Vascular endothelial growth factor receptor-2 in breast cancer

Biochimica et Biophysica Acta 1806 (2010) 108–121

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Biochimica et Biophysica Acta

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Review

Vascular endothelial growth factor receptor-2 in breast cancer

Shanchun Guo a, Laronna S. Colbert b, Miles Fuller a, Yuanyuan Zhang a, Ruben R. Gonzalez-Perez a,⁎a Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, USAb Clinical Medicine, Hematology/Oncology Section, Morehouse School of Medicine, Atlanta, GA 30310, USA

⁎ Corresponding author. Tel.: +1 404 752 1581.E-mail address: [email protected] (R.R. Gonzalez

0304-419X/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbcan.2010.04.004

a b s t r a c t
a r t i c l e i n f o
Article history:Received 28 January 2010Received in revised form 16 April 2010Accepted 21 April 2010Available online 11 May 2010

Keywords:VEGFVEGFR-2Tumor angiogenesisBreast cancerAutocrine/paracrine loopLeptin

Investigations over the last decade have established the essential role of growth factors and their receptorsduring angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family inmammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which aretransmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In theearly 1990s, the above VEGFR was structurally characterized by cDNA cloning. Among these three receptors,VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 isconsidered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulatestumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them arenow in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as anessential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark thecontinuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: theautocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumorangiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements ofpromoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In thisreview, we will focus on updated information regarding VEGFR-2 research with respect to the molecularmechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, andregulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimedat directly targeting cancer cell proliferation and survival.

-Perez).

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092. VEGFR-2: a committed pro-angiogenic receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093. Structure of VEGFR-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1094. VEGFR-2 signaling pathways and targeted effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1095. The autocrine/paracrine VEGF/VEGFR-2 loop: a cancer cell survival process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1106. Expression of VEGFR-2 in human breast cancer and other cancer types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1107. Regulation of VEGFR-2 levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

7.1. VEGFR-2 co-activators, cellular localization and trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117.2. Regulation of VEGFR-2 expression: cis-elements of VEGFR-2 promoter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1127.3. Epigenetic regulation of VEGFR-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137.4. The Notch signaling-VEGFR-2 link. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137.5. Estrogen-mediated regulation of VEGFR-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147.6. Cytokine and growth factor regulation of VEGFR-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

7.6.1. Leptin regulation of VEGFR-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147.7. Clinical significance of targeting VEGFR-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

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109S. Guo et al. / Biochimica et Biophysica Acta 1806 (2010) 108–121

1. Introduction

Formation of the primary vascular plexus in the embryo is drivenby vasculogenesis; additional blood vessels are generated byangiogenesis, which are progressively pruned and remodeled into afunctional adult circulatory system [1]. Vasculogenesis encompassesthe transformation of mesodermic angioblasts into endothelial cells(EC) and further development of new blood vessels. In contrast,angiogenesis is the sprouting of new capillaries from pre-existingblood vessels that involves endothelial cell differentiation, prolifera-tion, migration, cord formation and tubulogenesis [1]. Many endo-thelium-specific molecules can influence these processes, includingmembers of the VEGF, angiopoietin and ephrin families. In addition,non-vascular endothelium-specific factors contribute to blood vesselformation, i.e., platelet-derived growth factor, PDGF and transforminggrowth factor-β, TGF-β families [2].

Crucial factors for blood vessel formation are the VEGF membersshowing multiple interactions with receptor tyrosine kinases (RTK),namely VEGFR-1 (fms-like tyrosine kinase, Flt-1) [3], VEGFR-2 (fetalliver kinase, Flk-1 in mice or KDR in humans) [4–6], and VEGFR-3 (Flt-4)[7]. A fourth receptor, Flt-3/Flk-2, belonging to the RTK family, wasidentified, but it does not bind to VEGF [8]. Receptor Neuropilins 1 and2 (NRP-1/-2) are another class of high-affinity non-tyrosine kinasereceptors for VEGF on endothelial and neuronal cell surfaces [9].VEGF-A is the major form that binds and signals through VEGFR-2 todevelop blood vessels and maintain the vascular network [10]. In thisrespect VEGFR-2 is essential for life [11]. This is supported by the factthat homozygous-deficient Flk-1 mice die in the second week ofgestation as a consequence of insufficient development of hemato-poietic and EC [4]. In contrast, VEGF signals through VEGFR-1 (Flt-1)are also required for the organization of embryonic vasculature, butthey are not essential for endothelial cell differentiation. Indeed,homozygous mice for targeted mutation of VEGFR-1 gene produce ECfrom angioblasts, but develop non-functional blood vessels and die ataround 10 days of gestation [12]. VEGFR-2 mediates the major growthand permeability actions of VEGF [13], whereas VEGFR-1 may have anegative role, either by acting as a decoy receptor or by suppressingsignaling through VEGFR-2 [2]. VEGFR-3 binds VEGF-C and VEGF-D tomainly regulate lymphatic EC [9]. However, VEGFR-3 is also essentialfor early blood vessel development and plays a role in tumorangiogenesis [14,15]. The Flt-3/Flk-2 receptor (CD135) binds theFlt-3 ligand to promote the growth and differentiation of primitivehematopoietic cells. Flt-3/Flk-2 is expressed on CD34+ hematopoi-etic stem cells, myelomonocytic progenitors, primitive B-cell pro-genitors, and thymocytes and control differentiation of hematopoieticand non-hematopoietic cells [8,16].

2. VEGFR-2: a committed pro-angiogenic receptor

VEGFR-2 is a transmembrane receptor that plays an important rolein endothelial cell development [1,17] and is thought to mediate thekey effects of the endothelial-specific mitogen VEGF on cell prolifer-ation and permeability. Therefore, themajority of VEGFR-2 actions arerelated to angiogenesis [18,19].

3. Structure of VEGFR-2

VEGFR-2 was discovered before the identification of its ligand,VEGF [1,2]. The receptor tyrosine kinase named KDR in human [2,20],Flk-1 [4] or NYW/FLK-1 in mice [21] and TKr-11 in rat [22] was earlieridentified as a transducer of VEGF in EC [23]. VEGFR-2 consists of 1356and 1345 amino acids in humans and mice, respectively, and can beseparated into four regions: the extracellular ligand-binding domain,transmembrane domain, tyrosine kinase domain, and downstreamcarboxy terminal region [3–6]. The extracellular ligand-bindingdomain consists of seven immunoglobulin (Ig)-like domains with

the ligand-binding region being localized within the second and thirdIg domains [24–27]. Shinkai et al., further demonstrated that the thirdIg-like domain is critical for ligand binding, the second and fourthdomains are important for ligand association, and the fifth and sixthdomains are required for retention of the ligand bound to the receptormolecule [26]. Ligand-binding specificities of VEGFR-2 differ fromVEGFR-1; VEGFR-2 binds VEGF-A, VEGF-C, VEGF-D and VEGF-E [28–32], whereas VEGFR-1 binds VEGF-A, VEGF-B and PIGF [33–35].

The classic interpretation of ligand-binding specificities is cur-rently used for all aspects of vascular effects driven by VEGF–VEGFRs,which have helped in elucidating their function. However, thepotential formation of VEGFR heterodimers, i.e., VEGFR-2–VEGFR-1[36] and VEGFR-2–VEGFR-3 [37] has generally been understated. Itwas noticed that substantial differences in signal transduction occurupon distinct VEGFR heterodimers complex formation because kinasedomains and substrate specificities differ [33,36–39]. These com-plexes can also differentially interact with VEGF and PIGF [33,36]resulting in unique signaling pattern and likely different feedbackmechanisms [38].

