Endogenous Stimulators and Inhibitors of Angiogenesis in Gastrointestinal Cancers: Basic Science to Clinical Application

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1 GASTROENTEROLOGY 2005;129: SPECIAL REPORTS AND REVIEWS Endogenous Stimulators and Inhibitors of Angiogenesis in Gastrointestinal Cancers: Basic Science to Clinical Application MALIN SUND,* MICHAEL ZEISBERG,* and RAGHU KALLURI*,, *Center for Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts; and Harvard MIT Division of Health Sciences and Technology, Boston, Massachusetts Progression of cancer is dependent on acquisition of vascular networks within the tumor. Tumor angiogenesis is dependent on up-regulation of angiogenesis stimulators to overcome the endogenous anti-angiogenic barrier. Such disruption of angiogenesis balance to favor neovascularization is a key step for progression of tumor growth and metastasis. In this regard, the vascular basement membrane and the extracellular matrix have been found to be rich sources of angiogenesis stimulators and inhibitors that become bioavailable on proteolysis of the matrix by tumor microenvironment related enzymes. In this review the subgroup of endogenous angiogenesis stimulators and inhibitors is discussed, and their mechanism of action during tumor angiogenesis is evaluated. The role in regulating tumor growth and the possibility of using them as prognostic markers for human gastrointestinal cancers is discussed. Furthermore, we specifically address the role of vascular endothelial growth factor in human gastrointestinal cancers and discuss the development and use of bevacizumab (Avastin; anti vascular endothelial growth factor antibody [Genentech, CA]) in the treatment of colorectal and other gastrointestinal cancers. All normal tissues are dependent on oxygen delivery for survival, and it now is well established that tumor growth and survival is dependent on the recruitment of new blood vessels and capillaries. 1 3 These new vessels allow tumors to grow larger in size from a few millimeters by providing oxygen, nutrients, and waste disposal that cannot be achieved by simple diffusion. 1 3 In the process of tumor angiogenesis, new vessels are formed via sprouting events from pre-established vasculature. This requires the detachment of endothelial cells (EC) from pre-existing blood vessels, degradation of the vascular basement membrane (VBM), and migration of the liberated ECs into the extracellular space. The migrating ECs subsequently become surrounded by a provisional extracellular matrix (ECM), form a new endothelial lumen and VBM, and finally become enveloped by pericytes to form a mature blood vessel. 1,4 9 In recent years the role of circulating endothelial progenitor cells in the formation of tumor vasculature has raised a lot of interest because blocking these cells potentially could reduce tumor angiogenesis. 10 Initial high-profile reports indicated that a vast majority of endothelial cells in tumors potentially could be bonemarrow derived. 11 However, several other reports using both animal models and data derived from sexmismatched bone marrow transplantations in human beings who subsequently developed cancer have shown that the frequency of bone-marrow derived cells that incorporate into the newly formed tumor vasculature is much lower (average, 4.9%; range, 1% 12%) than initially described. 16,17 Interestingly, it does appear that there is incorporation of stem cells into the perivascular area, but these are not endothelial cells. 13,18 A reason for this discrepancy may be the use of different tumor models (subcutaneous vs orthotopic) and varying microscopy and staining conditions (eg, regular light microscopy vs confocal microscopy). 12,13 Blood vessels associated with tumors have been described as abnormal, having altered branching, displaying irregular diameters, and increased leakiness (Figure 1). 3,9,19 One of the structural reasons for the observed abnormalities in the tumor vasculature is caused presumably by incomplete pericyte coverage because these cells confer support to the blood vessel wall. 9,20 23 Interestingly, although tumor-associated blood vessels contain a Abbreviations used in this paper: EC, endothelial cell; ECM, extracellular matrix; 5-FU, 5-fluorouracil; IFL, irinotecan 125 mg/m 3, 5-fluorouracil 500 mg/m 3, and leucovorin 20 mg/m 3 ; MMP, matrix metalloproteinase; NC, noncollagenous; TSP, thromospondin; VBM, vascular basement membrane; VEGF, vascular endothelial growth factor by the American Gastroenterological Association /05/$30.00 doi: /j.gastro

