[CANCER RESEARCH 60, 4010–4015, August 1, 2000]

Advances in Brief

Hypoxia-inducible Factor-1a Is a Positive Factor in Solid Tumor Growth

Heather E. Ryan, Michelle Poloni, Wayne McNulty, David Elson, Max Gassmann, Jeffery M. Arbeit, andRandall S. Johnson1

Department of Biology, University of California, San Diego, California 92093 [H. E. R., M. P., W. M., R. S. J.]; Cancer Genetics Program, University of California, SanFrancisco Comprehensive Cancer Center, San Francisco, California 94143 [D. E., J. M. A.]; and Institute of Physiology, University of Zurich-Irchel, CH-8057 Zurich,Switzerland [M. G.]

Abstract

Deficiencies in oxygenation are widespread in solid tumors. The tran-scription factor hypoxia-inducible factor (HIF)-1 a is an important medi-ator of the hypoxic response of tumor cells and controls the up-regulationof a number of factors important for solid tumor expansion, including theangiogenic factor vascular endothelial growth factor (VEGF). We haveisolated two cell lines nullizygous for HIF-1a, one from embryos geneti-cally null for HIF-1 a, and the other from embryos carrying loxP-flankedalleles of the gene, which allows for cre-mediated excision. The loss ofHIF-1a negatively affects tumor growth in these two sets of H-ras-trans-formed cell lines, and this negative effect is not due to deficient vascular-ization. Despite differences in VEGF expression, vascular density is sim-ilar in wild-type and HIF-1 a-null tumors. The evidence from theseexperiments indicates that hypoxic response via HIF-1a is an importantpositive factor in solid tumor growth and that HIF-1 a affects tumorexpansion in ways unrelated to its regulation of VEGF expression.

Introduction

To understand the biology of tumor growth, it is critical to alsounderstand the cellular response to changes in oxygen tension. Theprocess of tumor expansion is characterized by rapid growth as atumor establishes itself in the host. Accompanying this rapid growthare alterations in the microenvironment of the tumor cells, typicallycaused by an inability of the local vasculature to supply enoughoxygen and nutrients to rapidly dividing tumor cells (1). Whereas thehypoxia that results may inhibit new cell division or even lead to celldeath (2–5), it can also lead to adaptive responses that will help thecells survive (6). These responses include an induction of angiogen-esis and a switch to anaerobic metabolism; in addition, hypoxia canact as a selective force within a tumor for cells that harbor mutations,further improving their chance for survival (7). Hypoxia thus repre-sents a paradox for those studying tumor growth: although oxygendeprivation can have negative effects on cell growth, the hypoxicresponse can mitigate those effects and even drive critical tumorigenicadaptations.

Control of the hypoxic response in mammalian cells occurs througha number of mechanisms, primarily transcriptional and posttranscrip-tional mechanisms (8). The transcription factor HIF-12 is one of themajor regulators of hypoxic response (reviewed in Ref. 9) and wasfirst identified by Semenza and colleagues (10–12) as a regulator ofhypoxia-induced erythropoietin expression. The HIF-1 binding sitewas then found on a wide range of promoter elements of genesup-regulated by hypoxia. This provided the first indication that there

was a common mechanism regulating hypoxic response via transcrip-tion. The activation of transcription by HIF-1 occurs through theoxygen-regulated stabilization of HIF-1a, followed by its dimeriza-tion with ARNT, a constitutively expressed protein. Two other hy-poxia-responsive homologues of the HIF-1a gene have been cloned,yet there appears to be little redundancy in hypoxic response (13–17).In cells examined thus far, the loss of HIF-1a results in a total loss ofbinding to HIF-1 response elements (18, 19).

The hypoxic response would appear to promote tumor growth bypromoting cell survival; this likely occurs through its induction ofangiogenesis and its activation of anaerobic metabolism. Initial datahave indicated that this is likely the case because loss of either HIF-1aor ARNT has been shown to retard tumor growth (19, 20). Themechanism for this retardation appears to be decreased vasculariza-tion accompanied by an increase in apoptosis. The decrease in vas-cularization presumably occurs in part through a loss of HIF-1-mediated expression of a critical effector of tumor angiogenesis,VEGF. These tumor data support a model in which the primary roleof tumor hypoxia and the hypoxic response is to promote tumorangiogenesis. The role of HIF-1a as a tumor-promoting factor hasbecome a controversial point, however, because recent work by onegroup has indicated that HIF-1a acts as a tumor suppressor, ornegative factor, in ES cell-derived tumors (21).

