Antiangiogenesis Agents
Cindy H. Chau
William Douglas Figg Sr.
INTRODUCTION
Blood vessels are indispensable for tumor growth and metastasis, and the formation of a new network of blood vessels from the existing vasculature, termed angiogenesis, is one of the essential hallmarks of cancer development.1 Indeed, it was over 70 years ago that the existence of tumor-derived factors responsible for promoting new vessel growth was postulated,2 and that tumor growth is essentially dependent on vascular induction and the development of a neovascular supply.3 By the late 1960s, Dr. Judah Folkman and colleagues4 had begun the search for a tumor angiogenesis factor. In the 1971 landmark report, Folkman5 proposed that inhibition of angiogenesis by means of holding tumors in a nonvascularized dormant state would be an effective strategy to treat human cancer, and hence laid the groundwork for the concept behind the development of antiangiogenesis agents. This fostered the search for angiogenic factors, regulators of angiogenesis, and antiangiogenic molecules over the next few decades and shed light on angiogenesis as an important therapeutic target for the treatment of cancer and other diseases.
A decade has passed since the regulatory approval of the first antiangiogenic drug bevacizumab, and while initial results were regarded as highly promising, clinical evidence indicated that antiangiogenic therapy also had limitations. Successful development and clinical translation of this novel class of agents depends on the complete understanding of the biology of angiogenesis and the regulatory proteins that govern this angiogenic process, topics that have been covered in greater detail in another section of this textbook. This chapter will briefly review the mechanisms underlying tumor angiogenesis followed by an in-depth discussion of antiangiogenic therapy, the modes of action of angiogenesis inhibitors, and the successes and challenges of this treatment modality.
UNDERSTANDING THE ANGIOGENIC PROCESS
Angiogenic Switch and Regulatory Proteins
Tumor development and progression depend on angiogenesis. Recruitment of new blood vessels to the tumor site is required for the delivery of nutrients and oxygen to the cancerous growths and for the removal of waste products.6 Cancer cells promote angiogenesis at an early stage of tumorigenesis, beginning with the release of molecules that send signals to the surrounding normal host tissue and stimulate the migration of microvascular endothelial cells (EC) in the direction of the angiogenic stimulus. These angiogenic factors not only mediate EC migration, but also EC proliferation and microvessel formation in tumors undergoing the switch to the angiogenic phenotype.7 Experimental evidence for this angiogenic switch was observed when hyperplastic islets in transgenic mice (RIP-Tag model) switch from small (<1 mm), white microscopic dormant tumors to red, rapidly growing tumors.7 Dormant tumors have been discovered during autopsies of individuals who died of causes other than cancer.8 These autopsy studies suggest that the vast majority of microscopic in situ cancers never switch to the angiogenic phenotype during a normal lifetime. Such incipient tumors are usually not neovascularized and can remain harmless to the host for long periods of time as microscopic lesions that are in a state of dormancy.9,10 These nonangiogenic tumors cannot expand beyond the initial microscopic size and cannot become clinically detectable, lethal tumors until they have switched to the angiogenic phenotype11,12,13 through neovascularization and/or blood vessel cooption.14 Depending on the tumor type and the environment, this switch can occur at different stages of the tumor progression pathway and ultimately depends on a net balance of positive and negative regulators. Thus, the angiogenic phenotype may result from the production of growth factors by tumor cells and/or the downregulation of negative modulators.
