[CANCER RESEARCH 60, 4010 – 4015, August 1, 2000]
Advances in Brief
Hypoxia-inducible Factor-1␣ Is a Positive Factor in Solid Tumor Growth
Heather E. Ryan, Michelle Poloni, Wayne McNulty, David Elson, Max Gassmann, Jeffery M. Arbeit, and
Randall 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, San
Francisco 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 transcription factor hypoxia-inducible factor (HIF)-1␣ is an important mediator of the hypoxic response of tumor cells and controls the up-regulation
of a number of factors important for solid tumor expansion, including the
angiogenic factor vascular endothelial growth factor (VEGF). We have
isolated two cell lines nullizygous for HIF-1␣, one from embryos genetically null for HIF-1␣, and the other from embryos carrying loxP-flanked
alleles of the gene, which allows for cre-mediated excision. The loss of
HIF-1␣ negatively affects tumor growth in these two sets of H-ras-transformed cell lines, and this negative effect is not due to deficient vascularization. Despite differences in VEGF expression, vascular density is similar in wild-type and HIF-1␣-null tumors. The evidence from these
experiments indicates that hypoxic response via HIF-1␣ is an important
positive factor in solid tumor growth and that HIF-1␣ affects tumor
expansion in ways unrelated to its regulation of VEGF expression.
Introduction
To understand the biology of tumor growth, it is critical to also
understand the cellular response to changes in oxygen tension. The
process of tumor expansion is characterized by rapid growth as a
tumor establishes itself in the host. Accompanying this rapid growth
are alterations in the microenvironment of the tumor cells, typically
caused by an inability of the local vasculature to supply enough
oxygen and nutrients to rapidly dividing tumor cells (1). Whereas the
hypoxia that results may inhibit new cell division or even lead to cell
death (2–5), it can also lead to adaptive responses that will help the
cells survive (6). These responses include an induction of angiogenesis and a switch to anaerobic metabolism; in addition, hypoxia can
act as a selective force within a tumor for cells that harbor mutations,
further improving their chance for survival (7). Hypoxia thus represents a paradox for those studying tumor growth: although oxygen
deprivation can have negative effects on cell growth, the hypoxic
response can mitigate those effects and even drive critical tumorigenic
adaptations.
Control of the hypoxic response in mammalian cells occurs through
a number of mechanisms, primarily transcriptional and posttranscriptional mechanisms (8). The transcription factor HIF-12 is one of the
major regulators of hypoxic response (reviewed in Ref. 9) and was
first identified by Semenza and colleagues (10 –12) as a regulator of
hypoxia-induced erythropoietin expression. The HIF-1 binding site
was then found on a wide range of promoter elements of genes
up-regulated by hypoxia. This provided the first indication that there
was a common mechanism regulating hypoxic response via transcription. The activation of transcription by HIF-1 occurs through the
oxygen-regulated stabilization of HIF-1␣, followed by its dimerization with ARNT, a constitutively expressed protein. Two other hypoxia-responsive homologues of the HIF-1␣ 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-1␣ results in a total loss of
binding to HIF-1 response elements (18, 19).
The hypoxic response would appear to promote tumor growth by
promoting cell survival; this likely occurs through its induction of
angiogenesis and its activation of anaerobic metabolism. Initial data
have indicated that this is likely the case because loss of either HIF-1␣
or ARNT has been shown to retard tumor growth (19, 20). The
mechanism for this retardation appears to be decreased vascularization accompanied by an increase in apoptosis. The decrease in vascularization presumably occurs in part through a loss of HIF-1mediated expression of a critical effector of tumor angiogenesis,
VEGF. These tumor data support a model in which the primary role
of tumor hypoxia and the hypoxic response is to promote tumor
angiogenesis. The role of HIF-1␣ as a tumor-promoting factor has
become a controversial point, however, because recent work by one
group has indicated that HIF-1␣ acts as a tumor suppressor, or
negative factor, in ES cell-derived tumors (21).
We have further explored this important issue through the generation of differentiated, genetically manipulated cell lines nullizygous
for HIF-1␣. We report here the generation of wild-type and HIF-1␣null H-ras- transformed mEFs. Our findings confirm that HIF-1␣ acts
as a positive regulator of tumor growth in this cell type as well.