We have further explored this important issue through the genera-tion of differentiated, genetically manipulated cell lines nullizygousfor HIF-1a. We report here the generation of wild-type and HIF-1a-null H-ras- transformed mEFs. Our findings confirm that HIF-1a actsas a positive regulator of tumor growth in this cell type as well.Surprisingly, we found no difference in vascular density betweenwild-type and null tumors, despite the fact that VEGF induction underhypoxia was significantly reduced bothin vitro and in vivo. Our datademonstrate that HIF-1a is a positive regulator of tumor growth afterH-ras transformation of fibroblasts and that the loss of HIF-1a altersVEGF expressionin vivo during solid tumor formation without aconcomitant effect on vascular density in null tumors. Furthermore,we demonstrate that the cre/loxP system will provide a useful way tounderstand the role of HIF-1a and the hypoxic response in otherprocesses.

Materials and Methods

Creation of Mice Carrying a loxP-flanked Allele of HIF-1 a. GenomicDNA was obtained as described previously (19). A loxP site was engineeredin the first intron through PCR, and a loxP-flanked neomycin resistancecassette was cloned into aSphI site in the second intron. The targeting vectorwas linearized, and 20mg of the vector were electroporated into R1 ES cells(22). The neomycin resistance cassette was removed by electroporation of 30mg of a cre-expressing plasmid into targeted cells [pML 78 (23, 24)]. PCR wasused to identify cell lines that had maintained loxP sites on either side of exon2. Chimeric mice were generated by injection of ES cells into C57Bl/6blastocysts (25).

Isolation of Wild-Type and HIF-1 a-null mEFs. Wild-type and HIF-1a-null embryos were harvested at embryonic day 9.5, dissociated by incubation

Received 4/9/00; accepted 6/14/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at University of California, SanDiego, Department of Biology, 9500 Gilman Drive, 0366, La Jolla, CA 92093-0366.Phone: (858) 822-0509; Fax: (858) 534-5831.

2 The abbreviations used are: HIF, hypoxia-inducible factor; VEGF, vascular endo-thelial growth factor; mEF, mouse embryonic fibroblast; ARNT, aryl hydrocarbon recep-tor nuclear translocator; ES, embryonic stem; TAg, T antigen.

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in 0.25% trypsin (Life Technologies, Inc.), and cultured. Embryos carrying twoloxP-flanked alleles of HIF-1a were harvested at embryonic day 13.5, disso-ciated by passage through an 18-gauge needle, and cultured. Cells wereimmortalized by stable transfection of SV40 large T antigen, using Super-fectamine (Qiagen) according to the manufacturer’s instructions and trans-formed by infection with a retrovirus expressing H-ras(26). The 1f/1f

ras/TAg cells were infected with adenovirus expressing eitherb-galactosidaseor cre recombinase.

The wild-type or null status of cells was confirmed by standard Southernblotting. Nuclear extracts were isolated from normoxic and hypoxic (4 h)cells by incubation in cell lysis buffer [10 mM Tris-HCl (pH 8.0), 1 mM

EDTA (pH 8.0), 150 mM NaCl, 0.5% NP40, 1mg/ml aprotinin, 1mg/mlpepstatin A, 1mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]and separation of the nuclei by centrifugation. Nuclei were lysed byincubation in a buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl,1 mM EDTA (pH 8.0), and 1 mM DTT. Extracts were analyzed by SDS-PAGE, electroblotting, and immunodetection with an anti-HIF-1a IgYantibody (27). Detection of HIF-1a was performed using a horseradishperoxidase-conjugated goat anti-IgY (Promega) secondary antibody andSuperSignal West Femto reagent from Pierce.

