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Angiogenesis Inhibitors: Current Strategies and Future Prospects

¹Molecular Pharmacology Section, Medical Oncology Branch and Affiliates, National Cancer Institute, National Institutes of Health, Bethesda, MD
²Molecular Pharmacology Section, Medical Oncology Branch and Affiliates, National Cancer Institute, National Institutes of Health, Bethesda, MD

Corresponding author: William D. Figg, PharmD, Molecular Pharmacology Section, Medical Oncology Branch and Af.liates, National Cancer Institute, National Institutes of Health, 10 Center Drive, 9000 Rockville Pike, Building 10, Room 5A01, Bethesda, MD 20892; wdfigg{at}helix.nih.gov

DISCLOSURES: The authors reported no conflicts of interest.

	Abstract

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

View larger version (39K): [in this window] [in a new window] [PowerPoint slide for Educational Purposes]   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-1, hypoxia inducible factor-1; VEGF, vascular endothelial growth factor.

View larger version (29K): [in this window] [in a new window] [PowerPoint slide for Educational Purposes]   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.

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 C (PLC)-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

View larger version (25K): [in this window] [in a new window] [PowerPoint slide for Educational Purposes]   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.

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- 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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 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-. In contrast to HIF-β, the levels of the HIF- subunits and their transcriptional activity are regulated by oxygen availability.

There are 3 related forms of human HIF- (HIF-1, HIF-2, and HIF-3), each of which is encoded by a distinct genetic locus. HIF-1 and HIF-2 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-3 is not fully understood, although a truncated form of murine HIF-3 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- and HIF-β subunits are produced constitutively, but in normoxia HIF-1 and HIF-2 are degraded by the proteasome in an oxygen-dependent manner. Hydroxylation of 2 prolines in HIF- enables HIF- to bind to the von Hippel-Lindau tumor suppressor protein (pVHL), which links HIF- to a ubiquitin ligase complex. The ubiquitin ligase catalyzes polyubiquitinylation of HIF-, targeting it for degradation by the proteasome. In addition, hydroxylation of an asparagine residue in HIF- disrupts the interaction between HIF- 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- stability.^83,84 In hypoxia, minimal to no hydroxylation occurs, enabling HIF- 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).

View larger version (38K): [in this window] [in a new window] [PowerPoint slide for Educational Purposes]   FIGURE 4 Both the Hypoxia Inducible Factor (HIF)- and HIF-β Subunits Are Produced Constitutively, But in Normoxia, the Subunit Is Degraded by the Proteasome in an Oxygen-Dependent Manner. Hydroxylation of 2 prolines in HIF- enables HIF- to bind to the von Hippel-Lindau tumor suppressor protein (pVHL), which links HIF- to a ubiquitin ligase complex. The ubiquitin ligase catalyzes polyubiquitinylation of HIF-, targeting it for degradation by the proteasome. In addition, hydroxylation of an asparagine residue in HIF- disrupts the interaction between HIF- 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 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.

	Antiangiogenesis Compounds

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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 p21^CIP/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- 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-, leading to lenalidomide and pomalidomide, the latter of which is 50,000-fold more potent than thalidomide at suppressing endotoxin-induced TNF- 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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- (IFN-), with sunitinib providing a statistically significant improvement in both the median PFS (47.3 weeks for sunitinib vs 24.9 weeks for IFN-) 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

	Monoclonal Antibodies Directed at EGFR

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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-1 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-1 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-1 caused by the inhibition of mTOR.^19,158–160 Although rapamycin inhibits HIF-1 in vitro, to our knowledge it is unknown to what degree the decrease in HIF-1 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions References

 
The MAPK signaling pathway is another pathway that can lead to increased angiogenesis and increased levels of HIF-1, 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-1 and targeting Ras has been shown to destabilize HIF-1 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-
IFN- 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- 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-. In 1989, IFN- 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-,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-1 protein levels by acting at the translational level without affecting rates of HIF-1 gene transcription or HIF-1 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-1.¹⁷⁵ 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-1 may provide new therapeutic avenues.

	HIF Pathways and Binding Partners

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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).

View larger version (30K): [in this window] [in a new window] [PowerPoint slide for Educational Purposes]   FIGURE 5 Examples of Inhibitors Known to Act on Hypoxia Inducible Factor (HIF) and/or HIF Regulatory Pathways Are Shown. HIF-1 indicates hypoxia inducible factor-1; 2ME2, 2-methoxyestradiol; HSP90, heat shock protein 90; HIF-1β, hypoxia inducible factor-1β; HRE, hypoxia response element.

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-1 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-1 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-1 or HIF-2 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- subunit binds, by chelating structural zincs bound to p300. The unstructured p300 can then no longer bind HIF-, 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-1, 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- degradation).¹⁸⁸ By inhibiting HSP90 from binding to HIF-1, the protein RACK1 is able to bind HIF-1, 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-1 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-1.¹⁹² Overexpression of Trx-1 has been shown to increase levels of HIF-1 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-1 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions 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.

