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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

Angiogenesis has become an attractive target for drug therapy because of its key role in tumor growth. An extensive array of compounds is currently in preclinical development, with many now entering the clinic and/or achieving approval from the US Food and Drug Administration. Several regulatory and signaling molecules governing angiogenesis are of interest, including growth factors (eg, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor), receptor tyrosine kinases, and transcription factors such as hypoxia inducible factor, as well as molecules involved in mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling. Pharmacologic agents have been identified that target these pathways, yet for some agents (notably thalidomide), an understanding of the specific mechanisms of antitumor action has proved elusive. The following review describes key molecular mechanisms and novel therapies that are on the horizon for antiangiogenic tumor therapy. CA Cancer J Clin 2010. © 2010 American Cancer Society, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

In 1971, Dr. Judah Folkman published a landmark article in the New England Journal of Medicine, with the hypothesis that solid tumors caused new blood vessel growth (angiogenesis) in the tumor microenvironment by secreting proangiogenic factors.1 This publication heralded the beginning of research on angiogenesis and hypoxia and their role in cancer. Over the last 4 decades, the discovery of a plethora of genes, signaling cascades, and transcription factors has revealed the complexity of the angiogenic process and furthered our understanding of this hypothesis.

Inhibition of Angiogenesis for Anticancer Purposes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

Because angiogenesis is a key process in tumor growth, and a limited process in healthy adults, developing angiogenesis inhibitors is a desirable anticancer target for which few side effects might be expected. Resistance to antiangiogenesis drugs is also unlikely to occur, or at least at a much lower rate than that observed with traditional cytotoxic chemotherapeutics, particularly if the genetically stable endothelial cells (ECs) are targeted.2 Selecting specifically for tumor ECs and vessels could be achieved by targeting their unique or unusual properties. Although physiologic angiogenesis is a tightly orchestrated process that is regulated by a balance of proangiogenic and antiangiogenic factors, tumor angiogenesis is erratic and irregular, with leaky vessels that are poorly formed.3–5 The tumor ECs divide more rapidly than nontumor ECs and also express markers that the normal ECs do not express.2 Because ECs line the blood vessels, they are also much more accessible to the circulation and therefore pharmacologic treatments than are the tumor cells themselves.

Advances in our understanding of the regulatory mechanisms that govern tumor angiogenesis continue to aid in drug development, particularly with the identification of new therapeutic targets. An understanding of how both newly developed and conventional anticancer compounds function to inhibit angiogenesis will help further our understanding of how tumor angiogenesis occurs and how it might be successfully limited to halt the growth and spread of a tumor. One interesting finding is that many conventional chemotherapeutics actually possess previously unknown antiangiogeneic activity. These include cytotoxic chemotherapeutic drugs, hormonal ablation therapies, and tyrosine kinase inhibitors.6

The following review will provide a broad overview of the key mechanisms involved in tumor angiogenesis and the various inhibitors that have demonstrated promise for cancer therapy.

Process of Carcinogenesis and Subsequent Tumor Angiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

The process of transformation from a normal cell into a cancer cell involves a series of complex genetic and epigenetic changes. In an influential article, Hanahan and Weinberg proposed that 6 essential “hallmarks” or processes were required for the transformation of a normal cell to a cancer cell.7 These processes include 1) self-sufficiency in growth signals; 2) insensitivity to antigrowth signals; 3) evasion of programmed death (apoptosis); 4) endless replication potential; 5) tissue invasion and metastasis; and, importantly, 6) sustained angiogenesis.7

Initially, the growth of a tumor is fed by nearby blood vessels. Once a certain tumor size is reached, these blood vessels are no longer sufficient and new blood vessels are required to continue growth. The ability of a tumor to induce the formation of a tumor vasculature has been termed the “angiogenic switch” and can occur at different stages of the tumor progression pathway depending on the type of tumor and the environment.4 Acquisition of the angiogenic phenotype can result from genetic changes or local environmental changes that lead to the activation of ECs.

One way for a tumor to activate ECs is through the secretion of proangiogenic growth factors, which then bind to receptors on nearby dormant ECs that line the interior of vessels (Fig. 1). At the time of EC stimulation, vasodilation and permeability of the vessels increase, and the ECs detach from the extracellular matrix and basement membrane by secreting proteases known as matrix metalloproteinases. The ECs then migrate and proliferate to sprout and form new branches from the pre-existing vasculature. The growth factors can also act on more distant cells recruiting bone marrow-derived precursor ECs and circulating ECs to migrate to the tumor vasculature.4, 8

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Figure 1. Tumor Cells Secrete Proangiogenic Growth Factors That Bind to Receptors on Dormant Endothelial Cells (ECs), Leading to Vasodilation and an Increase in Vessel Permeability. The ECs migrate and proliferate to form new branches from the pre-existing vasculature by detaching from the extracellular matrix and basement membrane. PI3K indicates phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; HIF-1α, hypoxia inducible factor-1α; VEGF, vascular endothelial growth factor.

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The proangiogenic growth factors may be overexpressed because of genetic alterations of oncogenes and tumor suppressors, or in response to the reduced availability of oxygen (Fig. 2). Tumor cell expression of many of the angiogenic factors, including vascular endothelial growth factor (VEGF), is regulated by hypoxia through the transcription factor hypoxia inducible factor (HIF).9 As the tumor cells proliferate, oxygen becomes depleted and a hypoxic microenvironment occurs within the tumor. HIF is degraded in the presence of oxygen, and therefore formation of hypoxic conditions leads to HIF activation and transcription of target genes. The strongest activation of HIF results from hypoxia, but several other factors can contribute to the increased expression and activity of HIF, including growth factors and cytokines such as tumor necrosis factor-α (TNF-α),10 interleukin-1β (IL-1β),11, 10 epidermal growth factor (EGF),12, 13 and insulin-like growth factor-1 (IGF-1),14 which lead to increased cell signaling. Along similar lines, oncogenes that trigger increased expression of growth factors and overactive signaling pathways can increase HIF expression and activity. For example, mutant Ras can contribute to tumor angiogenesis by enhancing the expression of VEGF through increased HIF activity.15, 16 The oncogenes v-Src17 and HER218 and dysregulated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways12–14, 19, 20 have also been shown to up-regulate HIF expression and HIF transcriptional activity.

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Figure 2. Pathways and Mechanisms That Can Lead to Increased Angiogenesis, Including Overexpression of Growth Factors and Receptor Tyrosine Kinase Dysregulation, Are Shown. HIF indicates hypoxia inducible factor.

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Receptor Tyrosine Kinase Signaling

Receptor tyrosine kinases (RTKs) are transmembrane proteins that mediate the transmission of extracellular signals (such as growth factors) to the intracellular environment, therefore controlling important cellular functions and initiating processes such as angiogenesis. Structurally, the RTKs generally are comprised of an extracellular ligand-binding domain, a single transmembrane domain, a catalytic cytoplasmic tyrosine kinase region, and regulatory sequences. RTKs are activated through the binding of a growth factor ligand to the extracellular domain, leading to receptor dimerization and subsequent autophosphorylation of the receptor complex by the intracellular kinase domain, using ATP.21 The phosphorylated receptor then interacts with a variety of cytoplasmic signaling molecules, leading to signal transduction and eventually angiogenesis, among other processes involved in cell survival, proliferation, migration, and differentiation of ECs.21, 22

RTKs that become dysregulated can contribute to the transformation of a cell. The dysregulation can occur through several different mechanisms, including 1) amplification and/or overexpression of RTKs; 2) gain of function mutations or deletions that result in constitutively active kinase activity; 3) genomic rearrangements that produce constitutively active kinase fusion proteins; 4) constant stimulation of RTKs from high levels of proangiogenic growth factors; and 5) retroviral transduction of a deregulating proto-oncogene that causes RTK structural changes, all of which lead to increased downstream signaling.21

The complex signaling network uses multiple factors to determine the biological outcome of the receptor activation. Although the pathways are often depicted as linear pathways for simplicity, they are actually a network of pathways with various cross-talk and overlapping functions, as well as distinct functions. Some of the known signaling cascades include the phospholipase Cγ (PLCγ)-protein kinase C (PKC)-Raf kinase-mitogen-activated protein kinase kinase (MEK)-MAPK and PI3K-AKT-mammalian target of rapamycin (mTOR) pathways, and activation of the Src tyrosine kinases.23–26 A detailed overview of the individual growth factors and their RTKs is beyond the scope of this review, but some of the main factors will be briefly covered.

