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Vascular Endothelial Growth Factor as a Target for Anticancer Therapy

Genentech, Inc., South San Francisco, California, USA

Correspondence: Napoleone Ferrara, M.D., Genentech, Inc., One DNA Way, South San Francisco, California 94080, USA. Telephone: 650-225-2968; Fax: 650-225-6443; e-mail: nf{at}gene.com

	LEARNING OBJECTIVES

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
After completing this course, the reader will be able to:

1. Explain the biology of angiogenesis.
   
2. Identify the role of VEGF in normal and tumor angiogenesis.
   
3. Describe the key ways in which VEGF has been targeted in cancer therapy.
   

	ABSTRACT

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
The development of a vascular supply is a critical factor in the growth and metastatic spread of malignant tumors. Of the multitude of growth factors that regulate physiological and pathological angiogenesis, vascular endothelial growth factor (VEGF) is believed to be the most important. There is evidence that overexpression of VEGF is correlated with an adverse prognosis, at least in some tumors. Tumor-expressed VEGF is particularly attractive as a target for anticancer therapy because its angiogenesis-promoting activity is at the level of the endothelial cell and, compared with agents that directly target tumor cells, tumor penetration is less critical for VEGF inhibitors. Moreover, recent work has shown that inhibiting tumor angiogenesis increases the effectiveness of coadministered chemotherapy and radiotherapy. This suggests that drugs that target VEGF or its receptors can be combined with traditional treatment modalities to ensure maximum effectiveness. A variety of agents aimed at blocking VEGF or its receptor-signaling system are currently being developed for the treatment of cancer. Of these, bevacizumab, a humanized monoclonal antibody directed at VEGF, is the most advanced in clinical development and has shown promising results in clinical trials.

Key Words. Vascular endothelial growth factor • Angiogenesis • Cancer • Monoclonal antibody • Bevacizumab

	INTRODUCTION

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
Chemotherapy has been the cornerstone of cancer treatment for several decades. However, chemotherapeutic agents have considerable nonspecific toxicities that limit the dosages that can be given. Moreover, development of resistance to treatment is common. Recent years have seen the advent of a new generation of agents that directly target either the malignant cell itself or cells supporting tumor growth.

Strategies targeting tumor angiogenesis have been a focus of intense research over the past decade, following observations that the growth and metastatic spread of tumors are dependent on their development of a vascular supply [1, 2]. This research effort has led to the isolation of an array of factors that mediate both positive and negative regulation of angiogenesis, with the most pivotal positive regulator being vascular endothelial growth factor (VEGF) [3, 4]. This review examines the central role that VEGF plays in angiogenesis, both in the normal physiological setting as well as in the context of cancer, with a particular focus on the application of current concepts to the development of new anticancer therapies.

	ANGIOGENESIS AND ITS ROLE IN TUMOR GROWTH

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
The vascular system is composed of two major cell types: vascular endothelial cells, lining the interior of blood vessels as a monolayer, and smooth muscle cells, which regulate intravascular pressure.

During embryogenesis, the formation of the cardiovascular system proceeds through an initial stage termed vasculogenesis, in which the endothelial cells differentiate from mesodermal precursors, followed by angiogenesis, the elaboration of the tubular network of endothelial cells. This process requires a series of steps, including endothelial cell activation, basement membrane degradation, cell migration, extracellular matrix invasion, endothelial cell proliferation, and capillary lumen formation. Subsequent stabilization of the vascular network involves the reversal of these processes, with the reestablishment of the basement membrane, cessation of cell proliferation, junctional complex formation, and recruitment of pericytes to support the vessel wall.

Angiogenesis continues throughout development, with new capillaries forming by sprouting or splitting from preformed vessels, followed by remodeling, as the organism grows to final form. In the adult, physiological angiogenesis continues, albeit at a much reduced level, and is associated primarily with maintenance of the vasculature, and with wound healing and menstrual cycling [3, 5–8].

