REVIEWS

TARGETING HIF-1 FOR

CANCER THERAPY
Gregg L. Semenza
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that are involved in crucial
aspects of cancer biology, including angiogenesis, cell survival, glucose metabolism and invasion.
Intratumoral hypoxia and genetic alterations can lead to HIF-1α overexpression, which has been
associated with increased patient mortality in several cancer types. In preclinical studies, inhibition
of HIF-1 activity has marked effects on tumour growth. Efforts are underway to identify inhibitors
of HIF-1 and to test their efficacy as anticancer therapeutics.

Interest in the role of hypoxia-inducible factor 1 (HIF-1)
in cancer biology has grown exponentially in the two
decades since its identification1, biochemical purifica-
tion2 and molecular characterization3. Much has been
learned recently about the cellular and molecular biol-
ogy of HIF-1 and its involvement in human cancer
progression, based on the analysis of human cancer
biopsies and experimental animal models. The con-
cept of intratumoral hypoxia as a driving force in can-
cer progression has been covered in a previous review 4,
and this review will focus on the potential of HIF-1 as
a therapeutic target.
Master regulator of oxygen homeostasis
The HIF-1 transcription factor mediates adaptive
responses to changes in tissue oxygenation. HIF-1 is a
heterodimer that consists of a constitutively expressed
HIF-1β subunit and a HIF-1α subunit, the expression
of which is highly regulated. As for any protein, the level
of HIF-1α expression is determined by the rates of pro-
tein synthesis and protein degradation. In the case of
HIF-1α, synthesis is regulated via O2-independent
mechanisms (FIG. 1), whereas degradation is regulated
primarily via O2-dependent mechanisms (FIG. 2), as
McKusick–Nathans Institute
of Genetic Medicine, described below.
The Johns Hopkins More than 60 putative direct HIF-1 target genes
University School of have been identified (FIG. 3) on the basis of one or
Medicine, Baltimore, more of the following: identification of a cis-acting
Maryland
21287-3914, USA. hypoxia-response element that contains a HIF-1
e-mail: gsemenza@jhmi.edu binding site1; loss of hypoxia-induced gene expression
doi:10.1038/nrc1187 in HIF1-α-null cells 5–7 or cells treated with small
interfering RNA that targets HIF1α mRNA8; increased
expression in von Hippel–Lindau (VHL)-null cells or
in cells transfected with a HIF-1α expression vector9.
The level of proof varies, especially in the present era
of global analysis by microarrays, in which the quan-
tity — but not the quality — of data has increased.
Analyses that do not involve the demonstration of a
functional HIF-1 binding site identify both direct and
indirect (secondary) targets of regulation by HIF-1.
Four groups of direct HIF-1 target genes that are par-
ticularly relevant to cancer encode angiogenic factors,
glucose transporters and glycolytic enzymes, survival
factors and invasion factors (FIG. 4). The products of
the genes that HIF-1 regulates act at several steps in
each of these processes, as shown by recent studies of
cancer-cell invasion8,10 (FIG. 5).
Expression of several HIF-1 target genes, such as
vascular endothelial growth factor (VEGF), is induced
by hypoxia in most cell types. However, for the major-
ity of HIF-1 target genes, expression is induced by
hypoxia in a cell-type-specific manner. HIF-1 activity
is induced by hypoxia in almost all cell types and
therefore HIF-1 alone cannot account for this cell-
type-specific gene expression. Rather, it is the func-
tional interaction of HIF-1 with other transcription
factors that determines the subgroup of HIF-1 target
genes that is activated in any particular hypoxic cell.
HIF-1 can be viewed as a messenger that is sent to the
nucleus to activate transcriptional responses to
hypoxia. The details of this response are determined by
the past (developmental) and present (physiological)

NATURE REVIEWS | C ANCER VOLUME 3 | OCTOBER 2003 | 7 2 1

REVIEWS
Summary
• Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric protein that consists of two
proteins — HIF-1α and HIF-1β. HIF-1 activates the transcription of many genes that
code for proteins that are involved in angiogenesis, glucose metabolism, cell
proliferation/survival and invasion/metastasis.
• HIF-1α protein synthesis is regulated by activation of the phosphatidylinositol
3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK) pathways. These
pathways can be activated by signalling via receptor tyrosine kinases, non-receptor
tyrosine kinases or G-protein-coupled receptors.
• HIF-1α protein degradation is regulated by O2-dependent prolyl hydroxylation,
which targets the protein for ubiquitylation by E3 ubiquitin-protein ligases. These
ligases contain the von Hippel–Lindau tumour-suppressor protein (VHL), which
binds specifically to hydroxylated HIF-1α. Ubiquitylated HIF-1α is rapidly degraded
by the proteasome.
• HIF-1α is overexpressed in human cancers as a result of intratumoral hypoxia as well
as genetic alterations, such as gain-of-function mutations in oncogenes (for example,
ERBB2) and loss-of-function mutations in tumour-suppressor genes (for example,
VHL and PTEN). HIF-1α overexpression is associated with treatment failure and
increased mortality.
• In xenograft assays, manipulation of HIF-1 activity by genetic or pharmacological
means has marked effects on tumour growth because of effects on angiogenesis,
glucose metabolism and/or cell survival.
• Screens are underway to identify small-molecule inhibitors of HIF-1 and to test
their efficacy as anticancer agents. These drugs might represent an important
component of novel combination therapies that are designed to target signalling
molecules in cancer cells.
programming of each cell. As a result, the total number
of HIF-1 target genes cannot be ascertained by analysis
of one or a few cell types. Perhaps 1–5% of all human
genes are expressed in response to hypoxia in one or
more cell types in a HIF-1-dependent manner.
Heterogeneity in target-gene expression is observed
even among cell lines that have been derived from can-
cers of the same histopathological type. Similar findings
have been reported for p53-dependent gene expres-
sion11. An additional complicating factor is the existence
of the related protein HIF-2α, which can also dimerize
with HIF-1β12–15. Heterodimers that contain HIF-1α or
HIF-2α seem to have overlapping but distinct specifici-
ties, with regard to physiological inducers and target-
gene activation16. A third related protein, HIF-3α, might
function primarily as an inhibitor of HIF-1α17.
Oxygen-dependent regulation of HIF-1
Cells transduce decreased O2 concentration into
increased HIF-1 activity via a novel O2-dependent post-
translational modification (FIG. 2). Three prolyl hydroxy-
lases — known as prolyl hydroxylase-domain protein
(PHD) 1–3, or, alternatively, as HIF-1 prolyl hydroxylase
(HPH) 1–3 — modify proline(Pro)-402 and -564 of
HIF-1α18,19. These proteins were originally designated
EGLN1–3 on the basis of sequence homology to EGL9,
the HIF-1 prolyl hydroxylase in Caenorhabditis
elegans19. Hydroxylation of Pro-402 and Pro-564 is
required for interaction of HIF-1α with the VHL
tumour-suppressor protein20–23. VHL is the recognition
component of an E3 ubiquitin-protein ligase that
targets HIF-1α for proteasomal degradation24–28. The
722 | OCTOBER 2003 | VOLUME 3
prolyl hydroxylases use molecular O2 and 2-oxoglutarate
(α-ketoglutarate) as substrates in a reaction that generates
prolyl-hydroxylated HIF-1α and succinate. Under physi-
ological conditions, O2 is a limiting substrate19, therefore
providing a mechanism for O2-dependent regulation of
HIF-1α expression29. Acetylation of HIF-1α at lysine-532
by the ARD1 acetyltransferase enhances the interaction
of VHL with HIF-1α, promoting its ubiquitylation
and degradation30.
Growth factor
Receptor tyrosine
kinase
PI3K RAS
AKT PTEN MEK
mTOR ERK
S6K MNK
4E-BPI
S6 P
40S
3′
60S eIF-4E
5′
HIF-1α protein
synthesis
HIF-1β
HIF-1 transcriptional activity
Figure 1 | Regulation of HIF-1α protein synthesis.
Growth-factor binding to a cognate receptor tyrosine kinase
activates the phosphatidylinositol 3-kinase (PI3K) and
mitogen-activated protein kinase (MAPK) pathways. PI3K
activates the downstream serine/threonine kinases AKT (also
known as protein kinase B (PKB)) and mammalian target of
rapamycin (mTOR). In the MAPK pathway, the extracellular-
signal-regulated kinase (ERK) is activated by the upstream
MAP/ERK kinase (MEK). ERK, in turn, activates MNK. ERK
and mTOR phosphorylate p70 S6 kinase (S6K) — which, in
turn, phosphorylates the ribosomal S6 protein — and the
eukaryotic translation initiation factor 4E (eIF-4E) binding
protein (4E-BP1). Binding of 4E-BP1 to eIF-4E inactivates the
latter, inhibiting cap-dependent mRNA translation.
Phosphorylation of 4E-BP1 prevents its binding to eIF-4E.
MNK phosphorylates eIF-4E and stimulates its activity
directly. The effect of growth-factor signalling is an increase in
the rate at which a subset of mRNAs within the cell (including
HIF-1α mRNA) are translated into protein.
www.nature.com/reviews/cancer
 REVIEWS

Degradation Ubiquitylation VHL

OH OAc OH OH

Normoxic PHD1–3 ARD1 PHD1–3 FIH-1

P402 K532 P564 N803

bHLH PAS
TAD-N TAD-C

p300/CBP
Hypoxic

Transcriptional
activation

Figure 2 | O2-dependent regulation of HIF-1 activity. O2 regulates the rate at which HIF-1α protein is degraded. In
normoxic conditions, O2-dependent hydroxylation of proline (P) residues 402 and 564 in HIF-1α by the enzymes PHD (prolyl
hydroxylase-domain protein) 1–3 is required for the binding of the von Hippel–Lindau (VHL) tumour-suppressor protein, which
is the recognition component of an E3 ubiquitin-protein ligase. VHL binding is also promoted by acetylation of lysine (K)
residue 532 by the ARD1 acetyltransferase. Ubiquitylation of HIF-1α targets the protein for degradation by the 26S
proteasome. O2 also regulates the interaction of HIF-1α with transcriptional co-activators. O2-dependent hydroxylation of
asparagine (N) residue 803 in HIF-1α by the enzyme FIH-1 (factor inhibiting HIF-1) blocks the binding of p300 and CBP to
HIF-1α and therefore inhibits HIF-1-mediated gene transcription. Under hypoxic conditions, the rate of asparagine and
proline hydroxylation decreases. VHL cannot bind to HIF-1α that is not prolyl-hydroxylated, resulting in a decreased rate of
HIF-1α degradation. By contrast, p300 and CBP can bind to HIF-1α that is not asparaginyl-hydroxylated, allowing
transcriptional activation of HIF-1 target genes. bHLH, basic helix–loop–helix; PAS, Per-Arnt-Sim; TAD-C, carboxy-terminal
transactivation domain; TAD-N, amino-terminal transactivation domain.