Target Gene Analysis.Cells were cultured for 0 or 8 h under hypoxia,and RNA was extracted with Trizol reagent (Life Technologies, Inc.)according to the manufacturer’s protocol. Approximately 15mg of totalRNA were loaded per lane, run on a 1% denaturing agarose gel, andhybridized with cDNA probes. Probes were generated as described in Ryanet al. (19).

Generation of Fibrosarcomas.A total of 1 3 107 cells were injected s.c.intrascapularly into immunocompromised mice, either RAG12/2 mice (28) or

nu/nu mice from Charles River. Tumors were harvested 16–18 days afterinjection, weighed, and processed for histology.

Tumor Histology. Sections were cut from frozen tissue and stained forCD31 as described previously. Vessel density in the most vascular regions ofthe tumor was determined with a Chalkley eyepiece graticule as described byFox et al. (29).

In situ hybridization was performed on paraformaldehyde-fixed, paraffin-embedded sections using35S-UTP-labeled riboprobes as described previously(30, 31).

Results

Creation of HIF-1a-null H-ras-transformed Cell Lines. Tocarry out initial characterization of differentiated cells that lack afunctional HIF-1a allele, we generated lines directly from HIF-1awild-type and null embryos. Embryos were harvested at embryonicday 9.5 and cells were immediately immortalized by stable trans-fection with SV40 large T antigen (32). The cells were transformedby infection with a retrovirus expressing the activated H-ras allele(26). Analysis of these cell lines by DNA and protein blottingconfirmed their wild-type or null status, and they are referred tohereafter as1/1 ras/TAg or 2/2 ras/TAg cell lines (Fig. 1,Cand D).

Homozygous deletion of HIF-1a results in embryonic death be-tween embryonic days 9 and 10 (18, 19). Cell lines isolated from thisearly stage of development might differ from standard mouse fibro-blasts in a number of respects. To control for these variables in this

Fig. 1. Generation of a loxP-flanked allele of HIF-1a. A, diagram of the strategy used to replace the endogenous HIF-1a locus with a loxP-flanked locus. Genomic structure aftertargeting and cre expression. A,AflIII; B, BamHI;Bg,BglII; P, PstI; X, XbaI.B, Southern blot analysis of the HIF-1a locus before cre expression showing aBamHI digest of genomicDNA and hybridization to a 59external probe.C, Southern blot analysis of genomic DNA from mEFras/TAg cell lines demonstrating either loss of the second exon (2f/2f, PstI/EcoRIdigest) or deletion of the second exon and intron (2/2,BamHI digest).D, Western blot analysis of mEFras/TAg cell lines showing induction of HIF-1a after 4 h of hypoxia (1%O2) in wild-type cells and loss of hypoxia-induced HIF-1a expression in the null cells.E, Northern blot analysis of 15mg of total RNA from wild-type and HIF-1a-null cells after0 or 8 h of hypoxia.Left, the cDNA probe used for hybridization.

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study, we also created HIF-1a-null cell lines via conditional targetingof the HIF-1a locus using the cre/loxP system (33).

We designed a conditional allele of HIF-1a in which the secondexon is flanked by loxP sites. The second exon encodes thehelix-loop-helix motif, which has been shown to be essential forHIF-1a dimerization with ARNT and subsequent transcriptionalactivation (34). The targeting vector contains a loxP site in the firstintron and a loxP-flanked neomycin resistance gene in the secondintron (Fig. 1A). Homologous recombination at the HIF-1a locusresults in an allele with a loxP site 59of exon 2 and the loxP-flanked neomycin resistance gene 39 of exon 2 (Fig. 1B). It ispossible that the presence of the neomycin resistance gene in thesecond intron could affect proper expression of HIF-1a; to safe-guard against this possibility, we transiently expressed cre recom-binase in the targeted ES cells. A fraction of the ES cells excisedthe neomycin resistance gene but retained 59 and 39 loxP sites,whose presence was confirmed by PCR (data not shown). These EScell clones were used for the generation of chimeras and mousestrains via blastocyst injection (25).

