REPORTLevels of Hypoxia-InducibleFactor-1� During BreastCarcinogenesis
Reinhard Bos, Hua Zhong, ColleenF. Hanrahan, Ellen C. M. Mommers,Gregg L. Semenza, Herbert M.Pinedo, Martin D. Abeloff, JonathanW. Simons, Paul J. van Diest, Elskenvan der Wall
Background: Hypoxia-inducible fac-tor-1 (HIF-1) is a transcription factorthat regulates gene expression in criti-cal pathways involved in tumor growthand metastases. In this report, we in-vestigated whether the level of HIF-1�is increased during carcinogenesis inbreast tissue and is associated withother tumor biomarkers. Methods: Par-affin-embedded clinical specimensfrom five pathologic stages of breast tu-morigenesis and from normal breasttissue were used. HIF-1� protein andthe biomarkers vascular endothelialgrowth factor (VEGF), HER-2/neu,p53, Ki-67, and estrogen receptor (ER)were identified immunohistochemical-ly, and microvessel density (a measureof angiogenesis) was determined. Asso-ciations among levels of HIF-1� andthese biomarkers were tested statisti-cally. All statistical tests are two-sided.Results: The frequency of HIF-1�-positive cells in a specimen increasedwith the specimen’s pathologic stage(P<.001, �2 test for trend) as follows:normal breast tissue (0 specimens with�1% HIF-1�-positive cells in 10 speci-mens tested), ductal hyperplastic le-sions (0 in 10), well-differentiated duc-tal carcinomas in situ (DCIS) (11 in 20),well-differentiated invasive breast can-cers (12 in 20), poorly differentiatedDCIS (17 in 20), and poorly differenti-ated invasive carcinomas (20 in 20). In-creased levels of HIF-1� were statisti-cally significantly associated with highproliferation and increased expressionof VEGF and ER proteins. In DCIS le-sions, increased levels of HIF-1� werestatistically significantly associatedwith increased microvessel density.HIF-1� showed a borderline associa-tion with HER-2/neu but no association
with p53. Conclusions: The level ofHIF-1� increases as the pathologicstage increases and is higher in poorlydifferentiated lesions than in the corre-sponding type of well-differentiated le-sions. Increased levels of HIF-1� areassociated with increased proliferationand increased expression of ER andVEGF. Thus, increased levels of HIF-1� are potentially associated with moreaggressive tumors. [J Natl Cancer Inst2001;93:309–14]
The role of hypoxia-inducible factor-1� (HIF-1�) is under increasing scrutinyby cancer researchers (1). HIF-1 bindsthe consensus sequence 5�-RCGTG-3�(where R is any purine) in the hypoxia-response elements of various target genes(2). HIF-1 activates the transcription ofmany genes controlling glucose transport-ers, glycolytic enzymes, gluconeogene-sis, high-energy phosphate metabolism,growth factors, erythropoiesis, heme me-tabolism, iron transport, vasomotor regu-lation, and nitric oxide synthesis (2,3)and, thus, may increase the survival oftumor cells under hypoxic conditions.Hypoxia influences the proliferation oftumor cells (4), the rate of apoptoticcell death (5), and metastasis (6). In ad-dition, by activating transcription of thevascular endothelial growth factor(VEGF) gene, HIF-1 is considered to be acentral initiator of angiogenic activity intumors (7–10).
The level of HIF-1� in cells is depen-dent on the intracellular oxygen concen-tration (11,12). When cells have normalconcentrations of oxygen, HIF-1� proteinis continuously degraded via the ubiquitinpathway. However, under low concentra-tions of oxygen (hypoxic conditions), theubiquitination of HIF-1� is blocked, theprotein is stabilized (11,13), and thus theprotein’s intracellular levels increase. Al-though the exact mechanism of oxygensensing remains to be elucidated, the cellprobably senses its oxygen concentrationthrough reactive oxygen species, so thatstabilization of the HIF-1� protein is saidto be redox induced (14).