4. VEGFR-2 signaling pathways and targeted effects

VEGFR-2 actions heavily depend on the specific activation oftyrosine amino acid residues within the intracytoplasmatic tail of thereceptor (for review see [40,41]). The majority of VEGFR-2 intracel-lular domains contain tyrosine residues that are involved inredundant actions on vasculogenesis or angiogenesis. VEGFR-2 signalsaffect vascular permeability and proliferation, survival and migrationof EC [42,43]. The upstream phosphorylation of a tyrosine residue(Y801) within the juxtamembrane domain of VEGFR-2 seems to berequired for the subsequent activation of the catalytic domain.However, the biological implications of these findings need to befurther investigated [44]. Specific serine residues in the VEGFR-2intracytoplasmatic region are targeted by protein kinase C (PKC) andinvolved in regulatory mechanisms of receptor levels (ubiquitinyla-tion and degradation) [45].

VEGF binding to VEGFR-2 triggers the specific activation oftyrosine amino acid residues within intracytoplasmatic tail of thereceptor inducing multiple signaling networks that result in endo-thelial cell survival, proliferation, migration, focal adhesion turnover,actin remodeling and vascular permeability [40]. Signaling throughmitogen-activated protein kinase (MAP; including MEK-p42/p44MAPK and p38 MAPK; linked to proliferation) [40,46] and phospha-tidylinositol 3′ kinase (PI-3K)/V-akt murine thymoma viral oncogenehomolog 1 (Akt1; linked to survival) [47] is a common receptortyrosine kinase activation pattern. Additional signaling pathways arealso triggered upon VEGFR-2 activation, i.e., phospholipase C gamma(PLCg)-protein kinase C (PKC; linked to proliferation) [48], focaladhesion kinase (FAK; linked to migration) [47] and T Cell-SpecificAdapter (TSAd)-Src kinase (linked to migration and vascularpermeability) [49] (see Fig. 1). Recent data suggest that over-expression of caveolin-1 in prostate cancer cells specifically inducephosphorylation of tyrosine 951 in the VEGFR-2 intracytoplasmatictail and increases angiogenesis [50]. On the other hand, tyrosine 1175mediates both PLCγ-1 and protein kinase A (PKA)-dependentsignaling pathways required for VEGF-induced release of von Will-ebrand factor from EC [51]. However, the involvement of specificVEGFR-2 intracytoplasmatic domains and associated activation ofspecific signaling pathways and driven functions is a point ofcontroversial discussion. Dayanir et al., found that activation of PI-3K/S6 but not Ras/MAPK kinase pathway is responsible for VEGFR-2-mediated cell growth [52]. Inhibition of p38 MAPK activity enhancesVEGF-induced angiogenesis in vitro and in vivo and prolonged ERK1/2 activation and increased endothelial survival, but abrogated VEGF-induced vascular permeability [53]. Intriguingly, VEGF-mediatedproliferation of VEGFR-2 transfected fibroblasts was slower and

Fig. 1. Schematic representation of how VEGFR-2 signaling pathways are linked to its main biological functions. Different VEGF isoforms can bind VEGFR-2 dimer. NRP-1/-2 are co-receptors that stabilize the VEGFR-2 dimer. Upon ligand binding to VEGFR-2 dimer several signaling pathways can be activated affecting diverse biological processes in endothelialand cancer cells.

110 S. Guo et al. / Biochimica et Biophysica Acta 1806 (2010) 108–121

weaker than in EC, suggesting the cell type-specific signalingmechanism(s) [46]. These results open the possibilities for differentialsignaling mechanisms/responses to VEGF via VEGFR-2 in cancercompared to EC. Inconsistent reports on VEGFR-2 signaling capabil-ities could be due to the complex interplay of signaling and inhibitingactions of other VEGF receptors. In addition, the activation andsignaling of VEGFR-2 could also be modified by the formation ofVEGFR-2 heterodimers exhibiting differential signaling potential asdescribed above.

5. The autocrine/paracrine VEGF/VEGFR-2 loop: a cancer cellsurvival process

Intensive research has been done on VEGF/VEGFR-2 roles invascular functions [40]. However, only a small number of reportshighlight a lesser known function of VEGF signaling that can directlyimpact cancer cell survival: the autocrine loop in cancer cells. Somereports suggest that a strictmolecular requirement for these autocrineactions of VEGF is the expression of VEGFR-1 as it was found in coloncarcinoma [54]. In line with these data,Wu et al., further reported thatselective signaling through VEGFR-1 on breast cancer cells supportstumor growth through downstream activation of the p44/42MAPK orAkt pathways [55]. However, in breast cancer cells, VEGFR-2 isoformwas not initially linked to cell survival [54,56]. The co-expression ofNRP-1 [57] and α6β4 integrin [56] but not VEGFR-2, was foundessential for the binding of VEGF and activation of the PI-3K survivalsignaling pathway in breast cancer cells. Moreover, it was suggestedthat breast cancer cells do not express VEGFR-2 [56,57]. In contrast,VEGF/VEGFR-2 was found to be essential to cell survival in eitherestrogen receptor positive (MCF-7) [58,59] or negative cells (MDA-MB-468) [59] after tamoxifen treatment. A signaling cascade fromVEGFR-2 via ERK1/2 to Ets-2 phosphorylationwas correlated to bettersurvival of untreated patients [59]. Moreover, a VEGF/VEGFR-2/p38kinase link was involved in poor outcome for tamoxifen-treatedpatients [58]. VEGF stimulation of Akt phosphorylation and activationof ERK1/2 correlated to VEGFR-2 expression and activation in variousbreast carcinoma cell lines and primary culture of breast carcinomacells [60].

Findings from our laboratory suggest that mouse (4T1, ER+) [61]and human breast cancer cells (MCF-7, ER+ and MDA-MB-231, ER-)express VEGFR-2 in vitro and in vivo [62]. Interestingly in these cells

the expression of VEGF and VEGFR-2 was linked to leptin signaling.Leptin is a small nonglycosylated protein (16 kDa) product of the obgene. White adipose tissue is the primary source of leptin in benigntissue but it is also expressed and secreted by cancer cells. Leptin is aknown mitogenic, inflammatory and angiogenic factor for manytissues [63] and increases the levels of VEGF/VEGFR-2 in cancer cells[61,62,64]. Therefore, leptin from corporal, mammary adipose tissueor cancer cells could affect cancer cells through autocrine andparacrine actions. Leptin increased the proliferation of 4T1 cells, buttreatment with anti-VEGFR-2 antibody resulted in a further increasein leptin induced VEGF concentrations in the medium. This suggests afeedback loop between VEGF expression and VEGFR-2 that could belinked to cell survival/proliferation [58]. Remarkably, inhibition ofleptin signaling mediated an impressive reduction of tumor growth inmice that paralleled a significant reduction of VEGF and VEGFR-2levels [61,62].

The endothelial-independent VEGF/VEGFR-2 autocrine loop wasfound essential for leukemia cell survival and migration in vivo [65].Results from these studies suggest that effective antiangiogenictherapies to treat VEGF-producing and VEGFR-expressing leukemiasmay require blocking both paracrine and autocrine VEGF/VEGFR-2angiogenic loops to achieve long term remission and cure [65]. On theother hand, stimulation of the proliferation of prostate cancer cells byVEGF depends on the presence of VEGFR-2 through autocrine loopsgenerated by IL-6 [66]. Overall these data suggest that targeting theVEGF/VEGFR-2 autocrine loop in cancer may be an innovative way totreat the disease. However, the relationships between activation ofspecific intracellular domains of VEGFR-2 and autocrine actions ofVEGF in cancer cells remains to be studied in detail.