Mice containing loxP-flanked alleles of HIF-1a were crossed, andmEFs were harvested from embryonic day 13.5 embryos that werehomozygous (1f/1f) for the conditionally targeted allele. The cellswere transformed with SV40 large T antigen and H-rasas describedabove.

We next generated HIF-1a-null cells by transiently expressing crerecombinase to delete the loxP-flanked second exon. We used ade-novirus to transiently express cre recombinase in1f/1f ras/TAg celllines. An adenovirus expressingb-galactosidase was also used toinfect 1f/1f ras/TAg cells. Theseb-galactosidase-infected cells werethen used as wild-type controls in all of the following experiments andare referred to as1f/1f ras/TAg cells. Cre-infected cells are referredto as2f/2f ras/Tag cells. Four to five days after infection, cells wereassayed for excision of the second exon. DNA analysis via Southernblotting (Fig. 1C) and PCR (data not shown) indicated completeexcision of the second exon in the entire cell population. This wasfurther confirmed by analysis of nuclear extracts from the condition-ally targeted cells, which showed an absence of HIF-1a protein underhypoxic conditions (Fig. 1D).b-Galactosidase-infected cells main-tained a wild-type Southern profile and expressed HIF-1a underhypoxia (Fig. 1,C and D). Both cell lines grew at similar rates inculture and formed similar numbers of colonies in soft agar assays(data not shown).

Loss of Hypoxia-mediated Transcriptional Induction in HIF-1a-null mEFs. HIF-1a regulates a wide array of genes in response tohypoxia. The loss of HIF-1a in ES cells leads to a reduction inhypoxic expression of VEGF and a number of other genes at themRNA level (18, 19). We assayed both of the HIF-1a-null mEF celllines for loss of target gene expression by Northern blotting (Fig. 1E).In the 2f/2f ras/TAg cell line, the absence of HIF-1a lessens thehypoxic induction of VEGF and completely inhibits that of the hy-poxia-responsive genes phosphoglycerate kinase, lactate dehydrogen-ase, and glucose transporter-1. The situation is similar in the2/2ras/TAg cell line.

HIF-1a Is a Positive Regulator of Tumor Growth. The wild-type and null transformed cell lines described above were used tocreate fibrosarcomas in immunocompromised animals. Cells wereinjected s.c., and at 16 –18 days after injection, the tumors wereharvested and weighed. In both sets of cell lines, the absence ofHIF-1a resulted in a significant decrease in tumor mass (Fig. 2).

Hypoxia is considered to be a major stimulus for tumor angiogen-esis, and VEGF has been shown to be crucial for this process (35–37).Considering both the role of HIF-1a in hypoxia-induced VEGF ex-pression and previously published tumor data, we looked at the degree

of vascularization within wild-type and HIF-1a-null tumors. Tumorsections were stained for the endothelial cell marker CD31. Thisrevealed no obvious difference in tumor vasculature between wild-type and null tumors (Fig. 3A). Quantification of vascular densitythrough Chalkley analysis confirmed that there was no significantdifference in vessel density between wild-type and HIF-1a-null tu-mors (Fig. 3B).

Because we did not observe any decrease in vascular density inthe null tumors, we analyzed VEGF expression in1f/1f ras/TAgand 2f/2f ras/TAg tumors byin situ hybridization (Fig. 4). Intumors derived from2f/2f ras/TAg cells, which show a signifi-cant difference in hypoxic regulation of VEGF under hypoxia inculture, there was a clear difference in the expression pattern ofVEGF. In wild-type tumors, high levels of VEGF are expressed inwide swaths throughout the tumor, similar to the expression pat-terns in solid tumors reported elsewhere for this gene (20). The nulltumors also have regions of very high VEGF expression, but it ismore circumscribed, in some cases to single cells as opposed tolarge numbers of cells in a single region (Fig. 4B, magnification,3400); this results in a more punctate pattern of VEGF expressionin the HIF-1a-null tumors.