GLYCOLYTIC METABOLISM
Two molecules of ATP and
NADH are generated by the
conversion of one molecule of
glucose to two molecules of
pyruvate. The NADH is then
used to reduce pyruvate to
lactate.
OXIDATIVE METABOLISM
Glucose is converted to
pyruvate, which is transported to
the mitochondria, converted to
acetyl coenzyme A and oxidized
to CO2 in the citric-acid cycle.
The NADH and FADH2
generated in this process provide
electrons to respiratory
cytochromes and, ultimately, to
O2 in the inner mitochondrial
membrane, generating ATP. The
complete oxidation of one
molecule of glucose results in
the production of 36 molecules
of ATP.
HIF-1α transactivation-domain function is also O2-
regulated31,32 via the action of FIH-1 (factor inhibiting
HIF-1)33. FIH-1 mediates this effect by hydroxylation of
asparagine(Asn)-803, which prevents the interaction of
HIF-1α with co-activators p300 and CBP34–36.
Structural analysis of the HIF-1α domains that interact
with VHL or p300/CBP has shown that hydroxylation
is a molecular switch that markedly alters the affinity of
these interactions37–40. Hydroxylation is similar to other
post-translational modifications (for example, phos-
phorylation) in that it functions to regulate
protein–protein interactions, but, unlike other modifi-
cations, it is an inherently O2-regulated process. HIF-2α
expression and activity are also regulated by proline
and asparagine hydroxylation.
Oxygen-independent regulation of HIF-1
Humans, like other metazoans, are constant and
obligate consumers of O2. The more cells that are pre-
sent in a tissue, the more O2 is consumed. So, when
one cell produces two daughter cells, O2 consump-
tion increases. It is not surprising that the main path-
ways that transduce proliferative and survival signals
from growth-factor receptors also induce HIF-1α
expression (FIG. 1) in what can be viewed as a pre-
emptive strategy for maintaining oxygen homeostasis.
Proliferating cells express VEGF, which stimulates
angiogenesis to provide the additional perfusion that
is required to maintain oxygenation of an increased
number of cells. In addition, proliferating cells prefer-
entially use GLYCOLYTIC rather than OXIDATIVE METABOLISM
to generate ATP 41. The concomitant induction of
angiogenesis and glycolysis with cell proliferation is
mediated partly by activating HIF-1 (REFS 42,43).
The increase in HIF-1α levels in response to
growth-factor stimulation differs in two important
respects from the increase in HIF-1α levels in
response to hypoxia. First, whereas hypoxia increases
HIF-1α levels in all cell types, growth-factor stimula-
tion induces HIF-1α expression in a cell-type-specific
manner. Second, whereas hypoxia is associated with
decreased degradation of HIF-1α, growth factors,
cytokines and other signalling molecules stimulate
HIF-1α synthesis via activation of the phosphatidyli-
nositol 3-kinase (PI3K) or mitogen-activated protein
kinase (MAPK) pathways 44–49 (FIG. 1). Pulse-chase
studies of MCF-7 breast cancer cells stimulated with
heregulin showed increased HIF-1α protein synthesis
that was inhibited by treatment with rapamycin — a
macrolide antibiotic that inhibits mammalian target of
rapamycin (mTOR; a kinase that functions down-
stream of PI3K and AKT)47. The effect of heregulin
was mediated via the 5′-untranslated region of HIF-1α
mRNA47. The known targets for phosphorylation by
mTOR are regulators of protein synthesis (FIG. 1).
However, it is not known whether phosphorylation of
these proteins by mTOR is necessary or sufficient for
increased HIF-1α synthesis. The translation of several
dozen different mRNAs is known to be regulated by
this pathway. Specific sequences in the 5′-untranslated
region could determine the degree to which the trans-
lation of any particular mRNA can be modulated by
mTOR signalling. HIF-1α protein expression is likely
to be particularly sensitive to changes in the rate of
synthesis because of its extremely short half-life under
non-hypoxic conditions. In addition to effects on
HIF-1α synthesis, activation of the RAF–MEK–ERK
signalling pathway has also been shown to stimulate

NATURE REVIEWS | C ANCER VOLUME 3 | OCTOBER 2003 | 7 2 3

REVIEWS

Cell proliferation Transcriptional regulation all cell types, the regulation of HIF-1 activity by
Cyclin G2 DEC1 growth-factor signalling is cell-type specific. For exam-
IGF2 DEC2
IGF-BP1 ETS-1
ple, in MCF-7 cells, heregulin induces HIF-1α protein
IGF-BP2 NUR77 synthesis but does not induce transactivation-domain
IGF-BP3 function47, whereas treatment of PC-3 prostate cancer
WAF1 pH regulation
TGF-α Carbonic anhydrase 9 cells with rapamycin inhibits HIF-1α protein stability
TGF-β3 and transactivation-domain function53. Oncogenic
Cell survival Regulation of HIF-1 activity mutations that activate signal-transduction pathways
ADM p35srj
induce HIF-1 activity via various mechanisms (TABLE 1).
EPO
IGF2 Epithelial homeostasis
Loss-of-function mutations in tumour-suppressor
IGF-BP1 Intestinal trefoil factor genes are also associated with increased HIF-1 activity.
IGF-BP2
IGF-BP3 Among these, the most marked effect is observed in
Drug resistance
NOS2
MDR1 clear-cell renal carcinomas (RCCs) and cerebellar
TGF-α
VEGF haemangiomas that have lost VHL function26,54. VHL
Nucleotide metabolism loss of function results in a marked increase in HIF-1
Apoptosis Adenylate kinase 3
NIP3 Ecto-5′-nucleotidase activity in non-hypoxic conditions because of impaired
NIX VHL-dependent ubiquitylation and proteasomal degra-
RTP801 Iron metabolism dation of HIF-1α and HIF-2α. Although the
Ceruloplasmin
Motility Transferrin O2-dependent regulation of transactivation function is
AMF/GPI HIF-1 Transferrin receptor still intact, FIH-1 might become limiting under condi-
c-MET
LRP1 Glucose metabolism
tions of HIF-1α and HIF-2α overexpression, leading to
TGF-α HK1 increased transcriptionally active HIF-1 under non-
HK2 hypoxic conditions in VHL-null cells. For several other
Cytoskeletal structure AMF/GPI
KRT14 ENO1 oncogenes and tumour-suppressor genes, mutation not
KRT18 GLUT1
KRT19 only has a marked effect on cancer progression, but also
GAPDH
VIM LDHA on HIF-1 activity (TABLE 1). Several growth factors, most
PFKBF3 notably insulin-like growth factor-2 (IGF2) and trans-
Cell adhesion PFKL
MIC2 PGK1 forming growth factor-α (TGF-α), are also HIF-1 target
Erythropoiesis
PKM genes8,55. Binding of these factors to their cognate recep-
TPI
EPO tors — the insulin-like growth factor 1 receptor
Angiogenesis Extracellular-matrix metabolism (IGF1R) and epidermal growth-factor receptor (EGFR),
EG-VEGF CATHD respectively — activates signal-transduction pathways
ENG Collagen type V (α1)
FN1 that lead both to HIF-1α expression (as described
LEP
LRP1 MMP2 above) and to cell proliferation/survival. HIF-1 there-
TGF-β3 PAI1
Prolyl-4-hydroxylase α (I) fore contributes to autocrine-signalling pathways that
VEGF
UPAR are crucial for cancer progression (FIG. 6). The mecha-
Vascular tone nisms that lead to increased levels of HIF-1α have been
α1B-adrenergic receptor Energy metabolism
ADM LEP elucidated primarily through experiments in cancer cell
ET1 lines. This work has been complemented by immuno-
Haem oxygenase-1 Amino-acid metabolism
NOS2 Transglutaminase 2 histochemical demonstration of HIF-1α overexpression
in human cancer biopsies.
Figure 3 | Genes that are transcriptionally activated by HIF-1. Genes that are involved in
many processes are transcriptionally activated by HIF-1. ADM, adrenomedullin; ALDA, aldolase HIF-1α expression in human cancers
A; ALDC, aldolase C; AMF, autocrine motility factor; CATHD, cathepsin D; EG-VEGF, endocrine-
gland-derived VEGF; ENG, endoglin; ET1, endothelin-1; ENO1, enolase 1; EPO, erythropoietin;
A common approach for analysing altered expression
FN1, fibronectin 1; GLUT1, glucose transporter 1; GLUT3, glucose transporter 3; GAPDH, of proteins in human cancers is to perform immuno-
glyceraldehyde-3-P-dehydrogenase; HK1, hexokinase 1; HK2, hexokinase 2; IGF2, insulin-like histochemistry on patient biopsy samples. Expression
growth-factor 2; IGF-BP1, IGF-factor-binding-protein 1; IGF-BP2, IGF-factor-binding-protein 2; can be characterized qualitatively (present or absent) or
IGF-BP3, IGF-factor-binding-protein 3; KRT14, keratin 14; KRT18, keratin 18; KRT19, keratin 19; quantitatively (for example, percentage of cells express-
LDHA, lactate dehydrogenase A; LEP, leptin; LRP1, LDL-receptor-related protein 1; MDR1, ing the protein). Statistical analyses are performed to
multidrug resistance 1; MMP2, matrix metalloproteinase 2; NOS2, nitric oxide synthase 2;
determine whether expression of the protein is corre-
PFKBF3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3; PFKL, phosphofructokinase
L; PGK 1, phosphoglycerate kinase 1; PAI1, plasminogen-activator inhibitor 1; PKM, pyruvate lated with the expression of other biomarkers (which
kinase M; TGF-α, transforming growth factor-α; TGF-β3, transforming growth factor-β3; TPI, could be located either upstream or downstream in a
triosephosphate isomerase; VEGF, vascular endothelial growth factor; UPAR, urokinase common pathway) or with clinical outcome. These
plasminogen activator receptor; VEGFR2, VEGF receptor-2; VIM, vimentin. studies provide associations that can engender mecha-
nistic hypotheses for testing in tissue culture or animal
models. In addition, immunohistochemistry might be
HIF-1α transactivation-domain function50,51. This useful in clinical trials for identifying target populations
effect is due at least in part to phosphorylation by ERK and assessing therapeutic responses.
of the co-activator p300, with which the transactiva- Protein-expression levels in tumours are usually
tion domains interact52. Unlike hypoxia, which induces compared with those of the surrounding normal tissue.
HIF-1α protein stability and transcriptional activity in In general, proteins that promote tumour progression