VEGF

VEGF and its RTK (VEGFR) play key roles in angiogenesis (Fig. 3).27–30 Although VEGF is actually a family of at least 7 members (Table 1), the term VEGF typically refers to the VEGF-A isoform, one of the most studied members and a major mediator of tumor angiogenesis. VEGF-A is a proangiogenic factor that plays important roles in cell migration, proliferation, and survival. Four spliced isoforms of VEGF-A are known (VEGF121, VEGF165, VEGF189, and VEGF206), with VEGF165 being the most predominant form.30 VEGF-A was initially identified for its ability to increase vascular permeability in guinea pigs and was termed vascular permeability factor (VPF)31; it was then separately identified for its ability to promote the growth of vascular ECs and was named VEGF.32 Cloning the VPF and VEGF genes revealed that they were actually the same.33, 34

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Figure 3. Vascular Endothelial Growth Factor (VEGF) Binds to the VEGF Receptor (VEGFR), a Receptor Tyrosine Kinase, Leading to Receptor Dimerization and Subsequent Auto Phosphorylation of the Receptor Complex. The phosphorylated receptor then interacts with a variety of cytoplasmic signaling molecules, leading to signal transduction and eventually angiogenesis. Examples of both preclinical and clinical compounds that inhibit the pathway are shown. PI3K indicates phosphoinositide 3-kinase; FARA-A, 9-β-D-arabinofuranosyl-2-fluoroadenine; mTOR, mammalian target of rapamycin; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinases.

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Table 1. Select Growth Factors and Receptors
GROWTH FACTORFUNCTION/ROLEMEMBERSRECEPTORS
  1. VEGF indicates vascular endothelial growth factor; PLGF, placental growth factors; VEGFR, vascular endothelial growth factor receptor; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; EGF, epidermal growth factor; TGF-α, tumor growth factor-α; HB-EGF, heparin-binding EGF-like growth factor; AR, amphiregulin; BTC, betacellulin; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; TGF-β, transforming growth factor-β; Ang, angiopoietin.

VEGFPhysiological blood vessel formation and pathological tumor angiogenesisVEGF-A, -B, -C, -D, and -E, and PLGF-1 and -2VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4)
PDGFCell growth and division, blood vessel formation, pericyte and smooth muscle recruitment and proliferationPDGF-A, -B, -C, and -D, which combine to form the 5 active homodimers and heterodimers: PDGF-AA, -AB, -BB, -CC, and -DDPDGF-α and -β
FGFVascular endothelial cell proliferation, migration, development, and differentiation23 members (FGF-1 through FGF-23)FGFR-1, -2, -3, and -4
EGFCell growth, proliferation, differentiation, angiogenesis, and survivalEGF, TGF-α, HB-EGF, AR, BTC, epigen, epiregulin, and neuregulins 1–4EGFR (ErbB1), HER2 (ErbB2/neu), HER3 (ErbB3), and HER4 (ErbB4)
TGF-βAngiogenesis, cell regulation and differentiation, embryonic development, wound healing, and growth inhibition propertiesTGF-β1, -β2, and -β3Type I, II, or III
AngiopoietinInitiation and progression of angiogenesis; vessel maintenance, growth, and stabilizationAng-1, -2, and -3/4TIE-1 and -2

When VEGF is secreted from tumor cells, it interacts with cell surface receptors, including VEGFR-1 and VEGFR-2, located on vascular ECs and bone marrow-derived cells. VEGFR-2 is believed to mediate the majority of the angiogenic effects of VEGF-A, whereas the role of VEGFR-1 is complex and not fully understood.30 A soluble form of VEGFR-1 can act as a decoy receptor, preventing VEGF-A from acting on VEGFR-2 and activating signaling pathways. However, there is also evidence indicating that VEGFR-1 plays an important role in developmental angiogenesis.30 A third receptor, VEGFR-3, is involved in lymphangiogenesis and does not bind VEGF-A.30

VEGF-A165 is commonly overexpressed by a wide variety of human tumors, and this overexpression has been correlated with progression, invasion and metastasis, microvessel density, and poorer survival and prognosis in patients.35–38 VEGF-A and VEGFR-2 are currently the main targets for antiangiogeneic efforts.27

Platelet-Derived Growth Factors

The family of platelet-derived growth factors (PDGFs) and receptors (PDGFRs) are involved in vessel maturation and the recruitment of pericytes.39 PDGF stimulates angiogenesis in vivo,40, 41 although to the best of our knowledge the role of PDGF in angiogenesis is not fully understood.42, 43 The family of PDGF ligands is comprised of 4 structurally related, soluble polypeptides that exist as 5 different homodimers and heterodimers (Table 1). There are 2 forms of the PDGF tyrosine kinase receptors: PDGFR-α and PDGFR-β.42 PDGF is expressed by ECs and generally acts in a paracrine manner, recruiting PDGFR-expressing cells, particularly pericytes and smooth muscle cells, to the developing vessels.43

Mutations involving up-regulation of PDGF and/or PDGFR have been described in human cancers, although to our knowledge the role of these mutations in cancer has not been fully characterized.43 Nearly all gliomas tested are positive for PDGF and PDGFR,44, 45 and overexpression of PDGFR has been associated with poor prognosis in patients with ovarian cancer,46 indicating a likely role for the PDGF pathway in human cancers.

Fibroblast Growth Factor

The mammalian fibroblast growth factor (FGF) family is comprised of 23 different proteins, which are classified into 6 different groups based on the similarity of their sequences.42, 47 The FGF ligands were among the earliest angiogenic factors reported and are involved in promoting the proliferation, migration, and differentiation of vascular ECs.48, 49 FGF ligands have a high affinity for heparin sulfate proteoglycans, which act as coreceptors by binding to both FGF and 1 of the 4 different FGF receptors (FGFRs) simultaneously.47 The FGF RTKs are widely expressed and are present on most, if not all, cell types, in which they act through a wide range of biological roles.42 FGFRs are often overexpressed in tumors, and mutations of the FGFR genes have been found in human cancers, making it particularly significant that FGFR activation in EC culture and animal models leads to angiogenesis.42, 47 Overexpression of various FGF ligands in different types of tumors has been documented.47 FGF-2 in particular has been shown to possess potent angiogenic activity,50 and is also commonly overexpressed in tumors and has been found to correlate with poor outcome in patients with non-small cell lung cancer (NSCLC) and bladder carcinoma.51, 52

EGF

The EGF family is comprised of 11 known members that bind to 1 of 4 EGF receptors (EGFRs).53 All of the receptors, except human epidermal growth factor receptor 3 (HER3), contain an intracellular tyrosine kinase domain.54 HER2 does not have any known ligands that bind with high affinity, despite it being a potent oncoprotein.53, 54 Activation of EGFR has been linked to angiogenesis in xenograft models,55 in addition to metastasis, cell proliferation, survival, migration, transformation, adhesion, and differentiation.54 Because activation of the EGFR pathway up-regulates the production of proangiogenic factors such as VEGF, it can be viewed as more of an indirect regulator of angiogenesis rather than a direct regulator, making the role of the EGF/EGFR system less important to angiogenesis than more direct regulators, such as the VEGF and PDGF systems.53, 54, 56, 57

Other Angiogenic Factors

Transforming Growth Factor-β

Transforming growth factor-β (TGF-β) and corresponding receptors are produced by nearly every cell type, although each of the 3 isoforms of TGF-β (TGF-β1, TGF-β2, and TGF-β3) demonstrates a different tissue expression pattern.58 TGF-β participates in angiogenesis, cell regulation and differentiation, embryonic development, and wound healing, and also has potent growth inhibition properties.58 The TGF-β receptors are classified as type I, II, or III. Type I and II receptors contain serine/threonine kinase domains in their intracellular protein regions, whereas type III does not possess kinase activity but is believed to participate in transferring TGF-β ligands to type II receptors.58 TGF-β ligands bind to and stimulate type II receptors that recruit, bind, and phosphorylate type I receptors, activating downstream signaling proteins known as SMADs, which are believed to be specific to the TGF-β family. Activated SMADs eventually move to the nucleus, where they can interact with different transcription factors, regulating gene expression in a cell-specific manner.58 SMADs have been found to be mutated at a high rate in pancreatic and colorectal cancer, and are found in other cancers as well, indicating that SMAD mutations and aberrant regulation likely contribute to the development of cancers.59 In addition to the SMADs, signaling mediated by TGF-β can involve activation of downstream targets such as MAPK and PI3K.59

