I. Introduction and literature review
1.3 VEGF and cancers
1.3.1 Vascular endothelial growth factor family, VEGF family
VEGF-A (commonly referred to as VEGF) was first identified by Dvorak et al. (2002) as a
vascular permeabilitity-inducing factor secreted by tumor cells, and thus referred to as vascular
permeability factor (VPF). Ferrara et al. (1996) later isolated and cloned VEGF-A as an endothelial
specific mitogen. VEGF is a major inducer of angiogenesis and it is structurally related to PlGF
(placenta growth factor), VEGF-B, VEGF-C, VEGF-D and Orf virus derived VEGF (also called
VEGF-E) (Ferrara and Davis-Smyth, 1997; Erikson and Alitalo, 1999; Persico et al., 1999; Achen
et al., 1998; Ogawa et al., 1998; Meyer et al., 1999). The biological functions of the VEGF family
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are mediated by activation of three structurally homologous tyrosine kinase receptors, VEGFR-1,
VEGFR-2, and VEGFR-3 (Neufeld et al., 1999; Veikkola et al., 2000; Aprelikova et al., 1992)
1.3.1.1 VEGF-A
VEGF-A is a 45-kDa homodimeric glycoprotein with a diverse range of angiogenic activities.
The VEGF-A gene undergoes alternative splicing to yield mature isoforms of 121, 165, 189, and
206 amino acids (Houck, 1991; Tischer et al., 1991). In addition, some less commonly expressed
variants have also been identified (VEGF145 and VEGF183) (Neufeld et al., 1999).
VEGF121 is freely secreted, whereas the largest isoforms (VEGF189 and VEGF206) are
sequestered in the extracellular matrix (ECM) and require cleavage by proteases for their activation
(Dvorak, 2002). VEGF165 exists in both a soluble and an ECM-bound form (Keyt et al., 1996). The
ECM-bound isoforms of VEGF-A, VEGF-C, and VEGF-D can be released in a diffusible form by
plasmin cleavage at the C-terminus, which generates a bioactive fragment (Park et al., 1993;
McColl et al., 2003). Alternatively, VEGF can be released from the ECM by MMP-9 to initiate the
angiogenic switch (Bergers et al., 2000). VEGF165 is the predominant isoform and is commonly
overexpressed in a variety of human solid tumors. In mice, homozygous or heterozygous deletion of
the VEGF gene is embryonically lethal, resulting in defects in vasculogenesis and cardiovascular
abnormalities, demonstrating that VEGF is essential for development (Carmeliet et al., 1996;
Ferrara, 1996). VEGF-A is also important to a number of postnatal angiogenic processes, including
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wound healing, ovulation, menstruation, maintenance of blood pressure, and pregnancy (Brown,
1992). VEGF-A has also been linked to several pathologic conditions associated with increased
angiogenesis, including arthritis, psoriasis, macular degeneration, and diabetic retinopathy (Ferrara
et al., 2003). In healthy conditions, endothelial cells survive for prolonged periods owing to the low
level of VEGFA expression by the vasculature, which acts as a survival signal. Hence, when
quiescent endothelial cells are deprived of this trophic VEGFA signal, they become dysfunctional
(pro-thrombotic), lose their vasorelaxing activity (nitric oxide release) or even disappear altogether
(which leads to bleeding) (Baffert et al., 2006; Gerber et al., 1999; Lee et al., 2007). Furthermore,
large amounts of VEGFA are produced in healthy conditions, which suggest that VEGFA is needed
for endothelial-cell homeostasis (Rudge et al., 2007). VEGFA binds to FLT1 with an affinity
(dissociation constant; Kd ~2–10 pM) that is much higher than for FLK1 (Sawano et al., 2001). Yet,
VEGFA induces weaker tyrosine-kinase activity in FLT1, possibly because of an inhibitory
sequence in the juxtamembrane domain that represses FLT1 activity (Seetharam et al., 1995;
Waltenberger et al., 1994; Gille et al., 2000). This weak tyrosine-kinase activity of FLT1 and its
high affinity for VEGFA have led to the development of a model in which FLT1 acts as a decoy
receptor and modulates angiogenesis through its ability to sequester VEGFA, and thereby reduces
signalling through FLK1 (Park et al., 1994). Additional functional diversity may occur through the
formation of dimers between VEGF family members. For example, VEGF-A may form
heterodimers with either PlGF (Cao et al., 1996) or VEGF-B (Olofsson et al., 1996). In humans,
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because of the alternative splice variants, this has the potential to create enormous diversity and
presents a challenge to assessing the functional consequences of heterodimerization (Birkenhager et
al., 1996). For example, the six human VEGF-A and four human PlGF isoforms could theoretically
form 24 VEGF-PlGF heterodimers, and VEGF-A could theoretically form 12 different
combinations with the two human VEGF-B variants.