Surprisingly, we found no difference in vascular density between
wild-type and null tumors, despite the fact that VEGF induction under
hypoxia was significantly reduced both in vitro and in vivo. Our data
demonstrate that HIF-1␣ is a positive regulator of tumor growth after
H-ras transformation of fibroblasts and that the loss of HIF-1␣ alters
VEGF expression in vivo during solid tumor formation without a
concomitant effect on vascular density in null tumors. Furthermore,
we demonstrate that the cre/loxP system will provide a useful way to
understand the role of HIF-1␣ and the hypoxic response in other
processes.
Materials and Methods
Creation of Mice Carrying a loxP-flanked Allele of HIF-1␣. Genomic
DNA was obtained as described previously (19). A loxP site was engineered
in the first intron through PCR, and a loxP-flanked neomycin resistance
cassette was cloned into a SphI site in the second intron. The targeting vector
Received 4/9/00; accepted 6/14/00.
was linearized, and 20 ␮g of the vector were electroporated into R1 ES cells
The costs of publication of this article were defrayed in part by the payment of page
(22). The neomycin resistance cassette was removed by electroporation of 30
charges. This article must therefore be hereby marked advertisement in accordance with
␮g of a cre-expressing plasmid into targeted cells [pML 78 (23, 24)]. PCR was
18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom requests for reprints should be addressed, at University of California, San
used to identify cell lines that had maintained loxP sites on either side of exon
Diego, Department of Biology, 9500 Gilman Drive, 0366, La Jolla, CA 92093-0366.
2. Chimeric mice were generated by injection of ES cells into C57Bl/6
Phone: (858) 822-0509; Fax: (858) 534-5831.
blastocysts (25).
2
The abbreviations used are: HIF, hypoxia-inducible factor; VEGF, vascular endoIsolation of Wild-Type and HIF-1␣-null mEFs. Wild-type and HIF-1␣thelial growth factor; mEF, mouse embryonic fibroblast; ARNT, aryl hydrocarbon recepnull embryos were harvested at embryonic day 9.5, dissociated by incubation
tor nuclear translocator; ES, embryonic stem; TAg, T antigen.
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HIF-1␣ PROMOTES TUMOR GROWTH
in 0.25% trypsin (Life Technologies, Inc.), and cultured. Embryos carrying two
loxP-flanked alleles of HIF-1␣ were harvested at embryonic day 13.5, dissociated by passage through an 18-gauge needle, and cultured. Cells were
immortalized by stable transfection of SV40 large T antigen, using Superfectamine (Qiagen) according to the manufacturer’s instructions and transformed by infection with a retrovirus expressing H-ras (26). The ⫹f/⫹f
ras/TAg cells were infected with adenovirus expressing either ␤-galactosidase
or cre recombinase.
The wild-type or null status of cells was confirmed by standard Southern
blotting. 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, 1 ␮g/ml aprotinin, 1 ␮g/ml
pepstatin A, 1 ␮g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]
and separation of the nuclei by centrifugation. Nuclei were lysed by
incubation 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 SDSPAGE, electroblotting, and immunodetection with an anti-HIF-1␣ IgY
antibody (27). Detection of HIF-1␣ was performed using a horseradish
peroxidase-conjugated goat anti-IgY (Promega) secondary antibody and
SuperSignal 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 15 ␮g of total
RNA were loaded per lane, run on a 1% denaturing agarose gel, and
hybridized with cDNA probes. Probes were generated as described in Ryan
et al. (19).
Generation of Fibrosarcomas. A total of 1 ⫻ 107 cells were injected s.c.
intrascapularly into immunocompromised mice, either RAG1⫺/⫺ mice (28) or
nu/nu mice from Charles River. Tumors were harvested 16 –18 days after
injection, weighed, and processed for histology.
Tumor Histology. Sections were cut from frozen tissue and stained for
CD31 as described previously. Vessel density in the most vascular regions of
the tumor was determined with a Chalkley eyepiece graticule as described by
Fox et al. (29).