724 | OCTOBER 2003 | VOLUME 3 www.nature.com/reviews/cancer


Intratumoral hypoxia Genetic alterations
* trast, associations between HIF-1α overexpression and
* was correlated with apoptosis in most tumours and the
Increased HIF-1α expression and HIF-1 activity
Metabolic adaptation Apoptosis resistance Angiogenesis Invasion/metastasis
ALDA, ENO1, GADPH, ADM, EPO, ET1, EG-VEGF, ENG, AMF, CATHD, CMET,
GLUT1, GLUT3, GPI, IGF2, NOS2, TGFA LEP, TGF-β3, FN1, KRT14, KRT18,
HK1, HK2, LDHA, VEGF, VEGFR2 KRT19, MMP2, UPAR,
PFKBF3, PFKL, PGK1, VIM
PGM, TPI
Figure 4 | Mechanisms and consequences of HIF-1 activity in cancer cells.
Immunohistochemical analysis of HIF-1α levels in two separate oropharyngeal cancers. The
biopsy section on the left shows HIF-1α protein (brown staining) in viable cancer cells surrounding
areas of necrosis (indicated by asterisks). The cancer cells that express the highest levels of HIF-1α
are at the greatest distance from a blood vessel (indicated by arrows) and are therefore the most
hypoxic. In the biopsy section on the right, there are no areas of necrosis and HIF-1α is detected
in cancer cells throughout the field, including cells that are immediately adjacent to a blood vessel
(arrows), indicating that increased HIF-1α levels are being driven by an O2-independent
mechanism, such as through genetic alteration. These two mechanisms are not mutually
exclusive — genetic alterations can amplify the response to hypoxia. In either case, increased
HIF-1 activity leads to upregulation of genes that are involved in many aspects of cancer
progression, including metabolic adaptation, apoptosis resistance, angiogenesis and metastasis.
See FIG. 3 for gene names. Photomicrographs reprinted with permission from REF. 81 © (2001)
American Association for Cancer Research.
(oncogene products) are usually found to be overex-
pressed, whereas proteins that inhibit progression
(tumour-suppressor gene products) are underexpressed,
or carry missense mutations that result in the accumula-
tion of a non-functional protein (for example, p53).
Although immunohistochemical analysis can be useful
in determining whether a specific protein is present at
higher levels in cancer cells compared with surrounding
normal tissue, it does not reveal whether the protein car-
ries any mutations or has been post-translationally mod-
ified, which could affect its function. Furthermore, the
effects of gain or loss of protein function can vary
according to the stage of cancer progression. For exam-
ple, oestrogen-receptor expression is increased in early-
stage breast cancer and lost in advanced stages. These
provisos notwithstanding, it is difficult to make the case
that a particular protein either promotes or inhibits can-
cer progression without corroborating evidence from
human cancer biopsies.
Immunohistochemical analyses using monoclonal
antibodies (FIG. 4) revealed that HIF-1α is overexpressed
in many human cancers54,56. Significant associations
between HIF-1α overexpression and patient mortality
have been shown in cancers of the brain (oligoden-
droglioma), breast, cervix, oropharynx, ovary and uterus
(endometrial) (TABLE 2). In some cancer types, such as
oropharyngeal cancer, the association was observed
NATURE REVIEWS | C ANCER
REVIEWS
among tumours from all patients, whereas in other can-
cer types the association was observed only in specific
subgroups, such as in early-stage cervical cancer. By con-
decreased mortality were reported for patients with head
and neck cancer and non-small-cell lung cancer57,58,
although other studies failed to replicate these results59,60.
In a study of ovarian cancer, HIF-1α overexpression
combination of apoptosis and HIF-1α overexpression
was associated with increased patient survival.
However, in ovarian cancers that overexpressed both
HIF-1α and p53 (mutant p53 has an increased half-
life), apoptosis levels were low and were associated with
a statistically significant decrease in overall patient sur-
vival61. In patients with early-stage oesophageal cancer,
the combination of HIF-1α overexpression and BCL2
overexpression was associated with a failure to respond
to photodynamic therapy 62. So, the effect of HIF-1α
overexpression is dependent on the cancer type and the
presence or absence of genetic alterations that affect the
balance between pro- and anti-apoptotic factors.
Xenograft studies have provided additional evidence in
support of these concepts.
Manipulating HIF-1 activity in vivo
The most common experimental method for demon-
strating that a particular gene product is involved in
tumour growth is the xenograft assay, which involves sub-
cutaneous injection of tumour cells into immunodefi-
cient mice. To show the role of a gene product, the cells
are transfected with expression vectors that encode a con-
stitutively active, dominant-negative or wild-type form of
the protein. To test for antitumour effects of a potential
therapeutic agent, the drug can be administered to mice
at various times after injection of the tumour cells. All of
these approaches have been used to study HIF-1 (TABLE 3).
The first cell lines used to investigate the role of Hif-1
were derived from the mouse hepatoma cell line
Hepa1. A Hif-1β-deficient subclone of Hepa1 — desig-
nated c4 — and spontaneous revertants of c4 were
analysed. Compared with wild-type Hepa1 cells, c4
cells showed markedly reduced xenograft growth and
angiogenesis and had increased areas of necrosis.
Revertants, on the other hand, regained the ability to
develop into tumours that were histologically similar to
wild-type Hepa1 cells43,63.
Hif1α –/– mouse embryonic stem (ES) cells have also
been analysed. ES cells are among the few non-
transformed cells that can grow as xenografts. Two inde-
pendently derived sets of Hif1α –/– ES cells were
markedly impaired with respect to xenograft vascular-
ization5,7. In one study, overall growth of xenografts that
were derived from Hif1α –/– ES cells was reported to be
increased, because of reduced apoptosis5, whereas in
another study xenograft growth was reported to be
decreased because of reduced angiogenesis7. These data
imply that differences in the genetic background of the
ES cells or of the xenograft recipients in the two studies
had a key role in determining the net effect of HIF-1α
deficiency on xenograft growth.
VOLUME 3 | OCTOBER 2003 | 7 2 5
REVIEWS