6. Expression of VEGFR-2 in human breast cancer and othercancer types

Because of the potential role of VEGFR-2 in tumor angiogenesis, itsexpression was investigated in breast carcinoma soon after it wasidentified as a tyrosine kinase-receptor for VEGF [23]. Upregulation ofVEGFR-2 mRNA was found earlier in invasive primary and metastaticbreast cancers [67]. Western blot and immunohistochemical analysesof endothelium and epithelium of mammary ducts in carcinomas,fibroadenomas and fibrocystic breast disease showed positiveexpression of VEGFR-2, which was also found in tumor stroma [68].


VEGFR-2 expression correlated with VEGF expression that suggestedthese molecules were co-expressed in breast cancer; however, thiswas not significantly associated with patient survival [69]. In addition,tumor-specific expression of VEGFR-2 correlated strongly withexpression of VEGF-A and progesterone receptor (PR) negativity,whereas VEGF-A was not associated with hormone receptor status[70].

In vitro studies demonstrated that VEGFR-2 receptors are largelyexpressed in breast cancer [58–62,64] and colon cancer cells as well asintra-tumoral vasculature [71] and VEGFR-2 mRNA is expressed inbreast cancer [64] as well as several pancreatic cancer cell lines [72]. Insitu hybridization analyses revealed that 85% of human ovarian cancerspecimens showed moderate to high VEGFR-2 expression [73] andsome western blotting in small cell lung cancer (SCLC) cells analysesdetected high VEGFR-2 [74].

Invasive and in situ breast cancers express many angiogenicfactors and this process was found throughout all tumor stages [75].Nakopoulou et al., detected VEGFR-2 in 64.5% (91/141) of invasivebreast carcinomas showing a widespread cytoplasmic expression inmost of the neoplastic cells [76]. VEGFR-2 expression was wellcorrelated with the nuclear grade of the invasive breast carcinoma(P=0.003), but demonstrated no correlation with histologic grade,stage, and patient survival [76] as was previously noticed [69]. Incervical adenosquamous carcinoma, VEGFR-2 positivity was observedin 22 of 30 cases (73.3%) but was significantly associated with lack ofmetastasis (P=0.038) [77].

In contrast, other studies suggest that not only the overexpressionof VEGFR-2 occurs in cancer, but also the expression of VEGFR-2relates to the disease stage, recurrence and worse outcome. VEGFR-2was found weakly expressed in normal tissues or cells, but VEGFR-2overexpression was reported in various cancers including lung, colon,uterus, ovarian cancer, as well as breast cancers [71]. In colon cancer,VEGFR-2 positive rate was found 46.7% [78]. In esophageal adenocar-cinoma and squamous-cell cancer, VEGFR-2 positive rate was 100%[79]. The VEGFR-2 protein-positive phenotype of kidney clear cellcarcinoma was relatively frequent (7/20, 35%), but was lost in bonemetastases (2/20, 10%) [80]. Increased VEGFR-2 expression correlateswith several features that predict progression of urothelial cancer,including disease stage and invasive phenotype. VEGFR-2 expressioncorrelated with disease stage (coefficient 0.23, P=0.05). In addition,VEGFR-2 expression increased with tumor invasion into the muscle(Pb0.01) [81]. Moreover, in primary glioblastomas amplification ofVEGFR-2 occurs frequently but also occurs in lower-grade gliomas andin their recurrent tumors [82]. Elevated expression of VEGFR-2 in HCC(hepatocellular carcinoma) was correlated with worse outcomefollowing liver transplantation. Vascular invasion was consistentlyassociated with HCC recurrence (Pb0.01) and overall mortality

Table 1VEGFR-2 expression in human tumors and tumor cell lines.

Tissue Subtype Positive rate Reference

Bladder Carcinoma 50% [81]Brain Glioma 6–17% [82]Breast Adenocarcinoma 64.5% [76]Cervix Adenosquamous carcinoma 73.3% [77]Colon Carcinoma 46.7% [78]Esophagus Carcinoma 100% [79]Kidney Clear cell cancer 35% [80]Lung Non-small cell carcinoma 54.2% [198]Oral Carcinoma ↑↑ [199]Ovary Carcinoma 100% [200]Pancreas Carcinoma 80% [201]Prostate Cancer* 100% [202]Skin Melanoma ↑↑ [203]

*Cell lines; ↑↑, overexpression of VEGFR-2 in cancer cells.

(Pb0.05). Subjects with VEGFR-2 overexpression in tumor arterioles(Pb0.01), venules (Pb0.05) had worse overall survival [83]. Table 1shows relative levels of expression of VEGFR-2 in different cancertypes.

Interestingly, in breast cancer VEGFR-2 expression was signifi-cantly correlated with two well-established proliferation indices, Ki-67 (P=0.037) and topo-IIα (P=0.009). This suggests VEGF mayexert growth factor activities on mammary cancer cells through itsreceptor VEGFR-2 [76]. Taken together, these data suggest that high-levels of VEGFR-2 may potentially serve as a biomarker in cancer.

7. Regulation of VEGFR-2 levels

Despite the essential role of VEGFR-2 in angiogenesis andcarcinogenesis the molecular mechanisms controlling its expressionare only partially known. Regulation of VEGFR-2 expression involves aseries of complex mechanisms, which include epigenetic changes,transcriptional regulation, cellular localization/trafficking, ligandbinding, co-activator activity, adhesion molecule expression, consti-tutive-embryonic derived signaling pathways and cytokine-growthfactor regulation. In addition, VEGFR-2 can assemble functionalcomplexes composed of homodimers and heterodimers with otherVEGF receptors and co-receptors that can bind VEGF or otherangiogenic ligands thereby affecting VEGFR-2 signaling capabilities(Fig. 2).

7.1. VEGFR-2 co-activators, cellular localization and trafficking

Heparin and heparan sulfates (components of proteoglycans,HSPGS) have affinity for VEGF165 (VEGF-A), the major isoform ofVEGF, promoting enhanced phosphorylation of VEGFR-2. This prob-ably occurs by enhancing ligand-binding capabilities to VEGFR-2.Therefore, HSPGs affect the localization, extent and intensity of VEGFRsignaling [84]. NRP-1 can stabilize the VEGF/VEGFR-2 complex andparticularly increase tumor angiogenesis [85]. Blood flow could likelyactivate VEGFR-2 through the formation of mechanosensory com-plexes [86]. Adhesion molecules (PECAM-1 and VE-cadherin) [86,87]and αvβ3 integrin [88] interplay with VEGFR-2 under diversebiological scenarios controlling VEGFR-2 expression and signaling.β3-integrin can limit the interaction of NRP-1 with VEGFR-2, thusnegatively affecting VEGF-mediated angiogenesis [89]. Invasive ductalcarcinoma has decreased expression of α6-integrin associated withhigher tumor angiogenesis presumably linked to VEGFR-2 expression.Indeed, loss of α6-integrin correlates to overexpression of activatedVEGFR-2 in murine melanoma and lung carcinoma in endothelial-specific α6-knockout mice [90]. However, the specific mechanismsinvolved in VEGFR-2 overexpression in tumors from these miceremains to be investigated. A negative regulator of angiogenesis isthrombospondin-1 (TSP-1 or CD36). This molecule negativelymodulates VEGF actions through a complex with β1-integrin andVEGFR-2 [91].