2 December 2005 GASTROINTESTINAL CANCER AND ANGIOGENESIS 2077 Figure 1. The characteristic features of normal blood vessels and tumor-associated blood vessels. The blood vessel wall consists of an EC lining, a VBM, and a supporting outer pericyte layer. Note that the pericytes can be in contact with the ECs through the BM, as determined by electron microscopy studies. Nevertheless, whether this actually happens in vivo still lacks solid experimental proof. The tumor vessels show irregularities in the vessel shape, loose EC connections, sparse pericyte coverage, and detached pericytes. In addition, many defects within the VBM, such as interruptions, multiple layers, and abnormal spikes, can be observed in blood vessels associated with tumors. VBM, there is evidence that these blood vessels are not without defects and show multiple layers and broad protein extensions from the vessel wall, leading to loose association with endothelial cells and pericytes (Figure 1). 24 Inai et al 25 have shown that the VBMs persist after the degeneration of the ECs when vascular endothelial growth factor (VEGF) is depleted. Such persisting VBM scaffolds provide a ghost-like blue-print of the destroyed vasculature. The persistence of the VBM after degeneration of tumor vessel ECs may provide a mold for quick vascular regrowth after termination of angiogenesis inhibition, as in the case of anti-vegf therapy. Therefore, it might be necessary to consider therapeutic strategies for targeting destruction of the VBM, after the ECs have been destroyed. 25 The view of the ECM has changed remarkably in recent years, from it being a mere structural scaffold that surrounds cells, to its active role in influencing cells through several outside-in signaling pathways, and by serving as a source for embedded growth factors. 4,26 The VBM is a specialized extracellular matrix that surrounds both normal and pathologic blood vessels of all sizes and constitutes a scaffold between the endothelial cells that line the vessels and smooth muscle cells/pericytes that form a peripheral supporting cell layer (Figure 1). 4,5 Interestingly, the VBM has been found to be a rich source of angiogenesis stimulators and inhibitors that become bioavailable on proteolysis of networks of matrix proteins by tumor microenvironment associated enzymes such as matrix metalloproteinases, cathepsins, and elastases. 4 Tumor angiogenesis likely is influenced by the physiologic balance conferred by the pro-angiogenic factors and the anti-angiogenic factors in the body and their local influence on the endothelium (Figure 2). 1 In addition to the total number of factors governing such balance, the concentration levels of each factor also might determine the physiologic status of the balance. If the balance is switched to favor pro-angiogenesis because of either increased angiogenic factors or a decrease in antiangiogenic factors, a new blood vessel/capillary will form. Therefore, one potentially can prevent tumor angiogenesis by decreasing the levels of angiogenic factors or by increasing the levels of anti-angiogenic factors (Figure 2). Altering the angiogenic balance thus offers a new way of influencing tumor growth and constitutes an important addition to the arsenal of cancer treatments currently being tested. There is also increasing evidence that certain anti-angiogenic treatment, such as anti- VEGF therapy, normalizes the tumor vasculature, thus increasing the effects of other treatments such as chemotherapy and radiotherapy. 27 Most of the parent molecules for the matrix-derived endogenous angiogenesis inhibitors are found in all basement membranes, such as type IV and XVIII collagens. 4,5 On the other hand, some are secreted proteins sequestered within the ECM that become bioavailable on degradation/turnover of the ECM and BM. 4,5 It is unknown which basement membrane is the source for the majority of the matrix-derived endogenous angiogenesis inhibitors. One possibility is that they are released during the angiogenic process from the vessel that is being remodeled (ie, the VBM). However, a significant amount also can be derived naturally from the epithelial BMs during tissue remodeling and matrix breakdown/turnover. Therefore, the matrix-derived inhibitors are molecules generated during the degradation/turnover of the ECM, which thus generate a bioactive ECM degradome. 4,5 An important advance in the anti-angiogenic field, namely the Food and Drug Administration approval of bevacizumab (Avastin; Genentech, San Francisco, CA) as a therapeutic agent for the treatment of metastatic colorectal cancer, is discussed in this review, as well as the future use of this drug in other gastrointestinal cancers. In addition, the current status of the matrix-derived