A nuclear localization signal at the C-terminal end of HIF-1� allows its trans-port from the cytoplasm to the nucleus,where it forms an active HIF-1 complexby binding to HIF-1�. HIF-1� can form
heterodimers with other proteins, such asthe aryl hydrocarbon receptor (15), and isconstitutively and abundantly present(11,16). Thus, the amount of HIF-1� pro-tein in the nucleus is rate limiting anddetermines the functional activity of theHIF-1 complex (2).
The level of the HIF-1� protein is in-versely related to the oxygen tension incultured cells (12) and in vivo (11). Hy-poxic oxygen tensions that induce HIF-1�levels have been demonstrated in tumorsin vivo and are associated with a poorclinical outcome (6,17,18). HIF-1 activityalso is associated with tumor progressionand angiogenesis in xenograft assays (19–21). These observations and the fact thatincreased levels of HIF-1� are found inmany common human cancers at diagno-sis (1,2) suggest that HIF-1 has an impor-tant role in cancer progression (22).
Since the discovery of HIF-1 (23), thecharacteristics of HIF-1 have been ex-plored in many studies using cultured celllines. In this study, we focused on the roleof HIF-1� in human breast carcinogene-sis. Because angiogenesis is necessary forhyperplastic epithelial cells to progress tomalignant cells (24) and because HIF-1may induce angiogenesis by activatingtranscription of the VEGF gene, we pro-pose that HIF-1 plays a role in breast car-cinogenesis. This hypothesis is supportedby previous findings that VEGF, one ofthe most potent molecules in angiogene-sis, is increased in ductal carcinoma insitu (DCIS) (25). We tested this hypoth-esis by evaluating the levels of HIF-1� innormal breast tissue and in different
Affiliations of authors: R. Bos, E. C. M. Mom-mers, P. J. van Diest (Department of Pathology), H.M. Pinedo, E. van der Wall (Department of MedicalOncology), Free University Hospital, Amsterdam,The Netherlands; H. Zhong, J. W. Simons, WinshipCancer Institute, Emory University School of Medi-cine, Atlanta, GA; C. F. Hanrahan (Brady Urologi-cal Institute), G. L. Semenza (Departments of Pedi-atrics and Medicine, Institute of Genetic Medicine),M. D. Abeloff (Oncology Center), The Johns Hop-kins University School of Medicine, Baltimore, MD.
Correspondence to: Elsken van der Wall, M.D.,Ph.D., Department of Medical Oncology, Free Uni-versity Hospital, P.O. Box 7057, NL-1007 MB Am-sterdam, The Netherlands (e-mail: [email protected]).
stages of breast cancer development(usual ductal hyperplasia, DCIS, and in-vasive breast cancer) and by determiningwhether the level of HIF-1� was associ-ated with proliferation (Ki-67), microves-sel formation, and/or the expression ofVEGF, HER-2/neu, p53, and the estrogenreceptor (ER).
MATERIALS AND METHODS
The level of HIF-1 was examined in randomlyselected samples of breast tissue from patients.These samples had been deposited in the breast can-cer tumor banks of the pathology departments of theFree University Hospital, Amsterdam, The Nether-lands, and the Gooi-Noord Hospital, Blaricum, TheNetherlands. Normal breast tissue was from reduc-tion mammoplasties performed on patients withoutproliferative breast disease. Specimens of pure duc-tal hyperplasia, pure DCIS, and invasive carcinomawere obtained from excision biopsy procedures ormastectomies. None of the patients with invasivebreast cancer had received any preoperative therapy.All specimens were fixed in neutral 4% bufferedformaldehyde. Informed consent to anonymouslyuse leftover patient material for scientific purposeswas a standard item in the treatment contract withthe patients.
We examined normal breast tissue (from 10 pa-tients), usual ductal hyperplasia (from 10 patients),well-differentiated DCIS (from 20 patients), poorlydifferentiated DCIS (from 20 patients), invasive car-cinoma grade 1 (from 20 patients), and invasive car-cinoma grade 3 (from 20 patients). Grading of DCISand of invasive cancer was done according to theprocedure of Holland et al. (26) and Elston and Ellis(27), respectively. The grading pathologist (P. J. vanDiest) was blinded in scoring all specimens for HIF-1� with respect to other biomarkers.
