nodal induces ERK12 activa-ion and that this ability of Nodal to activate ERK12 is mediated byLK47 activity Our results indicate there may be multiple points ofrosstalk between the SMAD23 and ERK12 pathways at the levelf receptor activation intracellular signalling and gene transcrip-ion (Fig 3) Importantly Nodal-induced ERK activation mediatesodal-induced EMT and invasion such that inhibition of this path-ay with U0126 (a MEK12 inhibitor) abrogates the effects of Nodal
n these phenotypes (Quail et al in press) Together these find-ngs indicate that putative Nodal-targeted therapies would likelyequire interference of both SMAD and ERK pathways
As described below Nodal plays paradoxical roles in humanluripotent stem cells maintaining pluripotency whilst initiat-
ng mesendoderm induction Such disparate functions suggest thatodal likely regulates chromatin accessibility by altering epige-
etic phenomena such as DNA or histone methylation Indeed
recent study by Massagueacute and colleagues (2011) showed thatodal signalling is capable of inducing the expression of differ-ntiation signals in human embryonic stem cells by activating
e recruitment of the demethylase Jmjd3 to the Nodal locus to lift polycomb-inducedst others such as hypoxia-induced HIF signalling) of how Nodal is re-activated in
ldquopoisedrdquo chromatin (Xi et al 2011) Poised chromatin markedby polycomb-mediated trimethylation of histone H3 at lysine 9(H3K9me3) and acetylation of lysine 18 (H3K18ac) ensures trans-criptional repression of embryonic genes however these genesare responsive to acute activation by developmental signals inthe environment Chromatin assumes a poised state in responseto pluripotency factors such as Oct4 and Nanog to ensure thetimely activation of differentiation cues during development In thisstudy NodalActivin signalling through phosphorylated SMAD23caused recruitment of TRIM33 to H3K9me3-K18ac at the promo-ters of genes associated with mesendoderm specification (such asNodal targets MixI1 and Gsc) Association of SMAD23 and TRIM33in these regions lifted methylations to activate poised chromatinrendering promoter regions accessible to SMAD23ndashSMAD4 trans-criptional complexes (Fig 2) This study is the first to propose amechanism of how Nodal mediates widespread differentiation cuesduring embryogenesis and lends insight into how other mediatorsof embryonic differentiation may work in a similar fashion
3 Nodal signalling in developmental models
31 Role of Nodal in mouse development
The first gradient that Nodal establishes during development isthe proximalndashdistal (PD) axis which later rotates to become the APaxis Nodal is activated through the developing epiblast by conver-tase enzymes that are secreted from the extraembryonic ectoderm(Beck et al 2002 Schier 2009) These convertases including Pace4and Furin cleave the pro-domain of Nodal to activate its signallingin the proximal epiblast adjacent to the extraembryonic ectoderm(Beck et al 2002 Schier 2003) Here Nodal is maintained and
concentrated either through autoregulation andor by activatingBMP in the extraembryonic ectoderm which signals back to theadjacent epiblast to induce Wnt (Vincent et al 2003) The Nodalgene contains a proximal epiblast enhancer (PEE) upstream of
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 889
Fig 3 Putative crosstalk between ERK and SMAD pathways during Nodal signalling and regulation of E-cadherin trafficking Studies from our laboratory and others suggestthere may be multiple points of crosstalk between the SMAD23 and ERK12 pathways We have recently shown that recombinant Nodal induces phosphorylation of ERK12This may be indirect through receptor phosphorylation of an upstream mediator of the ERK cascade (such as ShcA) or direct via receptor phosphorylation of ERK12 We havealso shown that MEK inhibition leads to a decrease in Nodal-induced phosphorylation of SMAD2 This may be due to altered phosphorylation of the SMAD23 linker regionby ERK MAPKs We have shown that MEK inhibition reduces transcription of Nodal-induced EMT transcription factors ERK may alter SMAD-mediated gene expression byregulating translocation of SMAD proteins to the nucleus or by interfering with gene transcription We also found that MEK inhibition rescued Nodal-induced mislocalizationo n thatp
ttbiivastedpShC
itmpiseNa(
left
f E-Cadherin In accordance with these observations previous reports have showlasma membrane after internalization during EMT
he transcription start site that is Wnt-responsive Activation ofhis enhancer by Wnt concentrates Nodal to the proximal epi-last (Fig 4A) (Brennan et al 2001) Activated Nodal signalling
n the proximal epiblast subsequently induces gene expression ofts endogenous inhibitors Lefty1 and Cerberus (CER1) in the distalisceral endoderm (DVE) to establish a Nodal PD gradient Lefty1nd CER1 subsequently restrict Nodal expression to the proximalide of the embryo where the primitive streak will form to ini-iate gastrulation The PD axis graded by Nodal expression willventually rotate to become the AP axis whereby the DVE will beisplaced to yield the AVE and the proximal epiblast will moveosteriorly (Fig 4B) (Collignon et al 1996 Constam 2009a 2009btrizzi et al 2008 Zhou et al 1993) It is not entirely understoodow this rotation is regulated however it has been shown to beripto-dependent (Ding et al 1998)
Following AP axis formation gastrulation occurs as the prim-tive streak elongates from the posterior to the anterior side ofhe embryo This process is orchestrated by ingression of proxi-
al posterior cells to form mesendoderm cells which elongate therimitive streak towards the anterior side of the embryo Nodal
s expressed in the primitive endoderm overlying the primitivetreak and is thought to co-ordinate its directional migration andlongation (Conlon et al 1994) Once gastrulation is completeodal expression becomes restricted to the periphery of the nodet the anterior end of the primitive streak hence the name ldquoNodalrdquoSchier 2003)
Following gastrulation the node is the central initiator of
eftndashright (LR) asymmetry (Schier 2003 Smith 1995 Takaokat al 2006 Zhou et al 1993) TGF--family members secretedrom the ventral node including Nodal and growth differen-iation factor 1 (GDF1) define the left side of the embryo
inhibition of the ERK pathway is required for relocalization of E-Cadherin to the
through interactions with mouse Cripto family member Cryp-tic co-receptors in the lateral plate mesoderm (Constam 2009a)Meanwhile a leftward flow induced by active cilia prevents Nodalfrom signalling on the right side of the embryo in conjunc-tion with the right side-specific expression of CER1 and Lefty1(Fig 4C) (Constam 2009a) Of note this is how Nodalrsquos endogenousinhibitor Lefty received its name It has been shown that murineembryos lacking node-specific Nodal expression are not capableof establishing LR asymmetry and exhibit asymmetric patterningdefects including random positioning of the stomach and abnormalbranching of lung lobes (Brennan et al 2002) Furthermore thesestudies have shown that short-range Nodal signalling may also playa role in establishing a midline between the left and right sides ofthe embryo (Brennan et al 2002)
Following axis patterning of the embryo somitogenesis occurswhereby cells condense into transient epithelial structures byundergoing mesenchymal-to-epithelial transition At the begin-ning of this process Nodal is restricted to mesoderm cells on the leftside of the embryo (Collignon et al 1996) Interestingly as Nodal isa critical regulator of gastrulation (one of the first major EMT eventsto occur during development) somitogenesis is accompanied byan abrupt silencing of Nodal signalling by 8 days postcoitum (dpc)(Collignon et al 1996) Until recently it was not completely under-stood how Nodal silencing during development occurred Howevernew investigations suggest that Nodal is silenced by polycomb-mediated methylation of histones in a reversible manner (Dahleet al 2010 Xi et al 2011) (Fig 2)
Several studies using genetically modified mice have been per-formed to assess the role of different aspects of the Nodal signallingpathway during development Murine Nodal null mutants areembryonic lethal as they are unable to initiate the primitive streak
890 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 4 Nodal morphogens pattern the murine embryo to establish multiple criticalaxes (A) Nodal is activated in the epiblast by convertase enzymes (ex Pace4 andFurin) secreted from the extraembryonic ectoderm (ExEct) Convertases cleave andactivate Nodal in the proximal epiblast where Nodal is maintained by autoregula-tion andor by activation of a BMPWnt signalling loop The Nodal gene containsa proximal epiblast enhancer (PEE) upstream of the transcription start site thatis Wnt-responsive activation of the PEE by Wnt concentrates Nodal to the prox-imal epiblast (B) Nodal subsequently induces gene expression of its endogenousinhibitors Lefty1 and CER1 in the distal visceral endoderm (DVE) to establish aproximal-distal (PD) gradient The PD axis graded by Nodal expression eventuallyrotates to become the anterior-posterior (AP) axis whereby the DVE is displacedto yield the anterior visceral endoderm (AVE) and the proximal epiblast movesposteriorly (C) During gastrulation Nodal is expressed in the primitive endodermoverlying the primitive streak and then becomes restricted to the node to initiateleftndashright (LR) asymmetry GDF1 and Nodal from the node define the left side of theedf
aat(amaoot
Fig 5 Comparison of Nodal expression patterns in early versus late gastrulationin mouse frog zebrafish and sea urchin development In mice Nodal is expressedin the node during early gastrulation and in the left lateral plate mesoderm dur-ing specification of LR asymmetry In the frog Xnr proteins are critical initiators ofgastrulation in the dorsal lip and become restricted to the lateral plate mesodermlater during neurulation In zebrafish Cyclops is expressed in the hypoblast of theembryonic shield and Squint is expressed in the dorsal forerunner cells during earlygastrulation During late gastrulation Southpaw induces the expression of Cyclopsin the left lateral plate mesoderm and the expression of Nodal inhibitors on theright side of the embryo (eg Lefty) In sea urchins Nodal is first expressed in the pre-sumptive ectoderm prior to gastrulation where it helps to establish the oralndashaboral
mbryo through interactions with Cryptic co-receptors in the lateral plate meso-erm Meanwhile a cilia leftward flow and LeftyCER1 inhibitors prevent Nodalrom signalling on the right side of the embryo
nd therefore do not gastrulate Cells within the epiblast proliferatend degenerate in these embryos rather than ingressing throughhe primitive streak and therefore gastrulation cannot occurConlon et al 1994 Zhou et al 1993) Nodal hypomorphs havelso been developed via insertion of a neomycin cassette Theseutants are viable however phenotypic analyses of these animals
re as of yet incomplete Given that the Nodal null mutant is embry-nic lethal attempts have been made to knock out other membersf the Nodal signalling pathway in murine embryos In most caseshese embryos recapitulate the effects observed in Nodal mutant
axis and is abruptly downregulated once gastrulation begins Following gastrula-tion Nodal is asymmetrically expressed on the right to restrict the development ofthe rudiment to the left side
embryos For example homozygous ActR1Bminusminus ActRIIBminusminus andSMAD2minusminus mice are embryonic lethal due to impaired embryonicpatterning and a subsequent inability to gastrulate (Brennan et al2001 Goumans and Mummery 2000 Nomura and Li 1998) Het-erozygote mutants are less consistent ActR1Bminus+ heterozygotes areviable and grossly normal whilst SMAD2minus+ heterozygotes exhibitgastrulation defects and usually die Interestingly some SMAD2minus+
embryos do survive however they have severe craniofacial defectswhich is an indication of deregulated neural crest delaminationfrom the dorsal neural tube (Thiery et al 2009)
32 Role of Nodal signalling in the development of non-murineorganisms
In addition to mice and humans Nodal is expressed in all othervertebrates as well as some invertebrates (including sea urchinsascidians and snails) where it appears to predominantly regulatechirality (Duboc and Lepage 2008 Grande and Patel 2009a 2009bSchier 2009) Generally whilst Nodal specifies mesendoderm invertebrates its expression is limited to ectoderm in invertebrateorganisms In all species Nodal proteins are expressed asymmetri-cally throughout the embryo where they play an important role inspecifying the left and right sides (Duboc et al 2005 Mohapatraet al 2009)
There are three Nodal-related proteins in zebrafish calledSquint Cyclops and Southpaw (Feldman et al 1998 Long et al2003 Schier 2005) During gastrulation Cyclops is expressed inthe hypoblast of the embryonic shield and Squint is expressed inthe dorsal forerunner cells of the developing zebrafish (Fig 5) Bothof these Nodal-related proteins are important for specificationof endodermal and mesodermal structures during gastrulation(Dougan et al 2003) Single mutant embryos for either Squint orCyclops are generally normal with only mild defects whilst doublemutant embryos for both Squint and Cyclops lack all endodermand most mesoderm tissue (Dougan et al 2003) This suggeststhat although Squint and Cyclops have distinct roles under normal
circumstances their capacity for redundancy ensures that propergastrulation occurs Following gastrulation Southpaw induces theexpression of Cyclops in the left lateral plate mesoderm and theexpression of Nodal inhibitors on the right side of the embryo
f Bioc
(2odeod
itisr(2futeterocrcuiTb(Xg
aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
rfft
ctiastNcopMaglltldbSvdpa(d
ppNppE2aaSdIcN(htAlcsoelsleApfiii2l
92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
es
4
weptsihietd2dse
4
oipeI(ooiemat
4
caab(Ldhobmbdsctls
5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
betiIaie2kvpsAHecatgh
5
tCtcCnipcegaN
5
cgarrceimcaero
5
Nia
94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
dkins HB Bianco C Schiffer SG Rayhorn P Zafari M Cheung AE et al Antibodyblockade of the Cripto CFC domain suppresses tumor cell growth in vivo Journalof Clinical Investigation 2003112575ndash87
l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
H
H
H
I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
T
T
T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 887
Table 1The pro- and anti-tumourigenic roles of Nodal in different types of cancer
Phenotype Pro-tumourigenic Anti-tumourigenic
Cancer types References Cancer types References
Invasion and migration Breast cancer glioma melanoma prostate cancerpancreatic cancer
Quail et al (2011 in press) Lee et al(2010) Postovit et al (2007b)Topczewska et al (2006) Lawrenceet al (2011) and Lonardo et al (2011)
Angiogenesis andvascular phenotypes
Melanoma breast cancer GBM Hardy et al (2010) McAllister et al(2010) Quail et al (2012c) and Huenget al (2011)
Proliferation andapoptosis
GBM endometrial cancer hepatocellular carcinomabreast cancer
De et al (2012) Papageorgiou et al(2009) Cavallari et al (2012) andQuail et al (2012b)
Ovarian cancerbreast cancerchoriocarcinoma
Xu et al (2004)Zhong et al (2009)Munir et al (2004)Fu and Peng (2011)
Stem cell phenotypes Self-renewal of GBM anchorage-independent growthof prostate cancer pancreatic spheroids and CSCsplasticity in breast cancer
De et al (2012) Lawrence et al (2011)Lonardo et al (2011) and Meyer et al(2009)
EMT Breast cancer choriocarcinoma Quail et al (in press)
Metastasis andtumourigenesisin vivo
Melanoma GBM prostate cancer hepatocellularcarcinoma pancreatic cancer breast cancer
Postovit et al (2007b) Topczewskaet al (2006) Lee et al (2010) Adkinset al (2003) Cavallari et al (2012)Lonardo et al (2011) and Quail et al(2012b in press)
Patient correlations Invasion and advanced stage in melanoma and breast Topczewska et al (2006) Hooijkaaset aet a(20
ra
2
2
cpc4wbIc(wtasNffiF2naaadA
namp
cancer MVD in breast cancer and glioma tumourgrade in GBM endometrial cancer and breast cancerlymph node status in breast cancer
eview the function and regulation of Nodal signalling in normalnd pathological conditions
Nodal signalling
1 Canonical SMAD-associated signalling
Like most TGF- family members Nodal is secreted from theell as a precursor (Fig 2) In order to form an active ligand therecursor protein must dimerize and the pro-domain must beleaved by substilisin-like pro-protein convertases such as Pace-
and Furin This event can occur in the extracellular space orithin the cell (Schier 2009) The active homodimer signals by
inding to activin-like kinase receptors type I (ALK47) and typeI (ActRIIB) Upon activating this receptor complex (ALK receptoromplex) mothers against decapentaplegicdrosophila homolog 2SMAD2) and possibly SMAD3 is phosphorylated and interactsith SMAD4 before translocating to the nucleus (Schier 2003) In
he nucleus transcription factors such as forkhead box HI (FOXH1)nd Mixer work with the activated SMADs to increase the expres-ion of Nodal responsive genes such as Goosecoid (Gsc) and Leftyodal also induces its own expression thereby creating a positive
