I Aguilar, M Mancuso and C Wisnieff
The Role of Hypoxia-Inducible Factors in Tumor Angiogenesis
Abstract
Although the vasculature of tumors differs from healthy tissue, tumor angiogenesis is triggered by many
of the same pathways used in development. Similar to embryonic growth, tumors upregulate vascular
endothelial growth factor (VEGF) and initiate important signaling cascades through Hypoxia-inducible
factors (HIFs), which respond to low oxygen environments [1-2]. Specifically, HIF-1 is important for
tumor growth because of its role as the proangiogenic master switch, which plays a critical role in the
upregulation of VEGF, cell proliferation, cellular survivability, invasion and migration [3]. HIF-1 is a
heterodimeric transcription factor composed of the two subunits, HIF-1α and HIF-1β, constitutively
expressed in the cytosol [4]. Oxygen sensing is mediated through HIF-1α, an oxygen sensitive protein
that stabilizes in hypoxic conditions and degrades in normoxic conditions. Downregulation of the
oxygen sensitive subunit HIF-1α has been shown to reduce the proliferation and survival of glioma stem
cells and shows potential as possible anticancer therapy [5]. Numerous studies have shown methods of
inhibition and point to possible treatment options through the use of commercially manufactured
pharmaceuticals [6-7]. In this work we review what is known about HIF’s role in tumor angiogenesis, its
structure, and its signaling pathways along with anti-cancer therapies focused on the inhibition of HIF-1
function.
Introduction
Perhaps the most well known part of the
vasculature recruitment cascade, Vascular
Endothelial Growth factor (VEGF) is ultimately
responsible for the recruitment of vessels during
development, after injury, and in several other
cases [8]. However, in areas of hypoxia, such as
those that occur during injury and the rapid celldivision associated with development, VEGF is
preceded by Hypoxia-inducible factors (HIFs)
which detect the low oxygen environment and up
regulate VEGF [4]. Through conformational
changes HIFs act as the body’s oxygen detectors
and are one of the primary signals for the
development of new vasculature.
HIF-α also takes three closely related forms, HIF1α, HIF-2α and HIF-3α, all of which behave
correspondingly in terms of transcription[3-4].
Both HIF-1α and HIF-2α are oxygen sensitive, but
each has distinct roles in the cellular response to
hypoxia [3]. Specifically, HIF-1α oxygen sensitivity
is responsible for the transcriptional response of
the hypoxia induced vascularization pathway
which the HIF-family is most prominently known
for.
During embryonic development HIF is
particularly important as it constantly signals the
growth of new vasculature as the embryo
progresses [4]. The growth of vasculature in
tumors is similar to that in embryos, triggered by
the rapidly dividing mass and high metabolic load.
HIFs are directly responsible for the growth of
tumor vasculature, and because of tumors
necessity for new vessels, represent interesting
targets in the repression of tumors. Here we take
a look at HIFs structures, their functions as oxygen
sensors, their functions as transcription factors,
HIF is composed of two subunits, HIF-1α
and HIF-1β, which ultimately form a heterodimeric
transcription factor [3-4, 9]. Both HIF-1α and HIF1β are constitutively expressed in the cytosol of
the cell [3]. HIF-1β is an aryl hydrocarbon receptor
nuclear translocator (ARNT), which can take one
of three forms all of which are capable of forming
a dimer with HIF-α subunits[4]. Similarly to HIF-β,
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I Aguilar, M Mancuso and C Wisnieff
and their role in inducing tumor angiogenesis.
Further, a look will be taken at how cancers
benefit from hypoxia and HIFs and how treatment
options might work by inhibition of the HIF
pathway.
Structure and Oxygen Sensitivity of HIF
The activation of HIF is a response to
hypoxia that activates a signaling cascade of
transcription
factors
which
promote
vascularization, ECM formation, and a number of
other changes necessary for the cell to change its
environment to a less hypoxic one. As mentioned
previously HIF is composed of two subunits which
must form a dimer to activate transcription of
target genes in the nucleus. Though each of HIFα’s homologs also have transcriptional effects,
HIF-1α’s role in hypoxia is more relevant to tumor
angiogenesis and will be reviewed more closely
here. HIF-1α is specifically important because of
its differential stability under normoxic and
hypoxic conditions which give it sensitivity to
surrounding oxygen levels.