Inverse regulation of VEGFR-1 and VEGFR-2 could play animportant role in controlling the growth and differentiation oftumor-associated EC. VEGF signaling through JNK/c-Jun pathwayinduces endocytosis, nuclear translocation and ubiquitin-mediateddownregulation of VEGFR-2 in human squamous-cell carcinomas[92]. A model recently proposed suggests that a negative feedbackloop regulates VEGFR-2 activities through the differential segrega-tion/localization of VEGFR-1 and VEGFR-2 [89,90]. The higher affinityof VEGFR-1 for VEGF blocks VEGFR-2 activation. In the model, Ca (2+)induces the translocation of VEGFR-1 from the trans-Golgi network tothe plasma membrane allowing preferential binding of VEGF. VEGFR-2 is degraded after activation by ubiquitin-mediated proteolysis thatis linked to ESCRT (endosomal sorting complex required fortransport) and to Rab GTPases [93]. This could also be stimulated byPKC pathway that requires the removal of VEGFR-2 carboxyl terminus

Fig. 2. Possible mechanisms involved in the regulation of VEGFR-2 levels in cancer cells. VEGF promotes tumor angiogenesis and upregulates VEGFR-2 in cancer cells increasingsurvival and proliferation through an autocrine/paracrine loop (MAPK/ERK1/2/p38 and PI-3K signaling pathways). VEGFR-2 can form homodimers or heterodimers (VEGFR-2/VEGFR-1 and VEGFR-2/VEGFR-3). PIGF binds with high affinity to VEGFR-1 but also to VEGFR-2/VEGFR-1 heterodimers. VEGFR-1, when activated by PIGF, switches onintermolecular cross talk, which transactivates VEGFR-2 and thereby enhances the response to VEGF [33]. NRP stabilizes the VEGF/VEGFR-2 complex but this could be hampered byβ3 integrin. The α6 integrin can decrease VEGFR-2 expression. HSPGS, PECAM-1 and VE-cadherin can regulate ligand binding and phosphorylation of VEGFR-2. Leptin and Jagged1–Notch signaling upregulates VEGFR-2 expression. In contrast, DII4–Notch signaling downregulates VEGFR-2 expression. Endocytosis and ubiquitin-mediated downregulation ofVEGFR-2 linked to ESCRT (endosomal sorting complex required for transport) is mediated by JNK/c-Jun and PKC signaling pathways activated by VEGF. Deacetylation/methylationof VEGFR-2 gene could also play a role in its epigenetic control.


[94]. Differential trafficking of VEGFR-2 is potentially due to theformation of complexes with diverse angiogenic regulators. Theseprocesses occur through the endosomal pathway controlling angio-genesis [95].

7.2. Regulation of VEGFR-2 expression: cis-elements of VEGFR-2promoter

The VEGFR-2 gene promoter is highly complex with multiple cis-elements [96]. Regulation of VEGFR-2 expression is dependent on anumber of factors, including cell context. VEGFR-2 gene promoter inhumans (KDR) and in mice (Flk-1) does not have a TATA box region,but does have several DNA binding sites for general and tissue-specifictranscription factors. Therefore, VEGFR-2 belongs to class II promoterstructure. This TATA-less gene contains four upstream Sp1 sites and asingle transcription start site [97] that binds multi-functionaltranscription factor TFII-I for gene expression [98]. Transfection ofVEGFR-2 promoter constructs into bovine aortic EC (BAECs) showedthat basal activity of VEGFR gene was primarily associated with theGC-rich−95 to−60 region of the promoter, which contains Sp, AP-2,and nuclear factor-κB (NFκB motifs) [96]. cAMP response elementbinding protein (CREB) and NFκB-related antigens bind specificsequences in the VEGFR-2 promoter of BAECs [98].

DNA footprinting studies also showed that protected sequencesbetween−110 and−25 in human umbilical vein EC (HUVECs) werecritical for VEGFR-2 promoter activity. However, comparable inter-actions were not observed in fibroblasts or HeLa cells [99]. Otherstudies also showed that the GC-rich −79 to −68 region of thepromoter was essential for activity in EC [100]. This region bound

both Sp1 and Sp3; however, their results suggested that Sp1expression enhanced VEGFR-2 expression but that Sp3 attenuatedthis response. In contrast, another report showed that basal and shearstress-induced activation of VEGFR-2 promoter constructs in HUVECswas primarily dependent on two more proximal GC-rich sites at −58and −44 bp [101]. Sp1-dependent DNA binding within −77 and−60 region of VEGFR-2 promoter seems to be essential for theregulation of both mRNA and protein in human EC by Rac-1 (a smallRho-GTPase) [102]. Consensus or nonconsensus estrogen receptorelement (ERE motifs) has not been found in 5′ VEGFR-2 promoterregion [96]. Results from chromatin immunoprecipitation assays(ChiPs) demonstrated that ERα is constitutively bound to the VEGFR-2 promoter and that these interactions are not enhanced aftertreatment with estradiol (E2), whereas ERα binding to the region ofthe pS2 promoter (a human breast cancer marker) containing an EREelement is enhanced by E2 [103]. Hormone-induced activation of theVEGFR-2 promoter constructs requires Sp3 and Sp4 but not Sp1,demonstrating that hormonal activation of VEGFR-2 involves a non-classical mechanism in which ERα/Sp3 and ERα/Sp4 complexesactivate GC-rich sites where Sp proteins, but not ERα, bind DNA[103]. Analysis of the mouse VEGFR-2 promoter sequence revealedthe presence of binding sites of additional transcriptional factors,which related to angiogenesis previously reported. Putative tran-scription factor binding sites are systematically listed in Table 2.Investigations using assays for transcription factor binding (EMSA,electrophoretic mobility shift assay, or others), ChiPs and luciferasereporter gene (containing binding site mutations) will be essential tofurther elucidate the molecular mechanisms involved in theregulation of VEGFR-2.

Table 2Putative regulatory elements in the 5′-flanking region of the mouse VEGFR-2 gene.

Factor Sequence Positiona Reference

IRF-1 TTTCCTCTT −932/−924 [204]ROR α1 TGAAAGGTCA −859/−850 [205]RXR-α GAAAGGTCA −858/−850 [206]RAR-α1 GAAAGGTCAG −858/−849 [207]Sp-3 CCAGCTCCCTG −712/−702 [100]FOXO3a TGGATAAG −565/−558 [208]Elf-1 AAAACAAAAC −497/−488 [209]NFκB TTGGGACTTTCA −385/−374 [210]AhR GGGGCGTGG −118/−108 [211]Pbx1b CGGACGCAGGG −108/−98 [212]PPAR-α TGACCCC −76/−70 [213]JunD TGACCCC −76/−69 [214]Sp1 ACCCCGCCCC −74/−65 [102]TFII-I CTGCCCTGAGTCC −34/−22 [98]P53 Not shown 21 sites [215]STAT5 Not shown 10 sites [216]STAT3 Not shown 6 sites [217]ER α Not shown 5 sites [135,136]

IRF-1: interferon regulatory factor I; RORα-1: the RAR-related orphan receptor orNR1F1 (nuclear receptor subfamily 1, group F, member 1); RXR-α: Retinoid X receptoralpha or NR2B1 (nuclear receptor subfamily 2, group B, member 1); FOXO3a: Forkheadbox receptor transcription factor 3a; Elf-1: Ets domain transcription factor; AhR: Arylhydrocarbon receptor; Pbx1b: Pre-B-cell leukemia transcription factor 1; PPAR-α:Peroxisome proliferator-activated receptor; JunD: Transcription factor.

a List of the putative regulatory elements corresponding to the consensus sequenceand their positions relatively to the transcriptional start site.


7.3. Epigenetic regulation of VEGFR-2

Growth of malignant cells including breast cancer and metastasisinvolves coordinating changes in expression programs of multiplegenes. Since genomic expression programs are regulated by theepigenome, it stands to reason that epigenomic changes plays acritical role in oncogenesis. Histone modifications could be promotedby several processes including methylation, acetylation, phosphory-lation, and ubiquitination that modulate binding interactions withspecific proteins and chromatin structure. Transcriptionally activeregions are generally hypoacetylated or hypermethylated. Mainmechanisms involved in epigenetic regulation of gene expressionare methylation of chromatin proteins and deacetylation of DNAnitrogen bases related mainly to methyltransferase and deacetylaseactivities, respectively [104].