	Footnotes

 
Available online at http://cajournal.org and http://cacancerjournal.org

Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Sydney, Australia

	References

Top Abstract Introduction Inhibition of Angiogenesis for... Process of Carcinogenesis and... Angiopoietins and TIE Receptors Delta/Jagged-Notch Signaling HIF Antiangiogenesis Compounds Inhibitors of Growth Factors,... Monoclonal Antibodies Directed... PI3K/AKT/mTOR Pathway Inhibitors MAPK-Farnesyltransferase Rho and... HIF Pathways and Binding... Known and Potential Side... Conclusions References

 

1. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182–1186.[Medline]
2. Kerbel RS. Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. Bioessays. 1991;13:31–36.[CrossRef][Medline]
3. Staton CA, Brown NJ, Reed MW. Current status and future prospects for anti-angiogenic therapies in cancer. Expert Opin Drug Discov. 2009;4:961–979.[CrossRef]
4. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer. 2003;3:401–410.[CrossRef][Medline]
5. Gasparini G, Longo R, Toi M, Ferrara N. Angiogenic inhibitors: a new therapeutic strategy in oncology. Nat Clin Pract Oncol. 2005;2:562–577.[CrossRef][Medline]
6. Kerbel RS, Viloria-Petit A, Klement G, Rak J. ‘Accidental’ anti-angiogenic drugs: anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples. Eur J Cancer. 2000;36:1248–1257.[CrossRef][Medline]
7. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.[CrossRef][Medline]
8. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–660.[CrossRef][Medline]
9. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003;9:677–684.[CrossRef][Medline]
10. Hellwig-Burgel T, Rutkowski K, Metzen E, Fandrey J, Jelkmann W. Interleukin-1beta and tumor necrosis factor-alpha stimulate DNA binding of hypoxia-inducible factor-1. Blood. 1999;94:1561–1567.[Abstract/Free Full Text]
11. Stiehl DP, Jelkmann W, Wenger RH, Hellwig-Burgel T. Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway. FEBS Lett. 2002;512:157–162.[CrossRef][Medline]
12. Jiang BH, Jiang G, Zheng JZ, et al. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ. 2001;12:363–369.[Abstract/Free Full Text]
13. Zhong H, Chiles K, Feldser D, et al. Modulation of hypoxia-inducible factor 1 expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 2000;60:1541–1545.[Abstract/Free Full Text]
14. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem. 2002;277:38205–38211.[Abstract/Free Full Text]
15. Chen C, Pore N, Behrooz A, Ismail-Beigi F, Maity A. Regulation of Glut1 MRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J Biol Chem. 2001;276:9519–9525.[Abstract/Free Full Text]
16. Rak J, Mitsuhashi Y, Bayko L, et al. Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. Cancer Res. 1995;55:4575–4580.[Abstract/Free Full Text]
17. Jiang BH, Agani F, Passaniti A, Semenza GL. V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression. Cancer Res. 1997;57:5328–5335.[Abstract/Free Full Text]
18. Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (Neu) signaling increases the rate of hypoxia-inducible factor 1 (HIF-1) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol. 2001;21:3995–4004.[Abstract/Free Full Text]
19. Land SC, Tee AR. Hypoxia-inducible factor 1 is regulated by the mammalian target of rapamycin (MTOR) via an MTOR signaling motif. J Biol Chem. 2007;282:20534–20543.[Abstract/Free Full Text]
20. Blancher C, Moore JW, Robertson N, Harris AL. Effects of ras and Von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1, HIF-2, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway. Cancer Res. 2001;61:7349–7355.[Abstract/Free Full Text]
21. Madhusudan S, Ganesan TS. Tyrosine kinase inhibitors in cancer therapy. Clin Biochem. 2004;37:618–635.[CrossRef][Medline]
22. Krause DS, Van Etten RA. Tyrosine kinases as targets for cancer therapy. N Engl J Med. 2005;353:172–187.[Free Full Text]
23. Engelman JA. Targeting PI3K signaling in cancer: opportunities, challenges and limitations. Nat Rev Cancer. 2009;9:550–562.[CrossRef][Medline]
24. Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov. 2009;8:627–644.[CrossRef][Medline]
25. Faivre S, Kroemer G, Raymond E. Current development of MTOR inhibitors as anticancer agents. Nat Rev Drug Discov. 2006;5:671–688.[CrossRef][Medline]
26. Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003;3:11–22.[CrossRef][Medline]
27. Grothey A, Galanis E. Targeting angiogenesis: progress with anti-VEGF treatment with large molecules. Nat Rev Clin Oncol. 2009;6:507–518.[CrossRef][Medline]
28. Ferrara N. Vascular endothelial growth factor. Arterioscler Thromb Vasc Biol. 2009;29:789–791.[Abstract/Free Full Text]
29. Ferrara N. VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer. 2002;2:795–803.[CrossRef][Medline]
30. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676.[CrossRef][Medline]
31. Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983–985.[Abstract/Free Full Text]
32. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun. 1989;161:851–858.[CrossRef][Medline]
33. Keck PJ, Hauser SD, Krivi G, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science. 1989;246:1309–1312.[Abstract/Free Full Text]
34. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989;246:1306–1309.[Abstract/Free Full Text]
35. Kaya M, Wada T, Akatsuka T, et al. Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res. 2000;6:572–577.[Abstract/Free Full Text]
36. Maeda K, Chung YS, Ogawa Y, et al. Prognostic value of vascular endothelial growth factor expression in gastric carcinoma. Cancer. 1996;77:858–863.[CrossRef][Medline]
37. Inoue K, Ozeki Y, Suganuma T, Sugiura Y, Tanaka S. Vascular endothelial growth factor expression in primary esophageal squamous cell carcinoma. Association with angiogenesis and tumor progression. Cancer. 1997;79:206–213.[CrossRef][Medline]
38. Ishigami SI, Arii S, Furutani M, et al. Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer. Br J Cancer. 1998;78:1379–1384.[Medline]
39. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277:242–245.[Abstract/Free Full Text]
40. Oikawa T, Onozawa C, Sakaguchi M, Morita I, Murota S. Three isoforms of platelet-derived growth factors all have the capability to induce angiogenesis in vivo. Biol Pharm Bull. 1994;17:1686–1688.[Medline]
41. Risau W, Drexler H, Mironov V, et al. Platelet-derived growth factor is angiogenic in vivo. Growth Factors. 1992;7:261–266.[Medline]
42. Cao Y, Cao R, Hedlund EM. Regulation of tumor angiogenesis and metastasis by FGF and PDGF signaling pathways. J Mol Med. 2008;86:785–789.[CrossRef][Medline]
43. Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 2008;22:1276–1312.[Abstract/Free Full Text]
44. Hermanson M, Funa K, Hartman M, et al. Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops. Cancer Res. 1992;52:3213–3219.[Abstract/Free Full Text]
45. Nister M, Libermann TA, Betsholtz C, et al. Expression of messenger RNAs for platelet-derived growth factor and transforming growth factor- and their receptors in human malignant glioma cell lines. Cancer Res. 1988;48:3910–3918.[Abstract/Free Full Text]
46. Dabrow MB, Francesco MR, McBrearty FX, Caradonna S. The effects of platelet-derived growth factor and receptor on normal and neoplastic human ovarian surface epithelium. Gynecol Oncol. 1998;71:29–37.[CrossRef][Medline]
47. Korc M, Friesel RE. The role of fibroblast growth factors in tumor growth. Curr Cancer Drug Targets. 2009;9:639–651.[CrossRef][Medline]
48. Maciag T, Mehlman T, Friesel R, Schreiber AB. Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain. Science. 1984;225:932–935.[Abstract/Free Full Text]
49. Abraham JA, Mergia A, Whang JL, et al. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science. 1986;233:545–548.[Abstract/Free Full Text]
50. Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest. 1990;85:2004–2008.[Medline]
51. Berger W, Setinek U, Mohr T, et al. Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells. Int J Cancer. 1999;83:415–423.[CrossRef][Medline]
52. Gazzaniga P, Gandini O, Gradilone A, et al. Detection of basic fibroblast growth factor MRNA in urinary bladder cancer: correlation with local relapses. Int J Oncol. 1999;14:1123–1127.[Medline]
53. Gullick WJ. The epidermal growth factor system of ligands and receptors in cancer. Eur J Cancer. 2009;45( suppl 1):205–210.[CrossRef][Medline]
54. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127–137.[CrossRef][Medline]
55. Perrotte P, Matsumoto T, Inoue K, et al. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin Cancer Res. 1999;5:257–265.[Abstract/Free Full Text]
56. van CH, Giaccone G, Hoekman K. Epidermal growth factor receptor and angiogenesis: opportunities for combined anticancer strategies. Int J Cancer. 2005;117:883–888.[CrossRef][Medline]
57. De LA, Carotenuto A, Rachiglio A, et al. The role of the EGFR signaling in tumor microenvironment. J Cell Physiol. 2008;214:559–567.[CrossRef][Medline]
58. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor β in human disease. N Engl J Med. 2000;342:1350–1358.[Free Full Text]
59. Bierie B, Moses HL. TGF–β and cancer. Cytokine Growth Factor Rev. 2006;17:29–40.[CrossRef][Medline]
60. Bernabeu C, Lopez-Novoa JM, Quintanilla M. The emerging role of TGF-beta superfamily coreceptors in cancer. Biochim Biophys Acta. 2009;1792:954–973.[Medline]
61. Bierie B, Moses HL. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer. 2006;6:506–520.[CrossRef][Medline]
62. Augustin HG, Young Koh G, Thurston G, Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-tie system. Nat Rev Mol Cell Biol. 2009;10:165–177.[CrossRef][Medline]