Changes in this angiogenic balance affecting the levels of activator and inhibitor molecules dictate whether an EC will be in a quiescent or an angiogenic state. Normally, the inhibitors predominate, thereby blocking growth. Once the balance shifts in favor of the angiogenic state, proangiogenic factors prompts the activation, growth, and division of vascular ECs, resulting in the formation of new blood vessels. Activated ECs produce and release matrix metalloproteinases (MMP) into the surrounding tissue to break down the extracellular matrix to allow the ECs to migrate and organize themselves into hollow tubes that eventually evolve into a mature network of blood vessels. Proangiogenic factors or positive regulators of angiogenesis include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (PlGF), platelet-derived growth factor (PDGF), placental growth factor, transforming growth factor-β, pleiotrophins, and others.15 Activation of the hypoxia-inducible factor 1 (HIF-1) via tumor-associated hypoxic conditions is also involved in the upregulation of several angiogenic factors.16 The angiogenic switch also involves the downregulation of angiogenesis suppressor proteins, which include endostatin, angiostatin, thrombospondin, and others.17,18 Most notably, however, is the link between many oncogenes and angiogenesis and the significant role oncogenes play in driving the angiogenic switch.19,20 These proangiogenic oncogenes not only induce the expression of stimulators, but may also downregulate inhibitors of angiogenesis.21
Endogenous Inhibitors of Angiogenesis
The infrequency of microscopic in situ tumors that actually undergo the angiogenic switch (<1%) suggests that naturally occurring endogenous inhibitors exist in the body to defend against the angiogenic switch in pathologic conditions and to limit physiologic angiogenesis.9 These circulating endogenous inhibitors could also prevent microscopic metastases from growing into visible tumors. Early studies by Langer et al.22,23 demonstrated the possible existence of such inhibitors through the extraction of a functional inhibitor from cartilage, a tissue that is poorly vascularized. Since then, dozens of endogenous angiogenesis inhibitors have been identified, some of which are listed in Table 28.1.17,18,24 Many of the endogenous inhibitors of angiogenesis that have been discovered to date are proteolytically cleaved fragments of larger proteins that are members of either the clotting/coagulation system
or members of the extracellular matrix family of glycoproteins. Endostatin is the most well-studied endogenous angiogenesis inhibitor.25,26 Other potent endogenous angiogenesis inhibitors include thrombospondin-127 and tumstatin.28 The discovery of vasohibin, an endogenous inhibitor that is selectively induced in ECs by proangiogenic stimulatory growth factors such as VEGF, demonstrated the existence of an intrinsic and EC-specific feedback inhibitor control mechanism,29,30 whereas most endogenous inhibitors of angiogenesis are extrinsic to ECs. More recently, a second endothelium-produced negative regulator of angiogenesis has been discovered, the Dll4-Notch signaling system.31,32 Both intrinsic factors have since been shown to control tumor angiogenesis by an autoregulatory or negative-feedback mechanism. The Dll4-Notch axis has emerged as a critical regulator of tumor angiogenesis, and inhibitors of this pathway (e.g., demcizumab, the anti-Dll4 monoclonal antibody) are currently being investigated in early phase trials of solid tumors.33
or members of the extracellular matrix family of glycoproteins. Endostatin is the most well-studied endogenous angiogenesis inhibitor.25,26 Other potent endogenous angiogenesis inhibitors include thrombospondin-127 and tumstatin.28 The discovery of vasohibin, an endogenous inhibitor that is selectively induced in ECs by proangiogenic stimulatory growth factors such as VEGF, demonstrated the existence of an intrinsic and EC-specific feedback inhibitor control mechanism,29,30 whereas most endogenous inhibitors of angiogenesis are extrinsic to ECs. More recently, a second endothelium-produced negative regulator of angiogenesis has been discovered, the Dll4-Notch signaling system.31,32 Both intrinsic factors have since been shown to control tumor angiogenesis by an autoregulatory or negative-feedback mechanism. The Dll4-Notch axis has emerged as a critical regulator of tumor angiogenesis, and inhibitors of this pathway (e.g., demcizumab, the anti-Dll4 monoclonal antibody) are currently being investigated in early phase trials of solid tumors.33