eedback loop (Schier 2009 Topczewska et al 2006) The speci-city of Nodal is established by the epidermal growth factor-criptoRL1 cryptic (EGF-CFC) family co-receptor Cripto-1 (Strizzi et al008 Topczewska et al 2006) Cripto-1 has an N-terminal sig-al peptide an EGF-like domain that directly interacts with Nodal
conserved cysteine-rich (CFC) domain that interacts with ALK4nd a hydrophobic C-terminal glycosyl-phosphatidylinositol (GPI)nchor (Minchiotti 2005) The adjacent positioning of the EGF-likeomain and the CFC domain helps bring Nodal into proximity withLK4 to facilitate enhanced binding (Minchiotti 2005)
Endocytosis is an important early step in Nodal-mediated sig-
al transduction (Blanchet et al 2008a 2008b) Specifically Nodalnd its co-receptor Cripto enter cells via a lipid-raft-associatedechanism (Blanchet et al 2008a) This NodalndashCripto com-
lex is subsequently sorted to the limiting membrane of early
l (2011) Lee et al (2010) Quaill (2012a) Papageorgiou et al09) and Strizzi et al (2012)
endosomes marked by the small GTPases Rab4 and Rab5 (Blanchetet al 2008b) These early endosomes also contain the ALK4receptor complex and the SMAD Anchor for Receptor Activation(SARA) a scaffold protein that couples the ALK receptors to effec-tor SMADs (Blanchet et al 2008b) Hence endosomes may act assignalling platforms where Nodal-activated receptors can phos-phorylate SMAD23
Nodal signalling is inhibited spatially and temporally dur-ing development by inhibitors such as Lefty A Lefty B andCerberus (Constam 2009b Schier 2009) These inhibitors aretranscribed in response to Nodal signalling and act as a nega-tive feedback mechanism to control Nodal localization and actionin the developing embryo (Schier 2003) Lefty in particular isalso regulated by alternate SMAD pathways canonical Wnt andOct34 and is up-regulated with differentiation (Tabibzadeh andHemmati-Brivanlou 2006) Lefty inhibits Nodal signalling throughinteractions with Nodal andor Cripto-1 that prevent activation ofthe ALK receptor complex (Schier 2003) Notably Nodal is uniqueamongst TGF- ligands in its requirement for the co-receptorCripto to signal effectively This dependence is also responsible forthe ability of Lefty proteins to inhibit Nodal in near exclusivityIndeed other TGF- members such as Activin signal in a Cripto-independent fashion and thus are not susceptible to inhibition byLefty (Cheng et al 2004) Interestingly whilst both Nodal and Leftyare expressed in most embryonic and adult stem cell contexts (asdescribed below) Lefty negative feedback is not present in cancercells (Hendrix et al 2007 Postovit et al 2008 Quail et al 2012a)
22 Non-canonical (non-SMAD) Nodal signalling
Non-SMAD pathway activation by Nodal is poorly understoodOne important exception is the requirement for p38 activationduring Nodal-induced anterior visceral endoderm (AVE) speci-
fication (Clements et al 2011) Activated p38 signalling in theAVE in turn amplifies Nodal signalling during anteriorndashposterior(AP) axis formation through phosphorylation of the SMAD2 linkerregion (which is receptive to phosphorylation by mitogen activated
888 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 2 The Nodal signalling pathway and mechanisms of regulation Nodal signals through activation of its receptor complex (ALK47 ActRIIB Cripto) and subsequentphosphorylation of SMAD23 SMAD23 forms a complex with SMAD4 and translocates to the nucleus to regulate target genes including Nodal (for autoregulation) andits endongenous inhibitor Lefty (for negative feedback) As embryogenesis proceeds Nodal signalling eventually activates differentiation programmes before it becomestranscriptionally silenced Nodal initiates differentiation via association of SMAD23 with TRIM33 and subsequent activation of poised chromatin in promoter regions ofgenes associated with mesendoderm specification Later Nodal is silenced possibly through polycomb-mediated trimethylation of histone H3 at Lysine 27 (H3K27me3) Thism ates thm amonga
pSsmtoIprpcNt
awptAcotNwoir
piNnaNe
ethylation signature is reversible as re-activated Nodal-SMAD23 signalling facilitethylations and re-activate transcription of Nodal This may be one mechanism (
berrant contexts
rotein kinase (MAPK) proteins) and subsequent enhancement ofMAD2 activity (Clements et al 2011) In keratinocytes of thekin SMAD and Extracellular Regulated Kinase (ERK) signalling areutually required for TGF--induced epithelial-to-mesenchymal-
ransition (EMT) as ERK substrates mediate nuclear translocationf SMADs and alter target gene expression (Davies et al 2005)n cancer non-SMAD pathway activation by other TGF--familyroteins has been characterized For instance the TGF- type 1eceptor is capable of activating MAPK signalling through phos-horylation of ShcA and subsequent interaction with the GRB2SOSomplex (Lee et al 2007) Given the similarities between TGF- andodal these studies lend insight into putative non-SMAD Nodal
argets during disease progressionWe recently determined that Nodal promotes cellular invasion
nd EMT-like phenomena via activation of the ERK12 MAPK path-ay in breast cancer and choriocarcinoma cells (Quail et al inress) Specifically we found that Nodal induces ERK12 activa-ion and that this ability of Nodal to activate ERK12 is mediated byLK47 activity Our results indicate there may be multiple points ofrosstalk between the SMAD23 and ERK12 pathways at the levelf receptor activation intracellular signalling and gene transcrip-ion (Fig 3) Importantly Nodal-induced ERK activation mediatesodal-induced EMT and invasion such that inhibition of this path-ay with U0126 (a MEK12 inhibitor) abrogates the effects of Nodal
n these phenotypes (Quail et al in press) Together these find-ngs indicate that putative Nodal-targeted therapies would likelyequire interference of both SMAD and ERK pathways
As described below Nodal plays paradoxical roles in humanluripotent stem cells maintaining pluripotency whilst initiat-
ng mesendoderm induction Such disparate functions suggest thatodal likely regulates chromatin accessibility by altering epige-
etic phenomena such as DNA or histone methylation Indeed
recent study by Massagueacute and colleagues (2011) showed thatodal signalling is capable of inducing the expression of differ-ntiation signals in human embryonic stem cells by activating
e recruitment of the demethylase Jmjd3 to the Nodal locus to lift polycomb-inducedst others such as hypoxia-induced HIF signalling) of how Nodal is re-activated in
ldquopoisedrdquo chromatin (Xi et al 2011) Poised chromatin markedby polycomb-mediated trimethylation of histone H3 at lysine 9(H3K9me3) and acetylation of lysine 18 (H3K18ac) ensures trans-criptional repression of embryonic genes however these genesare responsive to acute activation by developmental signals inthe environment Chromatin assumes a poised state in responseto pluripotency factors such as Oct4 and Nanog to ensure thetimely activation of differentiation cues during development In thisstudy NodalActivin signalling through phosphorylated SMAD23caused recruitment of TRIM33 to H3K9me3-K18ac at the promo-ters of genes associated with mesendoderm specification (such asNodal targets MixI1 and Gsc) Association of SMAD23 and TRIM33in these regions lifted methylations to activate poised chromatinrendering promoter regions accessible to SMAD23ndashSMAD4 trans-criptional complexes (Fig 2) This study is the first to propose amechanism of how Nodal mediates widespread differentiation cuesduring embryogenesis and lends insight into how other mediatorsof embryonic differentiation may work in a similar fashion
3 Nodal signalling in developmental models
31 Role of Nodal in mouse development
The first gradient that Nodal establishes during development isthe proximalndashdistal (PD) axis which later rotates to become the APaxis Nodal is activated through the developing epiblast by conver-tase enzymes that are secreted from the extraembryonic ectoderm(Beck et al 2002 Schier 2009) These convertases including Pace4and Furin cleave the pro-domain of Nodal to activate its signallingin the proximal epiblast adjacent to the extraembryonic ectoderm(Beck et al 2002 Schier 2003) Here Nodal is maintained and
concentrated either through autoregulation andor by activatingBMP in the extraembryonic ectoderm which signals back to theadjacent epiblast to induce Wnt (Vincent et al 2003) The Nodalgene contains a proximal epiblast enhancer (PEE) upstream of
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 889
Fig 3 Putative crosstalk between ERK and SMAD pathways during Nodal signalling and regulation of E-cadherin trafficking Studies from our laboratory and others suggestthere may be multiple points of crosstalk between the SMAD23 and ERK12 pathways We have recently shown that recombinant Nodal induces phosphorylation of ERK12This may be indirect through receptor phosphorylation of an upstream mediator of the ERK cascade (such as ShcA) or direct via receptor phosphorylation of ERK12 We havealso shown that MEK inhibition leads to a decrease in Nodal-induced phosphorylation of SMAD2 This may be due to altered phosphorylation of the SMAD23 linker regionby ERK MAPKs We have shown that MEK inhibition reduces transcription of Nodal-induced EMT transcription factors ERK may alter SMAD-mediated gene expression byregulating translocation of SMAD proteins to the nucleus or by interfering with gene transcription We also found that MEK inhibition rescued Nodal-induced mislocalizationo n thatp
ttbiivastedpShC
itmpiseNa(
left
f E-Cadherin In accordance with these observations previous reports have showlasma membrane after internalization during EMT
he transcription start site that is Wnt-responsive Activation ofhis enhancer by Wnt concentrates Nodal to the proximal epi-last (Fig 4A) (Brennan et al 2001) Activated Nodal signalling
n the proximal epiblast subsequently induces gene expression ofts endogenous inhibitors Lefty1 and Cerberus (CER1) in the distalisceral endoderm (DVE) to establish a Nodal PD gradient Lefty1nd CER1 subsequently restrict Nodal expression to the proximalide of the embryo where the primitive streak will form to ini-iate gastrulation The PD axis graded by Nodal expression willventually rotate to become the AP axis whereby the DVE will beisplaced to yield the AVE and the proximal epiblast will moveosteriorly (Fig 4B) (Collignon et al 1996 Constam 2009a 2009btrizzi et al 2008 Zhou et al 1993) It is not entirely understoodow this rotation is regulated however it has been shown to beripto-dependent (Ding et al 1998)
Following AP axis formation gastrulation occurs as the prim-tive streak elongates from the posterior to the anterior side ofhe embryo This process is orchestrated by ingression of proxi-
al posterior cells to form mesendoderm cells which elongate therimitive streak towards the anterior side of the embryo Nodal
s expressed in the primitive endoderm overlying the primitivetreak and is thought to co-ordinate its directional migration andlongation (Conlon et al 1994) Once gastrulation is completeodal expression becomes restricted to the periphery of the nodet the anterior end of the primitive streak hence the name ldquoNodalrdquoSchier 2003)
Following gastrulation the node is the central initiator of
eftndashright (LR) asymmetry (Schier 2003 Smith 1995 Takaokat al 2006 Zhou et al 1993) TGF--family members secretedrom the ventral node including Nodal and growth differen-iation factor 1 (GDF1) define the left side of the embryo
inhibition of the ERK pathway is required for relocalization of E-Cadherin to the
through interactions with mouse Cripto family member Cryp-tic co-receptors in the lateral plate mesoderm (Constam 2009a)Meanwhile a leftward flow induced by active cilia prevents Nodalfrom signalling on the right side of the embryo in conjunc-tion with the right side-specific expression of CER1 and Lefty1(Fig 4C) (Constam 2009a) Of note this is how Nodalrsquos endogenousinhibitor Lefty received its name It has been shown that murineembryos lacking node-specific Nodal expression are not capableof establishing LR asymmetry and exhibit asymmetric patterningdefects including random positioning of the stomach and abnormalbranching of lung lobes (Brennan et al 2002) Furthermore thesestudies have shown that short-range Nodal signalling may also playa role in establishing a midline between the left and right sides ofthe embryo (Brennan et al 2002)
Following axis patterning of the embryo somitogenesis occurswhereby cells condense into transient epithelial structures byundergoing mesenchymal-to-epithelial transition At the begin-ning of this process Nodal is restricted to mesoderm cells on the leftside of the embryo (Collignon et al 1996) Interestingly as Nodal isa critical regulator of gastrulation (one of the first major EMT eventsto occur during development) somitogenesis is accompanied byan abrupt silencing of Nodal signalling by 8 days postcoitum (dpc)(Collignon et al 1996) Until recently it was not completely under-stood how Nodal silencing during development occurred Howevernew investigations suggest that Nodal is silenced by polycomb-mediated methylation of histones in a reversible manner (Dahleet al 2010 Xi et al 2011) (Fig 2)
Several studies using genetically modified mice have been per-formed to assess the role of different aspects of the Nodal signallingpathway during development Murine Nodal null mutants areembryonic lethal as they are unable to initiate the primitive streak
890 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 4 Nodal morphogens pattern the murine embryo to establish multiple criticalaxes (A) Nodal is activated in the epiblast by convertase enzymes (ex Pace4 andFurin) secreted from the extraembryonic ectoderm (ExEct) Convertases cleave andactivate Nodal in the proximal epiblast where Nodal is maintained by autoregula-tion andor by activation of a BMPWnt signalling loop The Nodal gene containsa proximal epiblast enhancer (PEE) upstream of the transcription start site thatis Wnt-responsive activation of the PEE by Wnt concentrates Nodal to the prox-imal epiblast (B) Nodal subsequently induces gene expression of its endogenousinhibitors Lefty1 and CER1 in the distal visceral endoderm (DVE) to establish aproximal-distal (PD) gradient The PD axis graded by Nodal expression eventuallyrotates to become the anterior-posterior (AP) axis whereby the DVE is displacedto yield the anterior visceral endoderm (AVE) and the proximal epiblast movesposteriorly (C) During gastrulation Nodal is expressed in the primitive endodermoverlying the primitive streak and then becomes restricted to the node to initiateleftndashright (LR) asymmetry GDF1 and Nodal from the node define the left side of theedf
aat(amaoot
Fig 5 Comparison of Nodal expression patterns in early versus late gastrulationin mouse frog zebrafish and sea urchin development In mice Nodal is expressedin the node during early gastrulation and in the left lateral plate mesoderm dur-ing specification of LR asymmetry In the frog Xnr proteins are critical initiators ofgastrulation in the dorsal lip and become restricted to the lateral plate mesodermlater during neurulation In zebrafish Cyclops is expressed in the hypoblast of theembryonic shield and Squint is expressed in the dorsal forerunner cells during earlygastrulation During late gastrulation Southpaw induces the expression of Cyclopsin the left lateral plate mesoderm and the expression of Nodal inhibitors on theright side of the embryo (eg Lefty) In sea urchins Nodal is first expressed in the pre-sumptive ectoderm prior to gastrulation where it helps to establish the oralndashaboral
mbryo through interactions with Cryptic co-receptors in the lateral plate meso-erm Meanwhile a cilia leftward flow and LeftyCER1 inhibitors prevent Nodalrom signalling on the right side of the embryo
nd therefore do not gastrulate Cells within the epiblast proliferatend degenerate in these embryos rather than ingressing throughhe primitive streak and therefore gastrulation cannot occurConlon et al 1994 Zhou et al 1993) Nodal hypomorphs havelso been developed via insertion of a neomycin cassette Theseutants are viable however phenotypic analyses of these animals
re as of yet incomplete Given that the Nodal null mutant is embry-nic lethal attempts have been made to knock out other membersf the Nodal signalling pathway in murine embryos In most caseshese embryos recapitulate the effects observed in Nodal mutant
axis and is abruptly downregulated once gastrulation begins Following gastrula-tion Nodal is asymmetrically expressed on the right to restrict the development ofthe rudiment to the left side
embryos For example homozygous ActR1Bminusminus ActRIIBminusminus andSMAD2minusminus mice are embryonic lethal due to impaired embryonicpatterning and a subsequent inability to gastrulate (Brennan et al2001 Goumans and Mummery 2000 Nomura and Li 1998) Het-erozygote mutants are less consistent ActR1Bminus+ heterozygotes areviable and grossly normal whilst SMAD2minus+ heterozygotes exhibitgastrulation defects and usually die Interestingly some SMAD2minus+