a Normal epithelium To evaluate the effect of HIF-1α loss of function in

SV40 T-ANTIGEN
Large T-antigen — produced in
the early stage following
infection of cells with simian
virus 40 —promotes
transformation by binding to
and inactivating the host p53
and RB (retinoblastoma gene
product) proteins.
Basement
membrane
ECM
b ECM digestion
Cancer cell
Cathepsin D
uPAR
MMP2
Proteases
c New ECM production
Fibronectin 1
Fibronectin
c Mesenchymal transformation
Keratin 14,
18,19
Vimentin
AMF
TGF-α
c-MET
Figure 5 | HIF-1 target genes that encode invasion
factors. Invasion of the basement membrane is the defining
characteristic of epithelial cancers. a | Epithelial cells are
normally constrained by cell–cell contacts and by the
basement membrane. b | Cancer cells produce proteases,
including the urokinase-type plasminogen-activator receptor
(uPAR) and matrix metalloproteinase-2 (MMP2), which digest
the basement membrane/extracellular matrix (ECM).
c | Degraded ECM is replaced by fibronectin and other ECM
proteins that are recognized by integrins that are expressed on
cancer cells. d | An epithelial-to-mesenchymal transformation
occurs in which intermediate-filament production is switched
from keratin subtypes, which are characteristic of fixed
epithelial cells, to keratins and vimentin, which promote the
fluid structure that is required for motility and which is also
stimulated by expression of secreted factors such as autocrine
motility factor (AMF) and transforming growth factor-α
(TGF-α), and surface receptors such as the c-MET tyrosine
kinase. HIF-1 target genes that regulate invasion are listed.
transformed cells, fibroblasts were isolated from Hif1α –/–
embryos, immortalized by SV40 T-ANTIGEN expression, and
transformed by mutant HRAS expression. In these cells,
HIF-1α deficiency was associated with reduced
tumour mass 16–18 days after injection, although
angiogenesis was not affected64. In tissue culture, gly-
colysis and ATP levels under hypoxic conditions were
markedly reduced42. In xenograft assays, the Hif1α –/–
fibroblasts also manifested increased rates of apoptosis
in response to treatment with carboplatin and etopo-
side — agents that induce DNA double-strand breaks.
Sensitivity to agents that induce single-strand breaks
or inhibit DNA synthesis, however, was unchanged65.
These data indicate that HIF-1 regulates the expression
of one or more gene products that are required for the
repair of DNA double-strand breaks induced by
chemotherapy or radiation.
HIF-1 activity has also been manipulated in
human cancer cell lines. Expression of VEGF,
xenograft growth and angiogenesis were markedly
increased in HCT116 colon cancer cells that were
transfected with an expression vector that encoded
HIF-1α66. HIF-1α overexpression in PCI-10 pancre-
atic cancer cells was associated with an increased
frequency of xenograft growth after subcutaneous
injection without affecting tumour-microvascular
density 67. HIF-1α-overexpressing PCI-10 cells also
had an increased rate of survival when cultured
under conditions of glucose and oxygen deprivation.
As in the case of HRAS-transformed mouse embryo
fibroblasts described above, metabolic adaptation,
rather than angiogenesis, seems to be an important
mechanism by which HIF-1α overexpression
promotes PCI-10 xenograft growth.
Two strategies have been used to inhibit HIF-1
activity in human cancer cells. The first approach was
based on the demonstration that deletion of the
DNA-binding and transactivation domains results in
a dominant-negative form of HIF-1α that can bind
to HIF-1β, resulting in formation of an inactive het-
erodimer68. Overexpression of this dominant-
negative form of HIF-1α in PCI-43 pancreatic cancer
cells, which constitutively express high levels of HIF-1α,
led to an increase in the number of cells undergoing
apoptosis under conditions of glucose and oxygen
deprivation and a decreased ability to form tumours
in severe combined immunodeficiency (SCID) mice.
Tumour vascularization, however, was not affected69.
The second approach was based on the demonstra-
tion that HIF-1α contains two transactivation domains
— TAD-N and TAD-C31,32 (FIG. 2). TAD-C binding to its
co-activators, CBP and p300, is regulated by the
O2-dependent hydroxylation of Asn-803 by FIH-1 (REFS
33–36). A fusion protein that consists of GAL4 fused to
TAD-C inhibits the ability of HIF-1α to interact with
these co-activators, and therefore blocks HIF-1-
dependent transcription. Human breast (MDA-MB-435)
and colon (HCT116) cancer cells infected with a retro-
virus that encodes this fusion protein showed reduced
growth when injected into nude mice70. However, the

726 | OCTOBER 2003 | VOLUME 3 www.nature.com/reviews/cancer

 REVIEWS

IGF2 Renal carcinoma has been of particular interest to

Hypoxia
TGF-α investigators in the field, because the defining genetic
IGF1R EGFR lesion in RCC is loss of VHL function, resulting in high
levels of HIF-1α and HIF-2α protein that are not
O2 regulated. As a result, VEGF, GLUT1 and other HIF-1
target genes are constitutively expressed. In 786-O RCC
cells, only HIF-2α is expressed. When an expression vec-
tor encoding wild-type VHL was introduced into these
cells (which are designated 786-O/VHL), HIF-2α expres-
sion became O2 regulated and xenograft growth was
HIF-1 markedly reduced. To show that loss of O2-dependent
degradation of HIF-2α was necessary and sufficient
for the tumorigenic effect of VHL loss of function,
HIF1α 786-O/VHL cells were transfected with an expression
vector that encodes a form of HIF-2α containing a point
IGF2/TGF-α
mutation at the hydroxylation site (Pro531Ala). Ten
weeks after injection, these cells formed even larger
xenografts than 786-O cells, indicating that in this cell
line HIF-2α is a crucial downstream target of VHL71.
Similarly, overexpression in 786-O/VHL cells of a
fusion protein consisting of green fluorescent protein
Figure 6 | Involvement of HIF-1 in autocrine growth- (GFP) fused to the VHL-binding domain of HIF-2α —
factor stimulation of cancer cells. Binding of growth which blocks the interaction between HIF-2α and VHL
factors such as insulin-like growth factor-2 (IGF2) and
transforming growth factor-α (TGF-α) to their cognate
— leads to increased xenograft growth72. By contrast,
receptors — the IGF1 receptor (IGF1R) and the epidermal Vhl –/– ES cells manifest decreased xenograft growth in
growth-factor receptor (EGFR), respectively — stimulates nude mice73.
the expression of HIF-1α. This leads to increased HIF-1 Several important conclusions can be drawn from
transcriptional activity of target genes, which include those these studies. First, in all of the studies in which bona fide
that encode IGF2 and TGF-α. Alternatively, hypoxia can cancer cells were tested, increased levels of HIF-1α or
activate HIF-1, via increased expression of HIF-1α, and
HIF-2α were associated with increased tumour-
initiate autocrine signalling.
xenograft growth, whereas inhibition of HIF-1 activity
markedly impaired tumour growth. However, the spe-
cific consequences of increased HIF-1 activity differed
fusion protein also disrupts the ability of many other according to cell type. In pancreatic cancer cells, both
transcription factors to interact with CBP/p300, and gain- and loss-of-function experiments highlighted the
therefore limits the conclusions that can be drawn from role of HIF-1 activity in regulating glucose metabolism
these studies. and cell survival, whereas in colon cancer cells a correla-
tion between HIF-1α expression, angiogenesis and
tumour growth was observed that was not seen in pan-
Table 1 | Genetic alterations that increase HIF-1 activity creatic cancer cells. Second, HIF-1 activity was inversely
associated with tumour growth only in studies involving
Alteration in tumour Mechanism of HIF-1α induction References
ES cells that lacked the large complement of genetic
VHL loss of function Decreased ubiquitylation 26 alterations that are characteristic of cancer cells. These
p53 loss of function Decreased ubiquitylation 66 results emphasize that the consequences of HIF-1α over-
PTEN loss of function Increased synthesis 48,49 expression are dependent on the cellular context and
PI3K–AKT–mTOR signalling* Increased synthesis 47,48 therefore highlight the importance of using appropriate
MEK–ERK signalling* Increased synthesis 44
models to study the roles of HIF-1 in cancer biology. It
should be noted that all of the studies reported so far
ERBB2 gain of function Increased synthesis 47
involve injection of cells into the subcutaneous space and
EGFR signalling* Increased synthesis 48 therefore fail to replicate the stromal microenvironment
IGF1R signalling* Increased synthesis 44 in which cancer cells normally develop. The observation
PGE2 signalling* Increased synthesis 45,98 that altering HIF-1 activity did not affect the vasculariza-
SRC gain of function Increased synthesis 43 tion of pancreatic cancer xenografts might reflect the fact
ARF loss of function Decreased nucleolar sequestration 99 that those cells were growing in the subcutaneous space
rather than in the pancreas.
BCL2 overexpression Not determined 100
*Increased signalling could be due to genetic alteration in a component of the pathway or an
upstream activator. For example, AKT gain-of-function mutation or PTEN loss-of-function mutation
HIF-1 and clonal selection
induces PI3K–AKT–mTOR signalling; EGFR amplification or TGF-α overexpression induces EGFR Cancer progression involves the selection of cells that
(and PI3K–AKT–mTOR and MEK–ERK) signalling. EGFR, epidermal growth factor receptor; ERK, bear mutations that increase their rate of cell prolifer-
extracellular-signal-regulated kinase; IGFR1, insulin-like growth-factor-1 receptor; MEK, MAP/ERK
kinase; mTOR, mammalian target of rapamycin; PGE2, prostaglandin E2; PI3K, phosphatidylinositol ation and survival. These effects can be mediated by
3-kinase; VHL, von Hippel–Lindau protein. mutations that either directly target components of

NATURE REVIEWS | C ANCER VOLUME 3 | OCTOBER 2003 | 7 2 7

REVIEWS

Table 2 | Increased HIF-1α levels in human cancers* HIF-1 can induce apoptosis — for example, through
the stabilization of p53 (REF. 75) or through transacti-
Tumour type Association References
vation of BNIP3, which encodes a pro-apoptotic
Cervical, early stage Increased mortality 101
BCL2 family member76. In such cells, complementary
Cervical, RTX Increased mortality 102 mutations that inactivate p53 or activate BCL2
Lung, NSCLC Decreased mortality 58 expression could be necessary for the net effect of
Lung, NSCLC Increased mortality (HIF-2α) 59 increased HIF-1 activity to promote cancer-cell sur-
Breast, LN-positive Increased mortality 103 vival. In some cancers (for example, ovarian),
Breast, LN-negative Increased mortality 104
hypoxia-induced HIF-1-mediated apoptosis might
represent an important factor in the positive selection
Oligodendroglioma Increased mortality 105
of p53-null and BCL2-overexpressing cells77. In other
Oropharyngeal SCC Increased mortality, radiation resistance 81 cases, HIF-1α expression could be involved in the
Ovarian Increased mortality (with p53) 61 very early stages of cancer progression, as in the case
Oesophageal, early stage Resistance to PDT (with BCL2) 62 of VHL-null RCC (see later). HIF-1α levels are
Endometrial Increased mortality 106 increased in ductal carcinoma in situ — the pre-
Head and neck SCC, S/P surgery Decreased mortality 57
invasive stage of breast cancer — and are associated
with increased microvascular density 78, indicating
Head and neck SCC Increased mortality (HIF-2α) 60
that HIF-1α expression might have an important role
GI stromal tumour of stomach Increased mortality 107 early in breast cancer progression.
*Protein levels determined by immunohistochemical analysis of biopsy samples. GI, gastrointestinal; At each point in the temporal–spatial progression of
LN, lymph node; NSCLC, non-small-cell lung cancer; PDT, photodynamic therapy; RTX, radiation
therapy; SCC, squamous-cell carcinoma; S/P, status post. a cancer, there is a constant selection for cells bearing
particular (and changing) combinations of genetic and
epigenetic alterations that impart to those cells the
the proliferative/apoptotic machinery or indirectly greatest relative probability of survival and prolifera-
affect these processes; for example, by promoting tion. The ability to identify genetic alterations that are
angiogenesis. Many of these mutations also contribute selected during tumour progression provides a rational
to the invasive and metastatic properties of cancer approach to therapeutic target selection. However, in
cells74. Genetic alterations that activate oncogenes and addition to ‘primary’ targets of clonal selection (onco-
inactivate tumour-suppressor genes also result in genes and tumour-suppressor genes), candidates for
increased HIF-1α expression that might promote the therapeutic target selection should also include ‘sec-
survival of cells that bear these mutations and so con- ondary’ targets, the expression or activity of which are
tribute to their selection. As observed in xenograft affected as a result of genetic alterations involving
assays, the consequences of increased HIF-1 activity several ‘primary’ targets. HIF-1 represents a prime
are cell-type specific. In some cell types, increased example of such a ‘secondary’ target, whereas VEGF