TABLE 28.1 Examples of Endogenous Inhibitors of Angiogenesis | |
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Perhaps the most compelling genetic evidence that endogenous inhibitors suppress pathologic angiogenesis was observed in studies using mice deficient in tumstatin, endostatin, or thrombospondin 1 (TSP-1).34 These experiments demonstrate that normal physiologic levels of the inhibitors can retard the tumor growth and that their absence leads to enhanced angiogenesis and increased tumor growth by two- to threefold, strongly suggesting that endogenous inhibitors of angiogenesis can act as endothelium-specific tumor suppressors. The connection between a tumor suppressor protein and angiogenesis is best illustrated by the classic tumor suppressor p53. p53 inhibits angiogenesis by increasing the expression of TSP-135 by repressing VEGF36 and basic fibroblast growth factor-binding protein,37 and by degrading HIF-1,38 which blocks the downstream induction of VEGF expression. New evidence suggests that p53 also indirectly downregulates VEGF expression via the retinoblastoma pathway in a p21-dependent manner during sustained hypoxia.39 Furthermore, p53-mediated inhibition of angiogenesis may also occur in part via the antiangiogenic activity of endostatin and tumstatin.40 This landmark finding clearly demonstrates that p53 not only controls cell proliferation, but can also repress tumor angiogenesis through enzymatic mobilization of these endogenous angiogenesis inhibitor proteins to prevent ECs from being recruited into the dormant, microscopic tumors, thereby preventing the switch to the angiogenic phenotype.41 The discovery that these endogenous angiogenesis inhibitors can suppress the growth of primary tumors raises the possibility that such inhibitors might also be able to slow tumor metastasis. Indeed, the inhibition of angiogenesis by angiostatin significantly reduced the rate of metastatic spread.
DRUG DEVELOPMENT OF ANGIOGENESIS INHIBITORS
The first angiogenesis inhibitor was reported in 1980 and involved the low-dose administration of interferon α (IFN-α).42,43,44 Over the next decade, several compounds were discovered to have potent antiangiogenic activity, including protamine and platelet factor 4,45 trahydrocortisol,46 and the fumagillin analog TNP-470.47 The proof of concept that targeting angiogenesis is an effective strategy for treating cancer came with the approval of the first angiogenesis inhibitor, bevacizumab, by the U.S. Food and Drug Administration (FDA). Since then, several antiangiogenic agents have received FDA approval for cancer treatment (Table 28.2), and three additional agents (pegaptanib, ranibizumab, and aflibercept) are approved for the treatment of wet age-related macular degeneration.
Rationale for Antiangiogenic Therapy
Antiangiogenic therapy stems from the fundamental concept that tumor growth, invasion, and metastasis are angiogenesis dependent; thus, blocking blood vessel recruitment to starve primary and metastatic tumors is a rational approach. The microvascular EC recruited by a tumor has become an important second target in cancer therapy. Unlike the cancer cell (the primary target of cytotoxic chemotherapy), which is genetically unstable with unpredictable mutations, the genetic stability of ECs may make them less susceptible to acquired drug resistance.48 Moreover, ECs in the microvascular bed of a tumor may support 50 to 100 tumor cells. Coupling this amplification potential together with the lower toxicity of most angiogenesis inhibitors results in the use of antiangiogenic therapy, which should be significantly less toxic than conventional chemotherapy. However, the variable responses of antiangiogenic therapy observed in different tumor types and the fact that angiogenesis inhibitors have not delivered the benefits initially envisaged suggest that the precise mechanism of action of angiogenesis inhibitors is complex and remains incompletely understood.