embryos do survive however they have severe craniofacial defectswhich is an indication of deregulated neural crest delaminationfrom the dorsal neural tube (Thiery et al 2009)
32 Role of Nodal signalling in the development of non-murineorganisms
In addition to mice and humans Nodal is expressed in all othervertebrates as well as some invertebrates (including sea urchinsascidians and snails) where it appears to predominantly regulatechirality (Duboc and Lepage 2008 Grande and Patel 2009a 2009bSchier 2009) Generally whilst Nodal specifies mesendoderm invertebrates its expression is limited to ectoderm in invertebrateorganisms In all species Nodal proteins are expressed asymmetri-cally throughout the embryo where they play an important role inspecifying the left and right sides (Duboc et al 2005 Mohapatraet al 2009)
There are three Nodal-related proteins in zebrafish calledSquint Cyclops and Southpaw (Feldman et al 1998 Long et al2003 Schier 2005) During gastrulation Cyclops is expressed inthe hypoblast of the embryonic shield and Squint is expressed inthe dorsal forerunner cells of the developing zebrafish (Fig 5) Bothof these Nodal-related proteins are important for specificationof endodermal and mesodermal structures during gastrulation(Dougan et al 2003) Single mutant embryos for either Squint orCyclops are generally normal with only mild defects whilst doublemutant embryos for both Squint and Cyclops lack all endodermand most mesoderm tissue (Dougan et al 2003) This suggeststhat although Squint and Cyclops have distinct roles under normal
circumstances their capacity for redundancy ensures that propergastrulation occurs Following gastrulation Southpaw induces theexpression of Cyclops in the left lateral plate mesoderm and theexpression of Nodal inhibitors on the right side of the embryo
f Bioc
(2odeod
itisr(2futeterocrcuiTb(Xg
aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
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ctiastNcopMaglltldbSvdpa(d
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92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
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4
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4
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4
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5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
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5
tCtcCnipcegaN
5
cgarrceimcaero
5
Nia
94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
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vibavcrai(v(pc(abt
6
6
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DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
dkins HB Bianco C Schiffer SG Rayhorn P Zafari M Cheung AE et al Antibodyblockade of the Cripto CFC domain suppresses tumor cell growth in vivo Journalof Clinical Investigation 2003112575ndash87
l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
H
H
H
I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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V
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W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
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Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
888 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 2 The Nodal signalling pathway and mechanisms of regulation Nodal signals through activation of its receptor complex (ALK47 ActRIIB Cripto) and subsequentphosphorylation of SMAD23 SMAD23 forms a complex with SMAD4 and translocates to the nucleus to regulate target genes including Nodal (for autoregulation) andits endongenous inhibitor Lefty (for negative feedback) As embryogenesis proceeds Nodal signalling eventually activates differentiation programmes before it becomestranscriptionally silenced Nodal initiates differentiation via association of SMAD23 with TRIM33 and subsequent activation of poised chromatin in promoter regions ofgenes associated with mesendoderm specification Later Nodal is silenced possibly through polycomb-mediated trimethylation of histone H3 at Lysine 27 (H3K27me3) Thism ates thm amonga
pSsmtoIprpcNt
awptAcotNwoir
piNnaNe
ethylation signature is reversible as re-activated Nodal-SMAD23 signalling facilitethylations and re-activate transcription of Nodal This may be one mechanism (
berrant contexts
rotein kinase (MAPK) proteins) and subsequent enhancement ofMAD2 activity (Clements et al 2011) In keratinocytes of thekin SMAD and Extracellular Regulated Kinase (ERK) signalling areutually required for TGF--induced epithelial-to-mesenchymal-
ransition (EMT) as ERK substrates mediate nuclear translocationf SMADs and alter target gene expression (Davies et al 2005)n cancer non-SMAD pathway activation by other TGF--familyroteins has been characterized For instance the TGF- type 1eceptor is capable of activating MAPK signalling through phos-horylation of ShcA and subsequent interaction with the GRB2SOSomplex (Lee et al 2007) Given the similarities between TGF- andodal these studies lend insight into putative non-SMAD Nodal
argets during disease progressionWe recently determined that Nodal promotes cellular invasion
nd EMT-like phenomena via activation of the ERK12 MAPK path-ay in breast cancer and choriocarcinoma cells (Quail et al inress) Specifically we found that Nodal induces ERK12 activa-ion and that this ability of Nodal to activate ERK12 is mediated byLK47 activity Our results indicate there may be multiple points ofrosstalk between the SMAD23 and ERK12 pathways at the levelf receptor activation intracellular signalling and gene transcrip-ion (Fig 3) Importantly Nodal-induced ERK activation mediatesodal-induced EMT and invasion such that inhibition of this path-ay with U0126 (a MEK12 inhibitor) abrogates the effects of Nodal
n these phenotypes (Quail et al in press) Together these find-ngs indicate that putative Nodal-targeted therapies would likelyequire interference of both SMAD and ERK pathways
As described below Nodal plays paradoxical roles in humanluripotent stem cells maintaining pluripotency whilst initiat-
ng mesendoderm induction Such disparate functions suggest thatodal likely regulates chromatin accessibility by altering epige-
etic phenomena such as DNA or histone methylation Indeed
recent study by Massagueacute and colleagues (2011) showed thatodal signalling is capable of inducing the expression of differ-ntiation signals in human embryonic stem cells by activating
e recruitment of the demethylase Jmjd3 to the Nodal locus to lift polycomb-inducedst others such as hypoxia-induced HIF signalling) of how Nodal is re-activated in
ldquopoisedrdquo chromatin (Xi et al 2011) Poised chromatin markedby polycomb-mediated trimethylation of histone H3 at lysine 9(H3K9me3) and acetylation of lysine 18 (H3K18ac) ensures trans-criptional repression of embryonic genes however these genesare responsive to acute activation by developmental signals inthe environment Chromatin assumes a poised state in responseto pluripotency factors such as Oct4 and Nanog to ensure thetimely activation of differentiation cues during development In thisstudy NodalActivin signalling through phosphorylated SMAD23caused recruitment of TRIM33 to H3K9me3-K18ac at the promo-ters of genes associated with mesendoderm specification (such asNodal targets MixI1 and Gsc) Association of SMAD23 and TRIM33in these regions lifted methylations to activate poised chromatinrendering promoter regions accessible to SMAD23ndashSMAD4 trans-criptional complexes (Fig 2) This study is the first to propose amechanism of how Nodal mediates widespread differentiation cuesduring embryogenesis and lends insight into how other mediatorsof embryonic differentiation may work in a similar fashion
3 Nodal signalling in developmental models
31 Role of Nodal in mouse development
The first gradient that Nodal establishes during development isthe proximalndashdistal (PD) axis which later rotates to become the APaxis Nodal is activated through the developing epiblast by conver-tase enzymes that are secreted from the extraembryonic ectoderm(Beck et al 2002 Schier 2009) These convertases including Pace4and Furin cleave the pro-domain of Nodal to activate its signallingin the proximal epiblast adjacent to the extraembryonic ectoderm(Beck et al 2002 Schier 2003) Here Nodal is maintained and
concentrated either through autoregulation andor by activatingBMP in the extraembryonic ectoderm which signals back to theadjacent epiblast to induce Wnt (Vincent et al 2003) The Nodalgene contains a proximal epiblast enhancer (PEE) upstream of
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 889
Fig 3 Putative crosstalk between ERK and SMAD pathways during Nodal signalling and regulation of E-cadherin trafficking Studies from our laboratory and others suggestthere may be multiple points of crosstalk between the SMAD23 and ERK12 pathways We have recently shown that recombinant Nodal induces phosphorylation of ERK12This may be indirect through receptor phosphorylation of an upstream mediator of the ERK cascade (such as ShcA) or direct via receptor phosphorylation of ERK12 We havealso shown that MEK inhibition leads to a decrease in Nodal-induced phosphorylation of SMAD2 This may be due to altered phosphorylation of the SMAD23 linker regionby ERK MAPKs We have shown that MEK inhibition reduces transcription of Nodal-induced EMT transcription factors ERK may alter SMAD-mediated gene expression byregulating translocation of SMAD proteins to the nucleus or by interfering with gene transcription We also found that MEK inhibition rescued Nodal-induced mislocalizationo n thatp
ttbiivastedpShC
itmpiseNa(
left
f E-Cadherin In accordance with these observations previous reports have showlasma membrane after internalization during EMT
he transcription start site that is Wnt-responsive Activation ofhis enhancer by Wnt concentrates Nodal to the proximal epi-last (Fig 4A) (Brennan et al 2001) Activated Nodal signalling
n the proximal epiblast subsequently induces gene expression ofts endogenous inhibitors Lefty1 and Cerberus (CER1) in the distalisceral endoderm (DVE) to establish a Nodal PD gradient Lefty1nd CER1 subsequently restrict Nodal expression to the proximalide of the embryo where the primitive streak will form to ini-iate gastrulation The PD axis graded by Nodal expression willventually rotate to become the AP axis whereby the DVE will beisplaced to yield the AVE and the proximal epiblast will moveosteriorly (Fig 4B) (Collignon et al 1996 Constam 2009a 2009btrizzi et al 2008 Zhou et al 1993) It is not entirely understoodow this rotation is regulated however it has been shown to beripto-dependent (Ding et al 1998)
Following AP axis formation gastrulation occurs as the prim-tive streak elongates from the posterior to the anterior side ofhe embryo This process is orchestrated by ingression of proxi-
al posterior cells to form mesendoderm cells which elongate therimitive streak towards the anterior side of the embryo Nodal
s expressed in the primitive endoderm overlying the primitivetreak and is thought to co-ordinate its directional migration andlongation (Conlon et al 1994) Once gastrulation is completeodal expression becomes restricted to the periphery of the nodet the anterior end of the primitive streak hence the name ldquoNodalrdquoSchier 2003)
Following gastrulation the node is the central initiator of
eftndashright (LR) asymmetry (Schier 2003 Smith 1995 Takaokat al 2006 Zhou et al 1993) TGF--family members secretedrom the ventral node including Nodal and growth differen-iation factor 1 (GDF1) define the left side of the embryo
inhibition of the ERK pathway is required for relocalization of E-Cadherin to the
through interactions with mouse Cripto family member Cryp-tic co-receptors in the lateral plate mesoderm (Constam 2009a)Meanwhile a leftward flow induced by active cilia prevents Nodalfrom signalling on the right side of the embryo in conjunc-tion with the right side-specific expression of CER1 and Lefty1(Fig 4C) (Constam 2009a) Of note this is how Nodalrsquos endogenousinhibitor Lefty received its name It has been shown that murineembryos lacking node-specific Nodal expression are not capableof establishing LR asymmetry and exhibit asymmetric patterningdefects including random positioning of the stomach and abnormalbranching of lung lobes (Brennan et al 2002) Furthermore thesestudies have shown that short-range Nodal signalling may also playa role in establishing a midline between the left and right sides ofthe embryo (Brennan et al 2002)
Following axis patterning of the embryo somitogenesis occurswhereby cells condense into transient epithelial structures byundergoing mesenchymal-to-epithelial transition At the begin-ning of this process Nodal is restricted to mesoderm cells on the leftside of the embryo (Collignon et al 1996) Interestingly as Nodal isa critical regulator of gastrulation (one of the first major EMT eventsto occur during development) somitogenesis is accompanied byan abrupt silencing of Nodal signalling by 8 days postcoitum (dpc)(Collignon et al 1996) Until recently it was not completely under-stood how Nodal silencing during development occurred Howevernew investigations suggest that Nodal is silenced by polycomb-mediated methylation of histones in a reversible manner (Dahleet al 2010 Xi et al 2011) (Fig 2)
Several studies using genetically modified mice have been per-formed to assess the role of different aspects of the Nodal signallingpathway during development Murine Nodal null mutants areembryonic lethal as they are unable to initiate the primitive streak
890 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 4 Nodal morphogens pattern the murine embryo to establish multiple criticalaxes (A) Nodal is activated in the epiblast by convertase enzymes (ex Pace4 andFurin) secreted from the extraembryonic ectoderm (ExEct) Convertases cleave andactivate Nodal in the proximal epiblast where Nodal is maintained by autoregula-tion andor by activation of a BMPWnt signalling loop The Nodal gene containsa proximal epiblast enhancer (PEE) upstream of the transcription start site thatis Wnt-responsive activation of the PEE by Wnt concentrates Nodal to the prox-imal epiblast (B) Nodal subsequently induces gene expression of its endogenousinhibitors Lefty1 and CER1 in the distal visceral endoderm (DVE) to establish aproximal-distal (PD) gradient The PD axis graded by Nodal expression eventuallyrotates to become the anterior-posterior (AP) axis whereby the DVE is displacedto yield the anterior visceral endoderm (AVE) and the proximal epiblast movesposteriorly (C) During gastrulation Nodal is expressed in the primitive endodermoverlying the primitive streak and then becomes restricted to the node to initiateleftndashright (LR) asymmetry GDF1 and Nodal from the node define the left side of theedf
aat(amaoot
Fig 5 Comparison of Nodal expression patterns in early versus late gastrulationin mouse frog zebrafish and sea urchin development In mice Nodal is expressedin the node during early gastrulation and in the left lateral plate mesoderm dur-ing specification of LR asymmetry In the frog Xnr proteins are critical initiators ofgastrulation in the dorsal lip and become restricted to the lateral plate mesodermlater during neurulation In zebrafish Cyclops is expressed in the hypoblast of theembryonic shield and Squint is expressed in the dorsal forerunner cells during earlygastrulation During late gastrulation Southpaw induces the expression of Cyclopsin the left lateral plate mesoderm and the expression of Nodal inhibitors on theright side of the embryo (eg Lefty) In sea urchins Nodal is first expressed in the pre-sumptive ectoderm prior to gastrulation where it helps to establish the oralndashaboral
mbryo through interactions with Cryptic co-receptors in the lateral plate meso-erm Meanwhile a cilia leftward flow and LeftyCER1 inhibitors prevent Nodalrom signalling on the right side of the embryo
nd therefore do not gastrulate Cells within the epiblast proliferatend degenerate in these embryos rather than ingressing throughhe primitive streak and therefore gastrulation cannot occurConlon et al 1994 Zhou et al 1993) Nodal hypomorphs havelso been developed via insertion of a neomycin cassette Theseutants are viable however phenotypic analyses of these animals
re as of yet incomplete Given that the Nodal null mutant is embry-nic lethal attempts have been made to knock out other membersf the Nodal signalling pathway in murine embryos In most caseshese embryos recapitulate the effects observed in Nodal mutant
axis and is abruptly downregulated once gastrulation begins Following gastrula-tion Nodal is asymmetrically expressed on the right to restrict the development ofthe rudiment to the left side
embryos For example homozygous ActR1Bminusminus ActRIIBminusminus andSMAD2minusminus mice are embryonic lethal due to impaired embryonicpatterning and a subsequent inability to gastrulate (Brennan et al2001 Goumans and Mummery 2000 Nomura and Li 1998) Het-erozygote mutants are less consistent ActR1Bminus+ heterozygotes areviable and grossly normal whilst SMAD2minus+ heterozygotes exhibitgastrulation defects and usually die Interestingly some SMAD2minus+