1.3.1.2. VEGF-B and PlGF
VEGF-B and PlGF null mice display no defects in embryonic vasculogenesis or developmental
abnormalities, suggesting that the role of PlGF and VEGF-B may be redundant (Carmeliet, 2001).
However, loss of PlGF impairs angiogenesis, plasma extravasation, and collateral growth during
ischemia, inflammation, wound healing, and tumor growth, suggesting a role for PlGF in pathologic
states in the adult. VEGF-B exists as two isoforms (VEGF167 and VEGF 186) that bind to FLT1
and to neuropilin 1 (Neufeld, 2001). Neuropilin 1 is widely expressed in various tissues (Nash, 2006)
including endothelial and mural cells, brown fat, skin, lung, placenta, brain and retina, but it is
particularly abundant in the heart and skeletal muscle (Li, 2001). Despite some evidence for
VEGFB activity in vitro, genetic studies have revealed that VEGFB-deficient mice are healthy and
fertile, and do not display vascular defects, which indicates that VEGFB is redundant in
angiogenesis in the developing embryo and healthy adult in vivo (Li et al., 2008; Aase et al., 2001;
Bellomo et al., 2000). The role of VEGFB in tumor growth remains largely elusive. VEGFB has
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been detected in a wide range of tumors, including meningioma, and colorectal, lung and breast
cancer (Andre et al., 2000; Donnini et al., 1999; Niki et al., 2000). Moreover, VEGFB expression
correlates with microvascular density in oral squamous cell carcinoma (Shintani et al., 2004).
1.3.1.3 VEGF-C and VEGF-D
The VEGF homologs VEGF-C and VEGF-D play key roles during embryonic and postnatal
lymphangiogenesis. Homozygous deletion of the VEGF-C gene in mice is embryonically lethal and
heterozygous deletion results in postnatal defects associated with defective lymphatic development
(Karkkainen et al., 2004). Both factors induce lymphangiogenesis in transgenic mouse models
(Jeltsch et al., 1997; Veikkola et al., 2001). VEGF-C and VEGF-D may also play a role in new
blood vessel growth as well, especially during pathological states such as tumor growth.
1.3.1.4. VEGF-E
VEGF-E is not a mammalian VEGF homolog, but rather a viral protein encoded by the
parapoxvirus Orf virus, which preferentially utilizes kinase-insert domain-receptor (KDR)/fetal
liver kinase-1 (Flk-1) receptor and carries a potent mitotic activity without heparin-binding domain
(Ogawa et al., 1998). VEGF-E shares 22% sequence identity to VEGF-A.
1.3.2 VEGF receptors
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VEGF ligands mediate their angiogenic effects via several different receptors. Two receptors
were originally identified on endothelial cells and characterized as the specific tyrosine kinase
receptors VEGFR-1 (also referred to as fms-like tyrosine kinase 1 [Flt-1]) (Shibuya et al., 1990).
VEGFR-2 (also referred to KDR, (Terman, 1992) and the murine homologue, Flk-1) (Matthews et
al. 1991). These two receptors share 44% homology and possess a characteristic structure consisting
of seven extracellular immunoglobulin-like domains, a single transmembrane domain, and a
consensus tyrosine kinase domain interrupted by a kinase insert domain (Shibuya et al., 1990;
Terman, 1991). More recently, an additional tyrosine kinase receptor, VEGFR-3 (also referred to as
fms-like tyrosine kinase 4 [Flt-4]), was identified and has been found to be primarily associated
with lymphangiogenesis (Kaipainen et al., 1995; Paavonen et al., 2000). The various members of
the VEGF family have differing binding specificities for each of these receptors. All of the
VEGF-A isoforms bind to both VEGFR-1 and VEGFR-2, whereas PlGF-1, PlGF-2 and VEGF-B
are specific for VEGFR-1 binding and activation (Park et al., 1994; Olofsson et al., 1998; Silvestre
et al., 2003). Naturally occurring heterodimers of VEGF-A and PlGF have also been identified that
can bind to and activate VEGFR-2 (Cao et al., 1996; DiSalvo et al., 1995). VEGF-E specifically
interacts with VEGFR-2 whereas VEGF-C and VEGF-D interact with both VEGFR-3 and
VEGFR-2 (Shibuya et al., 2003; Achen et al., 1998; Joukov et al., 1996).
1.3.2.1 VEGF-R1
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VEGFR-1 is a receptor for VEGF-A and has the unique ability to bind VEGF-B and PlGF.