In situ hybridization was performed on paraformaldehyde-fixed, paraffinembedded sections using 35S-UTP-labeled riboprobes as described previously
(30, 31).
Results
Creation of HIF-1␣-null H-ras-transformed Cell Lines. To
carry out initial characterization of differentiated cells that lack a
functional HIF-1␣ allele, we generated lines directly from HIF-1␣
wild-type and null embryos. Embryos were harvested at embryonic
day 9.5 and cells were immediately immortalized by stable transfection with SV40 large T antigen (32). The cells were transformed
by infection with a retrovirus expressing the activated H-ras allele
(26). Analysis of these cell lines by DNA and protein blotting
confirmed their wild-type or null status, and they are referred to
hereafter as ⫹/⫹ ras/TAg or ⫺/⫺ ras/TAg cell lines (Fig. 1, C
and D).
Homozygous deletion of HIF-1␣ results in embryonic death between embryonic days 9 and 10 (18, 19). Cell lines isolated from this
early stage of development might differ from standard mouse fibroblasts in a number of respects. To control for these variables in this
Fig. 1. Generation of a loxP-flanked allele of HIF-1␣. A, diagram of the strategy used to replace the endogenous HIF-1␣ locus with a loxP-flanked locus. Genomic structure after
targeting and cre expression. A, AflIII; B, BamHI; Bg, BglII; P, PstI; X, XbaI. B, Southern blot analysis of the HIF-1␣ locus before cre expression showing a BamHI digest of genomic
DNA and hybridization to a 5⬘ external probe. C, Southern blot analysis of genomic DNA from mEF ras/TAg cell lines demonstrating either loss of the second exon (⫺f/⫺f, PstI/EcoRI
digest) or deletion of the second exon and intron (⫺/⫺, BamHI digest). D, Western blot analysis of mEF ras/TAg cell lines showing induction of HIF-1␣ after 4 h of hypoxia (1%
O2) in wild-type cells and loss of hypoxia-induced HIF-1␣ expression in the null cells. E, Northern blot analysis of 15 ␮g of total RNA from wild-type and HIF-1␣-null cells after
0 or 8 h of hypoxia. Left, the cDNA probe used for hybridization.
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study, we also created HIF-1␣-null cell lines via conditional targeting
of the HIF-1␣ locus using the cre/loxP system (33).
We designed a conditional allele of HIF-1␣ in which the second
exon is flanked by loxP sites. The second exon encodes the
helix-loop-helix motif, which has been shown to be essential for
HIF-1␣ dimerization with ARNT and subsequent transcriptional
activation (34). The targeting vector contains a loxP site in the first
intron and a loxP-flanked neomycin resistance gene in the second
intron (Fig. 1A). Homologous recombination at the HIF-1␣ locus
results in an allele with a loxP site 5⬘ of exon 2 and the loxPflanked neomycin resistance gene 3⬘ of exon 2 (Fig. 1B). It is
possible that the presence of the neomycin resistance gene in the
second intron could affect proper expression of HIF-1␣; to safeguard against this possibility, we transiently expressed cre recombinase in the targeted ES cells. A fraction of the ES cells excised
the neomycin resistance gene but retained 5⬘ and 3⬘ loxP sites,
whose presence was confirmed by PCR (data not shown). These ES
cell clones were used for the generation of chimeras and mouse
strains via blastocyst injection (25).
Mice containing loxP-flanked alleles of HIF-1␣ were crossed, and
mEFs were harvested from embryonic day 13.5 embryos that were
homozygous (⫹f/⫹f) for the conditionally targeted allele. The cells
were transformed with SV40 large T antigen and H-ras as described
above.
