60 Current Molecular Medicine 2008, 8, 60-67

Hypoxic Regulation of Metastasis via Hypoxia-Inducible Factors

Eelke H. Gort1, Arjan J. Groot1, Elsken van der Wall2, Paul J. van Diest1 and Marc A. Vooijs*,1

1
Department of Pathology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
2
Division of Internal Medicine and Dermatology, University Medical Center Utrecht, 3508 GA Utrecht, The
Netherlands
Abstract: Metastases formation is a major factor in disease progression and accounts for the majority of can-
cer deaths. The molecular mechanisms controlling invasion, dissemination to blood or lymphatic systems and
spread of tumor cells to distant organs are still poorly understood. Recent observations indicate that the meta-
static phenotype may already be present during the angiogenic switch of tumors. Intratumoral hypoxia corre-
lates with poor prognosis and enhanced metastases formation. The Hypoxia Inducible Factors (HIFs) are key
molecules in the hypoxic response and play critical roles during tumor cell expansion by regulating energy me-
tabolism and the induction of angiogenesis. Increasing evidence implicates HIF function in metastatic cell
characteristics, like epithelial to mesenchymal transition, cell detachment, invasion and tumor cell seeding.
Here, we review the link between tumor cell hypoxia and the acquisition of metastatic behavior. We hypothe-
size that polyclonal tumor selection by hypoxia enhances metastatic capacity by transcriptional control of key
regulators of metastasis. This polyclonal hypoxic gene profile potentially develops into a metastatic profile, driv-
ing metastasis formation. The hypoxic gene profile in primary tumors may therefore provide a prognostic indi-
cator in clinical decision-making.
Keywords: Hypoxia, cancer, metastasis, HIF, EMT, angiogenesis, polyclonality.

INTRODUCTION HYPOXIA AND HIF

Outgrowing metastases are the major cause of hu-
man cancer mortality. The acquisition of full metastatic
capacity of cancer cells requires several sequential
rate-limiting steps: i. cancer cells must detach from the
primary tumor, ii. invade into the surrounding stroma,
iii. intravasate into the blood or lymphatic vessels, iv.
spread into the capillary bed of distant organs, and v.
extravasate and colonize into the recipient organs [1]. It
is well established that tumor cell specific alterations
(cell autonomous) are not sufficient to drive the metas-
tatic cascade but that continuous interaction with the
microenvironment (non-cell autonomous) is needed to
permit tumor cell survival. One of crucial rate-limiting
events during human tumor growth is encountered
when tumors outgrow the pre-existing vasculature lead-
ing to hypoxia. Intratumoral hypoxia is a hallmark of
solid cancer. Hypoxic tumors have a higher tendency to
metastasize and are clinically characterized by therapy
resistance and a poor prognosis [2,3]. Tumor cell hy-
poxia affects various key steps during the metastatic
cascade and selects for metastatic variants [4]. Central
components in the cellular hypoxia response are the
Hypoxia-inducible Factors (HIFs). Here, we review how
hypoxia directly affects gene-expression programs driv-
ing metastatic spread. We summarize the increasing
evidence implicating HIFs in several key steps in the
metastatic cancer cascade. Finally, we formulate a
concept of how the early hypoxic microenvironment
drives the selection of polyclonal metastatic cells con-
tributing to more aggressive tumor behavior.
*Address correspondence to this author at the Department of Pathol-
ogy, University Medical Center Utrecht, 3508 GA Utrecht, The Neth-
erlands; Tel: +31-(0)88-7558316; Fax: +31 (0)30 2544990; E-mail:
m.vooijs@umcutrecht.nl
In mammalian cells, the HIF transcriptional complex
is a key regulator of the local and systemic responses
to hypoxia that occur during normal development and
(patho-) physiological processes [5]. Intratumoral hy-
poxia, defined roughly when intercapillary distances
exceed 140 micron, correlates with poor prognosis and
has become an important clinical parameter [6,7]. Can-
cer cells adapt to hypoxic environments by converting
to glycolytic energy metabolism, induction of angio-
genesis and cellular survival programs, and this acute
cellular response to hypoxia is mediated by the HIF
basic helix-loop-helix (bHLH) transcription factor family
[8]. HIF is a heterodimeric protein complex composed
of a constitutively expressed bèta subunit, HIF-1 or
aryl hydrocarbon receptor nuclear translocator (ARNT),
and an oxygen-regulated alpha subunit both of which
belong to the bHLH-PAS (Per, ARNT, SIM) protein
family [9]. Mammalian cells have three HIF
isoforms.
HIF-1
and HIF-2
have been studied best and will be
considered here. HIF
stability is tightly regulated at
the posttranslational level by oxygen concentration.
Under normal oxygen tension (normoxia), HIF
is con-
stitutively hydroxylated and acetylated at multiple
proline and lysine residues in the Oxygen Dependent
Degradation Domain (ODDD) and on asparagine resi-
dues in the c-terminal transactivation domain (N832).
These modifications enhance binding of HIF
to the
Von Hippel-Lindau (VHL) protein, the recognition com-
ponent of an E3 ubiquitin ligase complex and regulate
assembly of HIF
/- heterodimers with transcriptional
co-activators. Prolyl hydroxylation marks HIF
for
ubiquitination and proteosomal degradation (for a re-
view see [10]). Thus, under normoxia HIF
protein is
continuously degraded. Under hypoxic conditions the