63. Thomas M, Augustin H. The role of the angiopoietins in vascular morphogenesis. Angiogenesis. 2009;12:125–137.[CrossRef][Medline]
64. Shim WS, Ho IA, Wong PE. Angiopoietin: a TIE(d) balance in tumor angiogenesis. Mol Cancer Res. 2007;5:655–665.[Abstract/Free Full Text]
65. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997;277:55–60.[Abstract/Free Full Text]
66. Lobov IB, Brooks PC, Lang RA. Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci U S A. 2002;99:11205–11210.[Abstract/Free Full Text]
67. Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science. 1999;284:1994–1998.[Abstract/Free Full Text]
68. Winkler F, Kozin SV, Tong RT, et al. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell. 2004;6:553–563.[Medline]
69. Oliner J, Min H, Leal J, et al. Suppression of angiogenesis and tumor growth by selective inhibition of angiopoietin-2. Cancer Cell. 2004;6:507–516.[CrossRef][Medline]
70. Thurston G, Kitajewski J. VEGF and Delta-Notch: interacting signalling pathways in tumour angiogenesis. Br J Cancer. 2008;99:1204–1209.[CrossRef][Medline]
71. Thurston G, Noguera-Troise I, Yancopoulos GD. The Delta paradox: DLL4 blockade leads to more tumour vessels but less tumour growth. Nat Rev Cancer. 2007;7:327–331.[CrossRef][Medline]
72. Mailhos C, Modlich U, Lewis J, et al. Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis. Differentiation. 2001;69:135–144.[CrossRef][Medline]
73. Gale NW, Dominguez MG, Noguera I, et al. Haploinsufficiency of Delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Nat Acad Sci U S A. 2004;101:15949–15954.[Abstract/Free Full Text]
74. Noguera-Troise I, Daly C, Papadopoulos NJ, et al. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature. 2006;444:1032–1037.[CrossRef][Medline]
75. Patel NS, Li JL, Generali D, et al. Up-regulation of delta-like 4 ligand in human tumor vasculature and the role of basal expression in endothelial cell function. Cancer Res. 2005;65:8690–8697.[Abstract/Free Full Text]
76. Hainaud P, Contreres JO, Villemain A, et al. The role of the vascular endothelial growth factor-Delta-like 4 ligand/Notch4-ephrin B2 cascade in tumor vessel remodeling and endothelial cell functions. Cancer Res. 2006;66:8501–8510.[Abstract/Free Full Text]
77. Hellstrom M, Phng LK, Hofmann JJ, et al. Dll4 Signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 2007;445:776–780.[CrossRef][Medline]
78. Zeng Q, Li S, Chepeha DB, et al. Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of notch signaling. Cancer Cell. 2005;8:13–23.[CrossRef][Medline]
79. Sainson RCA, Johnston DA, Chu HC, et al. TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype. Blood. 2008;111:4997–5007.[Abstract/Free Full Text]
80. Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature. 2006;444:1083–1087.[CrossRef][Medline]
81. Funahashi Y, Hernandez SL, Das I, et al. A notch1 ectodomain construct inhibits endothelial notch signaling, tumor growth, and angiogenesis. Cancer Res. 2008;68:4727–4735.[Abstract/Free Full Text]
82. Weidemann A, Johnson RS. Biology of HIF-1. Cell Death Differ. 2008;15:621–627.[CrossRef][Medline]
83. Kaelin WG, Ratcliffe PJ. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell. 2008;30:393–402.[CrossRef][Medline]
84. Schofield CJ, Ratcliffe PJ. Signalling hypoxia by HIF hydroxylases. Biochem Biophys Res Commun. 2005;338:617–626.[CrossRef][Medline]
85. Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1 in common human cancers and their metastases. Cancer Res. 1999;59:5830–5835.[Abstract/Free Full Text]
86. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 and HIF-2 in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol. 2000;157:411–421.[Abstract/Free Full Text]
87. Birner P, Gatterbauer B, Oberhuber G, et al. Expression of hypoxia-inducible factor-1 alpha in oligodendrogliomas: its impact on prognosis and on neoangiogenesis. Cancer. 2001;92:165–171.[CrossRef][Medline]
88. Zagzag D, Zhong H, Scalzitti JM, et al. Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression. Cancer. 2000;88:2606–2618.[CrossRef][Medline]
89. Bos R, Zhong H, Hanrahan CF, et al. Levels of hypoxia-inducible factor-1 during breast carcinogenesis. J Natl Cancer Inst. 2001;93:309–314.[Abstract/Free Full Text]
90. Giatromanolaki A, Koukourakis MI, Sivridis E, et al. Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer. 2001;85:881–890.[CrossRef][Medline]
91. Birner P, Schindl M, Obermair A, et al. Overexpression of hypoxia-inducible factor 1 is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res. 2000;60:4693–4696.[Abstract/Free Full Text]
92. Aebersold DM, Burri P, Beer KT, et al. Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res. 2001;61:2911–2916.[Abstract/Free Full Text]