The promoter DNA methylation of specific genes is a well-knownmechanism of epigenetic gene silencing in oncogenesis [105]. DNAmethylation, especially in CpG-rich 5′ regions, inhibits transcriptionby interfering with the initiation or by reducing the binding affinity ofsequence-specific transcription factors. Yamada et al., reported thataberrant promoter methylation of VEGFR-1 (Flt-1) was found in 24(38.1%) of 63 primary local prostate cancer samples, while in all 13benign prostate samples it was not [106]. These findings suggest thatmethylation of VEGFR-1 is related to prostate carcinogenesis.Nevertheless, it is still unclear what role the VEGFR-1 plays in thedevelopment and progression of prostate cancer [106]. A putativeoncogenic enzyme, Smyd3, dimethylates VEGFR-1 at lysine 831(K831me2). However, the biological consequences on tumor growthof these epigenetic changes are unknown [107].

Scarce information on VEGFR-2 methylation is currently available.In a recent report, VEGFR-2 methylation was detected at asignificantly higher level in cancer tissues of stomach, colon andhepatocellular cancer [108]. It is not know whether VEGFR-2methylation also occurs in human breast cancer. As VEGFR-2 wasnot detected in some cases of invasive breast carcinomas, this mayimplicate hypermethylation of VEGFR-2 as a cause for this subset ofbreast cancer patients [76]. Results from our laboratories show thatthe inhibition of VEGFR-2 gene methylation using aza-2′-deoxycyti-dine (5-AZA) significantly increases the expression of VEGFR-2 mRNA

in mouse mammary cancer cells. These data support the hypothesisthat VEGFR-2 methylation could play an important role in regulatingVEGFR-2 expression in breast cancer (unpublished results).

Another epigenomic regulatory mechanism of VEGFR-2 is chro-matin modification. Chromatin packages the genetic information ineither accessible or inaccessible configurations [109,110]. Someextensively studied modifications are methylation and acetylation oflysines at the tails of H3 and H4 histones [111,112]. The state ofhistone acetylation is dynamic and is determined by equilibrium ofacetylation catalyzed by histone acetyltransferases (HAT) anddeacetylation catalyzed by histone deacetylases (HDAC) [113]. HATsand HDACs are recruited to target genes by either activator orrepressor complexes [113]. Deacetylation of VEGFR-2 gene could alsoplay a role in its epigenetic control. Trichostatin A (TSA, an inhibitor ofHDAC) upregulates the expression of VEGFR-1 and VEGFR-2, butdownregulated NRP-1 and NRP-2 in non-small cell lung cancer [114].Functional studies in BAECs demonstrate that cAMP represseswhereas tumor necrosis factor-α (TNF-α) an activator of NFκB,stimulates promoter activity. Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit ofNFκB, significantly increase VEGFR-2 promoter activity [98]. We haveused TSA to study VEGFR-2 gene transcription in mouse mammarycancer cell lines. TSA induced dramatic elevations of VEGFR-2 mRNAand protein levels in some cell lines. These results suggest that, inbreast cancer cells, histone acetylation does modulate VEGFR-2 genetranscription (unpublished data).

7.4. The Notch signaling-VEGFR-2 link

Notch signaling pathway functions in an enormous diversity ofdevelopmental processes and its dysfunction is implicated in manycancers. This evolutionarily conserved pathway in multicellularorganisms regulates embryonic and stem cell fate [115]. It is generallybelieved that tumor angiogenesis will not occur in absence of Notchsignaling. When a Notch decoy is introduced in place of a functionalNotch receptor at a tumor site in the skin during angiogenesis, cellproliferation stops and the development of the new blood vesselsceases. This suggests that the Notch protein has some significant rolein angiogenesis [116,117]. Indeed, active Notch1 [118] or Notch4[119] signaling are involved in breast cancer angiogenesis. Soares etal., first demonstrated that a cross talk between Notch and E2signaling occurs in breast cancer and EC. Notch gene expression wasrequired for tubule-like structure formation in EC. Notch geneexpression clustered with hypoxia inducible factor-1 alpha (HIF-1αand was upregulated by E2. Thus, Notch has significant role in humanbreast carcinogenesis and angiogenesis [120].

Notch is a family of four mammalian transmembrane proteins thatfunction as a receptor for membrane bound ligands. There are fourmammalian Notch genes, Notch1–Notch4, and five ligands, Jagged1and Jagged2 (homologs of Drosophila Serrate-like proteins) and Delta-like 1 (DLL1), DLL3 and DLL4. Notch receptors have an extracellulardomain made up of multiple epidermal growth factor (EGF) domain;yet, its intracellular domain is made up of many domain types [121].The Notch proteins have been proven to affect diverse cell programs(proliferation, differentiation, and apoptosis) and as result Notchinfluences organogenesis and morphogenesis [122]. The activation ofa Notch receptor is triggered by ligands expressed on adjacent Jaggedand Delta cells. However, it has been found that Dll4 and Jagged1-Notch signaling pathways have opposing effects on angiogenesis[123]. While Jagged1–Notch signaling serves as a pro-angiogenicregulator [123], Dll4–Notch signaling has been shown to significantlydecrease the expression of VEGFR-2 thus inhibiting the proliferationof angiogenic cells [124]. These two signals operate in equilibriumwith one another, so that as the concentration of one signal increasesthe other will decrease proportionally. As a result of the ligandsignaling competitive nature towards one another, it is plausible to


speculate that the two are used as antagonistic mechanisms to helpregulate the processes of angiogenesis [123,124]. In vitro, theactivation of Notch1 or Notch4 in EC induces the expression of theHESR-1 transcription factor (expressed in mature vasculature, butreduced in proliferating EC) that in turns downregulates VEGFR-2.Notch-mediated reduction in VEGFR-2 levels results in decreased ECproliferation. This Notch mechanism may be involved in thephenotypic changes during EC proliferation and migration to networkformation [125]. Overall, one could speculate that the two types ofNotch ligands operate to signal the VEGFR-2 to either continue topromote cell proliferation or move onward in the angiogenic processto EC differentiation. Activation of Notch signaling in ER-negativebreast cancer cells results in direct transcriptional upregulation of theapoptosis inhibitor and cell cycle regulator survivin (baculoviralinhibitor of apoptosis repeat-containing 5 or BIRC5). Survivin is highlyexpressed by cancer cells and binds and inhibits caspase-3, controllingthe checkpoint in the G2/M-phase of the cell cycle. Therefore, theNotch-survivin functional gene signature is a hallmark of basal breastcancer, and may contribute to disease pathogenesis [126,127]. Inaddition, Notch signaling modulates other pathways, such as PI-3K–Akt and NFκB, also activated by VEGFR-2 signaling as discussed above.Therefore, crosstalk between Notch and VEGFR-2 signaling may becrucial for angiogenic processes.

In human MCF-7 BC cells overexpression of the γ-secretase (theenzyme that catalyzes intramembrane cleavage of the Notch receptorupon ligand binding required for Notch activation) liberated Notchintercellular domain and increased HIF-1α protein levels by anunknown mechanism [126,127]. Notch1 signaling can promote NFκBtranslocation to the nucleus and DNA binding by increasing bothphosphorylation of the IαB kinase α/β complex (a repressor of NFκBactivation) and the expression of some NFκB family members [128].We have found that leptin (an adipocytokine) activated NFκB, Sp1 andHIF-1α and increased the expression of Notch mRNA and protein inbreast cancer cells under normoxic conditions. Remarkably, leptininduced the expression of Notch1–4, Jagged1 and VEGFR-2 in thesecells. In particular, leptin-mediated activation of NFκB increasedVEGFR-2 and Notch. Furthermore, leptin increased through severalsignaling pathways promoter activities of VEGFR-2-Luc transfected-cells. Interestingly, leptin effects on VEGFR-2 were abrogated by a γ-secretase inhibitor. Moreover, VEGFR-2 transcription and expressionwas heavily dependent on VEGFR-2 gene methylation and histoneacetylation that could be linked to leptin and Notch effects(unpublished data). However, the role of Notch-mediated regulationof VEGFR-2 and signaling crosstalk in cancer cells is so far unknown.