Table 3 | Effects of altered HIF-1 activity on tumour growth*

Cell type Manipulation Rate of Tumour Ex vivo‡ References
tumour growth angiogenesis
Decreased HIF-1 activity §
Hepa1 mouse hepatoma Hif-1β LOF – – – 43,63
Mouse ES Hif-1α KO – – – 7
Mouse ES Hif-1α KO + – + 5
MEF (T-Ag, HRASV12) Hif-1α KO – 0 – 42,64
HCT116, MDA-MB-435 HIF1α TAD – – – 70
PCI-43 (pancreatic CA) HIF-1α DN – 0 – 69
MEF (T-Ag, HRASV12) Hif-1α KO, chemo – ND – 65
5 human cancer cell lines YC-1 i.p. – – ND 83
Increased HIF-1 Activity §
HCT116 (colon CA) HIF-1α GOF + + ND 66
PCI-10 (pancreatic CA) HIF-1α GOF + 0 + 67
786-O/VHL HIF-2α (P531A) + + ND 71
786-O/VHL HIF-1α ODD + ND ND 72
Mouse ES Vhl KO – + ND 73
*All effects monitored by xenograft growth in nude mice. ‡Growth or survival of cells cultured under conditions of hypoxia or O2/glucose
deprivation ex vivo. §In this context, HIF-1 refers to both HIF-1α–HIF-1β and HIF-2α–HIF-1β heterodimers. Chemo, chemotherapy; DN,
dominant negative; ES, embryonic stem; GOF, gain of function; i.p., intraperitoneal injection; KO, knockout (homozygous for null allele);
LOF, loss of function; MEF, mouse embryonic fibroblast; ND, not determined; ODD, O2-dependent degradation domain; TAD,
transactivation domain; T-Ag, large T antigen; –, decreased; +, increased; 0, no effect.

728 | OCTOBER 2003 | VOLUME 3 www.nature.com/reviews/cancer


Table 4 | Novel therapeutic agents that inhibit HIF-1 activity
Agent(s)
Inhibitors of signal-transduction pathways
BAY 43-9006
CCI-779
Celebrex
PD98059
Trastuzumab (Herceptin)
ZD-1839 (Iressa), OSI-774
Imatinib (Glivec)
Small-molecule inhibitors of HIF-1 activity
2ME2
17-AAG
Camptothecin, Topotecan
Pleurotin, 1-methylpropyl
2-imidazolyl disulphide
YC-1
BCR–ABL, breakpoint-cluster-region–Abelson-leukaemia; COX2, cyclooxygenase 2; EGFR,
epidermal growth-factor receptor; HSP90, heat-shock protein 90; MEK, MAP/ERK kinase; mTOR,
mammalian target of rapamycin; PDGFR, platelet-derived growth-factor receptor.
NCI DIVERSITY SET
A group of approximately 2,000
compounds that is representative
of the complete chemical
repository of the National
Cancer Institute’s Developmental
Therapeutics Program.
TOPOISOMERASE I INHIBITORS
Drugs that inhibit an enzyme
that relaxes supercoiled DNA by
introducing a transient single-
strand break.
NATURE REVIEWS | C ANCER
Molecular target(s) Current status
RAF kinase Clinical trials
mTOR Clinical trials
COX2 Clinical trials
MEK Not in clinical use
ERBB2 receptor tyrosine kinase Approved agent
EGFR tyrosine kinase Clinical trials
BCR–ABL, PDGFR tyrosine kinases Approved agent
Microtubule polymerization Clinical trials
HSP90 Clinical trials
Topoisomerase I Approved agents
Thioredoxin 1 Not in clinical use
Not determined Not in clinical use
and its receptors represent examples of a ‘tertiary’ tar-
get. The hypothesis that increased HIF-1 activity con-
tributes to clonal selection and cancer progression is
supported by clinical and experimental data. The asso-
ciation between increased HIF-1α expression and
increased patient mortality establishes a necessary clini-
cal correlation, whereas experimental manipulation of
HIF-1 activity in tumour xenografts provides evidence
of causation, as described above.
HIF-1 targeted therapeutics
Drugs that specifically target tumour stromal-cell
responses represent an important new class of thera-
peutic agents. In particular, a large number of drugs
are in clinical trials at present as anticancer agents
based on their ability to inhibit angiogenesis79. One
concern about this strategy is that inhibition of
angiogenesis might select for cancer cells that are
adapted to hypoxic conditions, as these are the cells
that are most likely to survive a reduction in perfu-
sion. A large body of data has indicated that hypoxic
cancer cells are more likely to be resistant to radiation
and chemotherapy, and have increased potential for
invasion, metastasis and patient mortality 80. Recent
studies have provided evidence indicating that HIF-1
mediates resistance to chemotherapy and radia-
tion65,81. Inhibition of HIF-1 activity could therefore
represent an important component of combination
anti-angiogenesis therapies. In addition to drugs that
have been developed specifically as anti-angiogenesis
agents, it is clear that many novel therapeutic agents
that target signal-transduction pathways have anti-
angiogenic effects. This effect seems to be due in part
to the fact that inhibition of signal-transduction
pathways results in decreased levels of HIF-1α.
Several examples are provided in TABLE 4. Screens for
small-molecule inhibitors of HIF-1 are underway in
REVIEWS
university, government and industry laboratories and
several agents that inhibit HIF-1, angiogenesis and
xenograft growth have been identified (TABLE 4). A
preliminary screen of the NCI DIVERSITY SET of small-
molecule chemotherapeutic agents using a cell-based
assay revealed that TOPOISOMERASE I INHIBITORS block HIF-1α
expression via an undetermined mechanism82. The
small molecule YC-1 (3-(5′-hydroxy-methyl-2′-furyl)-
1-benzylindazole) was also shown to reduce HIF-1α
levels and xenograft growth83. YC-1 is known to stimu-
late soluble guanylate-cyclase activity, but this effect is
not required for inhibition of HIF-1α levels. The mech-
anism by which YC-1 reduces HIF-1α levels has not
been established. HIF-1α interacts with the chaperone
HSP90, and the HSP90 inhibitor 17-allyl-aminogel-
danamycin (17-AAG) induces HIF-1α degradation in
a VHL-independent manner 84– 86, indicating that
HSP90 is required for HIF-1α stability. The redox reg-
ulator thioredoxin-1 also exerts a positive effect on
HIF-1α expression. Thioredoxin inhibitors block
HIF-1α expression and xenograft growth87. Finally,
disruption of microtubule polymerization by
2-methoxyoestradiol (2ME2) has also been shown
to result in decreased HIF-1α levels and decreased
VEGF mRNA expression in cultured cells88. In vivo,
2ME2 decreases tumour-xenograft growth and
vascularization. YC-1, topoisomerase I inhibitors,
17-AAG, thioredoxin inhibitors and 2ME2 share in
common an ability to decrease HIF-1α levels,
inhibit the expression of VEGF and other HIF-1 tar-
get genes, and impair xenograft growth and vascu-
larization. Therefore, the anticancer effects of these
agents might be due, in part, to their inhibition of
HIF-1. On the other hand, it seems that none of
these drugs specifically target HIF-1. The lack of
selectivity increases the difficulty in correlating mol-
ecular and clinical responses in patients, but it does
not disqualify these drugs as potential anticancer
agents. Ongoing screens should lead to the identifi-
cation of more selective HIF-1 inhibitors in the near
future. Assuming that this prediction holds, in what
clinical contexts are HIF-1 inhibitors most likely to
have therapeutic efficacy?
Candidate patient populations
RCC that is associated with VHL mutations is a good
candidate for HIF-1 targeted therapy. Whereas several
RCC cell lines (for example, 786-O) express HIF-2α
but not HIF-1α, immunohistochemical analyses have
revealed that overexpression is seen in most primary
cancers54,56. All experimental data indicate that HIF-1 is