embryos do survive however they have severe craniofacial defectswhich is an indication of deregulated neural crest delaminationfrom the dorsal neural tube (Thiery et al 2009)
32 Role of Nodal signalling in the development of non-murineorganisms
In addition to mice and humans Nodal is expressed in all othervertebrates as well as some invertebrates (including sea urchinsascidians and snails) where it appears to predominantly regulatechirality (Duboc and Lepage 2008 Grande and Patel 2009a 2009bSchier 2009) Generally whilst Nodal specifies mesendoderm invertebrates its expression is limited to ectoderm in invertebrateorganisms In all species Nodal proteins are expressed asymmetri-cally throughout the embryo where they play an important role inspecifying the left and right sides (Duboc et al 2005 Mohapatraet al 2009)
There are three Nodal-related proteins in zebrafish calledSquint Cyclops and Southpaw (Feldman et al 1998 Long et al2003 Schier 2005) During gastrulation Cyclops is expressed inthe hypoblast of the embryonic shield and Squint is expressed inthe dorsal forerunner cells of the developing zebrafish (Fig 5) Bothof these Nodal-related proteins are important for specificationof endodermal and mesodermal structures during gastrulation(Dougan et al 2003) Single mutant embryos for either Squint orCyclops are generally normal with only mild defects whilst doublemutant embryos for both Squint and Cyclops lack all endodermand most mesoderm tissue (Dougan et al 2003) This suggeststhat although Squint and Cyclops have distinct roles under normal
circumstances their capacity for redundancy ensures that propergastrulation occurs Following gastrulation Southpaw induces theexpression of Cyclops in the left lateral plate mesoderm and theexpression of Nodal inhibitors on the right side of the embryo
f Bioc
(2odeod
itisr(2futeterocrcuiTb(Xg
aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
rfft
ctiastNcopMaglltldbSvdpa(d
ppNppE2aaSdIcN(htAlcsoelsleApfiii2l
92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
es
4
weptsihietd2dse
4
oipeI(ooiemat
4
caab(Ldhobmbdsctls
5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
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5
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5
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5
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94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
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B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
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l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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H
H
I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
T
T
T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898 889
Fig 3 Putative crosstalk between ERK and SMAD pathways during Nodal signalling and regulation of E-cadherin trafficking Studies from our laboratory and others suggestthere may be multiple points of crosstalk between the SMAD23 and ERK12 pathways We have recently shown that recombinant Nodal induces phosphorylation of ERK12This may be indirect through receptor phosphorylation of an upstream mediator of the ERK cascade (such as ShcA) or direct via receptor phosphorylation of ERK12 We havealso shown that MEK inhibition leads to a decrease in Nodal-induced phosphorylation of SMAD2 This may be due to altered phosphorylation of the SMAD23 linker regionby ERK MAPKs We have shown that MEK inhibition reduces transcription of Nodal-induced EMT transcription factors ERK may alter SMAD-mediated gene expression byregulating translocation of SMAD proteins to the nucleus or by interfering with gene transcription We also found that MEK inhibition rescued Nodal-induced mislocalizationo n thatp
ttbiivastedpShC
itmpiseNa(
left
f E-Cadherin In accordance with these observations previous reports have showlasma membrane after internalization during EMT
he transcription start site that is Wnt-responsive Activation ofhis enhancer by Wnt concentrates Nodal to the proximal epi-last (Fig 4A) (Brennan et al 2001) Activated Nodal signalling
n the proximal epiblast subsequently induces gene expression ofts endogenous inhibitors Lefty1 and Cerberus (CER1) in the distalisceral endoderm (DVE) to establish a Nodal PD gradient Lefty1nd CER1 subsequently restrict Nodal expression to the proximalide of the embryo where the primitive streak will form to ini-iate gastrulation The PD axis graded by Nodal expression willventually rotate to become the AP axis whereby the DVE will beisplaced to yield the AVE and the proximal epiblast will moveosteriorly (Fig 4B) (Collignon et al 1996 Constam 2009a 2009btrizzi et al 2008 Zhou et al 1993) It is not entirely understoodow this rotation is regulated however it has been shown to beripto-dependent (Ding et al 1998)
Following AP axis formation gastrulation occurs as the prim-tive streak elongates from the posterior to the anterior side ofhe embryo This process is orchestrated by ingression of proxi-
al posterior cells to form mesendoderm cells which elongate therimitive streak towards the anterior side of the embryo Nodal
s expressed in the primitive endoderm overlying the primitivetreak and is thought to co-ordinate its directional migration andlongation (Conlon et al 1994) Once gastrulation is completeodal expression becomes restricted to the periphery of the nodet the anterior end of the primitive streak hence the name ldquoNodalrdquoSchier 2003)
Following gastrulation the node is the central initiator of
eftndashright (LR) asymmetry (Schier 2003 Smith 1995 Takaokat al 2006 Zhou et al 1993) TGF--family members secretedrom the ventral node including Nodal and growth differen-iation factor 1 (GDF1) define the left side of the embryo
inhibition of the ERK pathway is required for relocalization of E-Cadherin to the
through interactions with mouse Cripto family member Cryp-tic co-receptors in the lateral plate mesoderm (Constam 2009a)Meanwhile a leftward flow induced by active cilia prevents Nodalfrom signalling on the right side of the embryo in conjunc-tion with the right side-specific expression of CER1 and Lefty1(Fig 4C) (Constam 2009a) Of note this is how Nodalrsquos endogenousinhibitor Lefty received its name It has been shown that murineembryos lacking node-specific Nodal expression are not capableof establishing LR asymmetry and exhibit asymmetric patterningdefects including random positioning of the stomach and abnormalbranching of lung lobes (Brennan et al 2002) Furthermore thesestudies have shown that short-range Nodal signalling may also playa role in establishing a midline between the left and right sides ofthe embryo (Brennan et al 2002)
Following axis patterning of the embryo somitogenesis occurswhereby cells condense into transient epithelial structures byundergoing mesenchymal-to-epithelial transition At the begin-ning of this process Nodal is restricted to mesoderm cells on the leftside of the embryo (Collignon et al 1996) Interestingly as Nodal isa critical regulator of gastrulation (one of the first major EMT eventsto occur during development) somitogenesis is accompanied byan abrupt silencing of Nodal signalling by 8 days postcoitum (dpc)(Collignon et al 1996) Until recently it was not completely under-stood how Nodal silencing during development occurred Howevernew investigations suggest that Nodal is silenced by polycomb-mediated methylation of histones in a reversible manner (Dahleet al 2010 Xi et al 2011) (Fig 2)
Several studies using genetically modified mice have been per-formed to assess the role of different aspects of the Nodal signallingpathway during development Murine Nodal null mutants areembryonic lethal as they are unable to initiate the primitive streak
890 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 4 Nodal morphogens pattern the murine embryo to establish multiple criticalaxes (A) Nodal is activated in the epiblast by convertase enzymes (ex Pace4 andFurin) secreted from the extraembryonic ectoderm (ExEct) Convertases cleave andactivate Nodal in the proximal epiblast where Nodal is maintained by autoregula-tion andor by activation of a BMPWnt signalling loop The Nodal gene containsa proximal epiblast enhancer (PEE) upstream of the transcription start site thatis Wnt-responsive activation of the PEE by Wnt concentrates Nodal to the prox-imal epiblast (B) Nodal subsequently induces gene expression of its endogenousinhibitors Lefty1 and CER1 in the distal visceral endoderm (DVE) to establish aproximal-distal (PD) gradient The PD axis graded by Nodal expression eventuallyrotates to become the anterior-posterior (AP) axis whereby the DVE is displacedto yield the anterior visceral endoderm (AVE) and the proximal epiblast movesposteriorly (C) During gastrulation Nodal is expressed in the primitive endodermoverlying the primitive streak and then becomes restricted to the node to initiateleftndashright (LR) asymmetry GDF1 and Nodal from the node define the left side of theedf
aat(amaoot
Fig 5 Comparison of Nodal expression patterns in early versus late gastrulationin mouse frog zebrafish and sea urchin development In mice Nodal is expressedin the node during early gastrulation and in the left lateral plate mesoderm dur-ing specification of LR asymmetry In the frog Xnr proteins are critical initiators ofgastrulation in the dorsal lip and become restricted to the lateral plate mesodermlater during neurulation In zebrafish Cyclops is expressed in the hypoblast of theembryonic shield and Squint is expressed in the dorsal forerunner cells during earlygastrulation During late gastrulation Southpaw induces the expression of Cyclopsin the left lateral plate mesoderm and the expression of Nodal inhibitors on theright side of the embryo (eg Lefty) In sea urchins Nodal is first expressed in the pre-sumptive ectoderm prior to gastrulation where it helps to establish the oralndashaboral
mbryo through interactions with Cryptic co-receptors in the lateral plate meso-erm Meanwhile a cilia leftward flow and LeftyCER1 inhibitors prevent Nodalrom signalling on the right side of the embryo
nd therefore do not gastrulate Cells within the epiblast proliferatend degenerate in these embryos rather than ingressing throughhe primitive streak and therefore gastrulation cannot occurConlon et al 1994 Zhou et al 1993) Nodal hypomorphs havelso been developed via insertion of a neomycin cassette Theseutants are viable however phenotypic analyses of these animals
re as of yet incomplete Given that the Nodal null mutant is embry-nic lethal attempts have been made to knock out other membersf the Nodal signalling pathway in murine embryos In most caseshese embryos recapitulate the effects observed in Nodal mutant
axis and is abruptly downregulated once gastrulation begins Following gastrula-tion Nodal is asymmetrically expressed on the right to restrict the development ofthe rudiment to the left side
embryos For example homozygous ActR1Bminusminus ActRIIBminusminus andSMAD2minusminus mice are embryonic lethal due to impaired embryonicpatterning and a subsequent inability to gastrulate (Brennan et al2001 Goumans and Mummery 2000 Nomura and Li 1998) Het-erozygote mutants are less consistent ActR1Bminus+ heterozygotes areviable and grossly normal whilst SMAD2minus+ heterozygotes exhibitgastrulation defects and usually die Interestingly some SMAD2minus+
embryos do survive however they have severe craniofacial defectswhich is an indication of deregulated neural crest delaminationfrom the dorsal neural tube (Thiery et al 2009)
32 Role of Nodal signalling in the development of non-murineorganisms
In addition to mice and humans Nodal is expressed in all othervertebrates as well as some invertebrates (including sea urchinsascidians and snails) where it appears to predominantly regulatechirality (Duboc and Lepage 2008 Grande and Patel 2009a 2009bSchier 2009) Generally whilst Nodal specifies mesendoderm invertebrates its expression is limited to ectoderm in invertebrateorganisms In all species Nodal proteins are expressed asymmetri-cally throughout the embryo where they play an important role inspecifying the left and right sides (Duboc et al 2005 Mohapatraet al 2009)
There are three Nodal-related proteins in zebrafish calledSquint Cyclops and Southpaw (Feldman et al 1998 Long et al2003 Schier 2005) During gastrulation Cyclops is expressed inthe hypoblast of the embryonic shield and Squint is expressed inthe dorsal forerunner cells of the developing zebrafish (Fig 5) Bothof these Nodal-related proteins are important for specificationof endodermal and mesodermal structures during gastrulation(Dougan et al 2003) Single mutant embryos for either Squint orCyclops are generally normal with only mild defects whilst doublemutant embryos for both Squint and Cyclops lack all endodermand most mesoderm tissue (Dougan et al 2003) This suggeststhat although Squint and Cyclops have distinct roles under normal
circumstances their capacity for redundancy ensures that propergastrulation occurs Following gastrulation Southpaw induces theexpression of Cyclops in the left lateral plate mesoderm and theexpression of Nodal inhibitors on the right side of the embryo
f Bioc
(2odeod
itisr(2futeterocrcuiTb(Xg
aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
rfft
ctiastNcopMaglltldbSvdpa(d
ppNppE2aaSdIcN(htAlcsoelsleApfiii2l
92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
es
4
weptsihietd2dse
4
oipeI(ooiemat
4
caab(Ldhobmbdsctls
5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
betiIaie2kvpsAHecatgh
5
tCtcCnipcegaN
5
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5
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94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
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l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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H
H
I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
890 DF Quail et al The International Journal of Biochemistry amp Cell Biology 45 (2013) 885ndash 898
Fig 4 Nodal morphogens pattern the murine embryo to establish multiple criticalaxes (A) Nodal is activated in the epiblast by convertase enzymes (ex Pace4 andFurin) secreted from the extraembryonic ectoderm (ExEct) Convertases cleave andactivate Nodal in the proximal epiblast where Nodal is maintained by autoregula-tion andor by activation of a BMPWnt signalling loop The Nodal gene containsa proximal epiblast enhancer (PEE) upstream of the transcription start site thatis Wnt-responsive activation of the PEE by Wnt concentrates Nodal to the prox-imal epiblast (B) Nodal subsequently induces gene expression of its endogenousinhibitors Lefty1 and CER1 in the distal visceral endoderm (DVE) to establish aproximal-distal (PD) gradient The PD axis graded by Nodal expression eventuallyrotates to become the anterior-posterior (AP) axis whereby the DVE is displacedto yield the anterior visceral endoderm (AVE) and the proximal epiblast movesposteriorly (C) During gastrulation Nodal is expressed in the primitive endodermoverlying the primitive streak and then becomes restricted to the node to initiateleftndashright (LR) asymmetry GDF1 and Nodal from the node define the left side of theedf
aat(amaoot
Fig 5 Comparison of Nodal expression patterns in early versus late gastrulationin mouse frog zebrafish and sea urchin development In mice Nodal is expressedin the node during early gastrulation and in the left lateral plate mesoderm dur-ing specification of LR asymmetry In the frog Xnr proteins are critical initiators ofgastrulation in the dorsal lip and become restricted to the lateral plate mesodermlater during neurulation In zebrafish Cyclops is expressed in the hypoblast of theembryonic shield and Squint is expressed in the dorsal forerunner cells during earlygastrulation During late gastrulation Southpaw induces the expression of Cyclopsin the left lateral plate mesoderm and the expression of Nodal inhibitors on theright side of the embryo (eg Lefty) In sea urchins Nodal is first expressed in the pre-sumptive ectoderm prior to gastrulation where it helps to establish the oralndashaboral
mbryo through interactions with Cryptic co-receptors in the lateral plate meso-erm Meanwhile a cilia leftward flow and LeftyCER1 inhibitors prevent Nodalrom signalling on the right side of the embryo
nd therefore do not gastrulate Cells within the epiblast proliferatend degenerate in these embryos rather than ingressing throughhe primitive streak and therefore gastrulation cannot occurConlon et al 1994 Zhou et al 1993) Nodal hypomorphs havelso been developed via insertion of a neomycin cassette Theseutants are viable however phenotypic analyses of these animals
re as of yet incomplete Given that the Nodal null mutant is embry-nic lethal attempts have been made to knock out other membersf the Nodal signalling pathway in murine embryos In most caseshese embryos recapitulate the effects observed in Nodal mutant
axis and is abruptly downregulated once gastrulation begins Following gastrula-tion Nodal is asymmetrically expressed on the right to restrict the development ofthe rudiment to the left side