7.5. Estrogen-mediated regulation of VEGFR-2

Estrogens exert important regulatory functions on vessel wallcomponents, which may contribute to the increased prevalence andseverity of certain chronic inflammatory, autoimmune diseases, aswell as tumor initiation, progression, particularly in tumors of thebreast, endometrium, ovary and prostate [129–131]. EC have alsobeen identified as targets for estrogens. ERs have been found in ECfrom various vascular beds. The regulatory functions of estrogen in ECresponses are relevant to vessel inflammation, injury, and repair. Inthese cells, estrogen affects nitric oxide production and release,modulates the expression of EC-adhesion molecules and regulatesangiogenesis [132–134].

The mechanisms through which estrogen regulates VEGFR-2 inangiogenesis are complex and may involve both genomic and non-genomic effects. It was earlier reported that estrogen stimulates ECgrowth as well as VEGF-dependent angiogenesis by the receptor-mediated pathway, especially ERα [135,136]. However, non-classicalmechanisms through ERα/Sp3 and ERα/Sp4 complexes were foundin some cancer cell lines, such as ZR-75 breast cancer cells. In thesecells E2 activates GC-rich sites where Sp proteins but not ER-α bind to

VEGFR-2 promoter to stimulate mRNA and protein expression [103].In contrast, in MCF-7 cells, the ERα/Sp protein–VEGFR-2 promoterinteractions involve the recruitment of the co-repressors SMRT(silencing mediator of retinoid and thyroid hormone receptor) andNCoR (nuclear receptor corepressor) resulting in decreased VEGFR-2mRNA levels [137].

7.6. Cytokine and growth factor regulation of VEGFR-2

VEGF is hypoxia inducible showing a temporal expression patternthat generally parallels VEGFR-2 expression. Contradictory data onthe direct role of hypoxia in the regulation of VEGFR-2 were reported.The differential and synergistic regulation of VEGF and VEGFR-2 byhypoxia in an organotypic cerebral slice culture system for EC waslinked to a direct induction of VEGF that subsequently upregulatesVEGFR-2 in EC. VEGF-induced VEGFR-2 upregulation was abrogatedby a neutralizing anti-VEGF antibody [138]. In contrast, it was laterreported that hypoxia upregulates VEGFR-2 in cultured cells by aposttranscriptional mechanism [139].

Inflammation and angiogenesis are frequently coupled in patho-logical situations like breast cancer. One of the hallmarks ofinflammation is an increase in vascular permeability frequentlydriven by an excess of VEGF and other mediators. Inflammationinduces EC activation and capillary sprouting [140]. Pathologicalangiogenesis is associated with the secretion of cytokines. However,the molecular and cellular mechanisms linking chronic inflammationto tumorigenesis remain largely unresolved. Many cytokines andgrowth factors are able to increase VEGF expression [141]. However, areduced number of these factors have been confirmed to regulateVEGFR-2 expression. The majority of inflammatory cytokines exertinflammatory effects through the induction of NFκB. a hallmark ofinflammatory responses. Activation of NFκB is essential for promotinginflammation-associated cancer [142]. VEGF [142] and VEGFR-2 [98]promoters have NFκB responsive cis-elements. Therefore, cytokine-activated NFκB increases angiogenesis by direct upregulation of pro-angiogenic genes. VEGFR-2 is associated with inflammatory breastcancer and is, therefore, a target for cancer prevention.

Cytokines have diverse effects on VEGFR-2. PIGF, erythropoietin orPDGFwere unable to upregulate VEGFR-2 [138]. Transforming growthfactor-beta (TGF-β) can downregulate VEGFR-2 [143] but discordantresults on TNF-α mediated downregulation [144] and upregulation[145] of VEGFR-2 have been reported. Moreover, TNF showedcontradictory effects on VEGFR-2 activity. TNF induced VEGFR-2, butblocked it signals, thus delaying the VEGF-driven angiogenic response[146]. Members of the chemokine family can also regulate VEGFR-2.CCL23 (also known as MPIF-1, MIP-3, or Ckb8) is a CC chemokineinitially characterized as a chemoattractant for monocytes anddendritic cells. In HUVEC, CCL23 mainly induced KDR/Flk-1 expres-sion at the transcriptional level. These effects were linked to CCL23-mediated phosphorylation of SAPK/JNK [147].

7.6.1. Leptin regulation of VEGFR-2Recently, leptin was added to the list of factors that upregulate

VEGF and VEGFR-2 [61,62,64]. Leptin actions are more often than notrelated to energy balance. However, leptin is also recognized for itscontributions to reproduction, angiogenesis, proliferation and inflam-mation. Leptin's actions are now being linked to the development andpathogenesis of cancer [148,149]. Higher levels of leptin are found infemale, post-menopausal women and obese individuals. The leptinlevels have been related to the incidence of various types of cancer,most notably breast cancer [150,151]. Leptin is a pleitropic adipocy-tokine, with mitogenic and angiogenic effects, that promotesanchorage, proliferation of breast cancer cells, microvessel andhematopoiesis and increase the levels of several factors includingcell cycle regulators [61,62,149]. Breast carcinoma cells express higherlevels of leptin and leptin receptor, OB-R, than normal mammary cells


and a significant correlation between leptin/OB-R levels withmetastasis and lower survival of breast cancer patients has beenfound [148,152,153]. A role for leptin in rodent mammary tumors hasbeen documented. Restriction of caloric intake (25%) reducesmammary tumor incidence and growth in rats treated with 7,12-dimethylbenz[α]anthracene (DMBA) either fed with low or high-fatdiets [154]. Moreover, studies on leptin (ob/ob) and OB-R (db/db)deficient mice have provided compelling data supporting a role forleptin in breast cancer development. Obese mice with deficiency inleptin signaling show a significantly lower incidence of mammarytumors than their lean littermates. MMTV (mouse mammary tumorvirus)-TGF-αmice have a proclivity to develop mammary tumors butwhen crossed with leptin/OB-R deficient mice, there is a reducedincidence of mammary tumors in their progeny [155,156].

Leptin acts through binding to specific membrane receptors ofwhich six isoforms (OB-R a–f) have been identified at the present time[157–159]. Binding of leptin to its receptor induces activation ofseveral canonical (JAK2/STAT; MAPK/ERK 1/2 and PI-3K/AKT1) andnon-canonical signaling pathways (PKC; stress-activated proteinkinase c-Jun N-terminal kinase, JNK and p38 MAP kinase) to exertits biological effects in food intake, energy balance, and adiposity aswell as in the immune and endocrine systems [160,161]. We havepreviously found that leptin signaling plays an important role in thegrowth of breast cancer that is associated with the regulation of pro-angiogenic, pro-inflammatory and pro-proliferative molecules [62].Leptin increases VEGFR-2 expression in endometrial cancer cells invitro [64] and in breast cancer cells in vitro and in vivo [61,62]. In 4T1cells leptin-mediated upregulation of VEGFR-2 was unaffected bytreatment with siRNA targeted against STAT3 whereas inhibition ofJAK2, PI-3K and MAPK cascades attenuated the leptin inducedincrease in VEGFR-2 [62]. In human breast cancer cells ER-positive(MCF-7) or ER-negative (MDA-MB-231), leptin in a dose–responsemanner significantly increased the levels of VEGFR-2 protein andmRNA [59]. However, the molecular mechanisms of how leptinsignaling regulates VEGFR-2 are largely unknown [162–164].