involved in the pathophysiology of this cancer, and the
upregulation of HIF-1 activity seems to occur in the
earliest detected neoplastic lesions89. HIF-1 target genes
that are activated in RCC include platelet-derived
growth factor-β (PDGFB)and TGF-α. These genes
encode proteins that activate the PDGF and EGF recep-
tors — two of the main receptor tyrosine kinases that
signal via the PI3K and MAPK pathways to stimulate
cell proliferation and survival. In addition, HIF-1
upregulates VEGF expression, leading to the abundant
VOLUME 3 | OCTOBER 2003 | 7 2 9
REVIEWS
PSEUDOPALISADING CELLS
Rows of viable cells surrounding
areas of necrosis that are a
histopathological characteristic
of glioblastoma multiforme.
ORTHOTOPIC
TRANSPLANTATION
The introduction of foreign
tumour cells into another
species at the site from which
they were derived. For example,
injection of human breast cancer
cells into the mouse mammary
fat pad.
730 | OCTOBER 2003 | VOLUME 3
vascularization that is a characteristic of RCCs. So,
increased HIF-1 activity in RCC has direct and marked
effects on both cancer and stromal cells.
Administration of a HIF-1 inhibitor, together with a
tyrosine kinase inhibitor that targets VEGF/PDGF
and/or EGFR, is one potential therapeutic regimen in
this disease.
Another candidate is glioblastoma multiforme
(GBM). Like RCC, GBM is an intractable disease, and
patients typically survive for less than 1 year. Among
gliomas, there is strong correlation between HIF-1α
expression, tumour grade and tumour vasculariza-
tion90. PTEN loss of function and EGFR gain of func-
tion are commonly observed in primary GBM, and
are known to increase HIF-1α levels48,49. GBMs are
highly vascularized tumours that are characterized by
areas of necrosis surrounded by PSEUDOPALISADING CELLS
that express high levels of HIF-1α protein and VEGF
mRNA. So, in GBM, hypoxia-induced HIF-1α
expression seems to also be involved in the disease
pathophysiology. GBM is the most highly invasive
cancer known and high levels of HIF-1α expression
have been detected at the leading edge of invading
cancer cells in both human biopsies and following
ORTHOTOPIC TRANSPLANTATION of labelled glioma cells into
the mouse brain 90. In patients with GBM, a HIF-1
inhibitor might be effective in combination with a
rapamycin derivative (that targets increased
PI3K–AKT–mTOR signalling induced by PTEN loss
of function) anti-angiogenic agents and/or tyrosine
kinase inhibitors.
A number of other tumour types have also been
found to upregulate HIF-1, as determined by immuno-
histochemical studies (TABLE 2). In patients with
oropharyngeal cancers, HIF-1α overexpression has
been associated with radiation resistance and increased
mortality, regardless of tumour grade, stage or other
biomarkers81. Importantly, HIF-1α overexpression in
the primary tumour was shown to predict radiation
resistance in lymph-node metastases. These tumours
might therefore be susceptible to HIF-1-targeted thera-
pies. The combination of HIF-1α overexpression and
(mutant) p53 overexpression also seems to define a
subset of ovarian cancers that might be susceptible to
treatment with HIF-1 inhibitors61.
Better predictors of clinical efficacy
The tumour-xenograft model represents the current
standard for preclinical testing of anticancer agents. This
model, however, has too many limitations — only a few
of which will be discussed here — to remain an accept-
able gateway to clinical trials. First, human cancers
develop by a stepwise process of mutation and selection.
Although clonal selection occurs, cancers are character-
ized by a tremendous degree of genetic heterogeneity.
Among the millions of cells in a tumour, there are likely
to exist subpopulations that are resistant to any single
therapeutic agent that might be administered and, for this
reason, multidrug regimens are likely to be necessary to
successfully treat advanced cancers. This degree of genetic
heterogeneity does not exist in a tumour xenograft. A
second key limitation of the xenograft model relates to
the crucial importance of the interactions that take place
between cancer cells and the stromal microenvironment.
The factors required for angiogenesis, invasion and
metastasis in one tissue differ significantly from those
required in another site. The subcutaneous space into
which xenografts are transplanted does not provide the
same microenvironment as in common sites of human
cancer such as the breast, colon and lung. Also, most
patients die of metastatic disease, which cannot be mod-
elled in encapsulated subcutaneous xenografts. Finally,
xenograft assays are performed in immunodeficient
(nude or SCID) mice, so the inflammatory cell popula-
tions that are involved in human cancer progression are
not represented in this model.
An incremental but significant advance that is rele-
vant to the issue of the tumour microenvironment is
the use of orthotopic transplantation. Glioblastoma
cells injected into the rodent brain form tumours that
share remarkable histological similarity to invasive
human glioblastomas90. Certain human breast cancer
cell lines, when injected into the mammary fat pad,
show a high incidence of metastasis91. These models
provide an opportunity to study important aspects of
cancer biology that cannot be addressed in subcuta-
neous xenografts, such as further analysis of the role
of HIF-1 in invasion and metastasis.
More elegant models of cancer have been developed
by engineering loss-of-function mutations in tumour-
suppressor genes or gain-of-function mutations in
oncogenes into the mouse genome. This approach can
model hereditary tumour-predisposition syndromes
in which a heterozygous mutation in a tumour-
suppressor gene such as TP53 is present in the
germline. However, for most oncogenes, mutations
have not been detected in the human germline, proba-
bly because they would result in developmental defects.
Novel approaches, such as the use of retroviral infec-
tion of the target tissue to induce oncogene expression,
might provide better mouse models of cancer92,93. A key
advantage of these models is that they reproduce the
heterogeneity of cancer progression. A drawback (espe-
cially as models for drug testing) is that they result in
only a limited number of animals that bear tumours of
a particular stage at any given time. The development
of in vivo tumour-imaging techniques94 could provide a
solution to this problem as tumour-bearing mice can
be identified by periodic screening and then started on
therapy. Testing of potential therapeutic agents (target-
ing HIF-1 or any other molecule) for efficacy in at least
one such model should be considered as a necessary
prerequisite to clinical trials.
Novel imaging techniques also provide a means to
monitor the response to therapy in patients 95–97.
In vivo imaging can be used to monitor tumour-cell
apoptosis and proliferation, angiogenesis, blood flow,
oxygenation and glucose metabolism. Developments
in magnetic resonance imaging and positron emis-
sion tomography should provide the means to moni-
tor any of these variables in patients in the near
future. Imaging can also be used to monitor the
www.nature.com/reviews/cancer