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Hypoxic Regulation of Metastasis via Hypoxia-Inducible Factors Current Molecular Medicine, 2008, Vol. 8, No. 1 61

activity of prolyl hydroxylases is attenuated since they

require ferrous-ions and O2 for their function, which
leads to HIF
stabilization. After nuclear translocation,
HIF
/ARNT heterodimers regulate transcription by
binding hypoxia responsive elements (HRE) [11].

ONCOGENIC REGULATION OF HIF

HIF protein stability and expression can also be in-
creased by tumor specific genetic alterations in onco-
genes or tumor suppressor genes. For example, in the
Von Hippel Lindau hereditary cancer syndrome the loss
of VHL leads to attenuated proteosomal degradation of
HIF
and predisposes to a variety of highly vascular-
ized malignant and benign neoplasms [12,13]. Also in
sporadic cancers deregulation of oncogenes and tumor
suppressor genes affects HIF signaling. For instance,
the phosphatidylinositol-3 kinase (PI 3-kinase)/Akt
kinase pathway directly impacts on HIF stability and
HER-2/neu overexpression or the tuberous sclerosis
complex gene TSC-2 (Tuberin) loss induce HIF-1

stabilization via an mammalian target of rapamycin
(mTOR)-dependent pathway [5]. Furthermore, HIF-1

stabilization depends on the TP53 tumor suppressor
protein [14]. Other pathways implicated in HIF regula-
tions are the mitogen-activated protein kinase (MAPK),
nuclear factor-kappaB (NF-B) and Notch signaling
pathways [15-17].

