The Biology of Angiogenesis

Angiogenesis is the process of new blood vessel growth. In malignant tumors this process is essential for the delivery of needed nutrients and oxygen for the continued growth and survival of cancer cells. Thus the process of angiogenesis and the subsequent development of therapies that inhibit the process have generated great interest since Judah Folkman's original hypothesis was presented over 3 decades ago. Folkman's studies in the 1970s sparked interest in the science of angiogenesis and led to the first specific therapy to inhibit angiogenesis, but it was not until 2004 that the first antiangiogenesis agent, bevacizumab (Avastin), was approved by the US Food and Drug Administration (FDA) in combination with chemotherapy. Since then, two multitargeted or dual action oral agents have been FDA-approved. Advances have also been made in understanding the science of antiangiogenesis, which has contributed to the design of agents as well as clinical trials in the treatment of several tumor types and is being studied actively in many others.

Antiangiogenesis therapy has emerged to become a significant component of cancer treatment today. Angiogenesis refers to new blood vessel growth stemming from an existing vasculature.[1] Folkman's research was the first to demonstrate that new blood vessel growth is critical to the growth and development of tumors and metastasis,[2] and that by blocking angiogenesis, tumors will have decreased growth.[3] This concept has been the focus of intense research for the past 3 decades, leading to the 2004 US Food and Drug Administration (FDA) approval of bevacizumab (Avastin), the first antiangiogenesis agent in monoclonal antibody form, in combination with chemotherapy. Subsequently, small-molecule inhibitor (oral) agents have been developed and research continues in this important area.

The vascular endothelial growth factor receptor (VEGFR) is an intriguing target in cancer therapy. Endothelial cells line the entire vascular system, both blood vessels and lymphatics, and together with smooth muscle cells, maintain intravascular pressure.[4] The physiologic role of angiogenesis is pivotal during embryogenesis, where vasculogenesis creates new vessel formation during embryo development.[5] Angiogenesis is also important in wound healing, muscle and bone growth, and menstruation; these effects are usually short-lived and linked to those physiologic events, although it does have a function in the maintenance of normal vasculature as well.[4,5] Angiogenesis is activated in pathologic conditions, including chronic inflammatory conditions and atherosclerosis.[5]

Angiogenesis and Cancer

In cancer, angiogenesis promotes the growth of tumors themselves and facilitates metastasis.[4] The vasculature is disorganized and highly permeable in tumor tissue compared to the organized appearance in normal tissue, which facilitates migration of endothelial cells.[6] VEGF can cause endothelial cells to accumulate, and it creates abnormalities in perivascular cells.[7] This leads to increased interstitial fluid pressure and, coupled with inefficient blood flow inside the tumor itself, can affect the efficacy of chemotherapy.[8] Hypoxia results from the slow and inconsistent blood flow in tumors, with resulting acidosis; this can add to chemotherapy and radiotherapy resistance by reducing available oxygen.[6] The presence of hypoxia continues to stimulate angiogenesis. The pericytes in tumor vasculature may be dysfunctional or even absent, and the endothelial cell basement membrane may be inconsistent.[6]

It is generally accepted that tumors cannot grow larger than 1 to 2 mm3 in size unless an adequate vascular supply is present.[2] Inactivation or inhibition of VEGFR signaling pathways has been shown to reduce the angiogenesis associated with specific tumor types, potentially leading to inhibition of tumor cell growth.[9]

Normally, there is a balance between proangiogenic and antiangiogenic factors in normal host cells (such as endothelial cells, pericytes, and other cells of the immune system).[10] When new blood vessels are needed, the "angiogenic switch" is turned on by increased proangiogenic factors. Endogenous proangiogenic factors include basic fibroblast growth factor (bFGF), transforming growth factors alpha and beta, VEGF, angiopoietins 1 and 2, as well as androgens, estrogens, interleukins, and proteinases. Antiangiogenic factors include angiostatin, endostatin, vasostatin, alpha- and beta-interferon, and possibly interleukin-12.[11]

In tumor cells, balance is shifted in favor of the proangiogenic factors so that the tumor can continue to proliferate and invade, and for metastatic sites to proliferate and disseminate. Tumors turn on the angiogenic switch with the release of tumor-related proangiogenic factors.[12]

The process for angiogenesis involves the vasodilation, permeability, and degradation of the stroma of the endothelial cell.[13] Once the VEGF network is activated, various signaling networks are activated that ultimately can promote survival of endothelial cells, mitogenesis, migration, and differentiation as well as increased vascular permeability.[14] This expansion of the permeability of the vascular tissue is responsible for the role of VEGF in inflammatory diseases as well as other pathologic conditions.[15] Both tumor cells and their stroma are sites of VEGF production.[16] Different malignant tumors may have upregulation of VEGF mRNA.[15]

Neovascularization refers to the release of multiple angiogenic ligands or growth factors that enter the microcellular arena, thus activating various receptors and stimulating the growth and proliferation of new capillary structures.[17] The physiologic changes in the tumor microenvironment by VEGF can affect drug delivery to tumor cells, particularly since the abnormal vasculature has increased permeability and changes in interstitial pressure.[6,18] The vasculature can also cause low or turbulent blood flow, which might cause decreased uptake of drug therapy by tumor cells themselves.