TGF-β is believed to have both proangiogenic and antiangiogenic properties, depending on the levels present. Low levels of TGF-β contribute to angiogenesis by up-regulating angiogenic factors and proteases, whereas high doses of TGF-β stimulate basement membrane reformation, recruit smooth muscle cells, increase differentiation, and inhibit EC growth.8 Tumor cells can also become resistant to TGF-β and will no longer respond to the growth-inhibiting properties, leading to tumor cell proliferation. Tumors that no longer respond to the growth inhibition signals from TGF-β can then exploit the abilities of TGF-β to regulate processes involved in angiogenesis, cell invasion, and tumor cell interactions.60

Overexpression of TGF-β1 has been reported in gastric, breast, colon, hepatocellular, lung, and pancreatic cancers and is correlated with tumor angiogenesis in addition to metastasis, disease progression, and poor prognostic outcome.61 High levels of endoglin, part of the TGF-β receptor complex, have also been detected in cancers and are associated with tumor metastasis.60

Angiopoietins and TIE Receptors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

The angiopoietin ligands and TIE RTKs play a regulatory role in vascular homeostasis and maintenance of quiescent ECs in adults and are also an essential component of embryonic vessel assembly and maturation.62 The angiopoietin (Ang) family of ligands (Ang-1, Ang-2, and Ang-3/-4) bind to the RTK TIE-2. To the best of our knowledge, no ligand has been identified for the TIE-1 receptor.62–64 Ang-1 behaves as an agonist, activating the TIE-2 receptor, whereas Ang-2 acts as an antagonist for TIE-2,65 although the role of Ang-2 in tumor angiogenesis is not fully understood and appears to be dependent on the environmental context. In the presence of VEGF-A, Ang-2 will promote angiogenesis, and in the absence of VEGF-A, Ang-2 will cause vessel regression.66, 67 Overexpression of Ang-2 has been found to correlate with increased angiogenesis, malignancy, and aggressive tumor growth in some cancers, and overexpression has reportedly led to decreased tumor growth and metastasis and vessel regression in other tumor types.62–64 The involvement of additional factors in Ang-2 function are likely the cause of conflicting data concerning Ang-2, indicating the need for further studies of the Ang/ TIE system.

Ang-1 overexpression leads to vasculature that is more mature and normal in appearance, explaining the vessel-normalization effect that results from anti-VEGF/VEGFR therapies because these effects are mediated through Ang-1.68 Most studies to date have shown that Ang-1 possesses mostly antitumorgenic effects, although some have indicated that Ang-1 can stimulate tumor growth.62

Although the angiopoietins and TIE receptors appear to play an important role during tumor angiogenesis, the specific mechanisms are controversial. A further understanding of the specific roles of the members of the Ang/TIE system may enable targeting of this system for antiangiogenic and anticancer purposes.

Attempts at targeting the TIE-2 pathway for angiogenesis inhibition have had more difficultly than some of the other angiogenesis targets, such as VEGF, in part because of the lack of understanding with regard to the agonistic and antagonistic roles of Ang-1 and Ang-2 on the TIE-2 receptor. However, there have been some efforts, and peptide-antibody fusions that bind and neutralize Ang-2 have been shown to decrease tumor growth and angiogenesis and suppress EC proliferation in preclinical models,69 demonstrating the feasibility of targeting Ang/TIE for antiangiogenic purposes.

Delta/Jagged-Notch Signaling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

The family of Notch receptors (Notch1-4) and their transmembrane ligands Delta-like (Dll1, Dll3, and Dll4) and Jagged (Jagged1 and Jagged2) play important roles in cells undergoing differentiation, acting primarily to determine and regulate cell fate, as well as playing a part in developmental and tumor angiogenesis. In healthy mice, Dll4 is required for normal vascular development and arterial formation, whereas in tumor angiogenesis, Dll4 and Notch signaling appear to play a role in regulating the cellular actions of VEGF.70, 71

Activation of Notch signaling is dependent on cell-to-cell interactions and occurs when the extracellular domain of the cell surface receptor interacts with a ligand found on a nearby cell. Lateral inhibition, which is one mechanism of Notch signaling, involves binding of a Notch ligand to a Notch receptor on an adjacent cell, which results in activation of the Notch signaling pathway in one cell and suppression in the other cell, resulting in 2 different fates for each cell.70 Notch receptors also participate in transcriptional regulation through a unique mechanism involving cleavage of the intracellular domain of the Notch receptor, which then translocates to the nucleus, in which it can participate in transcriptional regulation.71

Dll4 and Jagged1 in particular have been implicated in tumor angiogenesis, with strong expression of Dll4 noted in the endothelium of tumor blood vessels and much weaker expression observed in nearby normal blood vessels.72–75 The expression of Dll4 appears to be regulated directly by VEGF in the setting of tumor angiogenesis; increased levels of VEGF lead to increased expression of Dll4.75, 76 Dll4 then signals to the Notch receptor-expressing ECs to down-regulate VEGF-induced sprouting and branching.77 In this manner, Dll4 acts as a negative modulator of angiogenesis, regulating excessive VEGF-induced vessel branching, allowing vessel formation to occur at a productive and efficient rate.77 Overexpression of Jagged1, a Notch ligand, is dependent on MAPK signaling78 and has been associated with angiogenic ECs in vitro.79 Jagged1 is believed to promote angiogenesis, because overexpression in head and neck squamous cell carcinoma (SCC) cells leads to increased vascularization and tumor growth.78

Attempts to manipulate Notch signaling for anticancer purposes have been studied to date, particularly through inhibition of Dll4. It is interesting to note that inhibition of Dll4 leads to an increase in tumor vascular density; this increase is likely because of the lack of down-regulation of branching and sprouting caused by Dll4.74, 80 However, although an increase in vascularity is observed, the vascular network is very poorly formed and essentially nonfunctional (even more so than typical disorganized tumor vasculature), and a significant decrease in tumor size has been observed.74, 80 The decrease in tumor size was noted even in tumor models that are resistant to VEGF blockade, making inhibition of this pathway an attractive alternative for tumors that become resistant to VEGF inhibitors used in the clinic.74, 80 When Dll4 inhibition was combined with VEGF inhibition in tumors with no resistance, additional antitumor activity was observed compared with inhibition of either factor alone.80

Inhibition of Jagged1 has also been studied. Knockdown of Jagged1 expression in SCC cells was noted to inhibit proangiogenic effects of the cells in vitro, even when the cells were stimulated with growth factors.78 Another study examined inhibition of Notch receptor function, using a soluble Notch1 receptor decoy that prevented Dll1, Dll4, and Jagged1 from binding to Notch receptors.81 The decoy blocked angiogenesis in both in vitro and in vivo models, as well as causing a decrease in tumor growth using mammary xenografts.81

Inhibition of specific components of the Notch signaling pathway, such as Dll4 or Jagged1, or more broad inhibition of Notch signaling may prove to be effective for inhibiting functional angiogenesis, and neovascularization in tumors and some of the preclinical studies appear promising. However, further studies are needed to better understand the role that Notch signaling and its individual components play in tumor angiogenesis before these pathways can be exploited for clinical use.

HIF

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

HIF is a transcription factor involved in cellular adaptation to hypoxia. HIF transcriptional activity is regulated by the presence of oxygen and becomes active in low oxygen conditions (hypoxia). HIF controls a large number of genes involved in angiogenesis.9, 82 The active HIF complex is comprised of an α and β subunit in addition to coactivators including p300 and CREB binding protein (CBP). The HIF-β subunit (also known as ARNT) is a constitutive nuclear protein with additional roles in transcription not associated with HIF-α. In contrast to HIF-β, the levels of the HIF-α subunits and their transcriptional activity are regulated by oxygen availability.

