Vascular Endothelial Growth Factor Signaling Pathways:Therapeutic PerspectiveMarcin Kowanetz and Napoleone Ferrara

Abstract The establishment of a vascular supply is one of the earliest and most important events occurringduring embryonic development. Growth and maturation of a functional vascular network arecomplex and still incompletely understoodprocesses involvingorchestrated activationof vascularprogenitors in the early stages of embryonic development followedby vasculogenesis and angio-genesis.These processes require a tightly regulated activation of several growth factors and theirreceptors. The role of vascular endothelial growth factors (VEGF) and their receptors has beenstudied extensively due to their prominent role during blood vessel formation. Mice deficient invarious VEGF ligands or receptors show serious defects in vascular formation and maturation.Moreover, members of the VEGF family are involved in other significant biological processes,including lymphangiogenesis, vascular permeability, and hematopoiesis. Importantly, VEGF isreleased by tumor cells and induces tumor neovascularization. It is now well established that theVEGF axis represents an important target for antitumor therapy. AberrantVEGF signaling is also afeature of several other pathologic conditions, such as age-related macular degeneration andrheumatoid arthritis.

Components of theVascular Endothelial GrowthFactor Signaling Pathway

In mammals, the vascular endothelial growth factor (VEGF)family consists of five members: VEGF-A (thereafter calledVEGF), VEGF-B, VEGF-C, VEGF-D, and placental growth factor.In addition, alternative exon splicing results in generation offour main VEGF isoforms denoted VEGF121, VEGF165, VEGF189,and VEGF206 (1, 2). Moreover, VEGF165 can be cleaved byplasmin and various metalloproteinases at the COOH termi-nus, generating VEGF110 or VEGF113, two bioactive NH2-terminal fragments (3).Members of the VEGF family show different affinities for

one of the three VEGF tyrosine kinase receptors: VEGF receptor(VEGFR)-1, VEGFR-2, and VEGFR-3. Moreover, several core-ceptors, such as heparan sulfate proteoglycans and neuropilins,have been implicated in promoting activation of VEGFRs(reviewed in refs. 4, 5). VEGFR-1 is able to bind VEGF, VEGF-B,and placental growth factor; VEGFR-2 is activated primarily byVEGF but proteolytically cleaved forms of VEGF-C and VEGF-Dmay also activate this receptor; VEGFR-3 is activated only byVEGF-C and VEGF-D. Both VEGFR-1 and VEGFR-2 areexpressed in vascular endothelial cells. In addition, VEGFR-1is also expressed by monocytes and macrophages, hematopoi-etic stem cells, and certain nonendothelial cell types (6).VEGFR-2 is also expressed in hematopoietic stem cells (6).

Interestingly, subsets of liquid and solid tumor cells were foundto express VEGFR-1 and VEGFR-2 (7). VEGFR-3 regulateslymphangiogenesis, and its expression in the adult seems to belargely restricted to lymphatic endothelial cells (6).Gene targeting studies in mice have shown the importance

of VEGFs and VEGFRs in the development of the vascularsystem. VEGF seems particularly important because loss ofeven a single Vegf allele results in embryonic lethality at days11 to 12 (8, 9). Vegfr2�/� mice die at embryonic days 8.5 to9.5 due to defect in the development of hematopoietic andendothelial cells resulting in impaired vasculogenesis (10).Phenotypic analysis of animals lacking other VEGF-relatedligands and receptors has revealed their role at different stagesof vascular and lymphatic development (reviewed in ref. 11).As for other receptor tyrosine kinases, VEGFR activation is

dependent on availability of the ligand. Ligand binding to thereceptor leads to receptor homodimerization or heterodimeri-zation. However, the significance and signaling properties ofVEGFR heterodimers have not been extensively investigated(reviewed in ref. 12). Dimerization of receptors leads to theiractivation and subsequent autophosphorylation on certaintyrosine residues, which in turn triggers intracellular signalingcascade mediated by several effectors, which are able torecognize and dock at phosphorylated tyrosine residues of theactivated receptors (reviewed in ref. 11). These interactions aremediated by Src homology 2, phosphotyrosine-binding, andother domains of the signaling proteins (13).Despite a high degree of homology within the kinase