embryos For example homozygous ActR1Bminusminus ActRIIBminusminus andSMAD2minusminus mice are embryonic lethal due to impaired embryonicpatterning and a subsequent inability to gastrulate (Brennan et al2001 Goumans and Mummery 2000 Nomura and Li 1998) Het-erozygote mutants are less consistent ActR1Bminus+ heterozygotes areviable and grossly normal whilst SMAD2minus+ heterozygotes exhibitgastrulation defects and usually die Interestingly some SMAD2minus+
embryos do survive however they have severe craniofacial defectswhich is an indication of deregulated neural crest delaminationfrom the dorsal neural tube (Thiery et al 2009)
32 Role of Nodal signalling in the development of non-murineorganisms
In addition to mice and humans Nodal is expressed in all othervertebrates as well as some invertebrates (including sea urchinsascidians and snails) where it appears to predominantly regulatechirality (Duboc and Lepage 2008 Grande and Patel 2009a 2009bSchier 2009) Generally whilst Nodal specifies mesendoderm invertebrates its expression is limited to ectoderm in invertebrateorganisms In all species Nodal proteins are expressed asymmetri-cally throughout the embryo where they play an important role inspecifying the left and right sides (Duboc et al 2005 Mohapatraet al 2009)
There are three Nodal-related proteins in zebrafish calledSquint Cyclops and Southpaw (Feldman et al 1998 Long et al2003 Schier 2005) During gastrulation Cyclops is expressed inthe hypoblast of the embryonic shield and Squint is expressed inthe dorsal forerunner cells of the developing zebrafish (Fig 5) Bothof these Nodal-related proteins are important for specificationof endodermal and mesodermal structures during gastrulation(Dougan et al 2003) Single mutant embryos for either Squint orCyclops are generally normal with only mild defects whilst doublemutant embryos for both Squint and Cyclops lack all endodermand most mesoderm tissue (Dougan et al 2003) This suggeststhat although Squint and Cyclops have distinct roles under normal
circumstances their capacity for redundancy ensures that propergastrulation occurs Following gastrulation Southpaw induces theexpression of Cyclops in the left lateral plate mesoderm and theexpression of Nodal inhibitors on the right side of the embryo
f Bioc
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itisr(2futeterocrcuiTb(Xg
aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
rfft
ctiastNcopMaglltldbSvdpa(d
ppNppE2aaSdIcN(htAlcsoelsleApfiii2l
92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
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4
weptsihietd2dse
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xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
betiIaie2kvpsAHecatgh
5
tCtcCnipcegaN
5
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5
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94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
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l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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H
H
I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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T
U
V
V
V
V
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W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
f Bioc
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aicbiipaeeorfawrartNsg
3
apImsV
DF Quail et al The International Journal o
eg Lefty) (Duboc et al 2005 Long et al 2003 Toyoizumi et al005) Inhibition of Southpaw by in vivo injection of morpholinoligonucleotides demonstrated a critical role for this proteinuring LR asymmetry of the heart pancreas and brain (Longt al 2003) Together these studies demonstrate the importancef Nodal protein during gastrulation and LR asymmetry in theeveloping zebrafish
There are 5 Xenopus Nodal-related (Xnr) proteins with canon-cal functions including Xnr1 Xnr2 Xnr4 Xnr5 and Xnr6 Similaro the role of Nodal in mouse embryos Xnr proteins are criticalnitiators of Xenopus gastrulation they are expressed in the dor-al lip early in gastrulation (all except Xnr5) and then becomeestricted to the lateral plate mesoderm later during neurulationDuboc et al 2005 Osada and Wright 1999 Toyoizumi et al005) (Fig 5) In particular Xnr1 like mouse Nodal is requiredor LR asymmetry of the body plan as it is the only Xnr that isnilaterally expressed in the left lateral plate mesoderm (and nothe right lateral plate mesoderm) (Toyoizumi et al 2005) How-ver Xnr1 is dispensable for other Nodal-induced axes suggestinghat both redundant and non-redundant roles for the Xnr proteinsxist (Toyoizumi et al 2005) Indeed Xnrs do exhibit differentialegulation in some contexts For example goosecoid is capablef downregulating expression of Xnr2 but not Xnr5 or Xnr6 inell-dissociated embryos Instead Xnr5 and Xnr6 expression areegulated by a completely different mechanism that involves -atenin and maternal VegT (Takahashi et al 2000) Furthermorenlike other Nodal-related proteins Xnr5 and Xnr6 do not local-
ze to the Spemann organizer do not require SMAD signalling byGF- members for transcriptional activation and are not capa-le of rescuing embryonic axis formation following UV-irradiationTakahashi et al 2000) Taken together Nodal-related proteins inenopus have distinct yet overlapping roles and are essential forastrulation and LR asymmetry
Although a great deal is known about the establishment of LRsymmetry in vertebrate species this process is poorly understoodn invertebrates It is known that Nodal plays a role during this pro-ess in some invertebrates such as the sea urchin however it haseen reported that the pattern of Nodal expression is opposite to
ts expression in vertebrate embryos (Duboc et al 2005) Nodals first expressed in the presumptive ectoderm of the sea urchinrior to gastrulation where it helps to establish the oralndashaboral axisnd is abruptly downregulated once gastrulation begins (Duboct al 2005) This is in contrast to the mesodermendoderm-specificxpression during mouse frog and zebrafish embryogenesis Sec-nd following gastrulation Nodal is unilaterally expressed in theight side of the embryo rather than the left side as it is in mouserog and zebrafish In the developing sea urchin the tip of therchenteron buds to generate two symmetric coelomic poucheshich subdivide asymmetrically and eventually give rise to the
udiment on the left side It was previously unknown how rudimentsymmetry was established however a recent study showed thatight side-specific Nodal expression may play a role in mediatinghis event (Duboc et al 2005) (Fig 5) Taken together althoughodal seems to play a role in axis specification in sea urchins it
eems that its role is quite different from that in vertebrate embryo-enesis
3 Human pluripotent stem cells (hPSCs)
In a manner that recapitulates early development Nodal signalsre used to specify mesendoderm derivatives from human inducedluripotent stem cells and human embryonic stem cells (hESCs)
ndeed Nodal is an essential ingredient for the differentiation ofesoderm lineages such as cardiomyocytes and endoderm lineages
uch as pancreatic -cells (Cai et al 2012 Mfopou et al 2010allier et al 2009b) In an apparent paradox several studies with
hESCs have shown that Nodal signalling maintains pluripotencySpecifically Nodal signalling via SMAD23 induces the activationof Nanog transcription (Vallier et al 2009a) In turn Nanog inter-acts with SMAD23 to limit transcriptional activity of the Nodalsignalling pathway and inhibit endoderm differentiation (Vallieret al 2009a) Furthermore NodalActivin inhibition in hESCs withSB431542 (an ALK457 inhibitor) induces neuroectoderm specifi-cation (Patani et al 2009 Smith et al 2008 Vallier et al 2009a)Together these studies exemplify the role of Nodal in promot-ing mesendoderm induction whilst maintaining pluripotency byinhibiting differentiation This raises an important question Howcan one gene product have such seemingly contradictory func-tions It is likely that the ostensibly paradoxical roles of Nodalin the regulation of hPSCs are due to context and concentration-dependent differences in downstream signalling For exampleVallier and colleagues recently revealed that the ability of SMAD23(downstream of Nodal) to activate pluripotency versus endodermtranscriptional programmes is dependent upon the availabilityof binding partners such as Nanog (Brown et al 2011) IndeedSMAD23 associates with Nanog to promote the expression of keypluripotency factors such as Oct4 Tert and Myc In the absenceof Nanog SMAD23 can regulate endoderm-specific genes (Brownet al 2011) As a complement to this model Lee et al investigatedthe implications of graded NodalActivin signalling in mouse ESC(mESC Lee et al 2011) A twofold increase in SMAD2 phosphory-lation was enough to cause differentiation to mesoderm whereas atwofold decrease resulted in trophectoderm cell fates Importantlythey found that dose-dependent NodalActivin signalling dictateswith which genes pSMAD2 associates thereby eliciting differentoutcomes It is plausible that this type of dose-dependency alsoexists in hESCs
The role of Nodal in the regulation of pluripotency has been stud-ied to the greatest extent in human cells largely because mESCsare more reliant on LIF-regulated pathways Recent studies sug-gest that this may be due to differences in the stage at which thesecells are derived In support of this notion in back-to-back Naturepapers Brons (Brons et al 2007) and Tesar (Tesar et al 2007) inde-pendently reported on the establishment of mouse epiblast ES cell(EpiSC) lines that are strikingly similar to hESC Notably these EpiSCrequire ActivinNodal to remain undifferentiated and like hESCsthese cells are more glycolytic (Zhou et al 2012) Pig EpiSC alsorely on ActivinNodal signalling and have no requirement for LIF inmaintenance of pluripotency (Alberio et al 2010) Hence it appearsas though ActivinNodal may universally regulate pluripotency butthat the stage of the cells may dictate this dependence such thatlater more epiblast type cells are Nodal-dependent
4 Nodal signalling in adult and reproductive tissues
As outlined above Nodal plays a critical role during embry-onic development however recent studies have shown that thereare several non-embryonic tissues that exhibit Nodal expres-sion For example highly dynamic tissues that undergo necessarywidespread remodelling events such as the placenta the mam-mary gland and the cycling endometrium have been shown toexpress Nodal Furthermore subpopulations of cells within spe-cialized tissues such as pancreatic islets and liver cells expressNodal The following sections will outline the emergent novel rolesfor Nodal in non-embryonic contexts
41 Nodal signalling in the placenta
The role of Nodal in the placenta is controversial with someresearch pointing to beneficial functions and others pathogenic Itremains to be seen whether and how these divergent views can be
8 f Bioc
rfft
ctiastNcopMaglltldbSvdpa(d
ppNppE2aaSdIcN(htAlcsoelsleApfiii2l
92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
es
4
weptsihietd2dse
4
oipeI(ooiemat
4
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5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
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5
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94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
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A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
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l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
8 f Bioc
rfft
ctiastNcopMaglltldbSvdpa(d
ppNppE2aaSdIcN(htAlcsoelsleApfiii2l
92 DF Quail et al The International Journal o
econciled Considering the complexity of Nodal function and theeedback loops governing Nodal signalling as well as contributionsrom both the mother and the embryofoetus it may well be someime before these issues are resolved
In the developing mammalian blastocyst the trophectodermell layer surrounding the inner cell mass and blastocoel gives riseo the foetal portion of the placenta (Tanaka et al 1998) Uponmplantation foetal trophoblast cells invade the maternal deciduand spiral arteries in order to gain access to the maternal bloodupply A number of studies using mouse models have determinedhat Nodal supports normal placentation In mice insertional nullodal mutants exhibit disrupted embryonic development con-omitant with abnormal placentation featuring excessive numbersf trophoblast giant cells and a marked lack of diploid spongiotro-hoblasts (Iannaccone et al 1992 Ma et al 2001) For examplea et al confirmed the production of Nodal in the murine placenta
nd its presence in spongiotrophoblasts but not in differentiatediant cells (Ma et al 2001) They also determined that Nodal regu-ates differentiation of trophoblasts during placental developmentimiting the growth of giant cell and spongiotrophoblast layers suchhat the labyrinth may expand By promoting the expansion of theabyrinth layer it was concluded that Nodal facilitates the properevelopment and function of the nutrientwaste exchange systemetween mother and foetus (Ma et al 2001) Similarly SPC1 andPC4 double knockout mice (concomitant with reduced Nodal acti-ation) show accelerated differentiation of trophoblast stem cellsuring placentation (Guzman-Ayala et al 2004) In this case tro-hoblast cells in the extraembryonic ectoderm (Exe) secrete Pace4nd Furin proteases that activate Nodal which in turn induce Fgf4Guzman-Ayala et al 2004) Together they inhibit trophoblast cellifferentiation
Several studies suggest that Nodal is deleterious to humanlacentation and that it may contribute to pathologies such asre-eclampsia (PE) In the first study describing a negative role forodal in trophoblast cells Munir et al suggest that Nodal inhibitsroliferation and induces apoptosis causing G1 cell cycle arrest inart via upregulation of p27 (Munir et al 2004) p27 inhibits cyclinCdk2 thereby preventing cells from entering S phase (Munir et al004) In transfection experiments Nodal overexpressing cells or
constitutively active ALK7 decreased proliferation and increasedpoptosis via SMAD23 and dominant negative forms of ALK7 andMAD 23 reversed this effect In later studies this same groupescribed a pivotal role for Nodal in PE (Nadeem et al 2011)
n PE trophoblast cells exhibit restricted invasion and in severeases undergo increased apoptosis In this study confirmation ofodal and ALK7 expression in human placentae was presented
Roberts et al 2003) and the authors found that both proteins wereighly up-regulated in severe PE placentae as compared to con-rols In addition overexpression of Nodal or a constitutively activeLK7 both lead to decreased migration of HTR-8SVneo extravil-
ous cytotrophoblast cells (Nadeem et al 2011) Whilst the authorslaimed that increasing recombinant (r)Nodal decreased cell inva-ion this was only evident at concentrations of 100 ngml Nodalr higher Finally the authors have shown that inhibiting Nodalxpression in trophoblast cells results in a modest increase in cellu-ar invasion (Luo et al 2012 Nadeem et al 2011) In a more recenttudy this group has posited that high concentrations of Activin Aead to increased apoptosis of trophoblast cells by enhancing Nodalxpression and Nodal signalling through ALK7 (Yu et al 2012)t week 25 higher Activin A levels were evident in plasma andlacentae of severe PE patients In vitro increased apoptosis wasrst evident at doses of 25ndash100 ngml Activin A Transiently silenc-
ng ALK7 resulted in a 69 decrease in Nodal- and 74 decreasen Activin A-induced apoptosis in HTR8SVneo cells treated with50 ngml rNodal or 100 ngml Activin A This result was simi-
ar for transient knockdown of ALK4 thus suggesting that ALK4
hemistry amp Cell Biology 45 (2013) 885ndash 898
and ALK7 must contribute together to promote apoptosis throughNodal or Activin A signalling (though the authors only emphasizethe role of ALK7) Finally a recent paper has shown that miR-378a-5p increases the invasiveness and proliferation of trophoblastcells concomitant with reduced levels of Nodal (Luo et al 2012)Collectively these studies strongly suggest that Nodal decreasestrophoblast invasion and proliferation leading to diseases such asPE
In an apparent paradox several groups have demonstratedthat Nodal positively regulates embryo implantation associatedwith trophoblast invasion of the decidua The first piece of evi-dence linking Nodal to implantation and fertility involved in vivogene transfer experiments in mice showing that overexpression ofmaternal Lefty (a Nodal inhibitor) in endometrium reduces implan-tation (Tang et al 2005) As a corollary in humans Lefty (alsocalled EBAF or endometrial bleeding associated factor) is secretedat low levels during implantation in normal individuals How-ever aberrantly high expression of Lefty mRNA and abnormal Leftyprotein processing has been identified in endometria of infertilepatients during the implantation window (Ulloa et al 2001) Therole of Lefty in infertility is expertly reviewed in (Tabibzadeh2011)