One effect of anti-VEGF therapy seems to be the "normalization" of the vascular microenvironment (without significantly affecting the normal vasculature); this action may help facilitate the delivery of cancer drug therapy to the tumor cells themselves[18] (Figure 1). Blocking the signaling of VEGF seems to produce vasculature that is much less leaky and dilated, with more normal-appearing vessels that have more intact basement membrane and pericyte formation.[19] These physiologic changes in the vasculature also produce changes in how the vessels function and improve the interstitial fluid pressure and oxygenation for the tumor, which could lead to better delivery of chemotherapeutic drugs to tumors themselves.[19] Angiogenesis is crucial for tumor cells as the process provides oxygen, nutrients, and other growth factors facilitating survival,[14] and allows for removal of waste products. VEGF is the most potent factor identified to date and has a key role in the process of angiogenesis.[10]

The Role of Microvessel Density

Microvessel density (MVD), a surrogate for the extent of tumoral angiogenesis,[20] has been used to identify high-risk patients with specific tumor types, such as breast and prostate cancer.[20] Microvessel density can be used to approximate the extent of angiogenesis inside the tumor itself; in several studies high MVD has been shown to predict poor survival in breast cancer patients,[20] likelihood of local disease control in patients with head and neck cancer treated with radiotherapy,[21] and early recurrence in patients with hepatocellular cancer.[22] Thus MVD has the potential to help clinicians understand disease stage and tumor aggressiveness, as well as the potential for metastasis, recurrence, and survival.[23] However, there are negative studies that do not show a correlation between MVD and patient outcomes; thus, further study of the role of MVD is needed.[23]

The vegf Pathway

The VEGF family contains six glycoprotein growth factors: VEGF-A, VEGF-B, VEGF-C, VEGF-E, and placental growth factor (PIGF-1 and PIGF-2). The best-studied is the VEGF-A family.[14,24] There are three receptors in this family: VEGFR-1, VEGFR-2, and VEGFR-3; these receptors are transmembrane tyrosine kinases and are found mostly on endothelial cells (Figure 2).[14]

VEGF is the ligand that binds to VEGFR-1 and VEGFR-2, both receptor tyrosine kinases (RTKs). The receptors VEGFR-1 and VEGFR-2 have seven immunoglobulin-like domains extracellularly; both have a single transmembrane region (spanning the cellular membrane) and an intracellular tyrosine kinase (TK) domain (Figure 2).[15,24] Although VEGFR-3 is in the same family of RTKs, VEGF does not bind with this receptor. VEGFR-1 is found primarily on endothelial cells and monocytes and plays a role in cell motility while VEGFR-2 mediates the proliferative qualities of VEGF and vascular permeability.[25] VEGFR-1 has been found on specific tumor cell types, such as colorectal cancer, facilitating progression of disease and metastasis.[24] VEGFR-3 plays a role in lymphangiogenesis, possibly helping to promote tumor cell migration to regional lymph nodes.[24,25]

Another factor related to the VEGF family is platelet-derived growth factor (PDGF), which has angiogenesis properties and is critical to the function of pericytes. Pericytes, also called Rouget cells or periendothelial cells, exist in the basement membrane of capillaries and postcapillary venules and stabilize the blood vessel walls, helping to regulate blood flow in the microcirculation.[26] Platelet-derived growth factor B is expressed by endothelial cells while PDGFR-? is expressed by pericytes.[27] These two entities have a significant role in the maturation of blood vessels.[27]

The extracellular VEGF receptors act as signaling molecules in vascular development and the intracellular VEGF RTKs are vital parts of the signal transduction pathway for VEGF and the endothelial cell.[28] The receptors are activated by the binding of ligands. Ligands can be various growth factors or substances capable of binding to a receptor, but the primary ligand for this pathway is VEGF. Once the ligand binds to the receptor, the two receptors dimerize and subsequent autophosphorylation occurs.[28] The autophosphorylation is essentially a "switching" on of the intracellular pathway, sending the message downstream (signaling) to the cell nucleus, leading to endothelial cell proliferation and migration.