Leptin is able to induce the growth of breast cancer cells throughactivation of the JAK2/STAT3, ERK1/2, and/or PI-3K pathways, andcan mediate angiogenesis by inducing the expression of VEGF[163,165]. Leptin-mediated activation of mTOR (mammalian targetof rapamycin), mainly linked to MAPK, played a central role in leptinregulation of VEGF and VEGFR-2 in endometrial cancer cells [64]. Inaddition, leptin induces transactivation of the HER2/neu proto-oncogene (c-erbB-2), and interacts in triple negative breast cancercells with insulin like growth factor-1 (IGF-1) to transactivate theEGF-receptor (EGFR), thus promoting invasion and migration[166,167]. Leptin can also affect the growth of ER-positive breastcancer cells by stimulating aromatase expression and therebyincreasing estrogen levels through the aromatization of androgens,and by inducing MAPK-dependent activation of ER [163,168]. On theother hand, as it was mentioned in Section 7.4, leptin modulatesNotch expression in mouse mammary cancer cells. Therefore, leptin-mediated activation of PI-3K/Akt1/NFκB could be part of the crosstalklinking leptin and Notch signaling to regulate VEGFR-2.

In recent reports, the disruption of leptin signaling using pegylatedleptin peptide receptor antagonist (PEG-LPrA2)markedly reduced thegrowth of tumors and the expression of VEGF/VEGFR-2 in mousemodels of syngeneic and human breast cancer xenografts [61,62]. Themice treated with PEG-LPrA2 had diminished expression of VEGF/VEGFR-2, OB-R, leptin, IL-1R tI, PCNA and cyclin D1 [59]. PEG-LPrAtreatment did not affect mouse leptin levels in plasma suggesting thatthis compound did not interfere with the systemic leptin metabolism.Pharmacokinetic and toxicological studies suggested that the highmolecular weight PEG-LPrA2 derivative does not travel through theblood–brain barrier and therefore it is not bound to hypothalamic OB-R or accumulated in the central nervous system of mice. This suggestsPEG-LPrA2 may not interfere with leptin biological actions at

hypothalamic level. Indeed, no significant effects on body or carcassweight were found between mice treated with PEG-LPrA2 or control[59]. These data suggest that inhibition of leptin signaling may serveas a novel adjuvant for prevention and treatment of breast cancer. Thedramatic reduction of tumor growth could be directly related to thereduction of VEGF/VEGFR-2 levels and thereby to the abrogation ofthe VEGF/VEGFR-2 autocrine/paracrine cancer cell survival loop. Thiscould be especially important for populations under higher risk andexhibiting higher levels of leptin such as obese and post-menopausalwomen. The alarming increase of incidence of obesity in the Westerncountries emphasizes the importance of these findings [126,127].

7.7. Clinical significance of targeting VEGFR-2

Solid tumor malignancies, including breast, lung and prostatecarcinomas, are considered to be angiogenesis dependent. However,antiangiogenic therapies have shown varying results partly becauseeach tumor type secretes a distinct panel of angiogenic factors tosustain its own microvascular network. Additionally, recent evidencehas demonstrated that tumors develop resistance to antiangiogenictherapy by turning on alternate angiogenic pathways when onepathway is therapeutically inhibited. The redundancy of theseangiogenic pathways provides a plethora of targets for intervention.It is likely that successful complete inhibition of angiogenesis will relyon the use of combination and/or sequential therapies [169,170]. Inthis section we will briefly discuss experimental therapies forangiogenesis inhibition in breast cancer.

As already discussed, the role of angiogenesis in carcinogenesis iscomplex and mediated by many different factors. The central role ofthe Notch receptor, and its attendant Jagged and Delta cell ligands,and their role in angiogenesis has led to the development of Notchsignaling inhibition as a therapeutic intervention. The GSIs or smallmolecule inhibitors of γ-secretase have shown inhibition of tumor-igenesis. The main disadvantage with the use of GSIs is their non-specificity. Many physiological processes require Notch signaling,therefore, toxicity profiles may be profound. Additionally, subsequentdevelopment of antibodies directed specifically against the Notchreceptor or its ligands would offer another therapeutic alternative[171].

Tumor hypoxia occurs as tumors grow subsequently leading to anincrease in HIF-1α. Acetylation and deacetylation post-translationalmodifications are critical to HIF-1α signaling. As such, HDACinhibitors can be considered as a possible therapeutic interventionin breast cancer treatment [172]. The use of HDAC inhibitors in mousemodels in combination with VEGF receptor tyrosine kinase inhibitordecreases the expression of angiogenesis-related genes such asangiopoietin-2 and its receptor Tie-2, and survivin in EC. HDACinhibitors are already in clinical use in treating hematologicmalignanices such as myelodisplastic syndrome (MDS) and acutemyeloid leukemia (AML) [173].

Regulation of VEGFR-2 expression by Sp proteins has beenpreviously discussed in this paper (see Section 7.2). VEGFR-2 can betargeted by drugs that downregulate Sp proteins or block Sp-dependent transactivation [72]. Activation of VEGFR-2 via binding ofSp3 and Sp4 with ERα to promoter region of VEGFR-2 is enhanced byE2. It could be hypothesized that blocking secretion of E2 woulddownregulate this effect and hence inhibit angiogenesis [103,174].

Some antiangiogenic treatment strategies have entered the clinicto date. These agents include a humanized monoclonal antibodydirected to VEGF-A (Bevacizumab; Avastin; Genentech Inc, South SanFrancisco, CA), a chimeric monoclonal antibody directed to theVEGFR-2 (IMC-1C11), several small molecule inhibitors of theVEGFR-2 tyrosine kinase, and a nuclease-stabilized ribozyme (Angio-zyme), that specifically cleaves both Flt-1 and KDR mRNA [175]. Acompilation of antiangiogenetic agents targeting VEGF or VEGFRs nowbeing tested in clinical trials can be found in Table 3. Preclinical

Table 3Antiangiogenetic drugs targeting VEGF or VEGFRs in clinical trials.

Drug Target Clinical trial Reference

Bevacizumab VEGF/R FDA approved [179–182]IMC-1C11 VEGFR-2 Phases I and II [184–186]SU5416 VEGFR-2 Phases I and II [187,188]PTK 787 VEGFR-1/2 Phases I and II [190]ZD6474 VEGFR-2, EGFR Phases I and II [189]CP547632 VEGFR-2, PDGFR, FGFR Phase II [191]SU11248 VEGFR-1/2, PDGFR, Kit Phases I and II [218]SU6668 VEGFR-2, PDGFR Phase II [219,220]YM-359445 VEGFR-2 Phases I and II [221]AZD2171 VEGFR-2 Phases I and III [222–224]CT-322 VEGFR-2 Phases I and II [225]CEP-7055 VEGFR-2 Phase I [226,227]


models have shown regression of solid tumor growth and angiogen-esis with anti-VEGF monoclonal antibodies alone or in combinationwith chemotherapy [176–178]. Clinical benefit with bevacizumab hasbeen reported from clinically trials in metastatic colon, renal cell, andbreast cancer [179–182]. Bevacizumab was able to significantdecrease (66.7%) phosphorylated VEGFR-2 (Y951) in tumor cellsand increase in tumor apoptosis after one cycle of bevacizumab alone[181]. Applications of bevacizumab therapy are not confined tocancer. Macular degeneration, a disorder of the retina that isenhanced by age, is the most common cause of irreversible visionloss in older people. The disease is characterized by abnormal bloodvessels grow beneath the retina. Therefore, several antiangiogenictreatments are being tested in the clinic. Among these, anti-VEGFtherapies have potential successful applications. Investigations fromintravitreal bevacizumab (Avastin) therapy showed promising 6-month results in patients with neovascular macular degeneration[183].