In elegant experiments systematically removing uteri of non-pregnant pregnant and pseudopregnant Nodal-lacZ mice at variousearly time points (05ndash145 dpc) and examining them via immuno-histochemistry Park and Dufort documented Nodal expressionpatterns during pregnancy (Park and Dufort 2011) Whilst Nodalappears in the uterus at 05 dpc irrespective of successful fer-tilization its expression is only maintained after 35 days viasignals from the embryo Nodal can be identified in a clear band-ing pattern neatly demarking implantation sites Furthermore inembryo transfer experiments they discovered that the numberof non-staining bands correlated with the number of embryostransferred Refreshingly the authors also considered Lefty whilstLefty expression coincides with that of Nodal in the glandular andluminal epithelium of the inter-implantation space Lefty was alsoexpressed throughout the uterine horn during implantation Inan equally impressive study using a conditional Nodal knockoutmouse in which Nodal is absent only in the reproductive tract Parket al show that maternal Nodal is crucial for proper placentationintrauterine growth and full-term delivery (Park et al 2012) Theyfound that developmental defects in the placentae of these micewere due to increased apoptosis and decreased proliferation of thedecidua basalis leading to intrauterine growth restriction defec-tive maternalfoetal interface and loss of the foetus on day 175Hence it appears as though Nodal may be required for normalplacentation
42 Nodal signalling in the mammary gland
During pregnancy and lactation the terminal end buds ofthe mammary gland undergo proliferation and differentiationto form secretory milk-producing alveoli (Brisken et al 1998Ormandy et al 1997) Post-lactational involution reverts the mam-mary gland back to its pre-lactation state and is characterizedby widespread apoptosis of alveolar epithelial tissue stromalremodelling and adipocyte replenishment (Sutherland et al 2007Watson and Khaled 2008) Nodal and members of the Nodalsignalling pathway are cyclically expressed during mammary glandremodelling In particular one study found that Nodal CriptoALK-4 and SMAD4 were upregulated during lactation and down-
regulated during involution in BalbC mice (Bianco et al 2002Kenney et al 2004) These results suggest that Nodal signallingmay play a dynamic role during mammary gland remodellingwhereby Nodal upregulation correlates with proliferative alveolar
f Bioc
es
4
weptsihietd2dse
4
oipeI(ooiemat
4
caab(Ldhobmbdsctls
5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
betiIaie2kvpsAHecatgh
5
tCtcCnipcegaN
5
cgarrceimcaero
5
Nia
94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
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vibavcrai(v(pc(abt
6
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DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
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B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
dkins HB Bianco C Schiffer SG Rayhorn P Zafari M Cheung AE et al Antibodyblockade of the Cripto CFC domain suppresses tumor cell growth in vivo Journalof Clinical Investigation 2003112575ndash87
l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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L
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L
M
M
M
M
M
M
M
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N
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O
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DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
f Bioc
es
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weptsihietd2dse
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oipeI(ooiemat
4
caab(Ldhobmbdsctls
5
so
DF Quail et al The International Journal o
xpansion and Nodal downregulation correlates with apopto-is
3 Nodal signalling in the cycling endometrium
Nodal has also been studied in the human endometriumhich like the mammary gland undergoes many remodelling
vents during adulthood Endometrium cycling consists of threehases menstrual proliferative (re-epithelialization) and secre-ory (Papageorgiou et al 2009) Harrison and colleagues havetudied Nodal signalling in human endometrium during the var-ous phases of remodelling This group has shown that Lefty isighly expressed during late secretory and menstrual phases and
s downregulated during the proliferative phase (Papageorgiout al 2009) In contrast Nodal is highly expressed throughouthe proliferative phase and early secretory phase and is abruptlyownregulated by the mid-secretory phase (Papageorgiou et al009) Again although the endometrium harbours a completelyifferent microenvironment compared to the mammary gland iteems that Nodal expression correlates with proliferative epithelialxpansion during endometrial cycling
4 Nodal expression in human adult stem cells
Given that Nodal plays an important role in pluripotent embry-nic stem cells recent studies have tried to address whether Nodals likewise involved in the maintenance of human adult stem cellopulations Human adult liver stem cells have been shown toxpress Nodal and its co-receptor Cripto (Herrera et al 2006)nterestingly Lefty is also expressed by human adult liver stem cellsHerrera et al 2006) indicating that these proteins may keep eachther in check in an autocrine manner as they do in the embry-nic context Nodal and its receptors have also been detected innsulin-producing pancreatic -cells (Zhao et al 2012) Nodal over-xpression in these cells induces apoptosis in an ALK7-dependentanner (Zhang et al 2006 Zhao et al 2012) indicating that
utocrine regulation of this signalling pathway might also exist inhe pancreas
5 Nodal mutations in human disease
In humans mutations in the Nodal gene are associated withonditions that are dictated by improper establishment of LRsymmetry For example abnormal Nodal expression has beenssociated with nonsyndromic haloprosencephaly in which therain fails to divide into the left and right hemispheres normallySolomon et al 1993) and heterotaxy a disruption of normalR asymmetry of the thoracoabdominal cavity concomitant withefects in the heart and major vessels (Mohapatra et al 2009) Itas also been proposed that certain cancers more likely to arise onne side of the body may be regulated in part by Nodal For instancereast cancer is 5ndash10 more likely to occur in the left breast andelanomas are 10 more likely to develop on the left side of the
ody (Wilting and Hagedorn 2011) Indeed we and others haveemonstrated an important role for Nodal which specifies the leftide of the embryo during the progression of both of these can-er types (refer to Sections 51 and 58) However the concept thathe asymmetric roles of Nodal during embryonic development areinked to the emergence of asymmetric cancers later in adult life istill speculative
Nodal signalling in cancer
Nodalrsquos roles during embryogenesis dictate the pluripotenttate of embryonic stem cells and coincide with critical embry-nic EMT events (Mesnard et al 2006 Thiery et al 2009 Vallier
et al 2009b) Furthermore in non-embryonic tissue Nodal medi-ates highly dynamic and tightly regulated cellular behaviour(Bianco et al 2002 Nadeem et al 2011 Papageorgiou et al2009) One commonality amongst these contexts is the main-tenance or acquisition of cellular plasticity In the past decaderesearch has been focussed on identification of embryonic factorsthat are aberrantly expressed in cancer and that are responsi-ble for promoting cellular plasticity during cancer progressionHendrix and colleagues elegantly tackled this problem by usingthe zebrafish embryo as a functional in vivo reporter to gaininsight about what embryonic pathways might be activated incancer cells (Topczewska et al 2006) In their study aggres-sive melanoma cells (versus poorly aggressive melanoma cells)were injected into the animal pole of zebrafish embryos at theblastula stage and the effect of the melanoma cells on host devel-opment was observed after 6ndash8 h Interestingly the aggressivemelanoma cells were able to induce an almost-complete secondaryaxis but the poorly aggressive melanoma cells were not Thisnovel discovery was similar to the finding that injection of NodalRNA into zebrafish embryos could induce a secondary notochord(Toyama et al 1995) and that misexpression of a Nodal homo-logue in zebrafish embryos could induce an ectopic outgrowth(Thisse et al 2000) Together these data lead to the hypothesisthat Nodal might be a key mediator of melanoma tumourigene-sis
51 Melanoma
Hendrix and colleagues continued their investigation of Nodalin melanoma They found that aggressive melanoma cells seededon a hESC-conditioned matrix adopted a more melanocyte-likemorphology and less aggressive behaviour due to the secretionof Lefty by hESCs (Postovit et al 2008) In vivo transient inhibi-tion of Nodal in aggressive melanoma cell lines using morpholinooligonucleotides caused a significant reduction in tumourigen-esis and metastasis (Postovit et al 2007b Topczewska et al2006) In vitro Nodal receptor inhibition with a small moleculeinhibitor (SB431542 an ALK457 inhibitor) caused a transitiontowards a melanocyte-like differentiated state marked by upre-gulated Tyrosinase and downregulated VE-Cadherin and Keratin18 expression (Topczewska et al 2006) In human tissue sec-tions Nodal was expressed in vertical growth phase and metastaticmelanoma but was absent in normal skin (Topczewska et al 2006Yu et al 2010) This work has since been confirmed by othersusing a larger number of patient samples (Hooijkaas et al 2011)These latter studies indicate that Nodal is a common feature ofmore advanced (stages III and IV) melanomas but that it doesnot necessarily predict survival in these patients likely becausemany factors dictate progression at advanced stages Additionalstudies must be done to determine if the presence of Nodal inearly disease stages may predict progression Recent studies havealso linked Nodal to the ability of metastatic melanomas to co-opta vascular-like phenotype (Hardy et al 2010 Kirschmann et al2012 McAllister et al 2010) Indeed in situ hybridization hasshown that Nodal transcript localizes to melanoma cell-derivedvascular networks in melanomas Furthermore Hendrix and col-leagues have demonstrated that blocking Nodal signalling witha function-blocking antibody leads to a reduction in vasculo-genic mimicry by melanoma cells (Hardy et al 2010 McAllisteret al 2010) Together these findings demonstrate a role for Nodalduring invasion and aggressive behaviour of melanomas which
is not surprising given Nodalrsquos normal role in directing cellu-lar movements during development These findings also supportthe notion that cancer may be caused by deregulated embryonicsignals
8 f Bioc
5
betiIaie2kvpsAHecatgh
5
tCtcCnipcegaN
5
cgarrceimcaero
5
Nia
94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
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C
c
A
Nws8eBptP
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A
A
A
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B
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B
B
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96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
dkins HB Bianco C Schiffer SG Rayhorn P Zafari M Cheung AE et al Antibodyblockade of the Cripto CFC domain suppresses tumor cell growth in vivo Journalof Clinical Investigation 2003112575ndash87
l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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I
K
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L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
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metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
8 f Bioc
5
betiIaie2kvpsAHecatgh
5
tCtcCnipcegaN
5
cgarrceimcaero
5
Nia
94 DF Quail et al The International Journal o
2 Brain cancers
Several studies have revealed that Nodal may also promoterain cancer progression (De et al 2012 Hueng et al 2011 Leet al 2010) In one study Lee et al examined Nodal in the con-ext of glioblastoma multiforme (GBM) and reported that Nodals positively correlated with tumour grade in patient samplesn vitro Nodal overexpression in poorly invasive cell lines causedn increase in cellular invasion whilst Nodal knockdown in highlynvasive cell lines had the opposite effect (Lee et al 2010) Theffects of Nodal on invasion were shown to be mediated by MMP-
secretion and activation In vivo the authors showed that Nodalnockdown via shRNA reduced tumour burden and prolonged sur-ival in nude mice In accordance with these findings anotheraper by Peng and colleagues (2012) showed that Nodal promotespheroid formation and proliferation of U87 cells in vitro in anLK47-dependent manner (De et al 2012) Another paper byueng et al (2011) showed that Nodal correlated with vascularndothelial growth factor (VEGF) expression in human brain can-ers In this study Nodal expression in glioma cells was positivelyssociated with tumour size and vessel length in vivo Moreoverhis study suggested that Nodal may mediate its effects on tumourrowth and vascularization by increasing the normoxic levels ofypoxia-inducible factor-1 (HIF-1) (Hueng et al 2011)
3 Prostate and testicular cancers
Nodal has been reported to correlate with aggressive pheno-ypes in prostate and testicular cancer Nodal and its co-receptorripto have been shown to be overexpressed in tumourigenic tes-icular cancer cells such as NCCIT cells and their expression isorrelated with invasive phenotypes Interestingly inhibition ofriptondashNodal signalling reduces tumour growth of NCCIT cells inude mice (Adkins et al 2003) Nodal seems to have similar effects
n prostate cancer whereby Nodal is elevated in aggressive DU145rostate cancer cells compared to poorly aggressive LNCaP prostateancer cells (Lawrence et al 2011 Vo and Khan 2011) Nodal over-xpression in LNCaP cells causes elevated anchorage-independentrowth and invasion in vitro (Lawrence et al 2011) These resultsre similar to the effects of Nodal in glioma and melanoma wherebyodal mediates invasive and plastic phenotypes
4 Endometrial cancer
Nodal signalling has recently been implicated in endometrialancer (Papageorgiou et al 2009) This is particularly interestingiven the role of Nodal during normal endometrial cycling Duringdulthood the female endometrium undergoes a constant cycle ofemodelling events which include a menstrual phase a prolife-ative phase and a secretory phase Nodal has been shown to beyclically expressed during this process and is highest during thearly proliferative phase In contrast Nodalrsquos potent endogenousnhibitor Lefty reaches peak levels during the late secretory and
enstrual phase Interestingly patient biopsies of endometrial car-inoma showed a positive correlation between Nodal expressionnd grade and all samples exhibited a complete absence of Leftyxpression (Papageorgiou et al 2009) These results suggest thate-expression of Nodal might be governed in part by misexpressionf its endogenous inhibitors including Lefty
5 Ovarian cancer
Several studies conducted by Peng and colleagues have linkedodal with apoptosis in ovarian cancer Nodal overexpression
n ovarian cancer cells was associated with decreased metabolicctivity and proliferation (Xu et al 2004) Here Nodal was
hemistry amp Cell Biology 45 (2013) 885ndash 898
overexpressed in cancer cells that already express this morphogenhence it would be interesting to see the effects of Nodal inhibi-tion on advanced ovarian cancers Indeed based on its roles indevelopment it is possible that there is a concentration-dependentmulti-functionality of Nodal signalling and timing in metastaticprogression This would not be surprising since other membersof the TGF- superfamily tend to have variable concentration-dependent effects during embryology and cancer progressiondepending on temporal and spatial location (Bachman and Park2005 Hoffmann 1992 Meno et al 1996 Nishita et al 2000Soderberg et al 2009 Tian and Schiemann 2009)
56 Hepatocarcinoma
As previously mentioned the embryonic microenvironment iscapable of reprogramming aggressive cancer cells to become moreldquonormalrdquo Nodalrsquos endogenous inhibitor Lefty is a major mediatorof this effect as it is secreted by hESCs to abrogate the tumourgeniccapacity of Nodal-expressing cancer cells (Postovit et al 2008) Ofnote one paper by Cavallari et al (2012) found that conditionedmedia from human adult liver stem cells was capable of impair-ing tumourigenic phenotypes in Nodal-expressing HepG2 cells(Cavallari et al 2012) Interestingly HepG2 cells express Nodal butnot Lefty whereas human adult liver stem cells express both Nodaland Lefty proteins (Cavallari et al 2012) Accordingly this paperdemonstrated that the anti-tumourigenic effects of the liver stemcell conditioned media were due to the secretion of Lefty (Cavallariet al 2012) This article provides another excellent example of howcancer cells exhibit deregulated embryonic signals and in partic-ular how deregulated Nodal signalling in cancer can be controlledin embryonic environments
57 Pancreatic cancer