domain, the signaling properties of different VEGFRs greatlydiffer. Interestingly, VEGFR-1 binds VEGF with at least 10-foldhigher affinity than VEGFR-2, yet it becomes poorly activatedfor, thus far, unclear reasons (14). A study by Gille et al. (15) ofchimeric VEGFR-1 and VEGFR-2 revealed that the juxtamem-brane domain of VEGFR-1 plays an inhibitory role in VEGFR-1

Molecular Pathways

Authors’Affiliation:Genentech, Inc., South San Francisco, CaliforniaReceived 6/22/06; accepted 6/27/06.Requests for reprints: Napoleone Ferrara, Genentech, Inc., 1DNA Way, SouthSan Francisco, CA 94080. Phone: 650-225-2968; Fax: 650-225-6443; E-mail:nf@gene.com.

F2006 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-06-1520

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signaling pathways, although the precise mechanism requiresfurther investigation. The fact that VEGFR-1 is usually expressedat low levels has hampered progress in elucidating its signaltransduction pathways (Fig. 1). Overexpression experimentssuggested potential interacting partners of VEGFR-1, suchas phospholipase C-g, p85/phosphatidylinositol 3¶-kinase, Srchomology phosphatase-2, growth factor receptor-bound pro-tein 2 (Grb2), and Nck (11). Yet, it has been difficult tolink activation of VEGFR-1 to specific biological responses incells endogenously expressing this receptor. VEGFR-1 has beenshown to mediate monocyte migration (16), recruitment ofendothelial cell progenitors (17), hematopoietic stem cellsurvival (18), and release of growth factors from liverendothelial cells (19). Importantly, VEGFR-1 signaling hasbeen implicated in tumor metastasis. VEGFR-1-dependent

induction of matrix metalloproteinase-9 expression in preme-tastatic lung endothelial cells and macrophages has beenreported to promote lung metastasis (20). In addition, a recentstudy has shown that VEGFR-1-positive hematopoietic bonemarrow progenitors form cellular clusters at tumor-specificpremetastatic sites before the arrival of tumor cells and dictateorgan-specific tumor spread (21). Moreover, it has been shownthat VEGFR-1 activates extracellular signal-regulated kinase 1/2and stress-activated protein kinase/c-Jun NH2-terminal kinase(22) and Src family kinases (23) to mediate growth andmigration of human colorectal carcinoma cells. A recent studyhas shown that activation of VEGFR-1 in breast cancer cellssupports their growth and survival (24). However, the signalingproperties and biological functions of VEGFR-1 in cancer cellsrequire further investigation.

Fig. 1. Signal transduction and biological processes mediated byVEGFRs and various therapeutic strategies to inhibitVEGF signaling. Major tyrosine autophosphorylationsites (number) and intracellular effectors of activatedVEGFR-2. Signaling cascades triggered byVEGFR-1andVEGFR-3 are still incompletely understood. Inhibition ofVEGF signaling can be achieved by monoclonal antibodies targetingVEGF (bevacizumab and ranibizumab), aptamers that bind the heparin-binding domain ofVEGF165(pegaptanib), chimeric soluble receptors, such asVEGF-Trap, monoclonal antibodies targetingVEGFRs, various small-molecule tyrosine kinase inhibitors (sorafenib, sunitinib,and vatalanib), and antisense and short interfering RNA strategies (not shown).Top right inset, because pegaptanib binds toVEGF within heparin-binding domain(yellow),itinactivatesonlyVEGF165.Incontrast,bevacizumabandranibizumabinactivateallbiologicallyactiveformsofVEGF,suchasVEGF110.eNOS,endothelialnitricoxidesynthase;synthase; FAK, focal adhesion kinase; Grb2, growth factor receptor-bound protein 2;MAPK, mitogen-activated protein kinase;MEK, mitogen-activated proteinkinase/extracellular signal-regulated kinase kinase; PI-3K, phosphatidylinositol 3¶-kinase; PKB, protein kinase B; PKC, protein kinase C; PLC-c, phospholipase C-g;Shb, Src homology 2 and h cells; SHP-2, Src homology phosphatase-2;TSAd,T-cell-specific adapter; PlGF, placental growth factor.