CLINICAL SIGNIFICANCE OF HIF

Direct oxygen measurements into tumours has re-
vealed that areas of necrosis are surrounded by poor
oxygenated areas [18]. In those areas hypoxia-induced
nuclear HIF-1
protein can be readily detected by im-
munohistochemistry (Fig. (1)). Overexpression of HIF-
1
is associated with human cancer progression in
epithelial cancers of the head and neck, cervix, colon,
breast and lung (for a review see [19]). In breast can-
cer, HIF-1
expression is an independent predictor of
poor clinical outcome, with a greater predictive value
then other hypoxic markers like carbonic anhydrase IX
(CAIX) [2,20]. HIF-1
expression correlates with a
therapy resistance and primary tumor HIF-1
overex-
pression has been shown to predict presence of bone
marrow metastases in breast cancer patients [20,21].
Moreover, HIF-1
is not only expressed in primary tu-
mors, but also in metastatic lesions and HIF-1
ex-
pression predicts early relapse [22,3]. Gene expression
profiling has revealed a unique set of transcriptional
targets in hypoxic breast cancers [23]. These clinical
studies suggest an important role for HIF in disease
progression. We will now discuss the molecular evi-
dence supporting this hypothesis.
CANCER CELL DETACHMENT AND EMT
Cancer cell detachment is the initial event in meta-
stases formation of carcinomas and is marked by the
loss of cell adhesion molecules and the acquisition of
mesenchymal phenotypes [24]. This process is termed
epithelial to mesenchymal transition (EMT) although
Fig. (1). Peri-necrotic staining of HIF-1
. A) haematoxilin-
eosin staining and B) immunohistochemical staining for HIF-
1
in endometriod endometrial carcinoma. Note the nuclear
peri-necrotic staining of HIF-1
protein (black arrows). Ne-
crotic area indicated by N. Magnification 40 x.
this process is never complete and some epithelial
characteristics remain [25]. A hallmark of EMT is the
loss of E-Cadherin, an adhesion molecule frequently
lost in human cancer and causal in tumor formation
and metastasis [26,27]. Loss of E-Cadherin enhances
cancer cell motility and resistance to anoikis [26,28].
EMT is a crucial process during normal development of
multicellular organisms [29]. EMT is controlled by AKT-,
Wnt-, Notch-, and Hedgehog-signaling pathways and
can be induced by a number of growth factors, like
transforming growth factor  (TGF-), epidermal growth
factor (EGF), hepatocyte growth factor (HGF) and fi-
broblast growth factors (FGF) [29]. Several transcrip-
tion factors critical for mesoderm formation, like SNAI1
(SNAIL1), ZEB and certain bHLH factors, are known to
induce EMT through repression of E-Cadherin and in-
duction of mesenchymal gene expression [30]. Recent
findings implicate the HIF pathway in EMT induction,
acting through these mesodermal fate genes. In VHL-
deficient renal cell carcinomas, both HIF-1
and HIF-
2
are involved in the downregulation of E-Cadherin
62 Current Molecular Medicine, 2008, Vol. 8, No. 1
expression [31-33]. One of the EMT regulating factors
regulated by HIF signaling is SNAI1, a zinc-finger tran-
scription factor indispensable for mesoderm develop-
ment [33,34,31]. In addition to SNAI1, other known rep-
ressors of E-Cadherin, like TCF3 (E12/E47), ZEB1
(yEF1/ ZFHX1A), and ZEB2 (SIP1/ ZFHX1B), are
upregulated in VHL-deficient renal cell carcinomas
[31,32]. Using genetic screens in C. elegans, we re-
cently identified ceTWIST (helix-loop-helix-8 or HLH-8)
as a critical downstream target in hypoxia sensing [35].
TWIST1 is directly regulated by HIF-2
under hypoxia.