Modes of Action of Antiangiogenic Agents
Various strategies for the development of antiangiogenic drugs have been investigated over the years, with these agents being classified into several different categories depending on their modes of action. Some inhibit ECs directly, whereas others inhibit the angiogenesis signaling cascade or block the ability of ECs to break down the extracellular matrix. Inhibitors may block one main angiogenic protein, two or three angiogenic proteins, or have a broad-spectrum effect by blocking a range of angiogenic regulators that can be located in both the tumor and ECs.49 In some cases, the antiangiogenic activity is discovered as a secondary function after the drug has received regulatory approval for a different primary function. For example, bortezomib is a proteasome inhibitor that is approved for multiple myeloma and was later found to possess antiangiogenic activity via inhibiting VEGF. Some small-molecule drugs may display their antiangiogenic activity through inducing the expression of endogenous angiogenesis inhibitors such as celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, which inhibits angiogenesis by increasing levels of endostatin.25
Some drugs possess antiangiogenic properties but with mechanisms that are not completely understood, such as thalidomide and its analogs, lenalidomide and pomalidomide, referred to as immunomodulatory drugs. Thalidomide was originally shown to inhibit angiogenesis by D’Amato et al.50 in 1994 and this was subsequently confirmed in several different in vitro and ex vivo
assays.51,52,53,54 Interestingly, unlike other mechanisms of action, the antiangiogenic activity of thalidomide is believed to require enzymatic activation. The extent to which the antiangiogenic properties of thalidomide and its analogs play a role in its antimyeloma activity is not clearly understood. Several mechanisms have been proposed that involve the downregulation of cytokines in EC, the inhibition of EC proliferation, the decrease in the level of circulating ECs, or the modulation of adhesion molecules between the multiple myeloma cells and the endogenous bone marrow stromal cells, thereby decreasing the production of VEGF and interleukin 6 (IL-6).55,56,57,58,59 The immunomodulatory agents are discussed in greater detail in another section of this textbook. Examples of the various types of angiogenesis inhibitors are highlighted in Table 28.3.
assays.51,52,53,54 Interestingly, unlike other mechanisms of action, the antiangiogenic activity of thalidomide is believed to require enzymatic activation. The extent to which the antiangiogenic properties of thalidomide and its analogs play a role in its antimyeloma activity is not clearly understood. Several mechanisms have been proposed that involve the downregulation of cytokines in EC, the inhibition of EC proliferation, the decrease in the level of circulating ECs, or the modulation of adhesion molecules between the multiple myeloma cells and the endogenous bone marrow stromal cells, thereby decreasing the production of VEGF and interleukin 6 (IL-6).55,56,57,58,59 The immunomodulatory agents are discussed in greater detail in another section of this textbook. Examples of the various types of angiogenesis inhibitors are highlighted in Table 28.3.
TABLE 28.2 Antiangiogenic Agents that Have Received U.S. Food and Drug Administration Approval for Cancer Treatment | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Drugs with antiangiogenic activity may be classified as either direct or indirect angiogenesis inhibitors. A direct angiogenesis inhibitor blocks vascular ECs from proliferating, migrating, or increasing their survival in response to proangiogenic proteins. They target the activated endothelium directly and inhibit multiple angiogenic proteins. Examples of direct angiogenesis inhibitors include many of the endogenous inhibitors of angiogenesis, such as endostatin, angiostatin, and TSP-1. Indirect angiogenesis inhibitors decrease or block expression of a tumor cell product, neutralize the tumor product itself, or block its receptor on ECs. The limitation to indirect inhibitors is that, over time, tumor cells may acquire mutations that lead to increased expression of other proangiogenic proteins that are not blocked by the indirect inhibitor. This may give the appearance of drug resistance and warrants the addition of a second antiangiogenic agent, one that would target the expression of these upregulated proangiogenic proteins. Examples of drugs that interfere with the angiogenesis-signaling pathway include the anti-VEGF monoclonal antibodies and small-molecule tyrosine-kinase inhibitors. These drugs target the major signaling pathways in tumor angiogenesis: VEGF, PDGF, and their respective receptors, as well as other growth factors and/or signaling pathways.