We next generated HIF-1␣-null cells by transiently expressing cre
recombinase to delete the loxP-flanked second exon. We used adenovirus to transiently express cre recombinase in ⫹f/⫹f ras/TAg cell
lines. An adenovirus expressing ␤-galactosidase was also used to
infect ⫹f/⫹f ras/TAg cells. These ␤-galactosidase-infected cells were
then used as wild-type controls in all of the following experiments and
are referred to as ⫹f/⫹f ras/TAg cells. Cre-infected cells are referred
to as ⫺f/⫺f ras/Tag cells. Four to five days after infection, cells were
assayed for excision of the second exon. DNA analysis via Southern
blotting (Fig. 1C) and PCR (data not shown) indicated complete
excision of the second exon in the entire cell population. This was
further confirmed by analysis of nuclear extracts from the conditionally targeted cells, which showed an absence of HIF-1␣ protein under
hypoxic conditions (Fig. 1D). ␤-Galactosidase-infected cells maintained a wild-type Southern profile and expressed HIF-1␣ under
hypoxia (Fig. 1, C and D). Both cell lines grew at similar rates in
culture and formed similar numbers of colonies in soft agar assays
(data not shown).
Loss of Hypoxia-mediated Transcriptional Induction in HIF1␣-null mEFs. HIF-1␣ regulates a wide array of genes in response to
hypoxia. The loss of HIF-1␣ in ES cells leads to a reduction in
hypoxic expression of VEGF and a number of other genes at the
mRNA level (18, 19). We assayed both of the HIF-1␣-null mEF cell
lines for loss of target gene expression by Northern blotting (Fig. 1E).
In the ⫺f/⫺f ras/TAg cell line, the absence of HIF-1␣ lessens the
hypoxic induction of VEGF and completely inhibits that of the hypoxia-responsive genes phosphoglycerate kinase, lactate dehydrogenase, and glucose transporter-1. The situation is similar in the ⫺/⫺
ras/TAg cell line.
HIF-1␣ Is a Positive Regulator of Tumor Growth. The wildtype and null transformed cell lines described above were used to
create fibrosarcomas in immunocompromised animals. Cells were
injected s.c., and at 16 –18 days after injection, the tumors were
harvested and weighed. In both sets of cell lines, the absence of
HIF-1␣ resulted in a significant decrease in tumor mass (Fig. 2).
Hypoxia is considered to be a major stimulus for tumor angiogenesis, and VEGF has been shown to be crucial for this process (35–37).
Considering both the role of HIF-1␣ in hypoxia-induced VEGF expression and previously published tumor data, we looked at the degree
Fig. 2. Loss of HIF-1␣ leads to a reduction in tumor mass. A, analysis of tumor mass
from ⫹/⫹ ras/TAg (n ⫽ 10) and ⫺/⫺ras/TAg (n ⫽ 10) tumors. B, analysis of tumor mass
from ⫹f/⫹f ras/TAg (n ⫽ 20) and ⫺f/⫺f ras/TAg (n ⫽ 16) tumors. Statistical analysis
was performed using Statview (Abacus Software).
of vascularization within wild-type and HIF-1␣-null tumors. Tumor
sections were stained for the endothelial cell marker CD31. This
revealed no obvious difference in tumor vasculature between wildtype and null tumors (Fig. 3A). Quantification of vascular density
through Chalkley analysis confirmed that there was no significant
difference in vessel density between wild-type and HIF-1␣-null tumors (Fig. 3B).
Because we did not observe any decrease in vascular density in
the null tumors, we analyzed VEGF expression in ⫹f/⫹f ras/TAg
and ⫺f/⫺f ras/TAg tumors by in situ hybridization (Fig. 4). In
tumors derived from ⫺f/⫺f ras/TAg cells, which show a significant difference in hypoxic regulation of VEGF under hypoxia in
culture, there was a clear difference in the expression pattern of
VEGF. In wild-type tumors, high levels of VEGF are expressed in
wide swaths throughout the tumor, similar to the expression patterns in solid tumors reported elsewhere for this gene (20). The null
tumors also have regions of very high VEGF expression, but it is
more circumscribed, in some cases to single cells as opposed to
large numbers of cells in a single region (Fig. 4B, magnification,
⫻400); this results in a more punctate pattern of VEGF expression
in the HIF-1␣-null tumors.