93. Koukourakis MI, Giatromanolaki A, Skarlatos J, et al. Hypoxia inducible factor (HIF-1a and HIF-2a) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy. Cancer Res. 2001;61:1830–1832.[Abstract/Free Full Text]
94. Mabjeesh NJ, Amir S. Hypoxia-inducible factor (HIF) in human tumorigenesis. Histol Histopathol. 2007;22:559–572.[Medline]
95. Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature. 1990;348:555–557.[CrossRef][Medline]
96. Kusaka M, Sudo K, Fujita T, et al. Potent anti-angiogenic action of AGM-1470: comparison to the fumagillin parent. Biochem Biophys Res Commun. 1991;174:1070–1076.[CrossRef][Medline]
97. Sin N, Meng L, Wang MQW, et al. The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. Proc Natl Acad Sci U S A. 1997;94:6099–6103.[Abstract/Free Full Text]
98. Griffith EC, Su Z, Niwayama S, et al. Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2. Proc Natl Acad Sci U S A. 1998;95:15183–15188.[Abstract/Free Full Text]
99. Nahari D, Satchi-Fainaro R, Chen M, et al. Tumor cytotoxicity and endothelial Rac inhibition induced by TNP-470 in anaplastic thyroid cancer. Mol Cancer Ther. 2007;6:1329–1337.[Abstract/Free Full Text]
100. Zhang Y, Griffith EC, Sage J, Jacks T, Liu JO. Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1/CIP1. Proc Nat Acad Sci U S A. 2000;97:6427–6432.[Abstract/Free Full Text]
101. Mauriz JL, Gonzalez-Gallego J. Antiangiogenic drugs: current knowledge and new approaches to cancer therapy. J Pharm Sci. 2008;97:4129–4154.[CrossRef][Medline]
102. Benny O, Fainaru O, Adini A, et al. An orally delivered small-molecule formulation with antiangiogenic and anticancer activity. Nat Biotechnol. 2008;26:799–807.[CrossRef][Medline]
103. Kruger EA, Figg WD. TNP-470: an angiogenesis inhibitor in clinical development for cancer. Expert Opin Investig Drugs. 2000;9:1383–1396.[CrossRef][Medline]
104. Kudelka AP, Verschraegen CF, Loyer E. Complete remission of metastatic cervical cancer with the angiogenesis inhibitor TNP-470. N Engl J Med. 1998;338:991–992.[Free Full Text]
105. Kudelka AP, Levy T, Verschraegen CF, et al. A phase I study of TNP-470 administered to patients with advanced squamous cell cancer of the cervix. Clin Cancer Res. 1997;3:1501–1505.[Abstract]
106. Tran HT, Blumenschein GR, Lu C, et al. Clinical and pharmacokinetic study of TNP-470, an angiogenesis inhibitor, in combination with paclitaxel and carboplatin in patients with solid tumors. Cancer Chemother Pharmacol. 2004;54:308–314.[Medline]
107. Bhargava P, Marshall JL, Rizvi N, et al. A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer. Clin Cancer Res. 1999;5:1989–1995.[Abstract/Free Full Text]
108. Cretton-Scott E, Placidi L, McClure H, et al. Pharmacokinetics and metabolism of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470, AGM-1470) in rhesus monkeys. Cancer Chemother Pharmacol. 1996;38:117–122.[CrossRef][Medline]
109. Logothetis CJ, Wu KK, Finn LD, et al. Phase I trial of the angiogenesis inhibitor TNP-470 for progressive androgen-independent prostate cancer. Clin Cancer Res. 2001;7:1198–1203.[Abstract/Free Full Text]
110. Satchi-Fainaro R, Puder M, Davies JW, et al. Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470. Nat Med. 2004;10:255–261.[CrossRef][Medline]
111. Franks ME, Macpherson GR, Figg WD. Thalidomide. Lancet. 2004;363:1802–1811.[CrossRef][Medline]
112. McBride WG. Thalidomide and congenital abnormalities. Lancet. 1961;278:1358–1358.
113. Lenz W, Pfeiffer RA, Kosenow W, Hayman DJ. Thalidomide and congenital abnormalities. Lancet. 1962;279:45–46.
114. D'Amato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A. 1994;91:4082–4085.[Abstract/Free Full Text]
115. Kenyon BM, Browne F, D'Amato RJ. Effects of thalidomide and related metabolites in a mouse corneal model of neovascularization. Exp Eye Res. 1997;64:971–978.[CrossRef][Medline]
116. Bauer KS, Dixon SC, Figg WD. Inhibition of angiogenesis by thalidomide requires metabolic activation, which is species-dependent. Biochem Pharmacol. 1998;55:1827–1834.[CrossRef][Medline]
117. Bartlett JB, Dredge K, Dalgleish AG. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat Rev Cancer. 2004;4:314–322.[CrossRef][Medline]
118. Tabin CJ. A developmental model for thalidomide defects. Nature. 1998;396:322–323.[CrossRef][Medline]
119. Parman T, Wiley MJ, Wells PG. Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat Med. 1999;5:582–585.[CrossRef][Medline]
120. Cundari S, Cavaletti G. Thalidomide chemotherapy-induced peripheral neuropathy: actual status and new perspectives with thalidomide analogues derivatives. Mini Rev Med Chem. 2009;9:760–768.[CrossRef][Medline]
121. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature. 1993;362:841–844.[CrossRef][Medline]
122. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004;350:2335–2342.[Abstract/Free Full Text]