Heeschen and colleagues reported a correlation betweenNodalActivin signalling and pancreatic cancer Both Nodal andActivin were expressed at low levels in well-differentiated adher-ent pancreatic cancer cells and at high levels in non-adherentpancreatic spheroids that express elevated pluripotency markers(called ldquocancer stem cellsrdquo (CSCs) in this study) (Lonardo et al2011) Treatment of CSCs with rNodal was found to increasespheroid formation size and invasion in vitro (Lonardo et al 2011)Conversely ActivinNodal signalling inhibition in pancreatic cancercells with an ALK47 inhibitor eliminated the CSC subpopulationand rendered cells receptive to gemcitabine chemotherapy in vitroand in an orthotopic mouse model (Lonardo et al 2011) This grouphas further revealed that pancreatic stellate cells a prominent com-ponent of the tumour stroma secrete Nodal and Activin A Hencestellate-cell-derived Nodal at the tumourndashstromal interface can acton pancreatic cancer cells to enhance tumourigenicity and inva-sion (Lonardo et al 2012) Together the results from these studiesdemonstrate the robust effects of Nodal during disease progressionand in promoting stem cell-like phenotypes in pancreatic cancer
58 Breast cancer
Studies have demonstrated an important role for Nodal dur-ing breast cancer progression In a very recent article Nodal waspositively correlated with advanced breast tumour stage lymphnode status tumour grade and invasion in over 400 patient sam-ples (Strizzi et al 2012) Nodal was not correlated with oestrogenand progesterone receptor (ERPR) status or HER2 status in these
patients indicating that Nodal may be a marker for breast can-cer progression irrespective of sub-type and thus a therapeutictarget for triple negative disease This is exciting because tar-gets for this disease have remained elusive In this vein we and
f Bioc
ontaeNsBapmieotb2tiWbo
vibavcrai(v(pc(abt
6
6
tztRpridscpttNTet2
DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
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l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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H
H
I
K
K
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
T
T
T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
f Bioc
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vibavcrai(v(pc(abt
6
6
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DF Quail et al The International Journal o
thers have shown that Nodal inhibition in highly aggressive tripleegative breast cancer cell lines leads to a reduction in multipleumourigenic phenotypes including proliferation clonogenicityngiogenesis invasion and migration (Postovit et al 2008 Quailt al 2011 2012b 2012c in press Strizzi et al 2012) Given thatodal is an embryonic protein it is not surprising that its role in
ustaining the ldquoCSCrdquo signature in breast cancer has been exploredreast CSCs have been defined as CD44+CD24minus (and more recentlys expressing high levels of aldehyde dehydrogenase (ALDHhi)) theroportion of cells within a tumour population with this surfacearker expression is considered to be representative of tumour-
nitiating capacity (Al-Hajj et al 2003) In an elegant study Meyert al demonstrated that breast cancer subpopulations were capablef phenotypic switching whereby CD44+CD24minus isolated popula-ions were able to convert to CD44+CD24minus cells (and vice versa)oth in vitro and in mouse models of tumourigenesis (Meyer et al009) Interestingly this phenotypic switching was dependent onhe ActivinNodal pathway indicating that Nodal may play a rolen sustaining tumour-initiating cell populations in breast cancer
hether Nodal induces stem cell-like phenotypes in breast cancereyond this CD44+CD24minus signature such as during self-renewalr EMT has not been explored
We have recently shown that Nodal promotes breast cancerascularization (Quail et al 2012c) This was particularly surpris-ng since in its normal developmental context Nodal had nevereen reported to induce embryonic vasculogenesis Furthermorelthough Nodal has been correlated with VEGF expression andessel length in human gliomas (Hueng et al 2011) a functionalonnection between Nodal and vascular recruitment had not beeneported We discovered that Nodal is capable of recruiting a vascul-ture in vivo and that inducible inhibition of Nodal once a tumours already established results in collapse of the tumour vasculatureQuail et al 2012c) We also found that knockdown of Nodal pre-ented spontaneous metastasis of MDA-MB-231 cells to the liverQuail et al in press) Furthermore using nearly 100 patient sam-les we determined that Nodal protein expression is positivelyorrelated with microvascular density in primary breast cancersQuail et al 2012c) These are important findings as they implicate
role for Nodal in the vascularization and subsequent metastasis ofreast tumours and suggest that Nodal may be a tumour-specificarget for blocking angiogenesis and progression in patients
Regulation of Nodal expression
1 Silencing of Nodal expression in development
Maternal mRNAs are deposited into the egg prior to fertilizationo direct early embryonic development until the maternal-to-ygotic transition takes place In order for embryonic mRNAs toake over at this point maternal mRNAs need to be removedepression occurs via microRNA (miRNA) short pieces of RNA com-lementary to target mRNA sequences miRNA-430 (miR-430) isesponsible for clearance of maternal transcripts in the develop-ng zebrafish embryo (Giraldez et al 2006) Choi and colleagueseveloped a target protection approach designing morpholinospecific to miRNA binding sites in Lefty mRNAs to disrupt spe-ific miRNA-mRNA interactions (Choi et al 2007) Whilst Squintrotection resulted in stronger Nodal activation Lefty2 protec-ion resulted in increased Lefty signalling that in turn dampenedhat of Nodal Hence in zebrafish miRNAs regulate the balance ofodal and Lefty signals so that normal development can ensue
his miRNA is not expressed in humans however as describedarlier studies in trophoblast models suggest that miR-378a-5pargets the 3primeUTR of Nodal to inhibit protein translation (Luo et al012)
In mouse development Nodal is silenced at 8 dpc as stemcells differentiate along their designated lineages Interestinglymechanisms of Nodal silencingactivation have only recently beeninvestigated in this context Keuhn and colleagues (2010) describedone mechanism of Nodal silencing in mESC whereby Nodal geneexpression is repressed by polycomb-mediated trimethylation ofhistone H3 at Lysine 27 (H3K27me3) Interestingly this meth-ylation signature is reversible as re-activated Nodal-SMAD23signalling facilitates the recruitment of the demethylase Jmjd3 tothe Nodal locus to lift polycomb-induced methylations and activatetranscription of Nodal (Fig 2) Polycomb-induced transcriptio-nal repression was evident for other SMAD23 targets includingBrachyrury however transcriptional re-activation was specificallymediated by the Nodal-SMAD23 pathway (Dahle et al 2010)There is also a large CpG island surrounding the transcriptional startsite of Nodal Studies have shown that methylation in the regionis increased in cancer cells exposed to hESC-derived extracellu-lar matrices concomitant with reduced Nodal expression (Postovitet al 2007a) However these effects of the hESC-derived factorson Nodal expression appear to be transient and to date DNA meth-ylation has not been directly linked to the regulation of Nodalexpression Regardless these findings highlight the importanceof epigenetic regulation of Nodal transcription and how Nodalsignalling mediates other critical developmental signals
62 Re-expression of Nodal in cancer
The results from the developmental studies described abovesuggest that Nodal suppression after development is reversibleand lend insight into how Nodal expression might be reacti-vated in growing tumours Furthermore these studies demonstratehow developmental pathways impose transcriptional plasticity incells that allow for re-activation of developmental signals in non-developmental contexts like cancer It is likely that environmentalconditions such as pH or hypoxia also influence epigenetic reg-ulation of Nodal For example we have previously shown thathypoxia can induce Nodal signalling in poorly aggressive breastcancer and melanoma cells as well as in human ESCs through HIF-1-mediated activation of Nodal transcription (Quail et al 20112012a) Specifically we determined that under hypoxic conditionsHIF increases Notch signalling likely by stabilizing the intracel-lular domain Notch then activates the Nodal Specific Enhancercausing an up-regulation of Nodal Nodal then supports its ownexpression by initiating positive feedback loops In support of thisconcept Notch in particular Notch 4 has been shown to promoteNodal expression in melanoma cells and Nodal expression is dra-matically reduced when its signalling is blocked It is plausible thatthe induction of Nodal signalling initiates Jmjd3 recruitment andsubsequent demethylation of H3K27me3 in promoter regions asdescribed by Kuehn and colleagues (Dahle et al 2010) to induce arobust increase in Nodal autoregulation (Fig 2) This would not onlyexplain the striking induction of Nodal expression under hypoxicconditions but perhaps also how Nodal expression is sustainedafter re-oxygenation of cancer cells However regulation of Jmjd3by Nodal in the context of cancer has not yet been described
7 Conclusions
Nodal has recently emerged as an important pro-tumourigenicprotein Indeed we and others have determined that Nodal pro-motes tumour formation and progression in diseases as diverse
as melanoma breast cancer brain cancer and pancreatic cancer(Table 1) Nodalrsquos re-emergence in cancers originating from allthree germ layers is notable and likely due the expression of thisprotein in the early embryo before these layers are specified The
8 f Bioc
pifNainaFwdlstatm
C
c
A
Nws8eBptP
R
A
A
A
B
B
B
B
B
B
B
B
B
96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
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l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
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H
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L
L
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L
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L
M
M
M
M
M
M
M
M
M
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N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
T
T
T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
8 f Bioc
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C
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A
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R
A
A
A
B
B
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B
B
B
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96 DF Quail et al The International Journal o
resence of Nodal in advanced cancers as well as its relative absencen normal tissues make Nodal a potential therapeutic target Indeeduture studies are needed to develop targeted therapies againstodal or Nodal signalling and to test the safety and efficacy of suchpproaches in animals and then in humans As we move forward its also extremely important to understand how this morphogen sig-als in both normal and pathological contexts so that nuances suchs dose responses and tissue specific functions can be appreciatedinally as we forge ahead with regenerative medicine strategiese must be particularly cognisant of how altering Nodal signallinguring the expansion of hPSCs or the specification of mesendoderm
ineages may impact the epigenetic landscape and tumour formingusceptibility of cells used in replacement therapies Studies overhe next several years will deepen our understanding of Nodal as
morphogen with myriad functions and will determine whetherhis embryonic protein is a viable target for the treatment of the
ost advanced and life threatening cancers
onflict of interest statement
LMP is an inventor on 2 patents related to targeting Nodal inancers
cknowledgements
We would like to thank all contributors to the work done onodal thus far and to apologize to those whose investigations weere not able to include due to space restrictions This work was
upported by the Canadian Institutes for Health Research (MOP9714 MOP 119589 and PLS 95381) and the Cancer Research Soci-ty to LMP DQ was supported by fellowships from the Canadianreast Cancer Foundation and a CIHR training grant GMS is sup-orted in part by a Translational Breast Cancer Fellowship fromhe London Regional Cancer Program LMP is the recipient of theremier New Investigator Award from the CIHR
eferences
dkins HB Bianco C Schiffer SG Rayhorn P Zafari M Cheung AE et al Antibodyblockade of the Cripto CFC domain suppresses tumor cell growth in vivo Journalof Clinical Investigation 2003112575ndash87
l-Hajj M Wicha MS Ito-Hernandez A Morrison SJ Clarke MF Prospective identifi-cation of tumorigenic breast cancer cells Proceedings of the National Academyof Sciences of the United States of America 20031003983ndash8
lberio R Croxall N Allegrucci C Pig epiblast stem cells depend on activinnodalsignaling for pluripotency and self-renewal Stem Cells and Development2010191627ndash36
achman KE Park BH Duel nature of TGF-beta signaling tumor suppressor vs tumorpromoter Current Opinion in Oncology 20051749ndash54
eck S Le Good JA Guzman M Ben HN Roy K Beermann F et al Extraembry-onic proteases regulate Nodal signalling during gastrulation Nature Cell Biology20024981ndash5
ianco C Adkins HB Wechselberger C Seno M Normanno N De LA et al Cripto-1activates nodal- and ALK4-dependent and -independent signaling pathways inmammary epithelial cells Molecular and Cellular Biology 2002222586ndash97
lanchet MH Le Good JA Mesnard D Oorschot V Baflast S Minchiotti G et alCripto recruits Furin and PACE4 and controls Nodal trafficking during proteolyticmaturation EMBO Journal 2008a272580ndash91
lanchet MH Le Good JA Oorschot V Baflast S Minchiotti G Klumperman J et alCripto localizes Nodal at the limiting membrane of early endosomes ScienceSignaling 2008b1ra13
rennan J Lu CC Norris DP Rodriguez TA Beddington RS Robertson EJNodal signalling in the epiblast patterns the early mouse embryo Nature2001411965ndash9
rennan J Norris DP Robertson EJ Nodal activity in the node governs leftndashrightasymmetry Genes and Development 2002162339ndash44
risken C Park S Vass T Lydon JP OlsquoMalley BW Weinberg RA A paracrinerole for the epithelial progesterone receptor in mammary gland development
Proceedings of the National Academy of Sciences of the United States of America1998955076ndash81
rons IG Smithers LE Trotter MW Rugg-Gunn P Sun B Chuva de Sousa Lopes SMet al Derivation of pluripotent epiblast stem cells from mammalian embryosNature 2007448191ndash5
hemistry amp Cell Biology 45 (2013) 885ndash 898
Brown S Teo A Pauklin S Hannan N Cho CH Lim B et al ActivinNodal signalingcontrols divergent transcriptional networks in human embryonic stem cells andin endoderm progenitors Stem Cells 2011291176ndash85
Cai W Guzzo RM Wei K Willems E Davidovics H Mercola M A Nodal to TGF-beta cascade exerts biphasic control over cardiopoiesis Circulation Research2012488522ndash6
Cavallari C Fonsato V Herrera MB Bruno S Tetta C Camussi G Role of Lefty in theanti tumor activity of human adult liver stem cells Oncogene 2012 [Epub aheadof print]
Chen J Li Y Yu TS McKay RM Burns DK Kernie SG et al A restricted cell popu-lation propagates glioblastoma growth after chemotherapy Nature 2012488522ndash6
Cheng SK Olale F Brivanlou AH Schier AF Lefty blocks a subset of TGFbeta signalsby antagonizing EGF-CFC coreceptors PLOS Biology 20042E30
Choi WY Giraldez AJ Schier AF Target protectors reveal dampening and balancingof Nodal agonist and antagonist by miR-430 Science 2007318271ndash4
Clements M Pernaute B Vella F Rodriguez TA Crosstalk between Nodalactivin andMAPK p38 signaling is essential for anteriorndashposterior axis specification CurrentBiology 2011211289ndash95
Collignon J Varlet I Robertson EJ Relationship between asymmetric nodal expres-sion and the direction of embryonic turning Nature 1996381155ndash8
Conlon FL Lyons KM Takaesu N Barth KS Kispert A Herrmann B et al A primaryrequirement for nodal in the formation and maintenance of the primitive streakin the mouse Development 19941201919ndash28
Constam DB Riding shotgun a dual role for the epidermal growth factor-CriptoFRL-1Cryptic protein Cripto in Nodal trafficking Traffic 2009a10783ndash91
Constam DB Running the gauntlet an overview of the modalities of travel employedby the putative morphogen Nodal Current Opinion in Genetics and Develop-ment 2009b19302ndash7
Dahle O Kumar A Kuehn MR Nodal signaling recruits the histone demethylaseJmjd3 to counteract polycomb-mediated repression at target genes ScienceSignaling 20103ra48
Davies M Robinson M Smith E Huntley S Prime S Paterson I Induction of an epithe-lial to mesenchymal transition in human immortal and malignant keratinocytesby TGF-beta1 involves MAPK Smad and AP-1 signalling pathways Journal ofCellular Biochemistry 200595918ndash31
De ST Ye G Liang YY Fu G Xu G Peng C Nodal promotes glioblastoma cell growthFrontiers in Endocrinology (Lausanne) 2012359
Ding J Yang L Yan YT Chen A Desai N Wynshaw-Boris A et al Cripto is required forcorrect orientation of the anteriorndashposterior axis in the mouse embryo Nature1998395702ndash7