VEGFR-1 is critical for physiologic and developmental angiogenesis. VEGFR-1 null mice die in
utero between days 8.5 to 9.5 due to excessive hemangioblast proliferation and poor organization of
vascular structures (Fong et al., 1995). VEGFR-1 was initially thought to be a negative regulator of
VEGF activity either by acting as a decoy receptor for VEGF (Hiratsuka et al., 1998) or by
downregulating VEGFR-2–mediated signaling (Dunk et al., 2001). VEGF-mediated stimulation of
VEGFR-1 autophosphorylation and signaling in endothelial cells is weak when compared to
signaling through VEGFR-2 (Waltenberger et al., 1994). A repressor motif has been identified in
the juxtamembrane region of VEGFR-1 that impairs phosphatidylinositol 3-kinase (PI3K) signaling
and endothelial cell migration in response to VEGF stimulation (Gille et al., 2000; Zeng et al.,
2001). However, other studies have indicated that VEGFR-1 has a positive, functional role in
certain cell types, participating in monocyte migration (Barleon et al., 1996; Clauss et al., 1996),
recruitment of endothelial cell progenitors (Lyden et al., 2001) increasing the adhesive properties of
natural killer cells (Chen et al., 2002) and inducing growth factors from liver sinusoidal endothelial
cells (LeCouter et al., 2003). A recent study (Autiero et al, 2003) showed that activation of
VEGFR-1 by PlGF resulted in transphosphorylation of VEGFR-2 in endothelial cells co-expressing
these receptors. Furthermore, VEGF/PlGF heterodimers are capable of activating intramolecular
VEGFR cross talk through formation of VEGFR-1/VEGFR-2 heterodimers. Other recent studies
have shown that during pathologic conditions such as tumorigenesis, VEGFR-1 is a potent, positive
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regulator of angiogenesis (Hiratsuka et al., 2001). A naturally occurring, alternatively spliced
soluble form of VEGFR-1 (sVEGFR-1) also exists (Kendall et al., 1993). sVEGFR-1 may function
to reduce or modulate endogenous VEGF or PlGF activity.
1.3.2.2. VEGF-R2
VEGFR-2 mediates the majority of the downstream effects of VEGF-A in angiogenesis,
including microvascular permeability, endothelial cell proliferation, invasion, migration, and
survival (Zeng et al., 2001; Millauer et al., 1993). The importance of VEGFR-2 in vasculogenesis is
demonstrated by the fact that hetero and homozygous knockout mice die in utero of defects in blood
island formation and vascular development (Shalaby et al., 1995). VEGFR-2–mediated proliferation
of endothelial cells involves activation of a phospholipase C gamma-protein kinase C-Raf-MAP
kinase signaling pathway, whereas survival and migration is believed to involve PI3K and focal
adhesion kinase, respectively (Veikkola et al., 2000; Abedi et al., 1997).
1.3.2.3 VEGFR3
VEGFR-3 is a receptor tyrosine kinase originally cloned from a human leukemia cell line and
human placenta (Pajusola et al., 1993; Galland et al., 1993). VEGFR-3 preferentially binds
VEGF-C and VEGFD. VEGFR-3 is expressed throughout the embryonic vasculature, but during
development and in the adult, its expression is limited to lymphatic endothelial cells (Kaipainen et
al., 1995). Homozygous deletion of the VEGFR-3 gene in mice leads to embryonic death at day 10
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to 12.5, with an underdeveloped yolk sac, poor perineural vasculature, and pericardial fluid
accumulation (Dumont et al., 1998). VEGFR-3 activation and upregulation of its ligands have been
observed in certain neoplastic conditions, including breast cancer and melanoma with elevated
levels of VEGF-C or VEGF-D associated with lymph node metastasis in patients (Achen et al.,
2001; Valtola et al., 1999; Pepper et al., 2003).
1.3.3 Functions of VEGF
1.3.3.1 Vessels Permeability
VEGF is also termed vascular permeability factor (VPF) (Dvorak et al., 1995). In fact, VEGF
is one of the most potent inducers of vascular permeability known - 50,000-fold more potent than
histamine (Dvorak, 2002). This ability to enhance microvascular permeability is one of the most
important properties of VEGF, especially with regards to the hyperpermeability of tumor vessels
that is thought to be largely attributable to tumor cell expression of VEGF. It has been suggested
that the increase in permeability results in the leakage of several plasma proteins, including
fibrinogen and other clotting proteins. This can lead to the deposition of fibrin in the extravascular
space, which subsequently retards the clearance of edema fluid and transforms the normally
antiangiogenic stroma of normal tissues into a proangiogenic environment (Dvorak et al., 1995;
2002). VEGF increases permeability in a variety of vascular beds, including the skin, peritoneal
wall, mesentery, and diaphragm, and can lead to pathologic conditions such as malignant ascites
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and malignant pleural effusions (Yoshiji et al., 2001; Yuan et al., 1996).