123. Miller KD, Chap LI, Holmes FA, et al. Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol. 2005;23:792–799.[Abstract/Free Full Text]
124. Miller K, Wang M, Gralow J, et al. Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med. 2007;357:2666–2676.[Abstract/Free Full Text]
125. Johnson DH, Fehrenbacher L, Novotny WF, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol. 2004;22:2184–2191.[Abstract/Free Full Text]
126. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355:2542–2550.[Abstract/Free Full Text]
127. Database of Current Clinical Trials. 2009. Bethesda, MD: US National Library of Medicine; 2009. Available at: http://www.clinicaltrials.gov. Accessed September 2009.
128. Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002;99:11393–11398.[Abstract/Free Full Text]
129. Tew WP, Colombo N, Ray-Coquard I, et al. VEGF-Trap for patients (Pts) with recurrent platinum-resistant epithelial ovarian cancer (EOC): preliminary results of a randomized, multicenter phase II study. J Clin Oncol. 2007;25(18 suppl):5508.
130. Massarelli E, Miller VA, Leighl NB, et al. Phase II study of the efficacy and safety of intravenous (IV) AVE0005 (VEGF Trap) given every 2 weeks in patients (Pts) with platinum- and erlotinib-resistant adenocarcinoma of the lung (NSCLA). J Clin Oncol. 2007;25(18 suppl):7627.
131. Hobday TJ, Rowland K, Dueck A, et al. N0537: a North Central Cancer Treatment Group (NCCTG) phase II study of VEGF Trap in patients with metastatic breast cancer previously treated with anthracycline and/or taxanes [abstract]. 2008 Breast Cancer Symposium Meeting Abstracts. Available at: http://asco.org. Abstract 164.
132. Gan HK, Seruga B, Knox JJ. Sunitinib in solid tumors. Expert Opin Investing Drugs. 2009;18:821–834.[CrossRef]
133. Motzer RJ, Hutson TE, Tomczak P, et al. Phase III randomized trial of sunitinib malate (SU11248) versus interferon-alfa (IFN-) as first-line systemic therapy for patients with metastatic renal cell carcinoma (MRCC) [abstract]. J Clin Oncol. 2006;24(18 suppl). Abstract LBA3.
134. Casali PG, Garrett CR, Blackstein ME, et al. Updated results from a phase III trial of sunitinib in GIST patients (Pts) for whom imatinib (IM) therapy has failed due to resistance or intolerance [abstract]. J Clin Oncol. 2006;24(18 suppl). Abstract 9513.
135. Demetri GD, van Oosterom AT, Garrett C, et al. Improved survival and sustained clinical benefit with SU11248 (SU) in Pts with GIST after failure of imatinib mesylate (IM) therapy in a phase III trial. 2006 Gastrointest Cancers Symposium Category Esophagus and Stomach. 2006. Abstract 8. Available at: asco.org.
136. Wilhelm SM, Adnane L, Newell P, et al. Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther. 2008;7:3129–3140.[Abstract/Free Full Text]
137. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004;64:7099–7109.[Abstract/Free Full Text]
138. Ratain MJ, Eisen T, Stadler WM, et al. Final findings from a phase II, placebo-controlled, randomized discontinuation trial (RDT) of sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC) [Abstract]. J Clin Oncol, 2005 ASCO Annual Meeting Proceedings. 2005;23(16 suppl): Abstract 4544.
139. Escudier B, Szczylik C, Eisen T, et al. Randomized phase III trial of the Raf kinase and VEGFR inhibitor sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC) [abstract]. J Clin Oncol. 2005;23(16 suppl pt I of II).
140. Sakamoto KM. Semaxanib (SUGEN). IDrugs. 2001;4:1061–1067.[Medline]
141. Longo R, Sarmiento R, Fanelli M, et al. Anti-angiogenic therapy: rationale, challenges and clinical studies. Angiogenesis. 2002;5:237–256.[CrossRef][Medline]
142. Kuenen BC, Rosen L, Smit EF, et al. Dose-finding and pharmacokinetic study of cisplatin, gemcitabine, and SU5416 in patients with solid tumors. J Clin Oncol. 2002;20:1657–1667.[Abstract/Free Full Text]
143. Pore N, Jiang Z, Gupta A, et al. EGFR tyrosine kinase inhibitors decrease VEGF expression by both hypoxia-inducible factor (HIF)-1-independent and HIF-1-dependent mechanisms. Cancer Res. 2006;66:3197–3204.[Abstract/Free Full Text]
144. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol. 2005;23:5892–5899.[Abstract/Free Full Text]
145. Gatzemeier U, Pluzanska A, Szczesna A, et al. Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the Tarceva Lung Cancer Investigation Trial. J Clin Oncol. 2007;25:1545–1552.[Abstract/Free Full Text]
146. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med. 2005;353:123–132.[Abstract/Free Full Text]
147. Moore MJ, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 2007;25:1960–1966.[Abstract/Free Full Text]
148. Heath VL, Bicknell R. Anticancer strategies involving the vasculature. Nat Rev Clin Oncol. 2009;6:395–404.[CrossRef][Medline]
149. Steeghs N, Nortier J, Gelderblom H. Small molecule tyrosine kinase inhibitors in the treatment of solid tumors: an update of recent developments. Ann Surg Oncol. 2007;14:942–953.[CrossRef][Medline]