TWIST1, the Saethre-Chotzen syndrome susceptibility
gene, is a master regulator of mesodermal fate and a
bHLH repressor that like SNAI1, binds E-box contain-
ing promoters. In invasive lobular breast cancer,
TWIST1 contributes to metastasis by promoting EMT
through down regulation of E-Cadherin and high levels
of TWIST1 correlates with high disease recurrence in
breast and ovarian cancer [36-38]. Whereas SNAI1-
mediated repression of E-Cadherin indirectly leads to
activation of mesenchymal markers such as Vimentin
[39,40], TWIST1 can directly activate N-Cadherin ex-
pression, a marker for EMT progression [41]. Thus both
SNAI1 and TWIST1 appear to orchestrate the Cadherin
switch during EMT. A central role for hypoxia and HIF-
dependent regulation EMT provides an attractive model
in which hypoxia in early tumorigenesis triggers cancer
cell detachment and invasion.
EXTRACELLULAR MATRIX MODULATION
AND INVASION
Strong evidence for a role of hypoxia and HIF as
regulators of ECM degradation and tumor cell migration
comes from the analysis of Erler and collegues who
identified the ECM crosslinking enzyme lysyl oxidase
(LOX) as a direct HIF-1
target [42]. LOX is a copper-
dependent amine oxidase that plays a critical role in
the biogenesis of connective tissue matrices by
crosslinking elastin and collagen and has been shown
to enhance tumor cell migration [43]. LOX expression
in hypoxic cancers correlates with poor patient survival
and inhibition of LOX activity in a mouse model of meta-
static breast cancer significantly reduced liver/bone
metastases [42]. Although the mechanism whereby
LOX increases metastatic behavior is unresolved it
may directly affect motility by regulating FAK-
dependent adhesion to collagen. These data warrant
the development of LOX inhibitors as anti-metastatic
therapeutics. Interestingly, LOX homologues LOXL2
and LOXL3 interact and cooperate with SNAI1 in the
downregulation of E-cadherin in carcinoma progression
[44]. Regulation of matrix metalloproteinases (MMPs),
urokinase type plasminogen activator (uPAR) and
Cathepsin D by hypoxia may provide additional means
for hypoxic ECM modulation [5].
CELL MOTILITY AND INVASION
Hypoxic tumor cells adopt a multitude of strategies
in addition to neoangiogenesis to overcome oxygen
deprivation by inducing cell motility to colonize oxygen
Gort et al.
proficient areas. This process is thought to occur inde-
pendent of the angiogenic switch and has been termed
the “invasive switch” [45]. Scatter factor or hepatocyte
growth factor (HGF) is a pleiotropic cytokine which
stimulates proliferation and invasion through its recep-
tor, the tyrosine kinase proto-oncogene c-Met [46]. c-
Met is frequently overexpressed or mutated in human
cancers [47]. Hypoxia in a HIF-dependent manner di-
rectly promotes invasion by inducing c-Met transcrip-
tion and sensitizing cells to HGF stimulation. c-Met is
required and sufficient for hypoxia-induced invasive
growth [45,48,49]. Thus, hypoxia unleashes a positive
feedback loop between HIF and c-Met that fuels inva-
sive growth. c-Met-induced motility is also enhanced by
hypoxic modulation of cell surface integrin signaling.
Integrins are adhesion receptors with bidirectional sig-
naling between plasma membrane and ECM, that play
essential roles in motility and migration [50]. MG-63
human osteosarcoma cells exposed to the hypoxia-
mimicking agent CoCl2 upregulate both integrin
5 (the
fibronectin receptor) and integrin
2 (the alpha chain of
the collagen receptor), resulting in increased adhesion
[51]. Upregulation of the integrin
51 dimer via HIF-1