There are 3 related forms of human HIF-α (HIF-1α, HIF-2α, and HIF-3α), each of which is encoded by a distinct genetic locus. HIF-1α and HIF-2α have been the best characterized, possessing similar domain structures that are regulated in a related manner by oxygen, although each isoform does have distinct and separate roles. To our knowledge, the role of HIF-3α is not fully understood, although a truncated form of murine HIF-3α known as inhibitory Per/Arnt/Sim (PAS) domain protein (IPAS) has been found to act as an inhibitor of HIF via dimerization with HIF-β.83

Both the HIF-α and HIF-β subunits are produced constitutively, but in normoxia HIF-1α and HIF-2α are degraded by the proteasome in an oxygen-dependent manner. Hydroxylation of 2 prolines in HIF-α enables HIF-α to bind to the von Hippel-Lindau tumor suppressor protein (pVHL), which links HIF-α to a ubiquitin ligase complex. The ubiquitin ligase catalyzes polyubiquitinylation of HIF-α, targeting it for degradation by the proteasome. In addition, hydroxylation of an asparagine residue in HIF-α disrupts the interaction between HIF-α and the coactivator p300, through a process independent of proteasomal degradation, which leads to reduced HIF transcriptional activity. In this manner, asparaginyl hydroxylation acts as a regulatory switch controlling the activity and specificity of HIF gene expression, as opposed to the prolyl hydroxylations, which control HIF-α stability.83, 84 In hypoxia, minimal to no hydroxylation occurs, enabling HIF-α to avoid proteasomal degradation and dimerize with HIF-β and coactivators, forming the active transcription complex on the hypoxia response element (HRE) associated with HIF target genes (Fig. 4).

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Figure 4. Both the Hypoxia Inducible Factor (HIF)-α and HIF-β Subunits Are Produced Constitutively, But in Normoxia, the α Subunit Is Degraded by the Proteasome in an Oxygen-Dependent Manner. Hydroxylation of 2 prolines in HIF-α enables HIF-α to bind to the von Hippel-Lindau tumor suppressor protein (pVHL), which links HIF-α to a ubiquitin ligase complex. The ubiquitin ligase catalyzes polyubiquitinylation of HIF-α, targeting it for degradation by the proteasome. In addition, hydroxylation of an asparagine residue in HIF-α disrupts the interaction between HIF-α and the coactivator p300 through a process that is independent of proteasomal degradation, which leads to reduced HIF transcriptional activity. Hypoxic conditions prevent hydroxylation of the α subunit, enabling the active HIF transcription complex to form at the hypoxia response element (HRE) associated with HIF-regulated genes. PHD, prolyl hydroxylase; FIH, factor inhibiting HIF; OH, hydroxylated amino acid residues.

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Because HIF regulates genes that enable cell survival in a hypoxic environment, including those involved in glycolysis, angiogenesis, and expression of growth factors, it has significance in the biology and regulation of tumor growth. The central role of HIF in the activation of angiogenic-related genes makes it a promising target for the treatment of solid tumors, particularly because HIF-1α and/or HIF-2α is reported to be overexpressed in the majority of solid tumors.85, 86 HIF-1α (and occasionally HIF-2α) overexpression in tumors has been found to be positively correlated with angiogenesis, aggressiveness, metastasis, and resistance to radiation/chemotherapy and negatively correlated with disease progression, survival, and outcome.87–94

Antiangiogenesis Compounds

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

Fumagillin and TNP-470

The antiangiogenic activity of fumagillin was discovered when a dish of cultured ECs was accidentally contaminated with the fungus Aspergillus fumigatus Fresenius, a fumagillin-producing organism.95 The contaminated ECs stopped proliferating but demonstrated no outward signs of toxicity.95 After isolating fumagillin as the source of the activity, a series of synthetic analogues were produced that also inhibited EC growth and proliferation without cytotoxic effects in vitro, and limited tumor-induced angiogenesis in xenograft models.95 The most potent of the analogues, known as TNP-470, was found to be a significantly more potent antiangiogenic compound than fumagillin in 4 different angiogenesis assays.96 To the best of our knowledge, the means by which fumagillin and TNP-470 exert their antiangiogenic properties are not fully understood. Several mechanisms have been proposed, including inhibition of methionine aminopeptidase97 through covalent modification of a histidine98 and prevention of endothelial activation of Rac1.99 TNP-470 also affects the cell cycle through activation of p53, leading to an increase in the G1 cyclin-dependent kinase inhibitor p21CIP/WAF and subsequent growth arrest.100, 101

TNP-470 demonstrates broad-spectrum anticancer activity in animal models,102 and to our knowledge was one of the first antiangiogenesis drugs to undergo clinical trials.103 In early clinical trials, TNP-470 demonstrated antitumor activity as a single agent, causing tumor progression to slow or even regress in SCC of the cervix,104, 105 as well as demonstrating activity in combination with paclitaxel and carboplatin.106 Although TNP-470 appeared promising in terms of anticancer activity, it presented several obstacles to its further clinical development and use. The primary hurdles included dose-limiting toxicities and a very short plasma half-life.107, 108 The toxicities observed were mainly neurological, including problems with motor coordination, short-term memory and concentration, dizziness, confusion, anxiety, and depression.107, 109 Later studies indicated that neurological symptoms could be eliminated by conjugating TNP-470 to a polymer, thus preventing the drug from penetrating the blood-brain barrier.110 However, this formulation still had a short half-life and could not be administered orally.110

To improve on its short half-life and oral availability, TNP-470 was conjugated to a di-block copolymer, monomethoxy-polyethyleneglycol-polylactic acid (mPEG-PLA). The polymeric drug is amphiphilic, causing self-assembly into micelles with the TNP-470 tails in the center.102 The micelle formulation improved the properties of TNP-470 in several ways. The micelles protected TNP-470, particularly from the acidic environment of the stomach, making the new formulation, named lodamin, orally available.102 In addition, the micelles increased the half-life because the micelles hydrolyze over time, allowing for the slow release of lodamin. This property allowed the accumulation of lodamin in tumor tissue (likely due to the permeability of the tumor vasculature), but still prevented penetration of the blood-brain barrier, effectively overcoming several of the major hurdles to the clinical use of TNP-470.102 One interesting observation was that particularly high concentrations of lodamin accumulated in the liver, and therefore lodamin may prove to be especially effective against primary liver cancer or metastases within the liver.102 The preclinical results of lodamin have been promising to date and warrant further investigation. Additional studies on the safety of lodamin in nonhuman primates may lead to clinical trials in the future. Furthermore, second-generation conjugated TNP-470 is currently in preclinical development.

Thalidomide

Thalidomide was initially marketed as a safe, nontoxic sedative and antiemetic in the 1950s in Europe, Australia, Asia, and South America (but was not approved by the US Food and Drug Administration [FDA] because of safety concerns).111 In countries in which it was approved for use, thalidomide became a popular treatment for pregnancy-related morning sickness until 1961, when 2 groups of physicians, McBride from Australia112 and Lenz et al from Germany,113 noted the link between severe limb and other birth defects in babies whose mothers had taken thalidomide during pregnancy. The drug was rapidly removed from the market in Europe in 1961 and from Canada in 1962 due to its previously unknown teratogenic effects.111

Thalidomide was rediscovered in 1965 as a useful treatment of erythema nodosum leprosum (ENL), due to its immunomodulatory and anti-inflammatory properties; however, it did not actually obtain FDA approval for use in the treatment of ENL until 1998.111 The idea of using thalidomide for cancer treatment occurred when its antiangiogenic properties were discovered.