Under hypoxic conditions HIF-1α is not
readily degraded and instead accumulates,
associates with HIF-1β, and translocates to the
nucleus where it activates gene by binding to
promoter regions [3-4, 10]. Genes that are
activated by HIF carry a hypoxia responsive
element (HRE) in the promoter region of the gene
[9]. Before the biochemical responses discussed
here were discovered, early studies showed that
HIF-1’s structure and function correlated with
hypoxia related events [11]. From this and more
recent studies the responses elicited by hypoxia in
general include erythropoiesis, neovascularization
and glycolosis, which either increases the delivery
of oxygen or shifts to metabolic pathways that do
not require oxygen [4,11]. Early studies showed
the dependence of HIF-1 activity with oxygen
Figure 1: HIF-1α regulation in normoxic and hypoxic conditions. Under normoxic conditions HIF-1α is inhibited
by FIH activity which modifys HIF-1α and prohobits it from binding to CBP / p300. Further, because of this
modifcation HIF dimers are less stable and rapidly degrade. In hypoxic enviornments HIF-1α undergoes a
conformational change and is no longer susceptible to the activity of FIH. Instead it binds to CBP/ p 300, and continues on
2
to signal transcription. (3)
I Aguilar, M Mancuso and C Wisnieff
tension, 1% O2 induced HIF-1 DNA binding activity
and target gene activation, whereas at 20% O2
these increased protein levels would decay
rapidly[11]. It is this activity that permits HIF to
act as a switch in response to environmental
oxygen concentration. In a tumor environment
this response to hypoxia enables the development
of tumor vasculature.
In order to initiate transcription of target
genes the dimer must also bind CBP/p300 as
mentioned, which is mediated by the protein REF1. Figure 1 also shows a suggested possible
negative feedback loop for transcription through
the activity of P35srj, which competes with HIF-1α
for binding with CBP/p300 [3]. In addition, a
homolog of HIF-1α, HIF-3α is also capable of
competing with HIF-1α for association with HIF-1β
under hypoxic conditions [3].
Specific domains of HIF-1α control the
sensitivity of HIF to environmental oxygen tension.
Research has shown that HIF-α subunits carry an
oxygen dependent degradation (ODD) domain,
which makes the protein susceptible to rapid
ubiquination and degradation by proteosomes in
normoxic conditions [3-4].
Ubiquination is
mediated by the von Hippel-Lindau protein
(VHL)[4,9]. As shown in the figure above, under
normoxic conditions downstream gene targets for
transcription are repressed by not only the
degradation of HIF, but also several inhibitors that
prevent activation of the HIF dimer. An example,
factor inhibiting HIF-1 (FIH), is an iron dependent
enzyme that hydrolyzes its target on HIF-1, which
inhibits the ability of HIF to bind to its co-activator
CBP/p300; CBP/p300 is necessary for activating
the dimer HIF [3-9].
Once activated in the cytosol, HIF
translocates to the nucleus to promote
transcription of several gene targets. Gene targets
of HIF are involved in events such as cell
survival/death, metabolism, pH regulation,
adhesion, extracellular matrix remodeling,
migration and metastasis[10]. Many of these
activities are directly involved in the process of
angiogenesis, one gene factor of particular
interest is vascular endothelial growth factor,
VEGF, which plays a major role in angiogenesis
and is known to be upregulated in hypoxia; VEGF
is a gene that also carries an HRE is its promoter
region [3]. It has also been directly observed that
in hypoxic conditions ovarian tumor cells
significantly increase production of both HIF-1α
and VEGF [2]. It is this cluster of genes that is
crucial in tumorigenesis, which is initiates the
development of the tumor vascular network.
HIF Induced Transcription
Due to the oxygen sensitivity of HIF-1α,
oxygen concentrations greatly affect HIF
transcriptional activity; higher molecular levels of
oxygen inhibit HIF’s transcriptional ability[3].
However, when oxygen levels are low the
conformational changes in HIF-1α stabilize the
dimer and allow for transcription to occur.
Cellular responses to other signaling pathways can
also stabilize HIF-1α and lead to HIF activation,
activation of growth factor and cell adhesion
pathways such as growth factor induced activation
of receptor tyrosine kinases (RTKs)[3].
HIF and Cancer Vasculature
Cancer, or uncontrolled cell division,
requires vasculature for increased transport to
grow past a certain size. To obtain this, cancer
recruits vasculature by hijacking the HIF signal
pathway to initiate the necessary cellular
processes for angiogenesis. Tumor angiogenesis
results in leakier vasculature with weaker
intercellular junctions than healthy tissue due to a
lack of true epithelial cells, however, the signaling
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I Aguilar, M Mancuso and C Wisnieff
is very similar.
Under normal conditions,
vasculature is hierarchically organized where
vessels are in proximity to each other to ensure
the adequate nutrient and oxygen supplies to all
cells. On the contrary, tumor vessels are chaotic,
dilated, tortuous, and distant from each other
(Figure 2) [12]. Nevertheless, the presence of a
local vascular network that supplies both oxygen
and nutrients is essential for tumor growth.
Inhibition of HIF and Cancer Therapy
Angiogenesis is a key factor in the
progression of cancer and has been demonstrated
to strongly correlate with risk of invasion and
metastasis[10]. An example of this phenomenon
was observed in a histochemical endometrial
adenocarcinoma study where cancer metastasis
increased as the number of newly formed vessels
increased, indicated by CD34 staining [14].