There are multiple transduction pathways for VEGF involving the regulation of tumor cell survival, including the Raf/mitogen-activated protein kinase pathway and the phosphatidylinositol 3 kinase (P13K/Akt) pathway.[24] Effective signal transduction results in the message reaching the endothelial cell nucleus, activation of genes, and subsequent changes leading to tumor vascularization, migration, proliferation, and metastasis. These pathways provide a pharmacologic target starting with inhibition of the ligand (VEGF) so it cannot bind to the VEGFR1 receptor, to blocking of the signaling pathways in the intracellular TK region, disrupting the message so it does not reach the nucleus of the cell.

Benefit of a Multitargeted Approach: The EGFR Pathway

Research has shown that additional growth factor receptors play a role in angiogenesis and that VEGF shares common signaling pathways with the epidermal growth factor receptor (HER-1 or EGFR).[29] For example, there is evidence that VEGF can be downregulated by the inhibition of EGFR, and that inhibition of VEGF can affect EGFR signaling as well.[30]

The HER (human epidermal growth factor) family includes HER-1 (erbB1), HER-2 (erbB2), HER-3 (erbB3), and HER-4 (erbB4), and epidermal growth factor and at least 10 other separate ligands can bind to the EGFR.[31] The additional ligands include transforming growth factor alpha (TGF-α), heparin-binding EGF, amphiregulin, and betacellulin among others.[31] Epidermal growth factor is an important target for anticancer therapy as it is integral in proliferation, apoptosis, angiogenesis, and metastasis.[31]

The cell-signaling system for the EGFR is activated when a ligand binds to the receptor, starting the process of dimerization and autophosphorylation; once that occurs, cell signaling can occur downstream.[31] Although there are external ligand-binding domains for HER-1, HER-3 and HER-4, HER-2 has no available ligand and must pair up with other members of this family to begin the cell-signaling. The external domains can be targeted with monoclonal antibody therapy agents (which are large molecules), and tyrosine kinase inhibitor agents can affect the internal TK domains of HER-1, HER-2, and HER-4. These agents are low molecular weight vs their large-molecule monoclonal antibody counterparts. The TKI agents can target cancer and endothelial cells by inhibition of the signaling pathway or by blocking RTK activity; several have been approved as cancer therapies. Some of these therapies target multiple areas within the cell, as well as the RTK region.

RAS is also an important component of the cell signaling system and may have activity through VEGFR, PDGFR, and EGFR to the RAF/MEK/ERK pathway, which is also vital to tumor cell survival, proliferation, and angiogenesis.[32] This is a particularly active area of cancer research and additional agents are currently under study. Selected monoclonal antibody and small-molecule agents targeting VEGF will now be reviewed.

Mechanism of Action for Selected VEGF-Targeted Agents

Although there are a variety of antiangiogenesis agents being studied in selected tumor types, several are currently approved by the FDA. One of the agents prevents VEGF from binding to the VEGFR1 receptor (bevacizumab), while others block VEGFR1 inside the cell membrane (tyrosine kinase inhibition) as well as interfere with PDGFRs (sunitinib [Sutent] and sorafenib [Nexavar]); see Figure 3.

Bevacizumab

Approved in the treatment of metastatic colorectal cancer in 2004, bevacizumab directly inhibits VEGF binding to VEGFR. Subsequently, the indication for use in that disease has been expanded, and this agent is now approved in the treatment of lung cancer and is being studied in a wide variety of tumors as well.

Bevacizumab does not have activity against other growth factors, such as fibroblast growth factor, epidermal growth factor, or platelet-derived growth factor.[33] The mechanism of action for this humanized monoclonal antibody is not completely understood, but there are data that demonstrate its ability to prune tumor vessels and reduce tumor MVD in rectal cancer patients by 40% to 50% after one infusion.[34] The proposed mechanism of action is the inhibition of new vessel formation (preventing the delivery of oxygen and nutrients needed to facilitate continued growth, proliferation, and metastasis) and the normalization of the remaining vasculature with a decrease in permeability and subsequent improvement of blood perfusion.[35] Bevacizumab appears to be most effective in combination with chemotherapy, leading to a survival benefit in patients with cancer.[35]