3 2078 SUND ET AL GASTROENTEROLOGY Vol. 129, No. 6 Figure 2. The angiogenic balance. Angiogenesis stimulators such as VEGF, basic fibroblast growth factor, and platelet-derived growth factor, and angiogenesis inhibitors such as endostatin, tumstatin, TSP-1, and others maintain the angiogenic balance in the body. When the total activity of pro-angiogenic molecules exceeds that of the inhibitors, the anti-angiogenic balance is switched to favor new blood vessel formation. On the other hand, an excess of anti-angiogenic molecules would favor inhibition of angiogenesis. Whether the physiologic situation favors inhibition of angiogenesis or is in balance with angiogenesis stimulators is not known. endogenous inhibitors of angiogenesis as putative therapeutic agents in the clinic are evaluated. Matrix-Derived Endogenous Inhibitors of Angiogenesis Tumstatin, Arresten, and Canstatin From Type IV Collagen Type IV collagen is the main protein component of all BMs and is crucial for the stability and assembly of this specialized connective tissue structure. 28,29 Type IV collagen is composed of 6 different type IV collagen chains in mammals; the 1 and 2 chains are found in most BMs, whereas the other chains show more restricted expression patterns in various tissues. Each type IV collagen chain consists of 3 domains, an N-terminal 7S domain, a middle triple-helical collagenous domain, and the C-terminal globular noncollagenous (NC) domain. 30 The NC1 domain is considered to be important for the assembly of the type IV collagen trimer and the subsequent formation of the type IV collagen network. Mice genetically deficient in 1 and 2 chains of type IV collagen die during intrauterine development, 31 whereas mice lacking the 3 chain of type IV collagen initially

4 December 2005 GASTROINTESTINAL CANCER AND ANGIOGENESIS 2079 Table 1. Matrix-Derived Endogenous Inhibitors of Angiogenesis ECM degradome derived inhibitors Tumstatin Endostatin Arresten Canstatin Endostatin of type XV collagen 6(IV) NC1 domain Parent molecule 3(IV) collagen 1(XVIII) collagen 1(IV) collagen 2(IV) collagen 1(XV) collagen EC receptor EC proliferation EC migration EC apoptosis Tumor growth Metastasis v 3 integrin integrin, tropomyosin a NA 1 1 integrin NA NA NA 2 2 NA 2 NA 6(IV) NA 2 NA NA NA NA collagen Endorepellin Perlecan 2 1 integrin NA 2 NA NA NA Anastellin Fibronectin NA NA NA NA 2 2 Other ECMderived inhibitors TSP-1 CD NA TSP-2 NA NA Fibulin-5 NA 2 NA NA NA NA NA, not assessed; NC, noncollagenous domain; 1, increase; 2, decrease. a Mouse endostatin also has been shown to inhibit EC proliferation. survive, but later die of renal failure. 32 The severe phenotype in collagen IV deficient mice is not unexpected because type IV collagen has an important role in ensuring tissue integrity and function. Our laboratory used proteomic techniques on degraded VBM preparations using tumor-associated enzymes (matrix metalloproteinases [MMPs], elastases, cathepsins, and so forth), and discovered novel degradation fragments from type IV collagen and other VBM proteins with unique antiangiogenic activity (Table 1). 4,33 Tumstatin is the NC1 domain of the 3 chain of type IV collagen (Table 1). This 28-kilodalton molecule was produced recombinantly and found specifically to induce apoptosis of proliferating endothelial cells. 34 Tumstatin inhibits angiogenesis in several in vitro angiogenesis assays and suppresses tumor growth It also was shown recently that overexpression of the anti-angiogenic domain of tumstatin in B16F1 mouse melanoma cells significantly reduces the capacity of these cells to metastasize. 39 Integrins expressed on the cell surface of ECs are known to be important for the interactions between ECs and the ECM, and the v 3 integrin on the proliferating endothelial cells was shown to be a functional receptor for tumstatin Murine lung endothelial cells isolated from mice deficient in 3 integrin do not show decreased proliferation when treated with recombinant tumstatin, providing evidence for the requirement of this integrin for tumstatin action. 40 Furthermore, when analyzing VEGF-induced neovascularization of Matrigel (BD Biosciences, San Jose, CA) plugs in 3 integrin deficient mice, insignificant inhibition of vascularization was observed on tumstatin treatment. 