VEGF (also known as vascular permeability factor) is a potent proangiogenic growth factor and its expression is upregulated by most cancer cell types. It stimulates EC proliferation, migration, and survival as well as induces increased vascular permeability. The different forms of VEGF bind to transmembrane receptor tyrosine kinases (RTK) on ECs: VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1 or kinase insert domain receptor/fetal liver kinase 1), or VEGFR3 (Flt-4).60 This results in receptor dimerization, activation, and autophosphorylation of the tyrosine-kinase domain, thereby triggering downstream signaling pathways. Other signaling molecules that may represent attractive therapeutic targets include PDGF and the angiopoietins (Ang1, Ang2). PDGF-B/PDGF receptor (R)-β plays an important role in the recruitment of pericytes and maturation of the microvasculature.61 Ang2, which binds the Tie-2 receptor, is mostly expressed in tumor-induced neovasculature, whereby its selective inhibition results in reduced EC proliferation.62 The angiopoietins are also involved in lymphangiogenesis, the formation of new lymphatic vessels, which plays a key role in tumor metastasis. An increased Ang2/Ang1 ratio correlates with tumor angiogenesis and poor prognosis in many cancers, thus making the angiopoietins an attractive therapeutic target. Angiopoietin inhibitors are currently under investigation in the preclinical and clinical setting.
TABLE 28.3 Examples of Drugs that Possess Antiangiogenic Activity or Inhibit Angiogenesis as a Secondary Function | ||||||||||||||||||||||||
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Other strategies for targeting angiogenesis involve the tumor microenvironment. Breakdown of the extracellular matrix is required to allow ECs to migrate into surrounding tissues and proliferate into new blood vessels; thus, drugs that target MMPs, enzymes that catalyze the breakdown of the matrix, can also inhibit angiogenesis. However, clinical development of MMP inhibitors (MMPI) has yielded disappointing results.63,64,65,66
Integrins are cell surface adhesion molecules that play an essential role in cell-cell and cell-matrix adhesion as well as in transmitting signals important for cell migration, invasion, proliferation, and survival. The involvement of integrin in tumor angiogenesis was demonstrated in studies that show the β-4 subunit of integrin promoting endothelial migration and invasion.67 Agents that target integrins (inhibitors of αvβ3 and αvβ5) have been evaluated as potential therapeutic options and include etaracizumab, cilengitide, and intetumumab. However, all three integrin inhibitors have proven to be largely ineffective in various early and late stage cancer trials.68,69,70,71,72,73 In summary, the downstream effects of antiangiogenic agents, in addition to blocking angiogenesis, may involve inducing vessel regression, promoting sensitization to radiotherapy and chemotherapy by depriving ECs of VEGF’s prosurvival signals, and inhibiting the recruitment of proangiogenic bone marrow-derived cells as well as reducing the self-renewal capability of cancer stem cells.74
CLINICAL UTILITY OF APPROVED ANTIANGIOGENIC AGENTS IN CANCER THERAPY
The following section reviews the current FDA-approved angiogenesis inhibitors (Table 28.2). These agents include: (1) the monoclonal anti-VEGF antibodies (bevacizumab and ziv-aflibercept); (2) small-molecule tyrosine-kinase inhibitors (TKI) (sorafenib, sunitinib, pazopanib, vandetanib, axitinib, cabozantinib, and regorafenib); and (3) the mammalian target of rapamycin (mTOR) inhibitors (temsirolimus and everolimus), as examples of drugs that possess antiangiogenic activity. Other approved drugs that also inhibit angiogenesis as a secondary function, such as thalidomide, are discussed in greater detail in another section of this textbook and are presented in Table 28.3.