150. Kim R, Emi M, Arihiro K, et al. Chemosensitization by STI571 targeting the platelet-derived growth factor/platelet-derived growth factor receptor-signaling pathway in the tumor progression and angiogenesis of gastric carcinoma. Cancer. 2005;103:1800–1809.[CrossRef][Medline]
151. Kvasnicka HM, Thiele J, Staib P, et al. Reversal of bone marrow angiogenesis in chronic myeloid leukemia following imatinib mesylate (STI571) therapy. Blood. 2004;103:3549–3551.[Abstract/Free Full Text]
152. Li S, Schmitz KR, Jeffrey PD, et al. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell. 2005;7:301–311.[CrossRef][Medline]
153. Saltz LB, Meropol NJ, Loehrer PJ Sr, et al. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol. 2004;22:1201–1208.[Abstract/Free Full Text]
154. Saltz LB, Rubin M, Hochster H, et al. Cetuximab (IMC-C225) plus irinotecan (CPT-11) is active in CPT-11-refractory colorectal cancer (CRC) that expresses epidermal growth factor receptor (EGFR) [abstract]. Proc Am Soc Clin Oncol. 2001;20:Abstract 7.
155. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 2004;351:337–345.[Abstract/Free Full Text]
156. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–578.[Abstract/Free Full Text]
157. Fang J, Cao Z, Chen YC, Reed E, Jiang BH. 9-β-D-arabinofuranosyl-2-fluoroadenine inhibits expression of vascular endothelial growth factor through hypoxia-inducible factor-1 in human ovarian cancer cells. Mol Pharmacol. 2004;66:178–186.[Abstract/Free Full Text]
158. Hudson CC, Liu M, Chiang GG, et al. Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Mol Cell Biol. 2002;22:7004–7014.[Abstract/Free Full Text]
159. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of MTOR in kidney cancer. Nat Med. 2006;12:122–127.[CrossRef][Medline]
160. Wang W, Jia WD, Xu GL, et al. Antitumoral activity of rapamycin mediated through inhibition of HIF-1alpha and VEGF in hepatocellular carcinoma. Dig Dis Sci. 2009;54:2128–2136.[CrossRef][Medline]
161. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med. 2002;8:128–135.[CrossRef][Medline]
162. Mesa RA. Tipifarnib: farnesyl transferase inhibition at a crossroads. Expert Rev Anticancer Ther. 2006;6:313–319.[CrossRef][Medline]
163. Liu M, Bryant MS, Chen J, et al. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res. 1998;58:4947–4956.[Abstract/Free Full Text]
164. Delmas C, End D, Rochaix P, et al. The farnesyltransferase inhibitor R115777 reduces hypoxia and matrix metalloproteinase 2 expression in human glioma xenograft. Clin Cancer Res. 2003;9(16 pt 1):6062–6068.[Abstract/Free Full Text]
165. End DW, Smets G, Todd AV, et al. Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro. Cancer Res. 2001;61:131–137.[Abstract/Free Full Text]
166. Brouty-Boye D, Zetter BR. Inhibition of cell motility by interferon. Science. 1980;208:516–518.[Abstract/Free Full Text]
167. Dvorak HF, Gresser I. Microvascular injury in pathogenesis of interferon-induced necrosis of subcutaneous tumors in mice. J Natl Cancer Inst. 1989;81:497–502.[Abstract/Free Full Text]
168. Sidky YA, Borden EC. Inhibition of angiogenesis by interferons: effects on tumor-and lymphocyte-induced vascular responses. Cancer Res. 1987;47:5155–5161.[Abstract/Free Full Text]
169. Singh RK, Gutman M, Bucana CD, et al. Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas. Proc Nat Acad Sci U S A. 1995;92:4562–4566.[Abstract/Free Full Text]
170. White CW, Sondheimer HM, Crouch EC, Wilson H, Fan LL. Treatment of pulmonary hemangiomatosis with recombinant interferon alfa-2a. N Engl J Med. 1989;320:1197–1200.[Medline]
171. Ezekowitz RA, Mulliken JB, Folkman J. Interferon alfa-2a therapy for life-threatening hemangiomas of infancy. N Engl J Med. 1992;326:1456–1463.[Abstract]
172. Kaban LB, Troulis MJ, Ebb D, et al. Antiangiogenic therapy with interferon alpha for giant cell lesions of the jaws. J Oral Maxillofac Surg. 2002;60:1103–1111.[CrossRef][Medline]
173. Kaban LB, Mulliken JB, Ezekowitz RA, et al. Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alfa-2a. Pediatrics. 1999;103 (6 pt 1):1145–1149.[Abstract/Free Full Text]
174. Marler JJ, Rubin JB, Trede NS, et al. Successful antiangiogenic therapy of giant cell angioblastoma with interferon alfa 2b: report of 2 cases. Pediatrics. 2002;109:e37.[Abstract/Free Full Text]
175. Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell. 2003;3:363–375.[CrossRef][Medline]
176. Fotsis T, Zhang Y, Pepper MS, et al. The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth. Nature. 1994;368:237–239.[CrossRef][Medline]
177. Sutherland TE, Anderson RL, Hughes RA, et al. 2-Methoxyestradiol–a unique blend of activities generating a new class of anti-tumour/anti-inflammatory agents. Drug Discov Today. 2007;12:577–584.[CrossRef][Medline]