40 Subsequently, the binding of tumstatin to 3 integrin was shown to cause an EC-specific inhibition of CAP-dependent protein translation, leading to the observed effect on cell proliferation ,41 Tumstatin is found in the circulation at a physiologic level of approximately ng/ml, 40 and most likely is generated by MMP cleavage of BMs containing the 3 chain of type IV collagen as a part of the regular BM turnover process. 34,40 Mice with an inactivation of the gene for the 3 chain of type IV collagen, and thus also lacking tumstatin, show accelerated tumor growth, associated with enhanced pathologic angiogenesis, with no effect on physiologic angiogenesis. 40 However, when these mice are supplemented with recombinant tumstatin to restore normal physiologic circulating concentrations, the increased rate of tumor growth was reduced to wild-type levels. The tumor-associated proteolytic enzyme MMP-9 has been found to cleave tumstatin most efficiently from the type IV collagen 3 chain. 40 The in vivo importance of this cleavage was shown when mice deficient in MMP-9 showed decreased circulating levels

5 2080 SUND ET AL GASTROENTEROLOGY Vol. 129, No. 6 of tumstatin and subsequently also accelerated tumor growth. 40 These results provide genetic evidence that lack of tumstatin, or the enzyme that releases it (MMP- 9), or the absence of its functional receptor on endothelial cells ( 3 integrin), leads to a shift in the angiogenic balance toward increased tumor growth. Arresten is the NC1 domain of the 1 chain of type IV collagen and was isolated by degradation of a human placental BM preparation (Table 1). 42 Arresten is a 26- kilodalton molecule that inhibits endothelial cell proliferation, migration, tube formation, and neovascularization of Matrigel plugs. By administering arresten to mice injected with subcutaneous tumors, both the growth of the primary tumors and the development of tumor metastases could be inhibited. 42 The potential receptor of arresten on the proliferating endothelium is 1 1 integrin. 42 Canstatin is the NC1 domain of the 2 chain of type IV collagen. This 26-kilodalton molecule was purified, recombinantly produced, and shown to inhibit significantly endothelial cell proliferation, migration, tube formation, and to induce apoptosis of these cells (Table 1). 43,44 Canstatin also suppressed in vivo growth of large and small size tumors in 2 human xenograft mouse models. 43 Interestingly, the NC1 domain of the 6 chain of type IV collagen also has been described to possess anti-angiogenic activity. 44 Endostatin From Type XVIII Collagen and the Endostatin Homologue From Type XV Collagen Type XVIII collagen is a heparan sulphate proteoglycan found in most vascular and other BMs that contains large noncollagenous domains at the N-terminus and C-terminus of the molecule, and a middle collagenous domain with multiple interruptions. 45 Endostatin, a fragment of the C-terminal NC11 domain of this collagen, was isolated initially from conditioned medium of a mouse hemangioendothelioma (Table 1). 46 This anti-angiogenic domain can be cleaved from type XVIII collagen by cathepsin-l, elastase, and matrilysin, and is found in the circulation at physiologic levels of ng/ml. 45,50 Mice deficient in endostatin show increased tumor growth when implanted with cancer cells that do not produce type XVIII collagen, and overexpression of circulating endostatin in transgenic mice leads to reduced tumor growth and vascularization. 51 Endostatin affects tumor growth by several means, including the inhibition of endothelial cell proliferation and migration, induction of apoptosis, and by causing G1 arrest of endothelial cells. 46,52 54 Several cell surface receptors using in vitro techniques have been reported for endostatin, such as 5 1, v 3, v 5 integrins, and glypicans. 41,55,56 Our laboratory and others have identified the interaction of endostatin to 5 integrin, and have shown that this binding leads to the inhibition of the focal adhesion kinase/c-raf/mek1/2/p38/erk1 mitogen-activated pathway, with no effect on PI3-kinase/ Akt/mTOR/4E-BP1 and CAP-dependent translation. 41 Others have found that the interaction with 5 integrin causes disruption of focal adhesions and actin stress fibers by down-regulating RhoA. 57,58 Most importantly, these results show that although endostatin and tumstatin both are molecules that are cleaved from 2 BM collagens, the anti-angiogenic action associated with them is mediated through distinct pathways. Another mechanism that potentially contributes to the anti-angiogenic effect of endostatin is by blocking of the binding of VEGF isoforms VEGF 165 and VEGF 121 to the VEGF receptor-2, which affects the tyrosine phosphorylation of this receptor. 