response of a drug target to therapy. For example,
imaging techniques could be used to show inhibition of
receptor-tyrosine-kinase activity or gene transcription,
and to correlate this molecular effect with patient
1. Semenza, G. L. & Wang, G. L. A nuclear factor induced by
hypoxia via de novo protein synthesis binds to the human
erythropoietin gene enhancer at a site required for
transcriptional activation. Mol. Cell. Biol. 12, 5447–5454
(1992).
2. Wang, G. L. & Semenza, G. L. Purification and
characterization of hypoxia-inducible factor 1. J. Biol. Chem.
270, 1230–1237 (1995).
3. Wang, G. L., Jiang, B.-H., Rue, E. A. & Semenza, G. L.
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS
heterodimer regulated by cellular O2 tension. Proc. Natl
Acad. Sci. USA 92, 5510–5514 (1995).
4. Harris, A. L. Hypoxia — a key regulatory factor in tumor
growth. Nature Rev. Cancer 2, 38–46 (2001).
5. Carmeliet, P. et al. Role of HIF-1α in hypoxia-mediated
apoptosis, cell proliferation and tumour angiogenesis.
Nature 394, 485–490 (1998).
6. Iyer, N. V. et al. Cellular and developmental control of O2
homeostasis by hypoxia-inducible factor 1α. Genes Dev.
12, 149–162 (1998).
7. Ryan, H. E., Lo, J. & Johnson, R. S. HIF-1α is required for
solid tumor formation and embryonic vascularization.
EMBO J. 17, 3005–3015 (1998).
8. Krishnamachary, B. et al. Regulation of colon carcinoma cell
invasion by hypoxia-inducible factor 1. Cancer Res. 63,
1138–1143 (2003).
9. Wykoff, C. C. et al. Identification of novel hypoxia
dependent and independent target genes of the von Hippel-
Lindau (VHL) tumour suppressor by mRNA differential
expression profiling. Oncogene 19, 6297–6305 (2000).
10. Pennacchietti, S. et al. Hypoxia promotes invasive growth by
transcriptional activation of the met protooncogene. Cancer
Cell 3, 347–361 (2003).
11. Yu, J. et al. Identification and classification of p53-regulated
genes. Proc. Natl Acad. Sci. USA 96, 14517–14522 (1999).
12. Ema, M. et al. A novel bHLH-PAS factor with close
sequence similarity to hypoxia-inducible factor 1α regulates
the VEGF expression and is potentially involved in lung and
vascular development. Proc. Natl Acad. Sci. USA 94,
4273–4278 (1997).
13. Flamme, I. et al. HRF, a putative basic helix-loop-helix-PAS-
domain transcription factor is closely related to hypoxia-
inducible factor-1α and developmentally expressed in blood
vessels. Mech. Dev. 63, 51–60 (1997).
14. Hogenesch, J. B. et al. Characterization of a subset of the
basic-helix-loop-helix-PAS superfamily that interacts with
components of the dioxin signaling pathway. J. Biol. Chem.
272, 8581–8593 (1997).
15. Tian, H., McKnight, S. L. & Russell, D. W. Endothelial PAS
domain protein 1 (EPAS1), a transcription factor selectively
expressed in endothelial cells. Genes Dev. 11, 72–82 (1997).
16. Brusselmans, K. et al. Hypoxia-inducible factor-2α (HIF-
2α) is involved in the apoptotic response to hypoglycemia
but not to hypoxia. J. Biol. Chem. 276, 39192–39196
(2001).
17. Makino, Y. et al. Inhibitory PAS domain protein (IPAS) is a
hypoxia-inducible splicing variant of thehypoxia-inducible
factor-3α locus. J. Biol. Chem. 277, 32405–32408 (2002).
18. Bruick, R. K. & McKnight, S. L. A conserved family of prolyl-
4-hydroxylases that modify HIF. Science 294, 1337–1340
(2001).
19. Epstein, A. C. et al. C. elegans EGL-9 and mammalian
homologs define a family of dioxygenases that regulate HIF
by prolyl hydroxylation. Cell 107, 43–54 (2001).
20. Ivan, M. et al. HIFα targeted for VHL-mediated destruction
by proline hydroxylation: implications for O2 sensing.
Science 292, 464–468 (2001).
21. Jaakkola, P. et al. Targeting of HIF-α to the von Hippel-
Lindau ubiquitylation complex by O2-regulated prolyl
hydroxylation. Science 292, 468–472 (2001).
22. Masson, N. et al. Independent function of two destruction
domains in hypoxia-inducible factor-alpha chains activated
by prolyl hydroxylation. EMBO J. 20, 5197–5206 (2001).
23. Yu, F., White, S. B., Zhao, Q. & Lee, F. S. HIF-1α binding to
VHL is regulated by stimulus-sensitive proline hydroxylation.
Proc. Natl Acad. Sci. USA 98, 9630–9635 (2001).
24. Cockman, M. E. et al. Hypoxia inducible factor-α binding
and ubiquitylation by the von Hippel-Lindau tumor
suppressor protein. J. Biol. Chem. 275, 25733–25741
(2000).
NATURE REVIEWS | C ANCER
outcome. This will be particularly crucial for
inhibitors that target kinases or transcription factors
such as HIF-1 that regulate many key physiological
pathways in cancer cells.
25. Kamura, T. et al. Activation of HIF1α ubiquitination by a
reconstituted von Hippel-Lindau (VHL) tumor suppressor
complex. Proc. Natl Acad. Sci. USA 97, 10430–10435
(2000).
26. Maxwell, P. H. et al. The tumour suppressor protein VHL
targets hypoxia-inducible factors for oxygen-dependent
proteolysis. Nature 399, 271–275 (1999).
27. Ohh, M. et al. Ubiquitination of hypoxia-inducible factor
requires direct binding to the β-domain of the von Hippel-
Lindau protein. Nature Cell Biol. 2, 423–427 (2000).
28. Tanimoto, K., Makino, Y., Pereira, T. & Poellinger, L.
Mechanism of regulation of the hypoxia-inducible factor-1α
by the von Hippel-Lindau tumor protein. EMBO J. 19,
4298–4309 (2000).
29. Jiang, B.-H., Semenza, G. L., Bauer, C. & Marti, H. H.
Hypoxia-inducible factor 1 levels vary exponentially over a
physiologically relevant range of O2 tension. Am. J. Physiol.
271, C1172–C1180 (1996).
30. Jeong, J. W. et al. Regulation and destabilization of HIF-1α
by ARD1-mediated acetylation. Cell 111, 709–720 (2002).
31. Jiang, B.-H. et al. Transactivation and inhibitory domains of
hypoxia-inducible factor 1α: modulation of transcriptional
activity by oxygen tension. J Biol. Chem. 272, 19253–19260
(1997).
32. Pugh, C. W. et al. Activation of hypoxia-inducible factor-1;
definition of regulatory domains within the α subunit. J Biol.
Chem. 272, 11205–11214 (1997).
33. Mahon, P. C., Hirota, K. & Semenza, G. L. FIH-1: a novel
protein that interacts with HIF-1α and VHL to mediate
repression of HIF-1 transcriptional activity. Genes Dev. 15,
2675–2686 (2001).
34. Hewitson, K. S. et al. Hypoxia-inducible factor (HIF)
asparagine hydroxylase is identical to factor inhibiting HIF
(FIH) and is related to the cupin structural family. J. Biol.
Chem. 277, 26351–26355 (2002).
35. Lando, D. et al. Asparagine hydroxylation of the HIF
transactivation domain a hypoxic switch. Science 295,
858–861 (2002).
36. Lando, D. et al. FIH-1 is an asparaginyl hydroxylase enzyme
that regulates the transcriptional activity of hypoxia-inducible
factor. Genes Dev. 16, 1466–1471 (2002).
37. Dames, S. A. et al. Structural basis for HIF-1α/CBP
recognition in the cellular hypoxic response. Proc. Natl
Acad. Sci. USA 99, 5271–5276 (2002).
38. Freedman, S. J. et al. Structural basis for recruitment of
CBP/p300 by hypoxia-inducible factor-1α. Proc. Natl Acad.
Sci. USA 99, 5367–5372 (2002).
39. Hon, W. C. et al. Structural basis for the recognition of
hydroxyproline in HIF-1α by pVHL. Nature 417, 975–978
(2002).
40. Min, J. H. et al. Structure of an HIF-1α-pVHL complex:
hydroxyproline recognition in signaling. Science 296,
1886–1889 (2002).
41. Brand, K. A. & Hermfisse, U. Aerobic glycolysis by
proliferating cells: a protective strategy against reactive
oxygen species. FASEB J. 11, 388–395 (1997).
42. Seagroves, T. N. et al. Transcription factor HIF-1 is a
necessary mediator of the pasteur effect in mammalian
cells. Mol. Cell. Biol. 21, 3436–3444 (2001),
43. Jiang, B. H., Agani, F., Passaniti, A. & Semenza, G. L.
V-SRC induces expression of hypoxia-inducible factor 1
(HIF-1) and transcription of genes encoding vascular
endothelial growth factor and enolase 1: involvement of HIF-1
in tumor progression. Cancer Res. 57, 5328–5335 (1997).
44. Fukuda, R. et al. Insulin-like growth factor 1 induces
hypoxia-inducible factor 1-mediated vascular endothelial
growth factor expression, which is dependent on MAP
kinase and phosphatidylinositol 3-kinase signaling in
colon cancer cells. J. Biol. Chem. 277, 38205–38211
(2002).
45. Fukuda, R., Kelly, B. & Semenza, G. L. Vascular endothelial
growth factor gene expression in colon cancer cells
exposed to prostaglandin E2 is mediated by hypoxia-
inducible factor 1. Cancer Res. 63, 2330–2334 (2003).
46. Hellwig-Burgel, T., Stiehl, D. P. & Jelkmann, W. in Oxygen
Sensing: Responses and Adaptation to Hypoxia (eds Lahiri,
S., Semenza, G. L. & Prabhakar, N. R.) 95–108 (Marcel
Dekker, Inc., New York, 2003).
47. Laughner, E. et al. HER2 (neu) signaling increases the rate
of hypoxia-inducible factor 1α (HIF-1α) synthesis: novel
REVIEWS
mechanism for HIF-1-mediated vascular endothelial
growth factor expression. Mol. Cell. Biol. 21, 3995–4004
(2001).
48. Zhong, H. et al. Modulation of HIF-1α expression by the
epidermal growth factor/phosphatidylinositol
3-kinase/PTEN/AKT/FRAP pathway in human prostate
cancer cells: implications for tumor angiogenesis and
therapeutics. Cancer Res. 60, 1541–1545 (2000).
49. Zundel, W. et al. Loss of PTEN facilitates HIF-1-mediated
gene expression. Genes Dev. 14, 391–396 (2000).
50. Richard, D. E. et al. p42/p44 mitogen-activated protein
kinases phosphorylate hypoxia-inducible factor 1α (HIF-1α)
and enhance the transcriptional activity of HIF-1. J. Biol.
Chem. 274, 32631–32637 (1999).
51. Sodhi, A. et al. The Kaposi’s sarcoma-associated herpes
virus G protein-coupled receptor up-regulates vascular
endothelial growth factor expression and secretion through
mitogen-activated protein kinase and p38 pathways acting
on hypoxia-inducible factor 1α. Cancer Res. 60, 4873–4880
(2000).
52. Sang, N. et al. MAPK signaling up-regulates the activity of
hypoxia-inducible factors by its effects on p300. J. Biol.
Chem. 278, 14013–14019 (2003).
53. Hudson, C. C. et al. Regulation of hypoxia-inducible
factor 1alpha expression and function by the mammalian