Further studies using magnetic resonance imaging
(MRI) have shown that the pathologic features of
glioblastoma (GBMs), can trigger vasogenic edema
[15]. However, tumor development is faster than
this form of vascular recruitment and a hypoxic
environment is perpetuated, triggering HIFs. VEGF
is highly present in tumors cells such as glioma
stem cells (GSC). Research has shown that GSCs
may preferentially express HIFs [5]. GSCs promote
tumor maintenance through elevated VEGF, which
can be elevated through HIF activity. In a study
where the mRNA levels of HIF are measured it was
found that that some genes are expressed
differently. Analysis of VEGF demonstrated that
hypoxia induced a greater increase in VEGF levels
in GSCs than in non-stem cells [5]. In addition,
hypoxia induced angiogenesis is blocked by
inhibitors of oncogene signaling pathways, such as
agents that inhibit VEGF indicating the existence
of a crosstalk between oncogenic and hypoxia
response pathways [13].
While it is was known that VEGF was a target of
the HIF transcription pathway, Li et al determined
that HIFs are required for significant glioblastoma
stem and non-stem cell VEGF expression.
Knockdown of HIFs by a lentiviral shRNA in GSCs
under hypoxia significantly reduced VEGF
promoter activity[5]. Additionally, Burkitt et. al.
showed that by inhibiting HIFs further improved
tumor response to sunitinib therapy. This
hypothesis was tested by disrupting HIFs genes in
colon cancer cells. It was found that disruption of
HIFs, genes led to improved tumor response to
sunitinib. The enhanced response was mediated
by two potential mechanisms. First, tumor
angiogenesis and perfusion were almost
completely inhibited by sunitinib when HIFs genes
were disrupted. The enhanced inhibitory effect on
tumor angiogenesis was mediated by the
inhibition of multiple proangiogenic factors,
including VEGF, and the induction of the
antiangiogenic factor, thrombospondin. Second,
disruption of HIFs genes directly inhibited tumor
cell proliferation. These preclinical findings have
clinical implications and suggest novel clinical
trials [7].
There are several options for the
inhibition of HIF pathway in order to stop tumor
angiogenesis, including the knockdown of HIFs
expression
[5],
ascorbate
and
iron
supplementation of HIF-1[6], Nitric Oxide (NO)
[17], and inhibition of HIF-1 transcriptional activity
by Anthracyclines [18].
Further, ascorbate and iron supplementation can
be used to suppress HIF-1[6].The down-regulation
of HIF in oxygenated cells is by a series of Fe(II)
and oxoglutarate dependent deoxygenases that
hydroxylate specific residues in the regulatory HIF1α subunits. The effects of ascorbate on HIF in
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I Aguilar, M Mancuso and C Wisnieff
human cancer cells lines were analyzed due to the
fact that these enzymes require ascorbate for
activity in vitro. In a study by BerchnerPfannschmidt et al, it was shown that
physiological concentration of ascorbate (25µm)
suppressed
HIF-1α
levels
and
HIF-1α
transcriptional targets, particularly when the
system was oncogenically activated in normoxic
cells[6].Also, Nitric Oxide (NO) decreases HIF-1α
stabilization and HIF-1α transcriptional activation
[17]. Studies have shown that prolonged exposure
to NO or low doses of this radical reduced the
accumulation of HIF-1α even under hypoxic
conditions. NO regulates HIF-1α by modulating
the activity of the oxygen-sensor enzymes PHDs
and FIH-1[17]. Finally, it was demonstrated the
inhibition of HIF-1 transcriptional activity by all
anthracyclines, including DNR, DXR, EPI, and IDA.
The anticancer effect of these drugs when
administered episodically at the maximum
tolerated dose was attributed to their ability to
intercalate DNA and induce topoisomerase IImediated strand breaks [18].
Conclusion
HIF plays an important role in positive life
functions like development and injury repair as
well as in cancer vasculature.
Through
conformational changes in HIF-1α oxygen
sensitivity is mediated into a cell signaling
response. As cancer progresses hypoxic regions
form in the center of the tumor mass and the HIF
signaling pathway is adversely activated. HIF
induced transcription of VEGF follows and leads to
increased vascularization in cancer along with
increased metastasis. However, because of the
already compromised vasculature in cancer this
mechanism likely represents a unique target for
anti-cancer therapy. Experiments have shown
that HIFs are critical for the development of
cancer and that inhibition of their function can
lead to reduced cancer proliferation. Multiple
therapeutics have been demonstrated to have a
significant effect on tumor growth and likely
represent a feasible targeted treatment due to the
much slower revascularization in non-cancerous
tissue.
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