59 The blocking occurs through direct binding of endostatin to the VEGF receptor Endostatin also has been described to be involved in matrix remodeling by inhibiting MMP-2 activity. 60 This molecule furthermore was found to stabilize cell-cell and cell-matrix adhesions, thus inhibiting angiogenesis because loosening of these junctions is required for vascular sprout formation. 61 Endostatin has been studied extensively and is currently in clinical trials, 4,62 the results of which are discussed in detail later in this review. Type XV collagen is a chondroitin sulfate proteoglycan with high homology to type XVIII collagen, sharing a similar structure with noncollagenous domains at the N-terminus and C-terminus and a middle collagenous domain with multiple interruptions. 63,64 Interestingly, the highest homology between type XVIII and XV collagens is found in the C-terminal endostatin domain Type XV collagen is located at BM zones of many tissues, including the VBM. 65,68 71 The role of the endostatin domain of type XV collagen in the regulation of angiogenesis largely is unknown, although it also has been shown to possess anti-angiogenic activity (Table 1). 67,72 Type XV collagen also has been speculated to possess tumor-suppressor activity. 73 Endorepellin Endorepellin is the 81-kilodalton C-terminal V- domain of perlecan, a large BM heparan sulphate proteoglycan. Endorepellin inhibits endothelial cell migration and tube formation, with inhibition of blood vessel growth in the chorioallantoic membrane and in the Matrigel plug assays (Table 1). 74 Mice deficient of per-

6 December 2005 GASTROINTESTINAL CANCER AND ANGIOGENESIS 2081 lecan die early during fetal development and thus a lack of endorepellin in the alteration of the angiogenic balance cannot currently be evaluated. 75 It recently has been shown that 2 1 integrin is the receptor for endorepellin on endothelial cells. 76 The binding of endorepellin to the receptor triggers a signaling pathway, leading to an increase in cyclic adenosine monophosphate and the activation of protein kinase A, focal adhesion kinase, p38 mitogen-activated protein kinase, and heat shock protein 27, followed by a down-regulation of p38 mitogenactivated protein kinase and heat shock protein 27, and the disassembly of actin stress fibers and focal adhesions, which subsequently results in the inhibition of EC migration and angiogenesis. 76 The terminal laminin-like globular domain of endorepellin contains the active site for the anti-angiogenic activity. 77 Interestingly, this laminin-like globular fragment has been found in the urine of patients with end-stage renal disease and the amniotic fluid of pregnant women with premature rupture of fetal membranes, indicating that proteolytic processing of laminin-like globular/endorepellin can occur physiologically in the body. The enzymes suggested in the processing of laminin-like globular/endorepellin are the bone morphogenic protein (BMP)-1/Tolloid family of metalloproteinases. 77 Anastellin Anastellin is a 76 amino acid fibronectin fragment, the III1-C peptide from the first type III repeat, with anti-angiogenic activity. 78 Fibronectin is an abundant ECM protein produced by most cell types and forms an adhesive fibrillar meshwork. The first type III repeat contains a site important for fibronectin self-assembly. 78 In vitro assays with anastellin have shown that this molecule can polymerize both fibronectin and fibrinogen, and these polymerized products show potent antitumor and antimetastasis effects when injected into mice bearing subcutaneous tumors (Table 1). Injecting the anastellin fragment alone into these mice shows the same; thus anastellin currently is believed to form an active complex with soluble fibronectin and fibrinogen; inducing slower tumor growth owing to reduced vascularization. 78 It recently was shown that the anti-angiogenic effect of anastellin is dependent on soluble fibronectin; an insignificant effect on vascularization of Matrigel plugs could be observed when this molecule was injected into mice lacking soluble fibronectin. 79 Interestingly, endostatin activity also was found to be dependent on soluble fibronectin in this study. 79 Yi et al thus conclude that a possible unifying explanation for activity of other matrix-derived endogenous inhibitors of angiogenesis is that they might form complexes with adhesion proteins, such as fibronectin and vitronectin, and that these complexes induce anti-angiogenic effects through interactions with integrins on the ECs. 79 This hypothesis needs experimental proof. Thrombospondin-1 and Thrombospondin-2 Thrombospondins-1 and -2 (TSP-1 and TSP-2) are secreted glycoproteins found in the ECM and pericellular matrix (Table 1). 