Sunitinib

Sunitinib was approved in 2006 for the treatment of renal cell cancer (RCC) and for the treatment of patients with gastrointestinal stromal cancer (GIST) after progression on imatinib (Gleevec). Ongoing studies are evaluating this agent in other tumor types as well. Sunitinib is a dual-action oral inhibitor of the tyrosine kinases VEGFR and PDGFR.[36] Approximately 80% of patients with clear-cell RCC have inactivation of the von Hippel–Lindau (VHL) gene, which codes for a protein that regulates the production of VEGF, PDGF, and proteins capable of inducing hypoxia. When the VHL gene is inactivated, overexpression of the proteins for VEGFR and PDGFR occur, potentially stimulating tumor angiogenesis, growth, and metastasis.[36] Hypoxia and absence of VHL can cause the molecules to act in a paracrine loop, stimulating angiogenesis.[32] This may be the physiologic basis for the vascularity seen in renal cell carcinoma, explaining why antiangiogenesis agents are effective in the treatment of this disease.[32]

Sunitinib also has activity in the inhibition of RTK signaling associated with c-KIT, a marker of GIST. Approximately 85% to 90% of gastrointestinal tumors (a form of sarcoma) have mutations in the KIT gene, promoting activation of KIT kinase activity.[37] A smaller 5% of patients have mutations in PDGFRA (the gene for platelet-derived growth factor receptor alpha). Thus, in patients with GIST, sunitinib blocks both PDGFR and VEGF signaling.

Sorafenib

Sorafenib is the first oral agent to be approved in the treatment of metastatic RCC. As a multitargeted RTK inhibitor agent, the drug works on both the extracellular and intracellular pathways that affect tumor cell growth.[38] Targets for this therapy include VEGFR1, VEGFR2, and VEGFR3 as well as PDGF-b in the tumor vascular tissue, and when inhibited, new blood vessel formation is disrupted.[38] Additional activity includes the inhibition of the c-Raf and b-Raf kinase pathways in the intracellular region, which helps to decrease tumor cell proliferation. These kinases belong to the RAF/MEK/ERK intracellular signaling cascade that is a downstream effector of RAS. As discussed previously, when activated by RTK stimulation, RAS potentially has activity through VEGFR, PDGFR, and EGFR to the RAF/MEK/ERK pathway, which is vital to tumor cell survival, proliferation, and angiogenesis.[32] Thus the drug has dual action and therefore is both cytostatic and cytoreductive vs cytotoxic.[38]

Thalidomide

Thalidomide (Thalomid) is a unique agent with a different mechanism of action. Although its antiangiogenic action is not completely understood, it appears to have a dual ability and can serve as an immunomodulator as well as an antiangiogenic agent.[39] Thalidomide has been shown to have activity by depleting VEGF.[40]

Investigative Agents

There are many agents with indirect or direct antiangiogenesis activity under current investigation in different tumor types. Oral vatalanib affects VEGFR1, VEGFR2, and VEGFR3 as primary targets as well as secondary targets PDGFR-b and c-Kit and has shown activity in metastatic colorectal cancer.[35] Vandetanib targets both EGFR and VEGF, and has shown activity in patients with non–small-cell lung cancer.[41] Semaxinib also acts as a multitargeted agent, affecting VEGFR1, VEGFR2, and VEGFR3, with secondary activity on PDGFR-b and c-Kit in combination with chemotherapy for metastatic colorectal carcinoma but lack of survival benefit led to the abandonment of further study.[35] ZD6474, as an oral inhibitor of both VEGF and EGFR, has been studied in lung tumors, showing a favorable toxicity profile and synergism with chemotherapy and radiation therapy as well.[42] VEGF Trap is a chimeric molecule that contains extracellular portions of VEGFR1 and VEGFR2 with the Fc portion of immunoglobulin G1.[39] This molecule has the ability to bind and "trap" the VEGF and is currently under study. There are also multiple other second-generation tyrosine kinase inhibitor and dual inhibitor agents under investigation.[43]

Conclusion

The science of angiogenesis is intriguing; a number of agents targeting molecules in the angiogenic process have been approved by the FDA approved and numerous others are under study. The approval of bevacizumab for the treatment of patients with advanced metastatic colorectal cancer has led to the use of this anti-VEGF agent in the first- and second-line settings for this disease; bevacizumab is now approved for the treatment of patients with unresectable or advanced non–small-cell lung cancer and is under study for many other tumor types. As ongoing trials continue to determine the effectiveness of new agents or combinations, antiangiogenic agents will continue to be an important component of oncology treatment. Additional pathways, such as the EGFR pathway, have a role in angiogenesis as well, and although not fully reviewed in this article, research supporting the use of anti-EGFR agents in cancer therapy has made similar strides.

Strategies to enhance the effectiveness of anti-VEGF therapies include combination approaches, better management of toxicities, and methods to overcome resistance to therapies. In summary, angiogenesis has been recognized as an integral part of tumor survival, and therapies to overcome this process are an exciting part of cancer therapy and research.

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