178. Semenza GL. Evaluation of HIF-1 inhibitors as anticancer agents. Drug Discov Today. 2007;12:853–859.[CrossRef][Medline]
179. Kong D, Park EJ, Stephen AG, et al. Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity. Cancer Res. 2005;65:9047–9055.[Abstract/Free Full Text]
180. Gradishar WG, Vogelzang NJ, Kilton LJ, et al. A phase II clinical trial of echinomycin in metastatic soft tissue sarcoma. An Illinois Cancer Center Study. Invest New Drugs. 1995;13:171–174.[CrossRef][Medline]
181. Olenyuk BZ, Zhang GJ, Klco JM, et al. Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist. Proc Natl Acad Sci U S A. 2004;101:16768–16773.[Abstract/Free Full Text]
182. Nickols NG, Jacobs CS, Farkas ME, Dervan PB. Modulating hypoxia-inducible transcription by disrupting the HIF-1-DNA interface. ACS Chem Biol. 2007;2:561–571.[CrossRef][Medline]
183. Kung AL, Wang S, Klco JM, Kaelin WG, Livingston DM. Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nat Med. 2000;6:1335–1340.[CrossRef][Medline]
184. Kung AL, Zabludoff SD, France DS, et al. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell. 2004;6:33–43.[CrossRef][Medline]
185. Cook KM, Hilton ST, Mecinovic J, et al. Epidithiodiketopiperazines block the interaction between hypoxia inducible factor-1alpha (HIF-1alpha) and p300 by a zinc ejection mechanism. J Biol Chem. 2009;284:26831–26838.[Abstract/Free Full Text]
186. Solit DB, Chiosis G. Development and application of Hsp90 inhibitors. Drug Discov Today. 2008;13:38–43.[CrossRef][Medline]
187. Pick E, Kluger Y, Giltnane JM, et al. High HSP90 expression is associated with decreased survival in breast cancer. Cancer Res. 2007;67:2932–2937.[Abstract/Free Full Text]
188. Isaacs JS, Jung YJ, Mimnaugh EG, et al. Hsp90 regulates a Von Hippel Lindau-independent hypoxia-inducible factor-1 alpha-degradative pathway. J Biol Chem. 2002;277:29936–29944.[Abstract/Free Full Text]
189. Liu YV, Baek JH, Zhang H, et al. RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha. Mol Cell. 2007;25:207–217.[CrossRef][Medline]
190. Taldone T, Gozman A, Maharaj R, Chiosis G. Targeting Hsp90: small-molecule inhibitors and their clinical development. Curr Opin Pharmacol. 2008;8:370–374.[CrossRef][Medline]
191. Powis G, Montfort WR. Properties and biological activities of thioredoxins. Annu Rev Pharmacol Toxicol. 2001;41:261–295.[CrossRef][Medline]
192. Huang LE, Arany Z, Livingston DM, Bunn HF. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem. 1996;271:32253–32259.[Abstract/Free Full Text]
193. Welsh SJ, Bellamy WT, Briehl MM, Powis G. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1 protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res. 2002;62:5089–5095.[Abstract/Free Full Text]
194. Welsh SJ, Williams RR, Birmingham A, et al. The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation. Mol Cancer Ther. 2003;2:235–243.[Abstract/Free Full Text]
195. Ramanathan RK, Kirkpatrick DL, Belani CP, et al. A phase I pharmacokinetic and pharmacodynamic study of PX-12, a novel inhibitor of thioredoxin-1, in patients with advanced solid tumors. Clin Cancer Res. 2007;13:2109–2114.[Abstract/Free Full Text]
196. Blouw B, Song H, Tihan T, et al. The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell. 2003;4:133–146.[CrossRef][Medline]
197. Carmeliet P, Dor Y, Herbert JM, et al. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature. 1998;394:485–490.[CrossRef][Medline]
198. Mack FA, Rathmell WK, Arsham AM, et al. Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth. Cancer Cell. 2003;3:75–88.[CrossRef][Medline]
199. Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nat Rev Clin Oncol. 2009;6:465–477.[CrossRef][Medline]
200. Zangari M, Fink LM, Elice F, et al. Thrombotic events in patients with cancer receiving antiangiogenesis agents. J Clin Oncol. 2009;27:4865–4873.[Abstract/Free Full Text]
201. Higa GM, Abraham J. Biological mechanisms of bevacizumab-associated adverse events. Expert Rev Anticancer Ther. 2009;9:999–1007.[CrossRef][Medline]
202. Eikesdal HP, Kalluri R. Drug resistance associated with antiangiogenesis therapy. Semin Cancer Biol. 2009;19:310–317.[CrossRef][Medline]
203. Park JW, Kerbel RS, Kelloff GJ, et al. Rationale for biomarkers and surrogate end points in mechanism-driven oncology drug development. Clin Cancer Res. 2004;10:3885–3896.[Free Full Text]
204. Brown A, Citrin D, Camphausen K. Clinical biomarkers of angiogenesis inhibition. Cancer Metastasis Rev. 2008;27:415–434.[CrossRef][Medline]
205. Nieder C, Wiedenmann N, Andratschke N, Molls M. Current status of angiogenesis inhibitors combined with radiation therapy. Cancer Treat Rev. 2006;32:348–364.[CrossRef][Medline]

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