might explain HER-2/neu-dependent fibronectin liga-
tion, resulting in increased survival of mammary ade-
nocarcinoma cells, since HER-2/neu can regulate HIF
expression under normoxic conditions [52,53]. Con-
versely, integrin-dependent signaling affects HIF activ-
ity itself via integrin-linked kinase (ILK)-mediated AKT
activation [54]. Fibroblasts lacking HIF-1
fail to
upregulate the GTPase RhoA, a core transducer of
integrin signaling critical in stress fiber formation, dur-
ing hypoxia [55]. Thus hypoxia and HIF enhance motil-
ity by acting at multiple levels to facilitate migration to-
wards nutrient and oxygen rich areas within oxygen-
deprived tissues.
ACIDOSIS AND INVASION
Cancer cells starved for oxygen and nutrients switch
to anaerobic glycolysis (the Pasteur effect) as an
alternative energy source in a process that is
predominantly HIF dependent [56]. Anaerobic
respiration produces lactic acid and adenosine
triphosphate (ATP) from glucose, which results in
acidosis. Acidosis is a general feature of human tumors
and has been shown to promote tumor cell invasion by
destruction of adjacent non-cancerous tissue and ECM
degradation [56]. Importantly, tumor-induced acidosis
can negate the effect of anti-cancer therapeutics that
only function under physiological pH [57]. To prevent
intracellular acidosis, HIF induces the transcription of
carbonic anhydrase XI (CAIX) that leads to increased
-
HCO3 uptake, thereby neutralizing the intracellular pH
albeit at the expense of extracellular acidosis, which is
+
further increased by the H ions expelled out of the cell
by proton pumps [8]. In addition, HIF-1
induces the
expression of the glycolytic enzyme phosphoglucose
isomerase (PGI or autocrine motility factor (AMF)), a
major cell motility stimulating factor [58,59]. Hypoxia-
induced cell motility can be inhibited by PGI inhibition.
Clearly, the numerous metabolic changes induced by
hypoxia, in part dependent on HIF, can promote
Hypoxic Regulation of Metastasis via Hypoxia-Inducible Factors
pendent on HIF, can promote metastatic behavior. This
has led to the concept that combination therapies tar-
geting both angiogenesis as well as intracellular (pH)
metabolism/regulation may enhance therapeutic effi-
ciency in cancer [8].
ANGIOGENESIS AND INTRAVASATION
The production of pro-angiogenic factors by hypoxic
(cancer) cells that induce endothelial cell proliferation
and vessel sprouting is perhaps the best-studied func-
tion of HIF. The “angiogenic switch” is a hallmark of
human solid cancer progression [60,61]. In 1971, it was
already proposed that the capacity of tumor cells to
induce angiogenesis promotes tumor cell progression
and that anti-angiogenic therapy may improve clinical
outcome [62]. Key players in this process are vascular
endothelial growth factors (VEGF), and VEGF recep-
tors 1 (VEGFR1 or Flt-1), which are directly regulated
by HIF [63,64]. Significantly, VEGF-A activation was
found to be crucial for tumor angiogenesis in a mouse
model for multistage pancreatic cancer [65]. The VEGF
gradient secreted by tumor cells induces sprouting of
endothelial cells, which express VEGFR1 and VEGFR2
[66]. Loss of HIF-1
in endothelial cells (EC) impairs
hypoxia-induced angiogenesis via a VEGF-VEGFR
autocrine loop, showing that hypoxia also affects EC
function directly, perhaps by impaired VEGFR expres-
sion [67]. In addition to VEGF, other angiogenic factors
are regulated by hypoxia, like angiopoietin-1, -2 and -4
(ANGPT1, -2 and -4), placental growth factor, and
platelet-derived growth factor-B [68,69]. Altogether,
hypoxia-induced angiogenesis is emerging as an im-
portant mechanism controlling metastatic capacity by
recruitment of blood vessels and lymphatic circulation.
This has already resulted in therapeutic strategies, us-
ing anti-angiogenic agents like VEGF receptor blocking
®
agents (e.g. Bevacizumab or Avastin ) [70]. At face
value, anti-angiogenic treatment may also have tumor
promoting effects by inducing hypoxia and selection of
metastatic variants that use alternative strategies (i.e
migration) to overcome nutrient deficiency. Paradoxi-
cally pro-angiogenic treatment of tumors by activating
the Notch-Delta signaling pathway may also provide
therapeutic benefit [71]. Delta-like 4 Notch ligand
(DLL4) signaling through Notch1 and Notch4 receptors
is critical for embryonic vascular development [72,73].
VEGF produced by both hypoxic tumor cells and endo-
thelial cells upregulates DLL4 and Notch signaling in
normal and tumor endothelial cells [74,75]. Blocking
DLL4/Notch signaling using neutralizing antibodies
stimulates non-productive angiogenic sprouting and
branching in tumors induced by VEGF and leads to
increased tumor hypoxia and decreased tumor growth
[76,77]. Thus, a delicate balance between VEGF and
DLL4/Notch signaling is required for proper (tumor)
angiogenesis. Disruption of this signaling cascade re-
sults in a decrease of functional angiogenesis (VEGF)
or increase in non-functional angiogenesis (DLL4). In-
terestingly, anti-DLL4 blocking strategies also proved
effective in tumors resistant to anti-VEGF treatment,
providing rationale for new combination therapies tar-
Current Molecular Medicine, 2008, Vol. 8, No. 1 63
geting both DLL4 and VEGF. The recently discovered
link between HIF and Notch signaling might play an
important role in controlling this balance [15].
SURVIVAL
Cancer cells continuously enter the circulation yet
only few seed and produce deadly metastatic tumors.
Generally, loss of interaction with the environment trig-
gers apoptosis (anoikis). Cancer cells can escape
anoikis by repression of apoptosis. For instance, over-
expression of the neurotrophin receptor TrkB protects
from cell death induced by cell-matrix dissociation [78].
TrkB expression can be up regulated directly by HIF-1,
but not HIF-2, under hypoxic conditions [79]. Further-
more, TrkB can regulate VEGF expression via HIF-1
[80]. In addition, c-Met signaling confers resistance to
anoikis and supports anchorage independence [81].
Furthermore, SNAI1/2 and TWIST1 act on anti-
apoptotic and prosurvival pathways by antagonizing
p53- and c-Myc-induced apoptosis, respectively
[82,83]. Direct evidence for a role of hypoxia in resis-
tance to anoikis is lacking, but hypoxia does increase
metastatic potential by promoting cell survival in metas-
tatic colonies [84,85].
SEEDING
Secondary tumor formation follows arrest and seed-
ing. Cells arrest in narrow capillary beds of the organ
vasculature which is an active process of cell-cell/cell-
matrix interaction, involving among others, the G-
protein-coupled CXC chemokine receptor 4 (CXCR4)
and the CXCR4 ligand stromal cell-derived factor-1