VEGFPathways

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There is now much evidence that VEGFR-2 is the majormediator of VEGF-driven responses in endothelial cells and it isconsidered to be a crucial signal transducer in both physiologicand pathologic angiogenesis. Its signaling pathways arerelatively well understood. Y1175 in human VEGFR-2 has beenidentified as a major autophosphorylation site following VEGFbinding (25). It serves as a docking site for phospholipase C-g(25), which indirectly mediates activation of the mitogen-activated protein kinase pathway and thus regulates cellproliferation. Moreover, Y1175 is a binding site for otheradaptor molecules, such as Src homology 2 and h cells(Shb; ref. 26). Interaction between Shb and phosphorylatedY1175 of VEGFR-2 is required for VEGF-dependent phospha-tidylinositol 3¶-kinase activation, and it is important forendothelial cell migration (26). A second major autophosphor-ylation site in human VEGFR-2 is Y1214, which is involved inactivation of Cdc42 and p38 mitogen-activated protein kinase(27), a pathway regulating cell motility. Lastly, in the tumorvasculature, T-cell-specific adapter (TSAd) binds to phos-phorylated Y951. Subsequently, activated TSAd forms acomplex with Src (28), which regulates cell migration. Severalother signaling molecules are activated by VEGFR-2, and theseinclude focal adhesion kinase and its substrate paxillin (29) orIQGAP1 (30). These pathways regulate endothelial cellmotility. VEGFR-2-mediated activation of Src and Akt pathwaysalso leads to increased production of nitric oxide, and thus,it may regulate vascular tone and permeability (6).VEGFR-3 and its ligands, VEGF-C and VEGF-D, are key

players in the regulation of normal and tumor lymphangio-genesis (reviewed in ref. 31). Several tyrosine residues havebeen predicted to become autophosphorylated on activationand dimerization of VEGFR-3 (11). However, a limited numberof signaling effectors have been shown to act downstream ofthis receptor. Y1337 is required for Grb2 and SHC-mediatedtransforming capacity of VEGFR-3 (32). Moreover, VEGFR-3mediates antiapoptotic effects as well as proliferation andmigration of lymphatic endothelial cells through Akt andprotein kinase C-mitogen-activated protein kinase signalingcascade (33).

VEGFSignaling and Disease�TherapeuticApproaches

Early pioneers observed over a century ago that tumor growthis accompanied by increased proliferation of blood vessels, andlater on, a few investigators suggested that such vessels play acritical role in tumorigenesis because they may enable tumorcells to acquire a growth advantage relative to normal cells(reviewed in ref. 34). In 1971, Folkman (35) proposed thatantiangiogenesis may be a novel anticancer strategy. That wasfollowed by the discovery of several proangiogenic factors (acidicand basic fibroblast growth factors, epidermal growth factor,transforming growth factor-a, transforming growth factor-h,VEGFs, and several more; reviewed in ref. 34). This potentialredundancy raised the possibility that tumor angiogenesis mayrequire the action of numerous factors, such that blocking asingle angiogenic molecule might have little or no effect ontumor growth. However, in 1993, experiments with neutralizingantibodies showed that blockade of VEGF alone can substan-tially suppress tumor growth and angiogenesis in mouse models(36). Subsequent studies with a dominant-negative VEGFR-2