Immunohistochemistry
Table 1 presents all antibodies, dilutions, incuba-tion times, and antigen-retrieval methods used. Im-munohistochemistry was performed on 4-�m-thickslides. After deparaffinization and rehydration, en-dogenous peroxidase activity was blocked for 30minutes in methanol containing 0.3% hydrogen per-oxide. After antigen retrieval, a cooling-off period of20 minutes preceded the incubation of the primaryantibody. Thereafter, the catalyzed signal amplifica-tion system (DAKO, Glostrup, Denmark) was usedfor HIF-1� staining according to the manufacturer’sinstructions. All other antibodies were detected by astandard avidin–biotin complex method with a bio-tinylated rabbit anti-mouse antibody (DAKO) andan avidin–biotin complex (DAKO). All stainingswere developed with diaminobenzidine. Before theslides were mounted, all sections were counter-stained for 45 seconds with hematoxylin and dehy-drated in alcohol and xylene. Appropriate negativecontrols (obtained by omission of the primary anti-body) and positive controls were used throughout;for HIF-1�-negative and -positive controls, respec-tively, we used normoxic and hypoxic prostate can-cer TSU cells, which were embedded in paraffin andprovided by Dr. A. M. DeMarzo (The Johns Hop-kins University School of Medicine, Baltimore,MD).
Quantification
For HIF-1� staining, only cells with completelyand darkly stained epithelial nuclei were regarded aspositive, and this nuclear staining was interpreted asan increased level; cytoplasmic staining, observedoccasionally, was ignored because active HIF-1 islocated only in the nucleus. The fraction of nucleiwith an increased level of HIF-1� or p53 or Ki-67positivity was estimated visually by two observers(R. Bos and P. J. van Diest). In DCIS specimens, theangiogenic activity was assessed by scoring thepresence of a vascular rim around the ducts and byestimating the microvessel density in the surround-ing stroma as described previously by Guidi et al.(28). In invasive cancers, microvessels were manu-ally counted by use of an ocular grid at a magnifi-cation of ×400, following the criteria of Weidner etal. (29,30) as described previously (31). In short, infour adjacent fields of vision in the most vascular-ized area (“hot spot,” total area � 0.6 mm2), mi-crovessels were counted and expressed as the mi-crovessel density per millimeters square.
HER-2/neu staining was scored as negative orpositive (membrane staining). VEGF expressionwas scored as weakly or strongly positive. ER statuswas determined by the evaluation of the histoscoreas described previously (32); a histoscore of 100 ormore was regarded as positive.
Statistical Methods
To evaluate whether the frequency of cells withthe elevated levels of HIF-1� increased duringbreast carcinogenesis, we performed a �2 test fortrend, grouping the results as normal, hyperplasia,DCIS, or invasive cancer. Associations between in-creased levels of HIF-1� and the other biomarkerswere analyzed with Fisher’s exact test. The meanpercentages of HIF-1�-positive cells in the differenthistologic groups were compared with the Mann–Whitney test. All analyses were performed with theSPSS package of computer programs for Windows,version 9.0.1, 1999 (SPSS Inc., Chicago, IL). P val-ues of less than .05 were regarded as statisticallysignificant. All statistical tests are two-sided.
RESULTS
A summary of the HIF-1� nuclearstaining in normal breast tissue and tis-sues from five stages of breast carcino-genesis is provided in Table 2. Normalbreast tissue (n � 10) and hyperplasticlesions (n � 10) showed no detectableHIF-1�. In contrast, increased levels ofHIF-1� were found in well-differentiatedDCIS specimens (11 specimens had�1% immunopositive cells of 20 speci-mens tested), with the number of positivespecimens further increasing in well-differentiated invasive breast (12 of 20).This trend continued with poorly differ-entiated DCIS lesions (17 of 20) andpoorly differentiated invasive carcinomas(20 of 20). In 28 of 30 HIF-1�-positivespecimens that contained necrosis, re-gional HIF-1� positivity was especiallynoted around the areas of necrosis (Fig. 1,D). Occasionally, a few cells with in-creased HIF-1� levels were observed inductal hyperplastic areas adjacent to inva-sive cancer and in areas of atypical ductalhyperplasia.