Studies of the effects of thalidomide suggested that the birth defects of the limbs could be caused by inhibition of blood vessel growth in the limb buds of a developing fetus.114 Thalidomide displayed antiangiogenic properties in a rabbit cornea micropocket model, although it is interesting to note that thalidomide did not inhibit angiogenesis in a chicken chorioallantoic membrane (CAM) assay.114 It was proposed that the teratogenic activity of thalidomide may be the result of one of the many metabolites of the parent thalidomide model, explaining the lack of activity in the CAM assay.114, 115 Bauer et al examined this proposal using liver microsomes coincubated with thalidomide in angiogenesis assays.116 Coincubation with human or rabbit liver microsomes led to potent antiangiogenic activity, demonstrating that a metabolite of thalidomide is responsible for the antiangiogenic activity, and that the metabolite is not produced by rodents.116

Although to our knowledge the mechanism of thalidomide (and metabolites) is not fully understood, some of its properties and activities are beginning to be deciphered. Thalidomide inhibits the synthesis of TNF-α in monocytes, microglia, and Langerhans cells, which provides thalidomide with its anti-inflammatory properties.111, 117 The immunomodulatory and anti-inflammatory effects of thalidomide likely contribute to its antiangiogenic effects and add to its anticancer activity. Studies of thalidomide in rabbits, an animal species susceptible to thalidomide's teratogenic effects, noted that it caused inhibition of mesenchyme proliferation in the developing limb bud of a fetus,118 embryonic DNA oxidation, and teratogenicity.119

Unfortunately, the preclinical animal studies of thalidomide that led to its widespread use in humans throughout the world used rodents, which are resistant to the teratogenic effects of thalidomide. The human tragedies that resulted highlight the importance of selecting correct animal models, as well as testing in different species when examining new treatments for clinical use.119

Thalidomide analogues were developed with the goal of improving inhibition of TNF-α, leading to lenalidomide and pomalidomide, the latter of which is 50,000-fold more potent than thalidomide at suppressing endotoxin-induced TNF-α secretion in cell models.117 Lenalidomide and pomalidomide also lack some of the side effects noted with thalidomide, such as constipation, peripheral neuropathy, and the sedative effects.111, 117 In 2006, lenalidomide was approved by the FDA for use in patients with recurrent multiple myeloma in combination with dexamethasone, whereas pomalidomide is currently in clinical development.120

Despite the previous damage thalidomide caused (and even, perhaps due to thalidomide's mechanism of action that led to birth defects), thalidomide and analogues are currently in development for antiangiogenic and anticancer use. Although the complex metabolism of thalidomide causes many challenges to its the development, initial results with analogues appear to indicate that structural alterations can change the side effect profile, potentially eliminating them, while still maintaining the desired anticancer activity and demonstrating promising results in clinical trials.

Inhibitors of Growth Factors, RTKs, and Signaling Pathways

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

Because growth factors stimulate ECs, leading to angiogenesis, targeting the growth factors, receptors, and subsequent signaling cascades make for promising targets in angiogenesis inhibition. The growth factors and RTKs are particularly attractive targets because it is possible to target them in the extracellular environment, removing drug development hurdles such as permeability of the cellular membrane. Significant progress in targeting these pathways has been made, and several drugs have been approved by the FDA or are currently in clinical development.

Growth Factor Inhibitors

Bevacizumab is a humanized monoclonal antibody that binds to VEGF-A, preventing it from binding to receptors and activating signaling cascades that lead to angiogenesis. Initial proof of the concept that targeting VEGF-A could inhibit the growth of tumors (despite its having no effect on the growth rate of the tumor cells in vitro) was demonstrated in a mouse model in 1993 using a monoclonal antibody against VEGF-A,121 leading to the clinical development of bevacizumab.

Initial clinical trials in patients with colorectal cancer tested irinotecan, fluorouracil (5-FU), and leucovorin with or without bevacizumab.122 The addition of bevacizumab significantly increased the progression-free survival (PFS), as well as the median overall survival (OS),122 leading to FDA approval of bevacizumab as the first drug developed solely for antiangiogenesis anticancer use in humans.

The anticancer activity of bevacizumab across all tumor types has demonstrated some mixed results. Bevacizumab did not provide any benefit with regard to PFS or OS for patients with metastatic breast cancer when used in combination with capecitabine.123 Further studies in a phase 3 trial of patients with previously untreated metastatic breast cancer using paclitaxel with or without bevacizumab demonstrated that the addition of bevacizumab increased PFS (11.8 months vs 5.9 months without bevacizumab) and increased the overall response rates (36.9% vs 21.2% without bevacizumab).124 However, there was still no significant increase noted in OS, as had been observed previously with colorectal cancer122 and NSCLC.125, 126 A beneficial response may be masked by the lack of biomarker screening in patients in many of the clinical trials, because bevacizumab is specific for VEGF. By screening for tumors that overexpress VEGF and/or are highly dependent on VEGF signaling, the likelihood of a positive response to bevacizumab would most likely be increased. Targeted therapies may prove more effective when patients are screened for markers, ensuring that the proper subset of the population is treated with a particular targeted drug.

Bevacizumab is currently being tested in several hundred clinical trials in a variety of different tumor types127 and as of 2009, bevacizumab was approved for various indications in colorectal cancer, NSCLC, breast cancer, renal cell carcinoma (RCC), and glioblastoma.

Aflibercept (VEGF-Trap)

Aflibercept (VEGF-Trap, AVE0005) is a soluble fusion protein of the human extracellular domains of VEGFR-1 and VEGFR-2 and the Fc portion of human immunoglobulin (Ig) G. Aflibercept binds to both VEGF-A and PlGF with a higher affinity than monoclonal antibodies and essentially renders the VEGF-A and PlGF ligands unable to bind and activate cell receptors.128 Aflibercept was engineered to optimize pharmacokinetic properties while still maintaining the potent VEGF blocking activity compared with that demonstrated by other anti-VEGF antibodies. In vitro, aflibercept demonstrated significant antiproliferative activity and completely blocked VEGF-induced VEGFR-2 phosphorylation when added in a 1.5-M excess of VEGF.128 Aflibercept inhibited tumor growth in xenograft models and blocked nearly all tumor-associated angiogenesis, resulting in tumors that appeared nearly avascular.128

Aflibercept is currently in clinical trials, with some early results reported. In phase 2 trials in which it was used as a single agent in patients with ovarian cancer, 41% of patients had stable disease at 14 weeks.129 In addition, a reduction of 30% or more in tumor size was noted in 8% of patients.129 Another phase 2 trial of aflibercept in 33 patients with NSCLC demonstrated 2 partial responses; to our knowledge, interim analysis results are not yet available.130 In contrast, a phase 2 trial of aflibercept in patients with metastatic breast cancer demonstrated a response rate of 5% and the PFS rate at 6 months was 10%, rates that did not meet efficacy goals and were determined to be too low to continue.131 Additional clinical trials of aflibercept are currently ongoing in a variety of malignancies including prostate cancer, colorectal cancer, ovarian cancer, thyroid cancer, RCC, and brain cancer.

RTK Inhibitors

There is a wide range of RTK inhibitors currently in all stages of development (preclinical, clinical, and FDA-approved). RTK inhibitors are particularly useful in treating cancer because of the dual roles they inhibit; both oncoprotein signal transduction and the downstream angiogenic processes are blocked. They also often target more than one type of receptor and affect both ECs and cancer cells because the receptors are expressed on both types of cell.3 Because the target kinase specificity between inhibitors can vary, different compounds have demonstrated various levels of efficacy and activity between cancers, as well as different side effect profiles. Several approaches to targeting the growth factors and receptors have been undertaken; some of these include compounds that bind to the ATP binding site of the RTK, blocking receptor activation, or with antibodies that bind to the growth factors or their receptor, preventing binding and subsequent receptor activation.

Sunitinib (SU11248) is an orally available compound that inhibits the VEGFR, PDGFR, Flt-3, c-kit, RET, and colony-stimulating factor (CSF)-1R receptor tyrosine kinases.132 In a phase 3 clinical trial in patients with metastatic RCC, sunitinib was compared with interferon-α (IFN-α), with sunitinib providing a statistically significant improvement in both the median PFS (47.3 weeks for sunitinib vs 24.9 weeks for IFN-α) and the objective response rate (24.8% vs 4.9%).133 In addition, the interim analysis of a phase 3 trial of sunitinib in patients with gastrointestinal stromal tumors (GIST) revealed a significantly longer time to disease progression (27.3 weeks vs 6.4 weeks for placebo), and at 22 weeks, stable disease was noted in 17.4% of patients treated with sunitinib versus 1.9% of patients receiving placebo.134, 135 A partial response was noted in 6.8% of patients treated with sunitinib versus 0% of patients treated with placebo.134, 135 The results from these trials led to FDA approval of sunitinib in 2006 for GIST and advanced metastatic RCC. Further studies and clinical trials of sunitinib are currently being conducted in additional cancers.