Angiogenesis is essential for the growth of most primary tumors and their subsequent metastasis (Fig. 1). Tumors can absorb sufficient nutrients and oxygen by simple diffusion up to a size of 1–2 mm, at which point their further growth requires the elaboration of a vascular supply. This process is thought to involve recruitment of the neighboring host mature vasculature to begin sprouting new blood vessel capillaries, which grow toward, and subsequently infiltrate, the tumor mass [9]. In addition, both physiological and tumor angiogenesis involve the recruitment of circulating endothelial precursor cells from the bone marrow to promote neovascularization [10, 11].

View larger version (92K): [in this window] [in a new window]   Figure 1. Angiogenesis is a necessary condition for sustained tumor growth.

Both physiologic and tumor angiogenesis are regulated by a host of growth factors in the microenvironment (Table 1), some of which, such as VEGF, are highly specific for endothelial cells, while others, such as basic fibroblast growth factor (bFGF) and the matrix metalloproteinases (MMPs), have a much broader range of action. Activating factors can be produced by the tumors themselves, by the surrounding tissue, or by infiltrating macrophages and fibroblasts [8]. The majority of the activating compounds exert their actions through endothelial cell surface receptors, for which they serve as ligands, ultimately leading to secretion of additional angiogenic factors. In addition, hypoxia, hypoglycemia, and mechanical stress can serve as stimuli [8]. In the case of the matrix metalloproteinases, the stimulation is thought to reflect the proteolysis of basement membrane constituents, such as heparan sulfate proteoglycans, and the consequent release of sequestered growth factors [13].

View larger version (36K): [in this window] [in a new window]   Figure 2. Molecular events in cancer.

	VEGF IN NORMAL AND TUMOR ANGIOGENESIS

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
Families of VEGF-Related Compounds and Their Receptors
VEGF, which is also termed VEGF-A or vascular permeability factor, belongs to the VEGF-platelet-derived growth factor (PDGF) supergene family; other family members include VEGF-B, VEGF-C, VEGF-D, and VEGF-E (a virally encoded protein), all showing varying degrees of homology with VEGF. Alternative splicing of the VEGF gene yields four isoforms of 121, 165, 189, and 206 amino acids, and other less frequent splice variants. VEGF-165, a 45-kiloDalton (kD) homodimeric glycoprotein, is the dominant form and is, in part, secreted and, in part, matrix bound. Both VEGF-189 and VEGF-206 are basic, with a high affinity for heparin, and remain sequestered in the extracellular matrix, presumably to heparan sulfate proteoglycans. VEGF-121 is acidic, does not bind heparin, and is secreted. The matrix-sequestered forms may be released by enzymatic action, either through the action of heparinase or through cleavage by plasmin to release a diffusible fragment (VEGF-110). The actions of VEGF-165 involve the activation of proteinase cascades, including that leading to plasmin generation, so the consequent plasmin-mediated release of matrix-bound VEGF isoforms provides an amplification mechanism [21].

VEGF actions are mediated through binding to two receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR; whose murine form is known as Flk-1). Activation of these receptors by VEGF triggers the phosphorylation of a multitude of proteins that are active in signal transduction cascades [3]. Other members of the VEGF family show different receptor-binding specificities, with VEGF-B and placental growth factor (PIGF) binding and activating only VEGFR-1. VEGF-C and VEGF-D bind a third receptor, VEGFR-3 (Flt-4), through which they mediate lymphangiogenesis [22, 23], and also show some activity toward VEGFR-2. Both VEGFR-1 and VEGFR-2 are found predominantly on the surfaces of vascular endothelial cells, where they bind VEGF with high affinity. Although VEGFR-1 binds with higher affinity, it is believed to act primarily as a decoy receptor, modulating the availability of VEGF to VEGFR-2, the principal receptor for VEGF signaling [3, 24]. Further modulatory actions are exerted by a soluble form of VEGFR-1, which binds VEGF and can inhibit VEGF-induced mitogenesis, as well as the neuropilins, receptors for the collapsin/semaphorin family. Neuropilin-2 binds to VEGFR-1, while neuropilin-1 binds to VEGF-165 and increases its affinity for VEGFR-2 by approximately one order of magnitude [25].