Statistical analysis (�2 test) of the HIF-1� data showed a statistically significantincrease in the number of cells with in-creased levels of HIF-1� over the pro-gression spectrum (normal � 0 of 10; hy-perplasia � 0 of 10; DCIS � 28 of 40;invasive cancer � 32 of 40; P<.001).
More poorly differentiated (DCIS andinvasive cancer) lesions (37 of 40) thanthe corresponding well-differentiated le-sions (23 of 40) showed increased levelsof HIF-1� (P<.001). Well-differentiated
Table 1. Antibodies, dilution, incubation, and detection methods used*
Antibody Species Company DilutionIncubation
timeAntigen-retrieval
method Detection
HIF-1� Mouse MAb Abcam 1 : 1000 30 min,20 °C
WB, TRS, 95 °C,45 min
CSA
CD31 Mouse MAb DAKO 1 : 40 o/n, 4 °C MW, Citr., 95 °C,10 min
ABC
VEGF Goat poly R&D 1 : 50 o/n, 4 °C MW, Citr., 95 °C,10 min
ABC
Ki-67 Mouse MAb Immunotech 1 : 40 o/n, 4 °C MW, Citr., 95 °C,10 min
ABC
Her-2/neu Mouse MAb MvdVijver 1 : 10 000 o/n, 4 °C None ABCp53 Mouse MAb DAKO 1 : 50 o/n, 4 °C MW, Citr., 95 °C,
10 minABC
ER Mouse MAb DAKO 1 : 50 o/n, 4 °C MW, Citr., 95 °C,10 min
and poorly differentiated DCIS lesionshad statistically different levels of HIF-1�(P � .028), as did well-differentiated andpoorly differentiated invasive breast can-cers (P<.001). Also, the percentage ofcells with increased levels of HIF-1� var-ied with different pathology, with a sta-tistically significant higher (P<.001)percentage of positive cells in poorly dif-ferentiated specimens (Table 2).
Table 3 shows comparisons of in-creased levels of HIF-1� with variousbiomarkers. Even in this small dataset,VEGF expression (P � .001), Ki-67 ex-pression (P<.001) (10% threshold), andER status (P � .001) were highly posi-tively associated with increased levels ofHIF-1�. HER-2/neu showed a positive,but borderline, association (P � .053)with HIF-1�. However, p53 expression(5% threshold for positivity) was not as-sociated with increased levels of HIF-1�.In the invasive cancers, a nearly positiveassociation was found between HIF-1�and microvessel density (P<.120). Stro-mal vascular density in DCIS showed astatistically significant positive associa-tion with HIF-1� (P � .041), but vascu-lar rim formation did not (P � 1.00).
HIF-1� positivity was found in 29 of34 specimens containing necrosis com-pared with 31 of 46 specimens withoutnecrosis. The mean percentage of HIF-1�-positive cells was higher in specimenswith necrosis (P � .019), a trend alsovisible in the subgroups of DCIS (P �.08) and invasive lesions (P � .02).
DISCUSSION
The purpose of this study was to de-termine whether increased levels of HIF-1� could be detected during differentstages of carcinogenesis in human breasttissue. HIF-1� appears to be involved inangiogenesis during prostate carcinogen-esis (33), and recent data (34) show thatincreased levels of HIF-1� represent anunfavorable characteristic in early cervi-
Table 2. Increased hypoxia-inducible factor-1� (HIF-1�) level in tissues at different stages duringbreast carcinogenesis
No. HIF-1� positivity,* No.
% HIF-1�†
Mean Range
Normal breast tissue 10 0 0 0–0Usual hyperplasia 10 0 0 0–0DCIS, well differentiated‡ 20 11 3.5 0–15DCIS,‡ poorly differentiated 20 17 14.9 0–50Invasive, well differentiated 20 12 11.8 0–53Invasive, poorly differentiated 20 20 15.7 1–53
*HIF-1� positivity � 1% or higher positive cells (per tissue sample).†Mean percentage of positive cells (per tissue sample).‡DCIS � ductal carcinoma in situ.
Table 3. Increased hypoxia-inducible factor-1� (HIF-1�) level associated with HER-2/neu, vascular endothelial growth factor (VEGF),estrogen receptor (ER), p53, Ki-67, and microvessed density (MVD)
*DCIS � ductal carcinoma in situ.†Fisher’s two-sided exact test.