Sorafenib (BAY 43-9006) is an oral inhibitor of the intracellular Raf kinase (B-Raf, C-Raf), therefore targeting the MAPK and Raf/MEK/extracellular signal-regulated kinases (ERK) signaling pathways. Sorafenib also inhibits VEGFR (VEGFR-2 and VEGFR-3), PDGFR-β, and c-kit.136 In most tumor cell lines (colon, pancreatic, and breast), but not all (NSCLC), sorafenib potently inhibited the Raf kinase, and blocked phosphorylation of ERK 1/2, an indicator of MAPK pathway blockade.137 Sorafenib was also shown to possess significant antiangiogenesis activity in vitro.137

A phase 2 trial of sorafenib in patients with advanced RCC demonstrated an increased PFS rate after 12 weeks (50% with sorafenib vs 18% with placebo) and a significantly increased median PFS (23 weeks vs 6 weeks with placebo).138 In a phase 3 trial of RCC (769 patients), sorafenib was found to increase the median PFS from 12 weeks with placebo to 24 weeks and increased the PFS rate after 12 weeks (79% vs 50% with placebo).139 Sorafenib was subsequently approved by the FDA for the treatment of RCC in 2005 and for the treatment of unresectable hepatocellular carcinoma in 2007.

Semaxanib (SU5416) was to our knowledge the first tyrosine kinase inhibitor tested in humans and is an inhibitor of VEGFR.140 Semaxanib was tested in combination with 5-FU and leucovorin compared with 5-FU and leucovorin alone in a phase 3 trial in metastatic colorectal cancer (737 patients).141 The addition of semaxanib did not appear to improve clinical outcome and additional toxicities were noted in the semaxanib arm, including an increased risk of hematological and thromboembolic events.141 Semaxanib in combination with cisplatin and gemcitabine also was found to have unacceptable toxicity associated with it, particularly severe thromboembolic events,142 leading the clinical development of semaxanib to be stopped.5

Erlotinib (OSI-774) is an oral inhibitor of the EGFR/HER1 RTK. Erlotinib is believed to exert anticancer activity at least partially through the inhibition of expression of proangiogenic factors.143 Although phase 3 clinical trials reported some mixed results, with 2 trials indicating no benefit in treating previously nontreated NSCLC patients with chemotherapy and erlotinib,144, 145 one trial in NSCLC patients who had failed chemotherapy treatment demonstrated that erlotinib provided an increase in PFS (2.2 months vs 1.8 months with placebo), median duration of response (7.9 months vs 3.7 months with placebo), response rate (8.9% vs less than 1% with placebo), and overall survival (6.7 months vs 4.7 months with placebo).146 A phase 3 trial of erlotinib in combination with gemcitabine in patients with pancreatic cancer demonstrated significantly improved survival.147 The results of these trials led to erlotinib being approved by the FDA for NSCLC patients who have failed chemotherapy and for pancreatic cancer patients when used in combination with gemcitabine.

Cediranib, pazopanib, vandetanib, lapatinib, and motesanib are examples of additional RTK inhibitors that are currently in clinical trials for a variety of cancers (Table 2).148

Table 2. Examples of Kinase Inhibitorsa
INHIBITOROTHER NAMESINHIBITS
  • VEGFR indicates vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; CSF-1, colony-stimulating factor-1.

  • a

    Please note list is not exhaustive.

AxitinibAG013736VEGFR, PDGFR, and c-kit
CanertinibCI-1033EGFR, HER2, HER3, and HER4
CediranibRecentin, AZD2171VEGFR, PDGFR-β, and c-kit
DasatinibSprycel, BMS-354825Abl, Src, and Tec
ErlotinibTarceva, OSI-774EGFR/HER1
GefitinibIressaEGFR/HER1
ImatinibGleevec, STI571Abl, PDGFR, and c-kit
LapatinibTykerb, GW-572016EGFR and HER2
LeflunomideArava, SU101PDGFR (EGFR and FGFR)
MotesanibAMG 706VEGFR, PDGFR, and c-kit
NeratinibHKI-272EGFR and HER2
NilotinibTasignaAbl, PDGFR, and c-kit
PazopanibArmala, GW786034VEGFR, PDGFR-α and -β, and c-kit
RegorafenibBAY 73-4506VEGFR-2 and Tie-2
SemaxinibSU5416VEGFR
SorafenibNexavar, BAY 43-9006Raf, VEGFR-2 and -3, PDGFR-β, and c-kit
SunitinibSutent, SU11248VEGFR, PDGFR, Flt-3, c-kit, RET, and CSF-1R
TandutinabMLN518, CT53518PDGFR, Flt-3, and c-kit
Toceranib  
VandetanibZactima, ZD6474VEGFR-2, PDGFR-β, EGFR, and RET
VatalanibPTK787VEGFR, PDGFR-β, and c-kit

Imatinib (STI571) inhibits the cytoplasmic and nuclear protein tyrosine kinase, Abl, as well as the RTK PDGFR and c-kit.149 Imatinib was the first commercially available small molecule tyrosine kinase inhibitor and has been used extensively in the treatment of chronic myelogenous leukemia (CML) because the molecular pathogenesis of CML involves the Bcr-Abl protein and deregulated tyrosine kinase activity.149 Imatinib is reported to demonstrate antiangiogenesis activity in vitro, which is believed to occur through inhibition of PDGFR.150, 151

Monoclonal Antibodies Directed at EGFR

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

Cetuximab and panitumumab (ABX-EGF) are indirect RTKs, using a different approach than the above small molecule inhibitors. Cetuximab is a chimeric monoclonal antibody that binds to the inactive form of EGFR on the extracellular domain.101 Cetuximab essentially prevents the ligand from being able to bind to the receptor and therefore any downstream signaling activation.152 It received accelerated FDA approval in 2004 for use in patients with EGFR-expressing metastatic colorectal cancer in combination with irinotecan or as a single agent for irinotecan-intolerant patients, after cetuximab demonstrated activity in clinical trials as a single agent and even more activity when used in combination with irinotecan.153–155 Cetuximab was subsequently tested in combination with radiotherapy for the treatment of patients with unresectable head and neck SCC and indicated that the addition of cetuximab to radiotherapy significantly increased median survival compared with radiotherapy alone,156 leading to additional FDA approval for this use. With regard to the antiangiogenic activity of cetuximab, studies have shown that EGF/EGFR inhibitors appear to cause a reduction in the synthesis of proangiogenic cytokines, rather than a direct inhibition of angiogenesis.55 Panitumumab is also a monoclonal antibody that binds to EGFR, inhibiting the phosphorylation and activation of EGFR-associated kinases.151

PI3K/AKT/mTOR Pathway Inhibitors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

PI3K signaling contributes to many cell processes, including angiogenesis, cell proliferation, survival, and motility, and is initiated by RTK activation.23, 24 Up-regulation of the PI3K pathway can increase angiogenesis through multiple pathways, including increasing the levels of HIF-1α under normoxic conditions.12–15, 18–20

Initial evidence that PI3K and AKT were involved in the regulation of angiogenesis in vivo was obtained when constitutively active PI3K and AKT were shown to induce angiogenesis and increase levels of VEGF.12 Cancer genome studies have highlighted the importance of this finding, demonstrating that components of the PI3K pathway are often mutated in human cancers, increasing the likelihood of inhibitors of this pathway demonstrating efficacy in the clinic.24

Inhibitors of the PI3K pathway have been found to decrease tumor angiogenesis and demonstrate HIF inhibition, including LY294002 and wortmannin, 2 compounds that directly inhibit members of the PI3K family.11–15, 20 Both LY294002 and wortmannin demonstrated unacceptable levels of toxicity in animals and therefore were not developed clinically. However, a wortmannin analogue, PX-866, and a conjugate version of LY294002 are both currently being tested in phase 1 trials.24 9-β-D-arabinofuranosyl-2-fluoroadenine (FARA-A) is a nucleoside analogue that causes DNA damage in S-phase cells and has been found to inhibit AKT, thereby inhibiting the expression of HIF-1α and VEGF.157 Perifosine is a lipid-based phosphatidylinositol analogue that inhibits AKT by preventing translocation of AKT to the cell membrane.24 Perifosine is currently in clinical trials for several different cancers. Rapamycin (sirolimus) acts as an inhibitor of mTOR and was initially used as an immunosuppressive agent.25 Rapamycin and analogues including temsirolimus (CCI-779) and everolimus (RAD001) block tumor angiogenesis in vivo, in addition to inhibiting tumor growth.19, 158–161 The blocked angiogenesis is believed to be due at least partially to the inhibition of HIF-1α caused by the inhibition of mTOR.19, 158–160 Although rapamycin inhibits HIF-1α in vitro, to our knowledge it is unknown to what degree the decrease in HIF-1α actually affects antitumor activity, because the PI3K/AKT/mTOR pathway plays a role in many cell processes and the antitumor activity may stem from acting on multiple downstream targets.