Discussion
Varying reports in the literature have made it unclear as to what
role the HIF-1-mediated hypoxic response might have in tumor
growth (19 –21). Conflicting evidence from ES cell-derived tumors
has indicated, on one hand, that HIF-1␣ acts as a positive regulator
of tumor growth (19), most likely through its activation of VEGF,
and, on the other hand, that HIF-1␣ acts as a negative regulator of
tumor growth, possibly through its stabilization of p53 in hypoxic
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Fig. 3. Loss of HIF-1␣ does not affect vessel density
within tumors. A, CD31 staining of frozen tumor sections
showing no obvious difference in vessel density. Magnification, ⫻100. B, analysis of Chalkley scores confirming
no statistical difference in vessel density between wildtype and null tumors.
cells (21, 38). A primary point of contention is whether HIF-1␣
promotes or inhibits tumor growth. To address this issue, we have
generated a tumor model using H-ras-transformed fibroblasts from
which HIF-1␣ can be genetically removed. With this model, we
have demonstrated that HIF-1␣ clearly acts as a positive regulator
of tumor growth.
Data published previously on the mechanism by which HIF-1
positively regulates tumor growth have focused on angiogenesis (19 –
21, 39). The consensus has been that the absence of either HIF-1␣ or
its dimerization partner, ARNT, leads to reduced vascularization
within a tumor due to a reduced capacity to hypoxically induce VEGF
expression. We were therefore surprised to find that loss of HIF-1␣
did not alter tumor vascularization in H-ras-transformed fibrosarcomas. This is in contrast to experiments in which VEGF itself is deleted
from tumor cells, causing a large decrease in vascular density (35),
and in contrast to the experimental evidence from ES cell-derived
tumors, where significant, albeit subtle, differences in vascular density
are seen (19, 21).
We considered the possibility that our observation might be due to
differences between in vitro and in vivo VEGF expression, where the
tumor environment works in such a way as to make expression
differences seen in culture inconsequential in vivo. This may explain
our observations; however, within the tumors, in situ hybridization
demonstrates that there are clear differences in VEGF expression
between ⫹f/⫹f and ⫺f/⫺f tumors. This difference is best seen in the
higher magnification in Fig. 4B, which shows that VEGF expression
within these tumors is more punctate and restricted. A possible explanation for the different expression pattern seen in the ⫺f/⫺f tumors
is that a higher degree of hypoxia is required to stimulate VEGF
expression in these cells and that this more restricted expression
pattern is an indication of those regions. This high level of VEGF
expression from a smaller number of cells may be able to compensate
for a general reduction in VEGF expression, ultimately resulting in an
adequate degree of vascularization.
The work presented here has clearly demonstrated that HIF-1
acts to promote tumor growth. What has become less clear is the
exact mechanism by which HIF-1 functions in this capacity. Our
fibrosarcoma model has provided the first indication that hypoxic
induction of angiogenesis may play a smaller role in tumor growth
than previously thought and that HIF-1-mediated regulation of
VEGF is not crucial for tumor vascularization. With the multitude
of HIF-1 target genes, there are a number of possible mechanisms
yet to be investigated, and in all likelihood, the effect of HIF-1 on
tumor growth is complex and involves the activation of several
adaptive pathways. We also report for the first time the development of conditionally targeted mice and transformed cell lines
from which HIF-1␣ can be easily excised via cre recombinase
expression. These should prove invaluable reagents to investiga-
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HIF-1␣ PROMOTES TUMOR GROWTH
Fig. 4. Absence of HIF-1␣ affects VEGF expression within a tumor. In situ hybridization demonstrating the punctate pattern of VEGF expression in ⫺f/⫺f ras/TAg tumors. A,
dark-field view at a magnification of ⫻100 (right), alongside a bright-field image of the same region. Exposure times for the dark-field photos were identical. B, bright-field view at
a higher magnification (⫻400). Black dots are exposed silver grains.
tors wishing to study the role of HIF-1␣ during normal development and the role of hypoxic response during tumorigenesis.
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Acknowledgments
We thank members of the Johnson laboratory for their support and advice
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Robert Warren, Michael Karin, Gregg Semenza, Ronald Wisdom, and Frank
Giordano for reagents and helpful advice.
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Hypoxia-inducible Factor-1α Is a Positive Factor in Solid Tumor
Growth
Heather E. Ryan, Michelle Poloni, Wayne McNulty, et al.
Cancer Res 2000;60:4010-4015.
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