VEGF gene expression is upregulated by a host of stimuli, including estrogen, nitric oxide (NO), and a variety of growth factors, such as fibroblast growth factor-4, PDGF, tumor necrosis factor alpha (TNF-), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), keratinocyte growth factor, interleukin (IL)-6, IL-1ß, and insulin-like growth factor 1 (IGF-1) (Fig. 3) [3, 25, 26]. Consistent with its role in tumor angiogenesis, expression of VEGF is upregulated by the common genetic events leading to malignant transformation, including loss of tumor suppressor genes, such as p53, and activation of oncogenes, such as ras, v-src, and HER2 [15]. Moreover, VEGF expression is particularly sensitive to oxygen tension and is rapidly upregulated by the hypoxia that characterizes most tumors, which is due to the aberrant nature of their vascular supply [27]. Shweiki et al. noted that, in glioblastoma multiforme tumors, the maximal expression of VEGF mRNA was found in necrotic (and presumably hypoxic) regions [28].

View larger version (16K): [in this window] [in a new window]   Figure 3. VEGF: a central role in tumor vasculature development and maintenance. Abbreviations: DC = dendritic cells; tPA = tissue-type plasminogen activator; uPA = urokinase-type plasminogen activator; uPAr = urokinase receptor; eNOS = endothelic nitric oxide synthase; COX-2 = cyclooxygenase-2.

Actions of VEGF in Angiogenesis
VEGF is a growth factor that is essential for the regulation of both physiological and pathological angiogenesis. It also plays a pivotal role in embryonic vasculogenesis, to the extent that deleting only one allele results in death of the embryo [29]. As described above, angiogenesis is a multistep process, and VEGF acts at several stages (Fig. 4):

View larger version (45K): [in this window] [in a new window]   Figure 4. Multiple functions of VEGF. Abbreviations: EC = endothelial cell; EPC = endothelial progenitor cell. Reprinted from Toi et al. [24] with permission from Elsevier.

• VEGF is a potent mitogen for vascular endothelial cells, but, with a few exceptions, is not mitogenic for other cell types [21].
  
• VEGF mediates the secretion and activation of enzymes involved in degrading the extracellular matrix. Acting on endothelial cells, VEGF induces expression of plasminogen activator and its inhibitor [30], expression of the urokinase receptor [31], and expression of the matrix metalloproteinases interstitial collagenase and gelatinase A, while decreasing levels of tissue inhibitors of metalloproteinases 1 and 2 [32, 33].
  
• VEGF acts as a survival factor for endothelial cells through the inhibition of apoptosis [34]. This action is mediated through the induction of expression of the antiapoptotic proteins Bcl-2 and Bcl-A1, regulation of the phosphatidylinositol 3-kinase/Akt pathway, increased phosphorylation of focal adhesion kinase, and stimulation of endothelial cell production of NO and prostaglandin I2 [35, 36].
  
• VEGF is essential for the mobilization of bonemarrow-derived endothelial cell precursors in the promotion of vascularization [10].
  
• In addition to promoting division of endothelial cells, VEGF also has an important role in modulating their migration to sites of angiogenesis [37].
  

Other actions of VEGF include increasing vascular permeability [38], inhibition of dendritic cell differentiation [39], upregulation of hexose transport into endothelial cells [40], induction of tissue factor [41], and induction of monocyte migration [42].

Owing to the amount of VEGF that tumors produce, a positive feedback loop is created, wherein VEGF-induced promotion of angiogenesis allows for enhanced tumor growth, which in turn results in increased VEGF secretion. This type of amplification can be further enhanced through the VEGF-mediated upregulation of expression of the VEGF receptors in endothelial cells [43]. In addition, tumor angiogenesis recruits bone-marrow-derived endothelial precursor cells, a process that is also dependent on VEGF and that can be completely ablated if signaling through both VEGFR-1 and VEGFR-2 is inhibited [11].