IMC-1C11, a chimeric monoclonal antibody, binds specifically tothe EC-surface extracellular domain of VEGFR-2, blocks VEGF–VEGFR-2 interaction and prevents VEGFR-2 activation of the intracellulartyrosine kinase pathway [184,185]. The initial Phase I trial of IMC-1C11was carried out in patients withmetastatic colorectal carcinoma.This has provided evidence of the safety and low toxicity for antibodyblockade of VEGFR-2, as well as insight into dose and schedulerequirements [186]. A fully human anti-VEGFR-2 agent has beenproduced as a second-generation agent, which is anticipated to benonimmunogenic for chronic administration as a single agent and incombination with chemotherapy or radiation. Semaxanib (SU5416)was the first specific synthesized potent and selective inhibitor of theVEGFR tyrosine kinase that is presently under evaluation in Phase Iclinical studies for the treatment of human cancers [187]. SU5416 wasshown to induce growth inhibition in mouse xenotransplants ofhuman tumors. But in several Phase II trials, results were disappoint-ing, albeit providing a good security profile [188]. There otheradditional inhibitors of the VEGFR-2 tyrosine kinase that are currentlybeing examined in clinical trials, such as PTK 787, ZD6474, andCP547632, which have been selected on the basis of relativelyselective inhibition of the VEGFR-2 ATP binding site [189–191]. SCC-S2, a novel antiapoptotic molecule, has shown to decrease theproliferation and tumorigenicity of MDA-MB435 human breast cancercells [192]. Treatment of these cells with a cationic liposomalformulation of SCC-S2 antisense oligo correlated with decreasedexpression of VEGFR-2 in tumor cells as well as human lungmicrovascular EC and loss of cell viability [193]. Targeted therapieswith the introduction of adenoviral vector expressing inducibleCaspase-9, (iCaspase-9) under transcriptional regulation with EC-specific VEGFR-2 promoter induced apoptosis of proliferating humandermal microvascular EC (HDMECs) [194].

New compounds/technologies are being developed to targetVEGFR-2. Mice treated with VEGFR-2-based DNA vaccine showedsignificant reduction of renal carcinomas [195]. A series of dual c-Met/

VEGFR-2 kinase inhibitors were found to significantly affect growth ofhuman xenografts [196]. Medicinal plants could be new sources foranti-VEGFR-2 drugs. Acetyl-11-keto-beta-boswellic acid (AKBA)derived from Bowawellia serrata inhibits prostate tumor growth byblocking VEGFR-2 signaling [197]. Previous studies in our laboratoryhave demonstrated that leptin-signal inhibition resulted in decreasedgrowth of mammary tumors derived from mouse and humans. Theexpression of VEGF and VEGFR-2 was increased under leptin signalingin cell culture and decreased by the actions of leptin peptideantagonists in vitro and in vivo [62]. Thus, our data strongly suggestthat leptin signaling inhibition could serve as an additional preven-tative and/or therapeutic modality for breast cancer.

8. Conclusions

VEGFR-2 is an important factor for EC development and angio-genesis. Aberrant VEGFR-2 expression/signaling is found in cancer.Paracrine effects of VEGF and diverse cytokines/growth factorssecreted by cancer cells upregulated VEGFR-2 in EC. Cytokines/growth factors also orchestrate autocrine/paracrine upregulation ofVEGFR-2 in cancer cells that is essential for the survival/proliferationactions of VEGF/VEGFR-2 loop. Targeting VEGFR-2 overexpression inendothelial/malignant cells could be an effective way to treat breastcancer. Additional studies are needed to further test and developspecific anti-VEGFR-2 therapies. Deeper understanding of VEGFR-2regulation and signaling crosstalk mechanisms in cancer cells willlikely lead to the development of new therapeutic modalities.Translational studies are needed to test these agents for efficacy andtoxicity in the breast cancer patient population.

Acknowledgements

This work was supported in part by grants from NIH/NCI1SC1CA138658-01; NIH/UAB Breast SPORE Career DevelopmentAward, Susan Komen Foundation for the Cure, and the GeorgiaCancer Coalition Distinguished Cancer Scholar Award to R.R.G-P., andfacilities and support services at Morehouse School of Medicine (NIHRR03034 and 1C06 RR18386).

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Glossary

4T1 cells: mouse mammary cancer cell line5-AZA: aza-2′-deoxycytidineAhR: aryl hydrocarbon receptorAkt: protein kinase BAP-1/-2: the activator protein 1 and 2BAECs: bovine aortic endothelial cellsCdk-2: cyclin-dependent kinase 2

ChiPs: chromatin immunoprecipitation assaysCREB: cAMP response element binding proteinCyclin D1: kinase and regulator of cell cycle D1DLL1–4: Delta-like 1–4E2: estradiolEC: endothelial cellsEGFR: epidermal growth factor receptorElf-1: Ets domain transcription factorEMSA: electrophoretic mobility shift assayER: estrogen receptorERK 1/2: extracellular regulated kinase 1 and 2ESCRT: endosomal sorting complex required for transportEts: E-twenty six family of transcription factorFAK: focal adhesion kinaseFGFR: fibroblast growth factor receptorFOXO3a: forkhead box receptor transcription factor 3aHAT: histone acetyltransferasesHDAC: histone deacetylasesHIF-1α: hypoxia regulated factor-1 alphaHUVECs: human umbilical vein endothelial cellsIGF-1: insulin like growth factor-1IRF-1: interferon regulatory factor IJAK2: Janus kinase 2JNK: c-Jun N-terminal kinase or SAPK (stress-activated protein kinase)JunD: Transcription factor jun-DMAPK: mitogen activated protein kinaseMCF-7: ER positive human breast cancer cell lineMDA-MB231: ER negative human breast cancer cell lineMEK: mitogen-activated protein kinase/extracellular signal-regulated kinasemTOR: mammalian target of rapamycinNFκB: eukaryotic nuclear transcription factor kappa BNRP-1/-2: Neuropilins 1 and 2 receptorsOB-R: leptin receptorP38 kinase: extracellular regulated kinase 38Pbx1b: pre-B-cell leukemia transcription factor 1;PDGF: platelet-derived growth factorPIGF: placental growth factor or PGFPI-3K: phosphoinositide 3-kinasePKC: protein kinase CPLCg: phospholipase C gammaPPAR-α: Peroxisome proliferator-activated receptor alphaP53: tumor suppressor protein 53Rac-1: a small Rho-GTPaseRORα-1: the RAR-related orphan receptor or NR1F1 (nuclear receptor subfamily 1,

group F, member 1)RTK: receptor tyrosine kinaseRXR-α: Retinoid X receptor alpha or NR2B1 (nuclear receptor subfamily 2, group B,

member 1)Sp1–3: Specificity protein 1–3STAT3: signal transducer and activator of transcription 3TAM: tamoxifenTFII: COUP transcription factor 2 or NR2F2TGF-β: transforming growth factor-betaTNF-α: tumor necrosis factor alphaTSA: Trichostatin ATSAd: T Cell-Specific AdapterTSP-1: Thrombospondin-1VEGF: vascular endothelial growth factorVEGFR-1: vascular endothelial growth factor receptor 1 or Flt-1VEGFR-2: vascular endothelial growth factor receptor 2 or KDR or Flk-1VEGFR-3: vascular endothelial growth factor receptor 3 or Flt-4

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Vascular endothelial growth factor receptor-2 in breast cancer