Anti-VEGF Therapy
Bevacizumab
Bevacizumab is a recombinant humanized anti-VEGF-A monoclonal antibody that received FDA approval in February 2004 for use in combination therapy with fluorouracil-based regimens for
metastatic colorectal cancer. Bevacizumab binds VEGF and prevents the interaction of VEGF to its receptors (Flt-1 and KDR) on the surface of ECs. It is the first antiangiogenic agent clinically proven to extend survival following a large, randomized, double-blind, phase III study in which bevacizumab was administered in combination with bolus irinotecan, 5-fluorouracil, and leucovorin (IFL) as first-line therapy for metastatic colorectal cancer (CRC).75 In 2006, its approval extended to first- or second-line treatment of patients with metastatic carcinoma of the colon or rectum. This recommendation is based on the demonstration of a statistically significant improvement in overall survival (OS) in patients receiving bevacizumab plus FOLFOX4 (5-flourouracil, leucovorin, and oxaliplatin) when compared to those receiving FOLFOX4 alone. In January 2013, it was further approved to treat mCRC for second-line treatment when used with fluoropyrimidine-based (combined with irinotecan or oxaliplatin) chemotherapy after disease progression following a first-line treatment with a bevacizumab-containing regimen based on clinical benefits observed in the randomized phase III study (ML18147).76 Despite the benefit in the metastatic setting, the addition of bevacizumab did not improve clinical outcomes in the adjuvant setting in CRC.77,78 In 2006, bevacizumab received an additional approval for use in combination with carboplatin and paclitaxel, and is indicated for first-line treatment of patients with unresectable, locally advanced, recurrent, or metastatic nonsquamous, non-small-cell lung cancer (NSCLC) based on the demonstration of a statistically significant improvement in OS in patients in the bevacizumab arm compared to those receiving chemotherapy alone.79 In February 2008, the FDA granted a conditional, accelerated approval for bevacizumab to be used in combination with paclitaxel for the treatment of patients who have not received chemotherapy for metastatic human epidermal growth factor receptor 2 (HER2)-negative breast cancer. However, additional clinical trials were conducted and the new data showed only a small effect on progression free survival (PFS) without evidence of an improvement in OS or a clinical benefit to patients sufficient to outweigh the risks; thus, the FDA rescinded its approval and removed the breast cancer indication from the drug’s label in November 2011.80,81,82 This controversial decision continues to be debated with ongoing subgroup analyses to identify patients who would likely benefit from bevacizumab.
metastatic colorectal cancer. Bevacizumab binds VEGF and prevents the interaction of VEGF to its receptors (Flt-1 and KDR) on the surface of ECs. It is the first antiangiogenic agent clinically proven to extend survival following a large, randomized, double-blind, phase III study in which bevacizumab was administered in combination with bolus irinotecan, 5-fluorouracil, and leucovorin (IFL) as first-line therapy for metastatic colorectal cancer (CRC).75 In 2006, its approval extended to first- or second-line treatment of patients with metastatic carcinoma of the colon or rectum. This recommendation is based on the demonstration of a statistically significant improvement in overall survival (OS) in patients receiving bevacizumab plus FOLFOX4 (5-flourouracil, leucovorin, and oxaliplatin) when compared to those receiving FOLFOX4 alone. In January 2013, it was further approved to treat mCRC for second-line treatment when used with fluoropyrimidine-based (combined with irinotecan or oxaliplatin) chemotherapy after disease progression following a first-line treatment with a bevacizumab-containing regimen based on clinical benefits observed in the randomized phase III study (ML18147).76 Despite the benefit in the metastatic setting, the addition of bevacizumab did not improve clinical outcomes in the adjuvant setting in CRC.77,78 In 2006, bevacizumab received an additional approval for use in combination with carboplatin and paclitaxel, and is indicated for first-line treatment of patients with unresectable, locally advanced, recurrent, or metastatic nonsquamous, non-small-cell lung cancer (NSCLC) based on the demonstration of a statistically significant improvement in OS in patients in the bevacizumab arm compared to those receiving chemotherapy alone.79 In February 2008, the FDA granted a conditional, accelerated approval for bevacizumab to be used in combination with paclitaxel for the treatment of patients who have not received chemotherapy for metastatic human epidermal growth factor receptor 2 (HER2)-negative breast cancer. However, additional clinical trials were conducted and the new data showed only a small effect on progression free survival (PFS) without evidence of an improvement in OS or a clinical benefit to patients sufficient to outweigh the risks; thus, the FDA rescinded its approval and removed the breast cancer indication from the drug’s label in November 2011.80,81,82 This controversial decision continues to be debated with ongoing subgroup analyses to identify patients who would likely benefit from bevacizumab.
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