Fig. 1. Immunohistochemical analysis of hypoxia-inducible factor-1� (HIF-1�) in normal breast tissue (A)and in hyperplasia (B) shows no increase in HIF-1�. Well-differentiated ductal carcinoma in situ (DCIS) (C)and poorly differentiated DCIS (D) show a striking pattern of increased HIF-1� around necrosis (N). Awell-differentiated ductal carcinoma shows HIF-1� positivity (E). A poorly differentiated medullary breastcarcinoma shows increased regional levels of HIF-1� (F). Scale bar � 100 �m.
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cal cancer. In this study, we did not detectincreased levels of HIF-1� in specimensfrom normal breast and areas with ductalhyperplasia, but we did detect increasedlevels in the majority of DCIS and inva-sive cancer specimens. Levels of HIF-1�increased as the degree of malignancy in-creased, confirming earlier pilot data (1)suggesting that HIF-1� may be a biomar-ker of preinvasive human breast cancers.In addition, we observed that an increasedlevel of HIF-1� was associated with highproliferation, strong VEGF expression,and ER positivity as well as with angio-genesis but only in the subgroup of DCISlesions. To our knowledge, this is the firstreport to implicate increased levels ofHIF-1� in early human breast carcinogen-esis.
A statistically significant associationwas observed between increased levels ofHIF-1� and VEGF expression. Further-more, a positive association was observedbetween increased levels of HIF-1� andintratumoral microvessel density in DCIS,supporting a role for HIF-1� in angiogen-esis during breast carcinogenesis. Ourfindings are consistent with a previous re-port (4) in xenografts of animal tumorsand transgenic models, which showedHIF-1� involvement in the angiogenicphenotype of cancer.
It is interesting that increased levels ofHIF-1� were most pronounced in poorlydifferentiated lesions, which supports thepreviously proposed progression modelfor breast cancer (35,36). In this model,well-differentiated cancers arise fromwell-differentiated precursor lesions andpoorly differentiated cancers from poorlydifferentiated precursor lesions. Poorlydifferentiated lesions (in the preinvasiveand invasive states) are clinically moreaggressive. The observed increased levelsof HIF-1� in poorly differentiated DCISmay indicate a higher likelihood that thislesion will acquire invasive properties andthat the resulting poorly differentiated in-vasive lesions will have a poorer progno-sis.
The association between increased lev-els of HIF-1� and ER status at firstseemed surprising but was consistent withthe following data: HIF-1� is known tostimulate the production of VEGF (7),and the VEGF gene has functional ERresponse elements (37). Furthermore, es-trogen stimulation increases phosphati-dylinositol 3-hydroxykinase activity (38),which belongs to a signaling pathway thatmay play a role in HIF-1� activation (33).
The relationship of estrogen action, ERinteractions, and HIF-1�-driven genes inbreast cancer certainly merits further in-vestigation.
Several mechanisms, which are notmutually exclusive, that induce elevatedlevels of HIF-1� may be active in breastcarcinogenesis. First, neoplastic breast le-sions may be hypoxic so that HIF-1� isinduced by low oxygen tension, as dem-onstrated in cultured cells (39), animalmodels (16), and hypoxic myocardium(40). Such hypoxic conditions occur insolid cancers, but it is not yet knownwhether this holds true in DCIS (micro-hypoxia). It is interesting to note, how-ever, the presence of HIF-1�-positivecells around areas of necrosis. Cells withincreased levels of HIF-1� were alsofound regularly around areas of necrosisin invasive cancers. In poorly differenti-ated DCIS and invasive lesions, necrosisoccurred more frequently. The differencein levels of HIF-1� protein between well-differentiated and poorly differentiated le-sions may, therefore, also be hypoxia re-lated. Studies addressing the intratumoraloxygen level in human breast cancer arerequired to define this important epige-netic parameter, which may have func-tional effects on HIF-1-activated genes.