(SDF-1
or CXCL12), and integrin-fibronectin interac-
tion [1]. Expression of CXCR4 in lung-, breast-, ovar-
ian-, renal-, and prostate cancer has been implicated in
increasing metastatic potential and expression of SDF1
is found in target tissue of metastases [86]. Staller et al.
showed for the first time that CXCR4 expression is
regulated by hypoxia in a HIF-2
dependent manner
[87]. In addition, SDF-1 is also upregulated in VHL defi-
cient cells, suggesting an autocrine pathway [88]. In
non-small cell lung cancer cells, activation of the epi-
dermal growth factor receptor also results in up regula-
tion of CXCR4, which was dependent on PI 3-
kinase/AKT/mTOR-mediated activation of HIF-1

[89,90]. In addition, HIF-mediated induction of SDF-1

in endothelial cells functions to recruit circulating
CXCR4-positive tumor cells or endothelial progenitor
cells (EPC) to ischemic tissue [88,91]. This provides
three strategies by which the CXCR4-SDF-1 interaction
influences cancer cell seeding; i. hypoxic tumor cells
express CXCR4 and seed after SDF-1 recognition in
distant non-hypoxic tissue, ii. hypoxic tumor cells ex-
press SDF-1 which might recruit CXCR4 expressing
cells by chemotaxis, and iii. hypoxic cancer cells ex-
pressing both CXCR4 and SDF-1 establish an
autocrine signaling pathway, which results cancer cell
autonomy. These are interesting findings and indicate
that disruption of the CXCR4-SDF axis may be ex-
64 Current Molecular Medicine, 2008, Vol. 8, No. 1
ploited to target hypoxic tumor seeding, using blocking
antibodies [92].
POLYCLONAL SELECTION OF METASTA-
SIS BY HYPOXIA
Apart from the effects of oxygen deprivation on ge-
nomic stability and environmental selection, resulting in
progression of more aggressive malignancy, hypoxia
induces a gene expression program, which facilitates
metastatic spread (Fig. (2)). Molecules involved in an-
giogenesis, cell detachment, cell motility, invasiveness
and chemotaxis are under direct control of the hypoxia-
regulated HIF pathway. The long held paradigm and
working model that continuous selection of rare spon-
taneous variants within cancers emerge and drive tu-
mor formation and progression is under scrutiny [93].
Fidler and colleagues were among the first to demon-
strate that B16 melanoma cells have an intrinsic cellu-
lar heterogeneity with respect to subpopulations of
cancer cells with different metastatic capacity [94,95].
This has recently been validated by Minn et al. who
observed that pre-existing subpopulations of MDA-MB-
231 breast cancer cells determine bone or lung metas-
tasis [96]. Importantly, this polyclonal heterogeneity
stabilizes the metastatic capacity of clonal subpopula-
Fig. (2). Hypoxic regulation of genes involved in metastasis.
Genes are divided by functional implication in metastasis.
Furthermore, up or down regulation under hypoxia and in-
volvement of HIF-1 or -2 is indicated.
Gort et al.
tions within tumors suggesting that some form of coop-
erative interaction exists between subclones that de-
termines the frequency of metastases [94]. Support for
this has been obtained from the discovery that primary
breast cancers already harbor the metastasis (poor-
prognosis) gene expression signature, suggesting that
most primary tumor cells have the capacity to metasta-
size rather than evolve through the emergence of rare
metastatic variants [97,98]. Remarkably, this signature
seems applicable to epithelial tumors in general, sug-
gesting this is in part an intrinsic biological property of
cancer cell populations [99]. In retrospect it seems ob-
vious to propose that this polyclonality and heterogene-
ity is driven in part by the presence of cancer stem