	SIGNIFICANCE OF VEGF IN CANCER

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
VEGF expression is increased in the majority of cancers examined to date. The list is extensive and includes hematological malignancies [44]; colon and rectal cancers [45]; liver cancer [46]; lung, thyroid, breast, gastrointestinal tract, kidney, and bladder cancers; ovary and uterine cervix carcinomas; angiosarcomas; germ-cell tumors; and intracranial tumors [21]. The correlation is not absolute, however, and varies depending on the particular study. For instance, in patients with colorectal cancer, Lee et al. found that approximately half of their patients showed elevated VEGF expression levels [45], whereas Tokunaga et al. reported that all of the 61 patients they observed had tumors that expressed VEGF [46]. In several studies involving breast cancer, colorectal cancer, and ovarian cancer, elevated VEGF levels were monitored after tumor resection and declined dramatically, with levels tending to rise again if the cancers recurred [47].

Several factors combine to contribute to more serious disease in instances of elevated VEGF expression. First, the role of VEGF in promoting the development of tumor vasculature supports this essential component of tumor development. Moreover, many tumors express VEGF receptors, so that VEGF can act as a paracrine factor, leading to a feedback loop not only through the stimulation of vascularization, but also through direct action on the tumor cells themselves. In this context, it can both promote the growth of transformed cell lines in vitro [48] and act as a survival factor for cancer cells through enhanced expression of the antiapoptotic factors Bcl-2 [49] and survivin [50]. VEGF-mediated inhibition of dendritic cell differentiation [39] may underlie the deficit of dendritic-cell infiltration that has been observed in gastric carcinoma tissues, suggesting that VEGF causes reduced immune surveillance of tumors [51]. Perhaps most importantly, the report that VEGF is essential for vascularization at very early stages of tumor formation by transformed cells implies that this growth factor is a key promoter of metastasis [51].

Thus, it is not surprising that VEGF expression in the primary tumor has been correlated in many studies with a greater risk for recurrence and poor prognosis in a variety of human cancers, including acute myeloid leukemia, breast cancer, colon cancer, hepatocellular carcinoma, non-small cell lung cancer, and ovarian cancer [8]. High VEGF levels have been found in malignant effusions, and it is thought that the permeability-enhancing effects of VEGF may promote effusion [24]. Elevated VEGF levels may also contribute to increased resistance to chemotherapy or endocrine therapy in advanced breast cancer [52]. Accordingly, VEGF status has proved to be of value in predicting the effectiveness of radiotherapy, chemotherapy, and hormonal therapy, as well as the likelihood of relapse, in a variety of cancers [24, 47].

	TARGETING VEGF: IMPLICATIONS FOR THERAPY

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
The application of angiogenesis inhibitors as anticancer agents is in its early stages, but over 25 drugs are under study in clinical trials. These inhibitors fall into several functional categories, including inhibitors of growth factors, endothelial cell signal transduction, endothelial cell proliferation, matrix metalloproteinases, endothelial cell survival, and bone marrow precursor cells [53]. Experience from these trials has brought new principles and challenges, many of which are likely to be applicable to drugs targeting VEGF.

Given its central role in promoting many cancers, VEGF provides an attractive target for therapeutic intervention. Two considerations in particular deserve emphasis: VEGF has a strategic position in the regulation of tumor angiogenesis, as it serves as a point of integration of a variety of upstream and downstream signals (Fig. 3), leading ultimately to stimulation of the endothelial cell; and VEGF promotes angiogenesis by acting directly on the endothelial cell, a genetically stable entity, rather than on genetically labile tumor cells. Accordingly, it has been suggested that, in contrast to drugs that inhibit angiogenesis indirectly through the inhibition of tumor-derived angiogenic factors, drugs that act to inhibit VEGF signaling may be less susceptible to selection of mutations that confer resistance [18]. However, recent evidence suggests that tumors can become resistant to antiangiogenic drugs, possibly through genetic mutations and instabilities associated with tumor cells [54, 55].