Clinical trials of temsirolimus and everolimus as single agents demonstrated improved survival in patients with advanced RCC, leading to FDA approval for this indication. Results of activity in other tumors have initially indicated mixed results and are being tested further.25, 24

MAPK-Farnesyltransferase Rho and Ras Inhibitors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

The MAPK signaling pathway is another pathway that can lead to increased angiogenesis and increased levels of HIF-1α, making it a logical target for antiangiogenesis. One approach has been to inhibit Ras and Rho, which are activators of the MAPK pathway. During Ras activation, a farnesyl group is transferred onto a cysteine residue in the C-terminal end of Ras, enabling Ras to interact with intracellular membranes via the farnesyl group.26 Without farnesylation, Ras can no longer interact with regulatory and effector molecules in the cell membrane and no MAPK pathway activation occurs. Ras is also involved in stabilizing HIF-1α and targeting Ras has been shown to destabilize HIF-1α and decrease HIF transcriptional activity.15, 20 Two farnesyltransferase inhibitors are tipifarnib (R115777)162 and lonafarnib (SCH66336).163 To the best of our knowledge, tipifarnib has been the most studied farnesyltransferase inhibitor to date, with antiangiogenic, antiproliferative, and proapoptotic activity demonstrated in preclinical studies.164, 165 However, clinical trials of tipifarnib in multiple cancers failed to demonstrate significant anticancer activity.162 It remains to be determined whether inhibition of farnesylation will be an effective anticancer strategy. Sorafenib, mentioned previously in the section regarding tyrosine kinase inhibitors, also acts on the MAPK pathway through inhibition of Raf.136

IFN-α

IFN-α was first discovered to have antiendothelial activity in 1980, when experiments indicated that it inhibited the motility of ECs in vitro166 and inhibited angiogenesis in vivo.167, 168 Low doses of IFN-α have been shown to down-regulate FGF expression in cancer cells169 and is most likely one of the mechanisms behind the antiangiogenic effects of IFN-α. In 1989, IFN-α was first used in humans to treat a hemangioendothelioma. After a low-dose daily treatment for 7 months, complete regression of lesions and symptoms occurred.170 These results led to the successful treatment of hemangiomas in infants using IFN-α,171 in addition to the successful treatment of patients with angioblastomas and giant cell tumors.172–174

2-Methoxyestradiol

2-Methoxyestradiol (2ME2) is a human metabolite of estradiol that inhibits tubulin polymerization, destabilizing the microtubules and causing cell cycle arrest.175 2ME2 has also been found to decrease HIF-1α protein levels by acting at the translational level without affecting rates of HIF-1α gene transcription or HIF-1α proteasomal degradation through a mechanism dependent on the microtubule disrupting properties of 2ME2.175 2ME2 possesses potent antiangiogenic and proapoptotic properties and inhibits cell proliferation and migration both in vitro and in vivo.176 The antiangiogenic properties of 2ME2 appear to come from both direct inhibition of ECs and inhibition of HIF-1α.175 In clinical trials, little antitumor activity was observed in breast and prostate cancers, which may be due to the short half-life and poor bioavailability of 2ME2.177 Reformulation has improved on bioavailability, although the half-life is still suboptimal.177 The development of analogues with improved properties may increase the efficacy and potential clinical use of 2ME2. A more thorough understanding of the targets which 2ME2 acts on and the relation between microtubules and HIF-1α may provide new therapeutic avenues.

HIF Pathways and Binding Partners

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

The central role of HIF in the transcriptional activation of genes under hypoxic conditions, including those involved in angiogenesis and cell proliferation, makes it a promising target for cancer treatment. There is good evidence that treatments targeting HIF, or components of the HIF regulatory pathway, will result in a physiological effect in humans.178 Studies regarding some of the different approaches focusing directly on HIF and its binding partners are discussed in the following section (Fig. 5).

thumbnail image

Figure 5. Examples of Inhibitors Known to Act on Hypoxia Inducible Factor (HIF) and/or HIF Regulatory Pathways Are Shown. HIF-1α indicates hypoxia inducible factor-1α; 2ME2, 2-methoxyestradiol; HSP90, heat shock protein 90; HIF-1β, hypoxia inducible factor-1β; HRE, hypoxia response element.

Download figure to PowerPoint

Preventing DNA Target Sequence Binding

One method of preventing transcriptional activation of HIF (and other transcription factors) is to block binding to its target DNA site, the HRE, inhibiting gene expression of VEGFA and other HIF-regulated genes. The natural product echinomycin has been found to bind a region of the HRE sequence (5′-ACGT-3′), which is also shared with the consensus sequence of c-Myc, another cancer-related transcription factor.179 Echinomycin was found to inhibit HIF transcriptional activity and VEGF expression in tumor cells in vitro, without inhibiting other tested transcription factors.179 However, clinical trials using echinomycin produced disappointing results,180 which may be due in part to the unclear selectivity and specificity of echinomycin, which binds only a short region of DNA. Despite the lack of clinical success observed with echinomycin, inhibition of the HIF-HRE complex still holds promise as a selective HIF inhibitor.

In an effort to generate a more specific HRE binding inhibitor, a series of polyamides were designed to specifically bind the VEGFA promoter sequence 5′-WTWCGW-3′, which did lead to a decrease in VEGF-A mRNA and protein levels in vitro.181 Another model used hairpin polyamides designed to bind HRE sequences.182 Although the hairpin polyamides did not have as strong of an effect on HIF-regulated genes as a HIF-1α siRNA model, the results did suggest that polyamides could potentially be designed to affect a select subset of target genes by targeting particular HREs182 and further validated HRE sites as a molecular target for pharmacological manipulation.

Preventing Cofactor Binding

Another way of blocking HIF activation is preventing HIF from binding to essential transcriptional coactivators. The interaction between the HIF-1α CTAD domain and the CH1 domain of the coactivator p300 has been demonstrated to be a potential drug target using polypeptides corresponding to the CH1 or the CTAD domain, which led to attenuation of HIF transcriptional activity in cell-based models and antitumor activity in xenograft models.183

A small molecule, chetomin, found in a high-throughput screen, blocked binding of p300 to either HIF-1α or HIF-2α in both in vitro and in vivo assays.184 In xenograft models, systemic administration of chetomin attenuated HIF-1–mediated gene expression and caused a significant reduction in tumor size.184 Although high levels of necrosis in tumor tissues were observed, repeated injections led to localized toxicity and coagulative necrosis at sites of tail vein injection. Unfortunately, due to the toxicity, chetomin is unlikely to be pursued as a chemotherapeutic drug, but it did prove that inhibition of HIF:p300 had antitumor effects, establishing this as a potential drug target.

Recent structure activity studies on chetomin led to a series of analogues that have demonstrated some activity in vitro.185 Chetomin and these analogues were found to disrupt the structure of the CH1 domain of p300, to which the HIF-α subunit binds, by chelating structural zincs bound to p300. The unstructured p300 can then no longer bind HIF-α, thereby preventing transcriptional activation of HIF.185 Further studies of zinc chelation of p300 and downstream effects on HIF transcriptional activity could be used for drug development of this target.