Discussion

Varying reports in the literature have made it unclear as to whatrole the HIF-1-mediated hypoxic response might have in tumorgrowth (19 –21). Conflicting evidence from ES cell-derived tumorshas indicated, on one hand, that HIF-1a acts as a positive regulatorof tumor growth (19), most likely through its activation of VEGF,and, on the other hand, that HIF-1a acts as a negative regulator oftumor growth, possibly through its stabilization of p53 in hypoxic

Fig. 2. Loss of HIF-1a leads to a reduction in tumor mass.A, analysis of tumor massfrom 1/1 ras/TAg (n5 10) and2/2ras/TAg (n5 10) tumors.B, analysis of tumor massfrom 1f/1f ras/TAg (n 5 20) and2f/2f ras/TAg (n 5 16) tumors. Statistical analysiswas performed using Statview (Abacus Software).

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cells (21, 38). A primary point of contention is whether HIF-1apromotes or inhibits tumor growth. To address this issue, we havegenerated a tumor model using H-ras-transformed fibroblasts fromwhich HIF-1a can be genetically removed. With this model, wehave demonstrated that HIF-1a clearly acts as a positive regulatorof tumor growth.

Data published previously on the mechanism by which HIF-1positively regulates tumor growth have focused on angiogenesis (19–21, 39). The consensus has been that the absence of either HIF-1a orits dimerization partner, ARNT, leads to reduced vascularizationwithin a tumor due to a reduced capacity to hypoxically induce VEGFexpression. We were therefore surprised to find that loss of HIF-1adid not alter tumor vascularization in H-ras-transformed fibrosarco-mas. This is in contrast to experiments in which VEGF itself is deletedfrom tumor cells, causing a large decrease in vascular density (35),and in contrast to the experimental evidence from ES cell-derivedtumors, where significant, albeit subtle, differences in vascular densityare seen (19, 21).

We considered the possibility that our observation might be due todifferences betweenin vitro andin vivo VEGF expression, where thetumor environment works in such a way as to make expressiondifferences seen in culture inconsequentialin vivo. This may explainour observations; however, within the tumors,in situ hybridizationdemonstrates that there are clear differences in VEGF expression

between1f/1f and2f/2f tumors. This difference is best seen in thehigher magnification in Fig. 4B, which shows that VEGF expressionwithin these tumors is more punctate and restricted. A possible ex-planation for the different expression pattern seen in the2f/2f tumorsis that a higher degree of hypoxia is required to stimulate VEGFexpression in these cells and that this more restricted expressionpattern is an indication of those regions. This high level of VEGFexpression from a smaller number of cells may be able to compensatefor a general reduction in VEGF expression, ultimately resulting in anadequate degree of vascularization.

The work presented here has clearly demonstrated that HIF-1acts to promote tumor growth. What has become less clear is theexact mechanism by which HIF-1 functions in this capacity. Ourfibrosarcoma model has provided the first indication that hypoxicinduction of angiogenesis may play a smaller role in tumor growththan previously thought and that HIF-1-mediated regulation ofVEGF is not crucial for tumor vascularization. With the multitudeof HIF-1 target genes, there are a number of possible mechanismsyet to be investigated, and in all likelihood, the effect of HIF-1 ontumor growth is complex and involves the activation of severaladaptive pathways. We also report for the first time the develop-ment of conditionally targeted mice and transformed cell linesfrom which HIF-1a can be easily excised via cre recombinaseexpression. These should prove invaluable reagents to investiga-

Fig. 3. Loss of HIF-1a does not affect vessel densitywithin tumors.A, CD31 staining of frozen tumor sectionsshowing no obvious difference in vessel density. Magni-fication, 3100.B, analysis of Chalkley scores confirmingno statistical difference in vessel density between wild-type and null tumors.

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tors wishing to study the role of HIF-1a during normal develop-ment and the role of hypoxic response during tumorigenesis.

Acknowledgments

We thank members of the Johnson laboratory for their support and advicethroughout the course of this work and Keith Laderoute, Amato Giaccia,Robert Warren, Michael Karin, Gregg Semenza, Ronald Wisdom, and FrankGiordano for reagents and helpful advice.

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HIF-1a PROMOTES TUMOR GROWTH

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2000;60:4010-4015. Cancer Res   Heather E. Ryan, Michelle Poloni, Wayne McNulty, et al.   Growth

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