1.3.3.2 Endothelial cell activation
VEGF exerts a number of different effects which include changes in endothelial cell
morphology, cytoskeleton alterations, and stimulation of endothelial cell migration and growth.
VEGF causes increased expression of a variety of different endothelial cell genes, including
procoagulant tissue factor and fibrinolytic pathway proteins, such as urokinase, tissue-type
plasminogen activator, type 1 plasminogen activator inhibitor, and urokinase inhibitor; and matrix
metalloproteases; GLUT-1 glucose transporter; nitric oxide synthase; and integrins (Dvorak et al.,
2002; Zachary, 2001; Eliceiri et al., 2001; 1999; Brooks et al., 1994).
1.3.3.3 Endothelial cell survival
VEGF was first shown to act as a survival factor for retinal endothelial cells (Alon et al., 1995).
In vitro, VEGF has been shown to inhibit apoptosis by activating the PI3K-Akt pathway (Gerber et
al., 1998; Gerber et al., 1998; Thakker et al., 1999) in addition to upregulating antiapoptotic
proteins such as bcl-2 and A1. VEGF has also been shown to activate focal adhesion kinase (FAK)
and associated proteins that have been shown to maintain survival signals in endothelial cells
(Abedi et al., 1997; Zachary et al., 2001).
1.3.3.4 Endothelial cell proliferation
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VEGF is a mitogen for endothelial cells. This endothelial cell proliferation appears to involve
VEGFR-2–mediated activation of extracellular kinases Erk1/2 in addition to another member of the
MAP kinase family, JNK/SAPK (Zachary et al., 2001; Meadows et al., 2001).
1.3.3.5 Endothelial cell invasion and migration
Degradation of the basement membrane is necessary for endothelial cell migration and
invasion and is an important early step in the initiation of angiogenesis. VEGF induces a variety of
enzymes and proteins important in the degradation process, including matrix-degrading
metalloproteinases, metalloproteinase interstitial collagenase, and serine proteases such as
urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (TTPA)
(Zachary et al., 2001; Choong et al., 2003). Activation of these various compounds leads to a
prodegradative environment that facilitates migration and sprouting of endothelial cells (Ferrara et
al., 1997). The intracellular mechanisms by which VEGF leads to increased endothelial cell
migration are not entirely clear, but appear to involve FAK-associated signaling leading to focal
adhesion turnover and actin filament organization as well as p38 MAPK-induced actin
reorganization (Abedi et al., 1997; Zachary et al., 2001)
1.3.4 Expression of VEGF on tumor cells
Several studies have reported the presence of VEGFRs on liquid and solid tumor cells,
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including those of non–small cell lung carcinoma, melanoma, prostate carcinoma, leukemia,
mesothelioma, and breast carcinoma (Dias et al., 2001; Bellamy et al., 1999; Decaussin et al., 1999;
Dias et al., 2000; Ferrer et al., 1999; Hayashibara et al., 2001; Lacal et al., 2000; Price et al., 2001).
Various VEGF ligands support tumor growth, not only by inducing angiogenesis, but also by acting
directly through VEGFRs expressed by tumor cells. Moreover, since the vast majority of solid
tumors and a variety of hematologic malignancies have the capacity to express VEGF, expression
of VEGFRs by tumor cells implicates a potential role for VEGF/VEGFR autocrine loops in these
tumors.
1.3.5 Expression of VEGF on OSCCs
VEGF expression is up-regulated significantly during the transition from NOM, through
dysplasia to OSCC. For the dysplastic lesions, no correlation is found between VEGF expression
and grade of dysplasia (Johnstone et al., 2007). The head and neck squamous cell carcinoma
(HNSCC) tumor cells express VEGFR-1, VEGFR-2, and VEGFR-3 in all specimens evaluated.
Staining for all 3 receptors is also found on tumor associated macrophages and fibrobasts, except
that VEGFR-2 is not present on fibroblasts. Staining intensity for VEGFR-1 and VEGFR-2 is
significantly higher in tumor cells and macrophages than in vascular endothelial cells (VECs)
stained for the same receptor. In HNSCC, as well as other tumor systems, VEGF has been shown to
be expressed by tumor cells and to induce proliferation of adjacent VECs via a paracrine
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mechanism (Benefield et al., 1996; Petruzzelli et al., 1997). Recent evidence has demonstrated that
VEGF may also have a direct effect on tumor cell activity (Masood et al., 2001). These data suggest
a possible autocrine mechanism for VEGF-regulated vasculogenesis and tumorigenesis.