Second, activated oncogenes and loss-of-function mutations in tumor suppressorgenes, such as PTEN (phosphatase andtensin homolog deleted on chromosome10), VHL (von Hippel–Lindau tumor sup-pressor gene), and p53, have been shownto modulate HIF-1� levels in certain tu-mor types (2). For example, loss of p53function has been shown in vitro to aug-ment HIF-1 and VEGF expression (21)and, under hypoxic conditions, HIF-1 isphosphorylated by extracellularly regu-lated kinases (41,42), confirming the po-tential role of the PI(3)K and mitogen-activated protein kinase pathways andupstream oncogenes. HER-2/neu acti-vates both pathways, and mutations in thisgene are among the most common geno-mic alteration in DCIS and early breastcancer. In our study, HIF-1� levels andHER-2/neu status were indeed positivelyassociated. Increased levels of HIF-1�can also occur as an effect of growth fac-tor activation of the PI(3)K/AKT (proteinkinase B)/FRAP (FK506 binding protein[FKBP])-rapamycin-associated proteinpathway (33,43), as has been shown intumor types other than breast cancer.
Third, activated immune responsesduring inflammation by the proinflamma-
tory cytokines interleukin 1� and tumornecrosis factor-� (44) may also modulateHIF-1 activity in tumors. Both cytokinesare important in the growth and differen-tiation of human breast cancer (45,46).
Finally, this study suggests breast can-cer angiogenesis may be driven in part byHIF-1. We detected a statistically signifi-cant positive association between in-creased levels of HIF-1� protein andVEGF expression in human breast tissue.HIF-1� activates angiogenesis by stimu-lating VEGF transcription (7), and VEGFis induced by both oncogenic transforma-tion and hypoxia (21,47,48). Increasedexpression of VEGF and its receptorsFlt-1 and Flk-1 have been demonstratedin breast cancer (49,50). Increased VEGFexpression in primary breast cancer con-fers a poorer prognosis at clinical presen-tation. Increased angiogenesis has alsobeen reported in DCIS (29). In our study,increased levels of HIF-1� were posi-tively associated with both VEGF and mi-crovessel density. These data support anangiogenesis-inducing role for HIF-1�during breast carcinogenesis. HIF-1� im-munostaining may serve as a surrogatebiomarker of angiogenic potential ofbreast cancer and deserves further large-scale clinical investigations.
It was interesting to note occasionalnuclei with increased levels of HIF-1� inusual ductal hyperplasia next to invasivecancers and areas of atypical ductal hy-perplasia. Usual ductal hyperplasia (andeven normal lobules) next to invasivecancer has been shown to harbor morpho-logic (51) and genetic (52) changes and,thus, should be considered to be a moreadvanced lesion than pure ductal hyper-plasia. Atypical ductal hyperplasia shouldbe placed between usual ductal hyperpla-sia and well-differentiated DCIS in thebreast progression spectrum (36) and somay be the earliest pure preinvasivebreast lesion with increased levels ofHIF-1�.
Increased levels of HIF-1� proteinwere observed at the earliest pathologi-cally detectable stages of breast cancercarcinogenesis and were increased in de-differentiated malignant tissue. Thus, weurge that therapeutics specifically target-ing and inhibiting HIF-1 (33,53,54) be ra-tionally tested to prevent malignant pro-gression in early breast cancer.
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NOTES
Supported by the AEGON International Scholar-ship in Oncology (Public Health Service [PHS]grant CA58236 [to R. Bos] from the National Can-cer Institute [NCI], National Institutes of Health,Department of Health and Human Services); by theAvon Breast Cancer Crusade (H. Zhong, J. W. Si-mons); by grant DAMD 17–98-1–8475 from theU.S. Department of Defense; by the CaPCure Foun-dation; and by PHS grant CA58236 from the NCI.
We thank Dr. A. M. DeMarzo (Department ofPathology, The Johns Hopkins University School ofMedicine, Baltimore, MD) for providing the embed-ded HIF-1� TSU-expressing cell line, Dr. H.V. Stel(a pathologist at the Gooi-Noord Hospital, Blaricum,The Netherlands) for some tumor material, Mrs. P.van der Groep for technical assistance with the im-munostaining, and Mrs. K. Heaney for assistance inpreparing the manuscript for submission.
Manuscript received June 15, 2000; revised De-cember 12, 2000; accepted December 19, 2000.
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