target of rapamycin. Mol. Cell. Biol. 22, 7004–7014
(2002).
54. Zhong, H. et al. Overexpression of hypoxia-inducible factor
1α in common human cancers and their metastases.
Cancer Res. 59, 5830–5835 (1999).
55. Feldser, D. et al. Reciprocal positive regulation of hypoxia-
inducible factor 1α and insulin-like growth factor 2. Cancer
Res. 59, 3915–3918 (1999).
56. Talks, K. L. et al. The expression and distribution of the
hypoxia-inducible factors HIF-1α and HIF-2α in normal
human tissues, cancers, and tumor-associated
macrophages. Am. J. Pathol. 157, 411–421 (2000).
57. Beasley, N. J. et al. Hypoxia-inducible factors HIF-1α and
HIF-2α in head and neck cancer: relationship to tumor
biology and treatment outcome in surgically resected
patients. Cancer Res. 62, 2493–2497 (2002).
58. Volm, M. & Koomagi, R. Hypoxia-inducible factor (HIF-1)
and its relationship to apoptosis and proliferation in lung
cancer. Anticancer Res. 20, 1527–1533 (2000).
59. Giatromanolaki, A. et al. Relation of hypoxia inducible factor
1α and 2α in operable non-small cell lung cancer to
angiogenic/molecular profile of tumours and survival. Br. J.
Cancer 85, 881–890 (2001).
60. Koukourakis, M. I. et al. Hypoxia-inducible factor (HIF1A and
HIF2A), angiogenesis, and chemoradiotherapy outcome of
squamous cell head-and-neck cancer. Int. J. Radiat. Oncol.
Biol. Phys. 53, 1192–1202 (2002).
61. Birner, P. et al. Expression of hypoxia-inducible factor 1α in
epithelial ovarian tumors: its impact on prognosis and on
response to chemotherapy. Clin. Cancer Res. 7, 1661–1668
(2001).
62. Koukourakis, M. I. et al. Hypoxia inducible factor (HIF-1α
and HIF-2α) expression in early esophageal cancer and
response to photodynamic therapy and radiotherapy.
Cancer Res. 61, 1830–1832 (2001).
63. Maxwell, P. H. et al. Hypoxia-inducible factor-1 modulates
gene expression in solid tumors and influences both
angiogenesis and tumor growth. Proc. Natl Acad. Sci. USA
94, 8104–8109 (1997).
64. Ryan, H. E. et al. Hypoxia-inducible factor-1α is a positive
factor in solid tumor growth. Cancer Res. 60, 4010–4015
(2000).
65. Unruh, A. et al. The hypoxia-inducible factor-1α is a
negative factor for tumor therapy. Oncogene 22,
3213–3220 (2003).
66. Ravi, R. et al. Regulation of tumor angiogenesis by p53-
induced degradation of hypoxia-inducible factor 1α. Genes
Dev. 14, 34–44 (2000).
67. Akakura, N. et al. Constitutive expression of hypoxia-
inducible factor 1α renders pancreatic cancer cells resistant
to apoptosis induced by hypoxia and nutrient deprivation.
Cancer Res. 61, 6548–6554 (2001).
68. Jiang, B. H. et al. Dimerization, DNA binding, and
transactivation properties of hypoxia-inducible factor 1.
J. Biol. Chem. 271, 17771–17778 (1996).
VOLUME 3 | OCTOBER 2003 | 7 3 1
REVIEWS
69. Chen, J. et al. Dominant-negative hypoxia-inducible factor
1α reduces tumorigenicity of pancreatic cancer cells
through the suppression of glucose metabolism. Am.
J. Pathol. 162, 1283–1291 (2003).
70. Kung, A. L. et al. Suppression of tumor growth through
disruption of hypoxia-inducible transcription. Nature Med. 6,
1335–1340 (2000).
71. Kondo, K. et al. Inhibition of HIF is necessary for tumor
suppression by the von Hippel-Lindau protein. Cancer Cell
1, 237–246 (2002).
72. Maranchie, J. K. et al. The contribution of VHL substrate
binding and HIF-1α to the phenotype of VHL loss in renal
cell carcinoma. Cancer Cell 1, 247–255 (2002).
73. Mack, F. A. et al. Loss of pVHL is sufficient to cause HIF
dysregulation in primary cells but does not promote tumor
growth. Cancer Cell 3, 75–88 (2003).
74. Bernards, R. & Weinberg, R. A. Metastasis genes: a
progression puzzle. Nature 418, 823 (2002).
75. An, W. G. et al. Stabilization of wild-type p53 by hypoxia-
inducible factor 1α. Nature 392, 405–408 (1998).
76. Bruick, R. K. Expression of the gene encoding the
proapoptotic Nip3 protein is induced by hypoxia. Proc. Natl
Acad. Sci. USA 97, 9082–9087 (2000).
77. Graeber, T. G. et al. Hypoxia-mediated selection of cells with
diminished apoptotic potential in solid tumours. Nature 379,
88–91 (1996).
78. Bos, R. et al. Levels of hypoxia-inducible factor 1α during
breast carcinogenesis. J. Natl Cancer Inst. 93, 309–314
(2001).
79. Semenza, G. L. Angiogenesis in ischemic and neoplastic
disorders. Annu. Rev. Med. 54, 17–28 (2003).
80. Hockel, M. & Vaupel, P. Tumor hypoxia: definitions and
current clinical, biologic, and molecular aspects. J. Natl
Cancer Inst. 93, 266–276 (2001).
81. Aebersold, D. M. et al. Expression of hypoxia-inducible
factor 1α: a novel predictive and prognostic parameter in the
radiotherapy of oropharyngeal cancer. Cancer Res. 61,
2911–2916 (2001).
82. Rapisarda, A. et al. Identification of small molecule inhibitors
of hypoxia-inducible factor 1 transcriptional activation
pathway. Cancer Res. 62, 4316–4324 (2002).
83. Yeo, E. J. et al. YC-1: a potential anticancer drug targeting
hypoxia-inducible factor 1. J. Natl Cancer Inst. 95, 516–525
(2003).
84. Isaacs, J. S. et al. Hsp90 regulates a von Hippel-Lindau-
independent hypoxia-inducible factor-1α-degradative
pathway. J. Biol. Chem. 277, 29936–29944 (2002).
85. Mabjeesh, N. J. et al. Geldanamycin induces degradation of
hypoxia-inducible factor 1α protein via the proteosome
732 | OCTOBER 2003 | VOLUME 3
pathway in prostate cancer cells. Cancer Res. 62,
2478–2482 (2002).
86. Zagzag, D. et al. Geldanamycin inhibits migration of glioma
cells in vitro: a potential role for hypoxia-inducible factor
(HIF-1α) in glioma cell invasion. J. Cell. Physiol. 196,
394–402 (2003).
87. Welsh, S. J. et al. The thioredoxin redox inhibitors
1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit
hypoxia-induced factor 1α and vascular endothelial
growth factor formation. Mol. Cancer Ther. 2, 235–243
(2003).
88. Mabjeesh, N. J. et al. 2ME2 inhibits tumor growth and
angiogenesis by disrupting microtubules and dysregulating
HIF. Cancer Cell 3, 363–375 (2003).
89. Mandriota, S. J. et al. HIF activation identifies early lesions
in VHL kidneys: evidence for site-specific tumor
suppressor function in the nephron. Cancer Cell 1,
459–468 (2002).
90. Zagzag, D. et al. Expression of hypoxia-inducible factor 1α
in brain tumors: association with angiogenesis, invasion, and
progression. Cancer 88, 2606–2618 (2000).
91. Price, J. E., Polyzos, A., Zhang, R. D. & Daniels, L. M.
Tumorigenicity and metastasis of human breast
carcinoma cell lines in nude mice. Cancer Res. 50,
717–721 (1990).
92. Holland, E. C. Gliomagenesis: genetic alterations and
mouse models. Nature Rev. Genet. 2, 120–129
(2001).
93. Van Dyke, T. & Jacks, T. Cancer modeling in the modern
era: progress and challenges. Cell 108, 135–144
(2002).
94. Pomper, M. G. Can small animal imaging accelerate drug
development? J. Cell. Biochem. 39 (Suppl.), 211–220
(2002).
95. Artemov, D., Mori, N., Ravi, R. & Bhujwalla, Z. M. Magnetic
resonance molecular imaging of the her-2/neu receptor.
Cancer Res. 63, 2723–2727 (2003).
96. Bhujwalla, Z. M. et al. Reduction of vascular and permeable
regions in solid tumors detected by macromolecular
contrast magnetic resonance imaging after treatment with
antiangiogenic agent TNP-470. Clin. Cancer Res. 9,
355–362 (2003).
97. Mankoff, D. A. et al. Blood flow and metabolism in locally
advanced breast cancer: relationship to response to
therapy. J. Nucl. Med. 43, 500–509 (2002).
98. Liu, X. H. et al. Prostaglandin E2 induces hypoxia-inducible
factor-1α stabilization and nuclear localization in a human
prostate cancer cell line. J. Biol. Chem. 277, 50081–50086
(2002).
99. Fatyol, K. & Szalay, A. A. The p14ARF tumor suppressor
protein facilitates nucleolar sequestration of HIF-1α and
inhibits HIF-1 mediated transcription. J. Biol. Chem. 276,
28421–28429 (2001).
100. Iervolino, A. et al. Bcl-2 overexpression in human melanoma
cells increases angiogenesis through VEGF mRNA
stabilization and HIF-1-mediated transcriptional activity.
FASEB J. 16, 1453–1455.
101. Birner, P. et al. Overexpression of hypoxia-inducible factor 1α
is a marker for an unfavorable prognosis in early-stage
invasive cervical cancer. Cancer Res. 60, 4693–4696 (2000).
102. Burri, P. et al. Significant correlation of hypoxia-inducible
factor-1α with treatment outcome in cervical cancer treated
with radical radiotherapy. Int. J. Radiat. Oncol. Biol. Phys.
56, 494–501 (2003).
103. Schindl, M. et al. Overexpression of hypoxia-inducible factor
1α is associated with an unfavorable prognosis in lymph
node-positive breast cancer. Clin. Cancer Res. 8,
1831–1837 (2002).
104. Bos, R. et al. Levels of hypoxia-inducible factor-1α
independently predict prognosis in patients with lymph node
negative breast carcinoma. Cancer 97, 1573–1581 (2003).
105. Birner, P. et al. Expression of hypoxia-inducible factor-1α in
oligodendrogliomas: its impact on prognosis and on
neoangiogenesis. Cancer 92, 165–171 (2001).
106. Sivridis, E. et al. Association of hypoxia-inducible factors 1α
and 2α with activated angiogenic pathways and prognosis
in patients with endometrial carcinoma. Cancer 95,
1055–1063 (2002).
107. Takahashi, R. et al. Hypoxia-inducible factor-1α expression
and angiogenesis in gastrointestinal stromal tumor of the
stomach. Oncol. Rep. 10, 797–802 (2003).
Online links
DATABASES
The following terms in this article are linked online to:
Cancer.gov: http://cancer.gov/
brain cancer | breast cancer | cervical cancer | endometrial cancer |
head and neck cancer | oesophageal cancer | oropharyngeal
cancer | ovarian cancer | pancreatic cancer | prostate cancer
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
AKT | ARD1 | BCL2 | BNIP3 | CBP | FIH-1 | GLUT1 | HIF-1α |
HIF-2α | HIF-3α | HSP90 | IGF2 | mTOR | p53 | p300 | PDGF-β |
PI3K | RAF | TGF-α | VEGF | VHL |
OMIM: http://www.ncbi.nlm.nih.gov/omim/
glioblastoma multiforme
Access to this interactive links box is free online.
www.nature.com/reviews/cancer