Dougan ST Warga RM Kane DA Schier AF Talbot WS The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm Development20031301837ndash51
Driessens G Beck B Caauwe A Simons BD Blanpain C Defining the mode of tumourgrowth by clonal analysis Nature 2012488527ndash30
Duboc V Lepage T A conserved role for the nodal signaling pathway in theestablishment of dorso-ventral and leftndashright axes in deuterostomes Jour-nal of Experimental Zoology Part B Molecular and Developmental Evolution200831041ndash53
Duboc V Rottinger E Lapraz F Besnardeau L Lepage T Leftndashright asymmetry in thesea urchin embryo is regulated by nodal signaling on the right side Develop-mental Cell 20059147ndash58
Feldman B Gates MA Egan ES Dougan ST Rennebeck G Sirotkin HI et al Zebrafishorganizer development and germ-layer formation require nodal-related signalsNature 1998395181ndash5
Fu G Peng C Nodal enhances the activity of FoxO3a and its synergistic interactionwith Smads to regulate cyclin G2 transcription in ovarian cancer cells Oncogene201130(37)3953ndash66
Gilbertson RJ Graham TA Cancer resolving the stem-cell debate Nature2012488462ndash3
Giraldez AJ Mishima Y Rihel J Grocock RJ Van DS Inoue K et al ZebrafishMiR-430 promotes deadenylation and clearance of maternal mRNAs Science200631275ndash9
Goumans MJ Mummery C Functional analysis of the TGFbeta receptorSmad path-way through gene ablation in mice International Journal of DevelopmentalBiology 200044253ndash65
Grande C Patel NH Lophotrochozoa get into the game the nodal pathway andleftright asymmetry in bilateria Cold Spring Harbor Symposia on QuantitativeBiology 2009a74281ndash7
Grande C Patel NH Nodal signalling is involved in leftndashright asymmetry in snailsNature 2009b4571007ndash11
Guzman-Ayala M Ben-Haim N Beck S Constam DB Nodal protein processing andfibroblast growth factor 4 synergize to maintain a trophoblast stem cell microen-vironment Proceedings of the National Academy of Sciences of the United Statesof America 200410115656ndash60
Hardy KM Kirschmann DA Seftor EA Margaryan NV Postovit LM Strizzi L et alRegulation of the embryonic morphogen Nodal by Notch4 facilitates mani-festation of the aggressive melanoma phenotype Cancer Research 20107010340ndash50
Hendrix MJ Seftor EA Seftor RE Kasemeier-Kulesa J Kulesa PM Postovit LM Repro-
gramming metastatic tumour cells with embryonic microenvironments NatureReviews Cancer 20077246ndash55
Herrera MB Bruno S Buttiglieri S Tetta C Gatti S Deregibus MC et al Isolation andcharacterization of a stem cell population from adult human liver Stem Cells2006242840ndash50
f Bioc
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K
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L
L
L
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L
L
L
L
M
M
M
M
M
M
M
M
M
N
N
N
O
O
P
DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
8 f Bioc
T
T
T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
f Bioc
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DF Quail et al The International Journal o
offmann FM TGF-beta family factors in Drosophila morphogenesis MolecularReproduction and Development 199232173ndash8
ooijkaas AI Gadiot J van BH Blank C Expression of the embryological morphogenNodal in stage IIIIV melanoma Melanoma Research 201121491ndash501
ueng DY Lin GJ Huang SH Liu LW Ju DT Chen YW et al Inhibition of Nodal sup-presses angiogenesis and growth of human gliomas Journal of Neuro-Oncology201110421ndash31
annaccone PM Zhou X Khokha M Boucher D Kuehn MR Insertional mutation of agene involved in growth regulation of the early mouse embryo DevelopmentalDynamics 1992194198ndash208
enney Adkins NJ Sanicola HB Nodal M Cripto-1 embryonic pattern formationgenes involved in mammary gland development and tumorigenesis Journal ofMammary Gland Biology and Neoplasia 20049133ndash44
irschmann DA Seftor EA Hardy KM Seftor RE Hendrix MJ Molecular pathwaysvasculogenic mimicry in tumor cells diagnostic and therapeutic implicationsClinical Cancer Research 2012182726ndash32
awrence MG Margaryan NV Loessner D Collins A Kerr KM Turner M et alReactivation of embryonic nodal signaling is associated with tumor progressionand promotes the growth of prostate cancer cells Prostate 2011711198ndash209
ee CC Jan HJ Lai JH Ma HI Hueng DY Gladys Lee YC et al Nodal promotes growthand invasion in human gliomas Oncogene 2010293110ndash23
ee KL Lim SK Orlov YL Yit LY Yang H Ang LT et al Graded NodalActivinsignaling titrates conversion of quantitative phospho-Smad2 levels into qual-itative embryonic stem cell fate decisions PLoS Genetics 20117e1002130
ee MK Pardoux C Hall MC Lee PS Warburton D Qing J et al TGF-beta activatesErk MAP kinase signalling through direct phosphorylation of ShcA EMBO Journal2007263957ndash67
onardo E Hermann PC Mueller MT Huber S Balic A Miranda-Lorenzo I et alNodalActivin signaling drives self-renewal and tumorigenicity of pancreaticcancer stem cells and provides a target for combined drug therapy Cell StemCell 20119433ndash46
onardo E Frias-Aldeguer J Hermann PC Heeschen C Pancreatic stellate cells forma niche for cancer stem cells and promote their self-renewal and invasivenessCell Cycle 2012111282ndash90
ong S Ahmad N Rebagliati M The zebrafish nodal-related gene southpaw isrequired for visceral and diencephalic leftndashright asymmetry Development20031302303ndash16
uo L Ye G Nadeem L Fu G Yang BB Honarparvar E et al MicroRNA-378a-5ppromotes trophoblast cell survival migration and invasion by targeting NodalJournal of Cell Science 20121253124ndash32
a GT Soloveva V Tzeng SJ Lowe LA Pfendler KC Iannaccone PM et al Nodal reg-ulates trophoblast differentiation and placental development DevelopmentalBiology 2001236124ndash35
cAllister JC Zhan Q Weishaupt C Hsu MY Murphy GF The embryonic morphogenNodal is associated with channel-like structures in human malignant melanomaxenografts Journal of Cutaneous Pathology 201037(Suppl 1)19ndash25
eno C Saijoh Y Fujii H Ikeda M Yokoyama T Yokoyama M et al Leftndashrightasymmetric expression of the TGF beta-family member lefty in mouse embryosNature 1996381151ndash5
esnard D Guzman-Ayala M Constam DB Nodal specifies embryonic visceral endo-derm and sustains pluripotent cells in the epiblast before overt axial patterningDevelopment 20061332497ndash505
eyer MJ Fleming JM Ali MA Pesesky MW Ginsburg E Vonderhaar BK Dynamicregulation of CD24 and the invasive CD44posCD24neg phenotype in breastcancer cell lines Breast Cancer Research 200911R82
fopou JK Chen B Sui L Sermon K Bouwens L Recent advances and prospects in thedifferentiation of pancreatic cells from human embryonic stem cells Diabetes2010592094ndash101
inchiotti G Nodal-dependant Cripto signaling in ES cells from stem cells to tumorbiology Oncogene 2005245668ndash75
ohapatra B Casey B Li H Ho-Dawson T Smith L Fernbach SD et alIdentification and functional characterization of NODAL rare variants in het-erotaxy and isolated cardiovascular malformations Human Molecular Genetics200918861ndash71
unir Xu S Wu G Yang Y Lala B Peng PK et al ALK7 inhibit proliferation andinduce apoptosis in human trophoblast cells Journal of Biological Chemistry200427931277ndash86
adeem L Munir S Fu G Dunk C Baczyk D Caniggia I et al Nodal signals throughactivin receptor-like kinase 7 to inhibit trophoblast migration and invasionimplication in the pathogenesis of preeclampsia American Journal of Pathology20111781177ndash89
ishita M Hashimoto MK Ogata S Laurent MN Ueno N Shibuya H et al Interactionbetween Wnt and TGF-beta signalling pathways during formation of Spemannrsquosorganizer Nature 2000403781ndash5
omura M Li E Smad2 role in mesoderm formation leftndashright patterning and cra-niofacial development Nature 1998393786ndash90
rmandy CJ Binart N Kelly PA Mammary gland development in prolactinreceptor knockout mice Journal of Mammary Gland Biology and Neoplasia19972355ndash64
sada SI Wright CV Xenopus nodal-related signaling is essential for mesendo-dermal patterning during early embryogenesis Development 1999126
3229ndash40
apageorgiou I Nicholls PK Wang F Lackmann M Makanji Y Salamonsen LA et alExpression of nodal signalling components in cycling human endometriumand in endometrial cancer Reproductive Biology and Endocrinology 20097122
Park CB DeMayo FJ Lydon JP Dufort D NODAL in the uterus is necessary for properplacental development and maintenance of pregnancy Biology of Reproduction201286194
Park CB Dufort D Nodal expression in the uterus of the mouse is regulatedby the embryo and correlates with implantation Biology of Reproduction2011841103ndash10
Patani R Compston A Puddifoot CA Wyllie DJ Hardingham GE Allen ND et alActivinNodal inhibition alone accelerates highly efficient neural conversionfrom human embryonic stem cells and imposes a caudal positional identityPLoS ONE 20094e7327
Postovit LM Costa FF Bischof JM Seftor EA Wen B Seftor RE et al The common-ality of plasticity underlying multipotent tumor cells and embryonic stem cellsJournal of Cellular Biochemistry 2007a101908ndash17
Postovit LM Margaryan NV Seftor EA Kirschmann DA Lipavsky A Wheaton WWet al Human embryonic stem cell microenvironment suppresses the tumori-genic phenotype of aggressive cancer cells Proceedings of the National Academyof Sciences of the United States of America 20081054329ndash34
Postovit LM Seftor EA Seftor RE Hendrix MJ Targeting Nodal in malig-nant melanoma cells Expert Opinion on Therapeutic Targets 2007b11497ndash505
Quail DF Taylor MJ Walsh LA Dieters-Castator D Das P Jewer M et al Low oxygenlevels induce the expression of the embryonic morphogen Nodal MolecularBiology of the Cell 2011224809ndash21
Quail DF Taylor MJ Postovit LM Microenvironmental regulation of cancerstem cell phenotypes Current Stem Cell Research amp Therapy 2012a7197ndash216
Quail DF Zhang G Walsh LA Siegers GM Dieters-Castator DZ Findlay SD et alEmbryonic morphogen nodal promotes breast cancer growth and progressionPLoS ONE 2012b7e48237
Quail DF Walsh LA Zhang G Findlay SD Moreno J Fung L et al Embry-onic protein nodal promotes breast cancer vascularization Cancer Research2012c723851ndash63
Quail DF Zhang G Findlay SD Hess DA Postovit LM Nodal promotes invasive phen-otypes via a Mitogen Activated Protein Kinase-dependent pathway Oncogenein press
Roberts HJ Hu S Qiu Q Leung PC Caniggia I Gruslin A et al Identification of novelisoforms of activin receptor-like kinase 7 (ALK7) generated by alternative splic-ing and expression of ALK7 and its ligand Nodal in human placenta Biology ofReproduction 2003681719ndash26
Schier AF Nodal signaling in vertebrate development Annual Review of Cell andDevelopmental Biology 200319589ndash621
Schier AF Axis formation squint comes into focus Current Biology200515R1002ndash5
Schier AF Nodal morphogens Cold Spring Harbor Perspectives in Biology20091a003459
Shen MM Nodal signaling developmental roles and regulation Development 2007Smith JC Mesoderm-inducing factors and mesodermal patterning Current Opinion
in Cell Biology 19957856ndash61Smith JR Vallier L Lupo G Alexander M Harris WA Pedersen RA Inhibition of
ActivinNodal signaling promotes specification of human embryonic stem cellsinto neuroectoderm Developmental Biology 2008313107ndash17
Soderberg SS Karlsson G Karlsson S Complex and context dependent regulationof hematopoiesis by TGF-beta superfamily signaling Annals of the New YorkAcademy of Sciences 2009117655ndash69
Solomon BD Gropman A Muenke M Holoprosencephaly overview 1993Strizzi L Hardy KM Margaryan NV Hillman DW Seftor EA Chen B et al Potential
for the embryonic morphogen Nodal as a prognostic and predictive biomarkerin breast cancer Breast Cancer Research 201214R75
Strizzi L Postovit LM Margaryan NV Seftor EA Abbott DE Seftor RE et al Emergingroles of nodal and Cripto-1 from embryogenesis to breast cancer progressionBreast Disease 20082991ndash103
Sutherland KD Lindeman GJ Visvader JE Knocking off SOCS genes in the mammarygland Cell Cycle 20076799ndash803
Tabibzadeh S Isolation characterization and function of EBAFLEFTY B rolein infertility Annals of the New York Academy of Sciences 2011122198ndash102
Tabibzadeh S Hemmati-Brivanlou A Lefty at the crossroads of ldquostemnessrdquo and dif-ferentiative events Stem Cells 2006241998ndash2006
Takahashi S Yokota C Takano K Tanegashima K Onuma Y Goto J et al Two novelnodal-related genes initiate early inductive events in Xenopus Nieuwkoop cen-ter Development 20001275319ndash29
Takaoka K Yamamoto M Shiratori H Meno C Rossant J Saijoh Y et al The mouseembryo autonomously acquires anterior-posterior polarity at implantationDevelopmental Cell 200610451ndash9
Tanaka S Kunath T Hadjantonakis AK Nagy A Rossant J Promotion of trophoblaststem cell proliferation by FGF4 Science 19982822072ndash5
Tang M Mikhailik A Pauli I Giudice LC Fazelabas AT Tulac S et al Decidual differ-entiation of stromal cells promotes proprotein convertase 56 expression andlefty processing Endocrinology 20051465313ndash20
Tesar PJ Chenoweth JG Brook FA Davies TJ Evans EP Mack DL et al New cell linesfrom mouse epiblast share defining features with human embryonic stem cells
Nature 2007448196ndash9
Thiery JP Acloque H Huang RY Nieto MA Epithelial-mesenchymal transitions indevelopment and disease Cell 2009139871ndash90
Thisse B Wright CVE Thisse C Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo Nature 2000403425ndash8
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metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
8 f Bioc
T
T
T
T
U
V
V
V
V
V
W
W
metabolism during ESC-to-EpiSChESC transition EMBO Journal 201231
98 DF Quail et al The International Journal o
ian M Schiemann WP The TGF-beta paradox in human cancer an update FutureOncology 20095259ndash71
opczewska JM Postovit LM Margaryan NV Sam A Hess AR Wheaton WW et alEmbryonic and tumorigenic pathways converge via Nodal signaling role inmelanoma aggressiveness Nature Medicine 200612925ndash32
oyama R OlsquoConnell ML Wright CV Kuehn MR Dawid IB Nodal induces ectopicgoosecoid and lim1 expression and axis duplication in zebrafish Development1995121383ndash91
oyoizumi R Ogasawara T Takeuchi S Mogi K Xenopus nodal related-1 isindispensable only for leftndashright axis determination International Journal ofDevelopmental Biology 200549923ndash38
lloa L Creemers JW Roy S Liu S Mason J Tabibzadeh S Lefty proteins exhibitunique processing and activate the MAPK pathway Journal of Biological Chem-istry 200127621387ndash96
allier L Mendjan S Brown S Chng Z Teo A Smithers LE et al ActivinNodalsignalling maintains pluripotency by controlling Nanog expression Develop-ment 2009a1361339ndash49
allier L Touboul T Chng Z Brimpari M Hannan N Millan E et al Early cell fatedecisions of human embryonic stem cells and mouse epiblast stem cells arecontrolled by the same signalling pathways PLoS ONE 2009b4e6082
incent SD Dunn NR Hayashi S Norris DP Robertson EJ Cell fate decisions withinthe mouse organizer are governed by graded Nodal signals Genes and Develop-ment 2003171646ndash62
isvader JE Lindeman GJ Mammary stem cells and mammopoiesis Cancer Research2006669798ndash801
o BT Khan SA Expression of nodal and nodal receptors in prostate stem cellsand prostate cancer cells autocrine effects on cell proliferation and migrationProstate 2011711084ndash96
atson CJ Khaled WT Mammary development in the embryo and adult a journeyof morphogenesis and commitment Development 2008135995ndash1003
ilting J Hagedorn M Leftndashright asymmetry in embryonic development andbreast cancer common molecular determinants Current Medicinal Chemistry2011185519ndash27
hemistry amp Cell Biology 45 (2013) 885ndash 898
Xi Q Wang Z Zaromytidou AI Zhang XH Chow-Tsang LF Liu JX et al Apoised chromatin platform for TGF-beta access to master regulators Cell20111471511ndash24
Xu G Zhong Y Munir S Yang BB Tsang BK Peng C Nodal induces apopto-sis and inhibits proliferation in human epithelial ovarian cancer cells viaactivin receptor-like kinase 7 Journal of Clinical Endocrinology and Metabolism2004895523ndash34
Yu L Harms PW Pouryazdanparast P Kim DS Ma L Fullen DR Expression of theembryonic morphogen Nodal in cutaneous melanocytic lesions Modern Pathol-ogy 2010231209ndash14
Yu L Li D Liao QP Yang HX Cao B Fu G et al High levels of activin a detectedin preeclamptic placenta induce trophoblast cell apoptosis by promotingnodal signaling Journal of Clinical Endocrinology and Metabolism 201297E1370ndash9
Zhang N Kumar M Xu G Ju W Yoon T Xu E et al Activin receptor-like kinase7 induces apoptosis of pancreatic beta cells and beta cell lines Diabetologia200649506ndash18
Zhao F Huang F Tang M Li X Zhang N Amfilochiadis A et al Nodal inducesapoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 beta-cells American Journal of Physiology Endocrinology and Metabolism2012303E132ndash43
Zhong Y Xu G Ye G Lee D Modica-Amore J Peng C Nodal and activin receptor-likekinase 7 induce apoptosis in human breast cancer cell lines Role of cas-pase 3 International Journal of Physiology Pathophysiology and Pharmacology20091(1)83ndash96
Zhou W Choi M Margineantu D Margaretha L Hesson J Cavanaugh Cet al HIF1alpha induced switch from bivalent to exclusively glycolytic
2103ndash16Zhou X Sasaki H Lowe L Hogan BL Kuehn MR Nodal is a novel TGF-beta-like
gene expressed in the mouse node during gastrulation Nature 1993361543ndash7