It is difficult for conventional therapies to gain adequate access to tumor tissue, first, because of the aberrant vasculature—highly tortuous with many blind ends—that characterizes most tumors [56], and second, because drug penetration into tumor tissue is diminished because interstitial pressure is elevated in most tumors, owing to severe leakage from the vasculature [57]. This latter effect is probably attributable to the actions of VEGF in enhancing vascular permeability. These factors are compounded by the secondary effects of radiotherapy and chemotherapy, which lead to local regions of hypoxia and induction of VEGF expression. The overproduction of VEGF not only exacerbates the high tumor interstitial pressure, but also protects tumor cells against the apoptosis that is normally induced by conventional therapies [49]. In principle, angiogenesis inhibitors, by targeting the vasculature rather than the tumor cells, should be able to circumvent the problem posed by high interstitial pressures in the tumors, since they do not require access to the tumor tissue.

Perhaps the most dramatic observation from recent studies has been the finding that coadministration of angiogenesis inhibitors with chemotherapy and radiotherapy can enhance the efficacy of these standard treatments. While it might be expected that drugs that target the tumor blood supply would exacerbate these difficulties, making the tumor cells even less accessible as the vasculature retracts, the reverse has been found in practice. Teicher et al. found that coadministration of the angiogenesis inhibitor TNR-470, a fumagillin analog, both enhanced both the permeability of murine tumors and led to a dramatic increase in cyclophosphamide-induced tumor cell killing [58]. Jain argued that this reflects a normalization of the tumor vasculature by angiogenesis inhibitors, so that some of the aberrant tumor blood vessels are pruned away as a result of endothelial cell death [56].

Moreover, many chemotherapy and radiotherapy regimens induce VEGF expression, which may contribute to tumor resistance to apoptosis-inducing modalities. Inhibition of VEGF or its receptors potentiates radiation-mediated killing of cancer cells, presumably by inhibiting the radiation-induced increase in VEGF and subsequent hypoxia [59, 60]. Indeed, an anti-VEGF antibody augmented the tumor response under normoxic and hypoxic conditions [59].

A variety of therapeutic strategies aimed at blocking VEGF or its receptor signaling system are currently being developed for the treatment of neoplastic diseases. VEGF/VEGFR blockade by monoclonal antibodies and inhibition of receptor signaling by tyrosine kinase inhibitors are the best studied approaches. VEGFR-1 ribozymes, VEGF toxin conjugates, and soluble VEGF receptors are also being investigated. Of these, bevacizumab (rhuMAb VEGF; Avastin^TM; Genentech, Inc.; South San Francisco, CA), a humanized monoclonal antibody directed at VEGF, is the most advanced in clinical development and has shown promising results in clinical trials.

	CONCLUSIONS

Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 
The ongoing development of angiogenesis inhibitors as cancer therapies is a further example of the principle that a fundamental understanding of physiological processes can lead to new therapies. VEGF has been shown to have central roles in key signaling pathways that mediate angiogenesis and in tumor growth and metastasis. Accordingly, therapies directed against VEGF or its receptors hold great promise in cancer treatment.

Key advantages of targeting VEGF include: by targeting the endothelial cell, rather than the tumor itself, issues of accessibility to the tumor interior are avoided and drug resistance may be less likely; given the central role of VEGF in both tumor growth and the establishment of metastases, its inhibition should prove effective at all stages of cancer, with the prevention of metastasis being a particular benefit; coadministration of therapies against VEGF with chemotherapy or radiotherapy is likely to lead to greater efficacies of these traditional therapies by preventing VEGF-mediated potentiation of cell survival, and given that targeting angiogenesis avoids the nonspecific toxicities associated with chemotherapy, there is ample scope for empirical combination, both with other antiangiogenesis therapies and with traditional chemotherapy and radiotherapy, to provide maximal flexibility in tailoring treatment options for the individual patient.

Most recently, this modality has received validation in a large, phase III clinical trial in metastatic colorectal cancer patients. Bevacizumab plus chemotherapy resulted in a highly significant longer time to progression and greater survival than chemotherapy alone [61].

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Top Learning Objectives Abstract Introduction Angiogenesis and Its Role... VEGF in Normal and... Significance of VEGF in... Targeting VEGF: Implications for... Conclusions References

 

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