(37) confirmed that vascularization and growth of a glioma isdependent on secretion of VEGF. These early encouraging fin-dings were confirmed and extended to a variety of tumormodels,including genetic models (reviewed in ref. 38). Importantly, inadults, VEGF-mediated angiogenesis occurs rarely (except duringwound healing and in the female reproductive cycle). Therefore,targeting VEGF is expected to have relatively modest effects onphysiologic processes. Several pharmacologic approaches toinhibit the VEGF axis have been described (39).The first antiangiogenic agent approved by the Food and

Drug Administration is bevacizumab (Avastin, Genentech, Inc.,South San Francisco, CA), a humanized version (40) of an anti-VEGF monoclonal antibody used in early proof-of-conceptstudies (36). In 2004, bevacizumab was approved for thetreatment of previously untreated metastatic colorectal cancerin combination with 5-fluorouracil-based chemotherapy regi-mens (41). Bevacizumab is presently being tested in severalphase III studies in combination with chemotherapy, includingrelapsed metastatic colorectal cancer, non–small cell lungcancer, and previously untreated or relapsed metastatic breastcancer. Preliminary evidence of clinical benefit has beenobserved in all cases, except in relapsed breast cancer incombination with capecitabine (reviewed in ref. 41). Althoughbevacizumab was generally well tolerated, some significanttoxicities were infrequently observed, including hypertension,gastrointestinal perforation, and arterial thromboemboliccomplications (reviewed in refs. 3, 41, 42).Currently, other anti-VEGF agents are at various stages of

clinical development (43). These include the following: VEGF-Trap (Regeneron, Tarrytown, NY), a soluble receptor targetingVEGF, VEGF-B, and placental growth factor; an antisenseoligonucleotide VEGF-AS (VasGene Therapeutics, Inc., LosAngeles, CA) targeting VEGF, VEGF-C, and VEGF-D; and others.Inhibition of VEGFR kinase activity is another approach

to reduce VEGF signaling. Several small-molecule tyrosinekinase inhibitors are at various stages of clinical development(39, 42, 43). Due to the large number of molecules undergoingdevelopment, only those in advanced clinical trials will bementioned here. Sunitinib (Sutent, Pfizer, New York, NY) isa small-molecule receptor tyrosine kinase inhibitor that, inaddition to VEGFR-2, blocks activity of platelet-derived growthfactor receptor-h, c-Kit receptor, and fms-related tyrosinekinase 3 and has been recently approved by the Food and DrugAdministration to treat patients with gastrointestinal stromaltumors. Sorafenib (Nexavar, Bayer, Leverkusen, Germany andOnyx Pharmaceuticals, Emeryville, CA) is a novel inhibitor ofRaf kinase and VEGFR-2 tyrosine kinase that was shown to blockcancer cell proliferation and angiogenesis in several differenttumors. Sorafenib has been recently approved by the Food andDrug Administration for metastatic renal cell carcinoma and isundergoing clinical testing in other malignancies. Vatalanib(PTK787/ZK222584, Novartis, Basel, Switzerland) blockskinase activity of all three VEGFRs as well as platelet-derivedgrowth factor receptor-h and c-Kit. Clinical trials of vatalanibin combination with chemotherapy in metastatic colorectalcancer patients did not meet their end points.Neutralizing monoclonal antibodies against VEGFRs repre-

sents an additional strategy to inhibit VEGF signaling. Recentstudies with anti-VEGFR-1 and anti-VEGFR-2 antibodies haveshown the ability of both reagents to decrease primary andmetastatic tumor growth in some mouse models (7, 24).