cells. Importantly, a significant fraction of signature
genes are stromal-derived suggesting a critical role for
tumor-host microenvironment in driving tumor progres-
sion and metastasis [100]. This is underscored by the
analysis that tumor-derived stromal expression signa-
tures might provide equally strong clinical classifiers as
tumor cell signatures [101].
We would like to extend the polyclonal selection
working model by proposing that intratumoral hypoxia
is a driving force behind the polyclonal expansion of
metastatic subpopulations within primary tumors (Fig.
(3)). This can explain both the clonality of secondary
tumors, as well as early on-set whole tumor population
malignancy, because gene signatures of multiple
clones add up to constitute the poor-prognosis gene-
signature. Intratumoral hypoxia is not only restricted to
necrotic areas but occurs throughout tumors and,
therefore affects the tumor population as a whole. We
have screened both breast and endometrial carcino-
mas that display high HIF levels for activating HIF mu-
tations but have found none to date (Horree et al., ac-
cepted) [102]. This is consistent with a model for poly-
clonal adaptation rather than monoclonal selection of
metastatic clones. Indeed, deletion of HIF-1
in a
mouse model for breast cancer demonstrates that HIF
is critical for tumor progression and metastasis but not
for tumor initiation [103]. Further, breast cancers with
bone marrow metastatic cells had higher HIF-1
ex-
pression on the RNA and protein level than those with-
out metastatic cells in the bone marrow [21]. Hypoxia
(through HIF) regulates a myriad of metastagenic
genes. The hypoxia-response signature predicts prog-
nosis independently and contributes to the poor-
prognosis gene signature in breast cancer
[2,23,104,105]. Identification of intratumoral hypoxia
might serve as a prognostic factor in clinical decision-
making.
CONCLUSION
In conclusion, the direct effect of hypoxia on cell
adhesion molecules and matrix degrading enzymes
provides a framework for testable hypotheses in mouse
models and cell systems. From a clinical point of view,
identification of the molecular networks regulating en-
hanced metastatic behavior provide important anti-
cancer targets that may aid in the diagnosis and man-
Hypoxic Regulation of Metastasis via Hypoxia-Inducible Factors Current Molecular Medicine, 2008, Vol. 8, No. 1 65

Fig. (3). The hypoxic gene profile versus the metastatic gene profile. On the left, the hypoxic gene profile is illustrated. The oxy-
gen gradient is indicated from red (normal oxygen levels) to blue (hypoxia). Cells that lie in the periphery of the blood supply
become hypoxic and start expressing genes involved in metastasis as indicated by different colors. On the right, a metastatic
primary tumor is depicted, containing cells that express genes involved in each sequential step in metastasis formation, thereby
constituting the metastatic gene profile. More blood vessels are present here because of the stimulatory effects of hypoxia on
angiogenesis. Cells expressing gene products functionally involved in the different steps of metastasis formation are depicted in
concordance with these function; i.e. angiogenesis, cell detachment, invasion and seeding. The hypoxic gene profile might de-
velop into metastatic profile, and therefore provide a predictive value in clinical decision-making.

agement of metastatic disease. Further elucidation of
the hypoxia/HIF-mediated transcriptional networks will
provide exciting insights into cancer biology and high-
light diagnostic and therapeutic avenues to be explored
in the near future.
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