Inhibition of Heat Shock Protein 90

The chaperone heat shock protein 90 (HSP90) possesses a wide range of functions, assisting in folding and stabilizing many cellular proteins.186 Client proteins of HSP90 include oncoproteins and/or angiogenic-related proteins (eg, HIF-1α, AKT, and mutant EGFR186) making HSP90 a regulatory component of many oncogenic processes. In addition, HSP90 is often overexpressed in cancer cells, can contribute to malignant transformation of cells, and has been associated with decreased survival in patients with breast cancer.187 It was initially believed that inhibition of HSP90 may not demonstrate selectivity for cancerous cells because there are high levels of HSP90 present in nearly all tissues, and HSP90 interacts with a large number of important cellular proteins in healthy cells; however, experimental evidence revealed that cancer cells were actually more sensitive to HSP90 inhibitors than healthy cells.186

In vitro evidence has demonstrated that inhibitors of HSP90, such as geldanamycin (GA) and its analogues 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminomethylamino-17-demethoxygeldanamycin (17-DMAG), do act on client proteins (eg, GA and 17-AAG promote HIF-α degradation).188 By inhibiting HSP90 from binding to HIF-1α, the protein RACK1 is able to bind HIF-1α, recruiting a ubiquitin ligase complex, inducing ubiquitination, and leading to proteasomal degradation.189 To our knowledge, 17-AAG was the first inhibitor to enter the clinic and demonstrated limited success; subsequent alterations to the formulation and delivery have reportedly improved on the efficacy.186 Clinical trials of several HSP90 inhibitors are currently ongoing. Although 17-DMAG was halted for clinical development in 2008 because of unfavorable toxicity, 17-AAG is currently in clinical trials for anticancer uses, and the effects on HIF-1α and angiogenesis are of interest.190

Thioredoxin Inhibitors

The thioredoxins (Trx) are redox proteins that function to reduce oxidized cysteines in proteins through a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reaction. One member, thioredoxin-1 (Trx-1), is overexpressed in many human tumors and has been associated with decreased patient survival.191 Trx-1 participates in the regulation of transcription factors, including HIF-1α.192 Overexpression of Trx-1 has been shown to increase levels of HIF-1α protein and VEGF expression in vitro, and increase angiogenesis in vivo, making it an attractive target for HIF and angiogenesis inhibition.193 The inhibition of Trx-1 by PX-12 and pleurotin prevents the accumulation of HIF-1α protein in hypoxic conditions, as well as decreases HIF-regulated gene expression in vitro and in vivo.194 PX-12 became the first Trx-1 inhibitor to enter a phase 1 trial of 38 patients with various types of solid tumors. PX-12 demonstrated some preliminary antitumor activity in the phase 1 trial,195 and as of mid-2009 was being tested in 2 phase 2 trials for patients with advanced/metastatic cancer and advanced pancreatic cancer.

Known and Potential Side Effects From the Inhibition of Angiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

Although many of the molecular targets proposed for the inhibition of angiogenesis appear promising, particularly through targeting HIF and related pathways, caution should always be used when applying findings from in vitro studies to clinical applications. As with most studies using isolated systems and molecular targets, many of the results obtained in preclinical studies have yet to be verified as relevant in a clinical setting. Even the significance of molecular targets such as HIF is still unknown in a clinical setting, and many of the preclinical studies have found conflicting results. For example, studies using embryonic stem cell tumors found that inhibition of HIF increased tumor growth,196, 197 and that activation of HIF led to a slower growth rate than the wild-type cells,198 indicating that the biology of HIF is still not completely understood. Caution should be exercised when drawing conclusions as to the role of the HIF system in cancer from results obtained using a limited number of cell types. Similar conclusions apply to other molecular targets in angiogenesis, particularly those that have not yet been targeted in humans, because there is still a significant knowledge gap in our understanding between preclinical and clinical studies.

In addition, as the use of angiogenesis inhibitors such as bevacizumab becomes widespread, the potential side effects that occur in the short-term or long-term use of angiogenesis inhibitors are becoming apparent. Some of these side effects include gastrointestinal perforations, impaired wound healing, bleeding, hypertension, proteinuria, and thrombosis.199–201 Many of the side effects are actually due to the direct effects of the drugs; cardiovascular complications are believed to be caused by the direct effects of angiogenesis inhibitors on the nontumor-associated ECs.200 To our knowledge, to date, the likelihood of these occurrences has been unpredictable and further studies are needed to measure the risk for patients, understand the cause of complications, and find prophylactic measures to minimize risk. The effects of the long-term administration of angiogenesis inhibitors are not fully known, because the majority of long-term studies have not yet been conducted due to the recent development of these drugs.101

It has also been found that most tumors develop mechanisms of resistance to antiangiogenic agents,202 and this may be a potential consequence of the long-term administration of antiangiogenesis inhibitors. Because multiple signaling pathways are involved in angiogenesis and more than one pathway is often dysregulated in human tumors, there is a certain level of signaling redundancy, and blocking a single pathway may not be highly effective and/or can lead to resistance when the tumor cells develop other angiogenesis mechanisms.202 By targeting multiple pathways, resistance may be able to be overcome or delayed, making combination drug therapies important in the design of future clinical trials.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References

The complex molecular pathways that govern tumor angiogenesis are logical targets for pharmacological manipulation given the important role they play in the growth and development of cancers. Initial trials of putative antiangiogenesis inhibitors have shown some promise in cancer, although this has not always translated to the clinic. A lack of validated biomarkers and patient screening restricts our ability to tailor specific drugs to patient cohorts, and might be viewed as one of the largest barriers to success in angiogenesis inhibition. As cancer pharmacology moves away from cytotoxic to so-called molecularly targeted drugs that are expected to have minimal side effects and toxicity, predictive biomarkers could be used to screen for patients likely to demonstrate a clinical response. Biomarkers also need to be validated for use as objective response measurements, because antiangiogenic monotherapies may only act as cytostatic agents, making objective response measurements such as tumor shrinkage less useful for determining the efficacy of a drug. The long time period required for an observable response also demonstrates the need for a rapid biomarker so that response to a treatment can be measured. Although some clinical trials have demonstrated that particular surrogate biomarkers of angiogenesis, such as circulating VEGF and microvessel density, have value as prognostic markers, more validated biomarkers need to be found so that antiangiogenic agents can be properly used and evaluated in the clinic.203, 204

The end goals of antiangiogenesis inhibitors also need to be determined. It is still not understood whether angiogenesis inhibitors will eliminate or shrink tumors or simply inhibit further growth and spread. If angiogenesis inhibitors are used as cytostatic agents, then further studies are needed to examine the effect of vessel normalization, whereby the tumor vessels become more organized and blood flow is improved, and whether improved tumor delivery of chemotherapeutics could be achieved. This means more studies using combination therapies with angiogenesis inhibitors and determining whether angiogenesis inhibitors are more effective in combination with chemotherapy or as single agents. In vitro studies have found that combination with traditional chemotherapies and/or radiotherapy increases the antitumor efficacy of kinase inhibitors,149 and this appears to be true with many of the antiangiogenesis inhibitors.205 It is also believed that combination therapies could be used to provide maximum antitumor effect and minimal side effects if a lower dose of each drug were to be used. Treatments could be tailored to target the specific altered pathways in a tumor with a combination of drugs to minimize resistance and provide a more effective combined treatment. Future clinical studies may provide the information needed to find the best combination of treatments for maximum anticancer effect and minimal side effects.

The research within the past decade has led to major advances in understanding the molecular pathways involved in tumor angiogenesis. This basic research has led to the identification of new targets associated with angiogenesis, leading to the development of an extensive number of preclinical, antiangiogenesis agents. Ongoing studies of different approaches currently are evaluating some of the molecular targets and agents, with some even in clinical trials, and data regarding efficacy and safety are currently emerging.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Inhibition of Angiogenesis for Anticancer Purposes
  5. Process of Carcinogenesis and Subsequent Tumor Angiogenesis
  6. Angiopoietins and TIE Receptors
  7. Delta/Jagged-Notch Signaling
  8. HIF
  9. Antiangiogenesis Compounds
  10. Inhibitors of Growth Factors, RTKs, and Signaling Pathways
  11. Monoclonal Antibodies Directed at EGFR
  12. PI3K/AKT/mTOR Pathway Inhibitors
  13. MAPK-Farnesyltransferase Rho and Ras Inhibitors
  14. HIF Pathways and Binding Partners
  15. Known and Potential Side Effects From the Inhibition of Angiogenesis
  16. Conclusions
  17. References