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Cancer is not the only pathologic condition to which anti-VEGF treatment can be applied. The neovascular form of age-related macular degeneration is a leading cause of blindnessamong the elderly (44). Choroidal neovascularization results inreduction or loss of central vision and legal blindness. Thecurrent Food and Drug Administration–approved age-relatedmacular degeneration treatments involve verteporfin (Visudyne,Novartis AG) photodynamic therapy (45) and pegaptanib(Macugen, OSI Pharmaceuticals, Melville, NY, and Pfizer), ananti-VEGF aptamer that inactivates the most abundant VEGF165isoform through binding to its exon 7–encoded heparin-binding domain (46). Both treatments were shown to slowthe progression of disease, although vision improved only in asmall group of patients. Ranibizumab (Lucentis, Genentech,Inc.) is an affinity-matured Fab variant derived from bevacizu-mab-Fab (3). Unlike pegaptanib, which selectively neutralizesintact VEGF165, ranibizumab neutralizes the biological activitiesof all human VEGF isoforms and bioactive proteolytic frag-ments. Preliminary analysis of randomized phase III trialsindicates that approximately one third of patients experiencedan improvement in visual acuity following treatment for 1 yearwith monthly intravitreal injections of ranibizumab. On June30, 2006, the Food and Drug Administration approvedranibizumab for the treatment of patients with the neovascularform of age-related macular degeneration. Several other agentstargeting the VEGF signaling pathways in age-related maculardegeneration have been developed and are in early stages ofclinical tests. These include the VEGF-Trap and short interferingRNA molecules Cand5 and Sirna-027 (47, 48), which inhibitexpression of VEGF and VEGFR-1, respectively.Altered VEGF signaling occurs in several other diseases, such

as inflammatory disorders, and in some pathologic conditionsof female reproductive tract (38). Elevated levels of VEGF werefound in rheumatoid arthritis patients, and administration ofsoluble VEGFR-1 attenuated the disease process in an animalmodel of rheumatoid arthritis (49). Increased VEGF levels havealso been reported in polycystic ovary syndrome, one of theleading causes of female infertility (50).

Concluding Remarks

There is now proof that antiangiogenic therapy using VEGFinhibitors results in a clinical benefit in cancer patients.

However, there are still some significant challenges to beovercome before the field may advance in a significant manner.One of the most important questions is why all patients donot respond to the anti-VEGF treatment. Therefore, a betterunderstanding of signaling pathways in tumor angiogenesisbesides VEGF and its receptors may help achieve an even moreeffective anticancer therapy. Furthermore, it would be desirableto have reliable markers to identify the patients who are mostlikely to respond to anti-VEGF treatment.

The biological functions of VEGFR-1 remain incompletelyunderstood. The recent finding that certain tumors expressVEGFR-1 (reviewed in ref. 7) implies that cancer cells may takeadvantage of autocrine VEGF signaling loops to survive andrapidly grow. Moreover, some tumors may also engage theVEGF-C/VEGFR-3 axis to promote migration and invasion (51).In addition, high levels of VEGF-C (as well as VEGF-D) activatelymphatic endothelial cells and enhance lymphatic metastasis(reviewed in ref. 52). Therefore, combining anti-VEGF agentswith inhibitors of VEGF-C or VEGFR-3 may provide a powerfulanticancer approach.

In addition to VEGF, tumor cells may secrete other membersof the VEGF family and express combination of several VEGFRs,raising the possibility that they contribute to tumor growth andmetastasis by autocrine and paracrine mechanisms. Althoughthis hypothesis has not been rigorously tested yet, it argues for abetter understanding of the biological responses mediated byVEGF-related growth factors.

Importantly, blocking VEGF signaling represents a promis-ing therapy not only for cancer patients. VEGF inhibitorshave been shown to slow down visual loss and, in somecases, even improve vision in age-related macular degenera-tion patients. Furthermore, several nonneoplastic disordersmight benefit from treatment with VEGF inhibitors. Elevatedlevels of VEGF have been found in synovial fluids ofrheumatoid arthritis patients (53). However, the function ofVEGF in rheumatoid arthritis is still not completelyunderstood. Recently, it has been suggested that VEGFR-1mediates proliferation of bone marrow hematopoietic cellsand immunity of monocytes/macrophages and promoteschronic inflammation in a murine model of rheumatoidarthritis (54). This suggests that therapeutic suppression ofVEGFR-1 activity might be beneficial for rheumatoid arthritispatients.

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