80,81 TSP-1 directly interacts with a variety of extracellular matrix proteins, influences the levels of extracellular proteases, and activates transforming growth factor. 80 It also has been shown that the expression of the oncogene Ras simultaneously up-regulates VEGF and down-regulates TSP-1 and TSP-2 expression in tumors. 82 As a result of these Ras-regulated changes in gene expression, the tumor cells can stimulate angiogenesis and promote remodeling of the extracellular matrix. 82 Many cell-surface receptors have been shown to bind TSP-1 to mediate signals between the extracellular space and ECs, 80 although CD36 on the endothelial cells is believed to be a major receptor for the antiangiogenic effect of TSP-1. 80,81 When tumors were implanted on TSP1-deficient mice, an increased growth rate and vascular density in tumors was observed. 51,83 On the other hand, overexpression of TSP-1 in human squamous cell carcinoma cell lines resulted in reduced angiogenesis of subcutaneous tumors. 84 Interestingly, when TSP1-deficient mice were crossed with p53-deficient mice, decreased survival was observed, highlighting the importance of hostderived factors for tumor progression. 83 The active site responsible for the anti-angiogenic effect of TSP-1 has been shown to be located in the N-terminal heparinbinding domain of this large protein. 85 In recent years several mimetics to this active region have been developed and tested for antitumor activity because of the limited potential clinical use of TSP-1 as a result of its large size. The phase I clinical trials using one such mimetic are discussed in detail later in this review. TSP-2 also possesses anti-angiogenic activity, and tumors implanted on TSP-2 deficient mice show significantly increased tumor vascularization. 86 Increasing the circulating level of TSP-2, by injecting fibroblasts overexpressing TSP-2 into mice, leads to an inhibition of tumor growth and angiogenesis in subcutaneous tumors of several different cancer cell lines. 87 The mechanism for this anti-angiogenic activity is believed to be inhibition of EC migration and tube formation, and increased ECspecific apoptosis mediated by the 80-kilodalton fragment in the N-terminal region of TSP-2. 88

7 2082 SUND ET AL GASTROENTEROLOGY Vol. 129, No. 6 Fibulin-5 Fibulin-5 is a member of the calcium-binding fibulin family of ECM proteins. Fibulin-5 is expressed strongly in large arteries and mediates adhesion of ECs to ECM by scaffolding cells to elastic fibers. Mice deficient in fibulin-5 are viable but show major defects in the organization of elastic fibers, resembling the human disorder cutis laxa. 89,90 Cell adhesion by fibulin-5 is mediated through interaction with v 3, v 5, and 9 1 integrins. 91 It recently has been described that this protein has anti-angiogenic activity in vitro, as overexpression of fibulin-5 in endothelial cells caused an inhibition of endothelial cell proliferation, perturbed angiogenic sprouting, and reduced invasion through Matrigel matrices 92 (Kalluri laboratory, unpublished data) (Table 1), but tumor growth in the absence of this molecule has not been studied in fibulin-5 deficient mice. Fibulin-5 also enhances the expression of TSP-1 from ECs, thus further shifting the angiogenic balance toward reduced vascularization. 92 VEGF as Matrix-Associated Endogenous Stimulator of Angiogenesis The angiogenic balance is determined by proangiogenic stimulators on one side and the activity of the endogenous angiogenesis inhibitors on the other side (Figure 2). Several proteins are described as pro-angiogenic, such as basic fibroblast growth factor, plateletderived growth factor, VEGF, angiogenin, angiopoietins, transforming growth factor and, tumor necrosis factor, and various interleukins and chemokines. 93 The VEGF family of growth factors, which consists of VEGF -A, -B, -C, -D, and placental growth factor, are the best characterized among the pro-angiogenic factors (Table 2). These molecules bind to the receptor tyrosine kinases VEGF receptor-1, -2, and -3, and neuropilin-1 and -2, on the EC to initiate a cascade of effects, leading to increased proliferation, migration, and reduced apoptosis with the endothelium of both blood and lymphatic vessels (Table 2). 93,94 Many isoforms of VEGF (VEGF 121, VEGF 145, VEGF 165, VEGF 183, VEGF 189, and VEGF 206 ) are found as a result of alternative splicing. The isoforms VEGF 121, VEGF 165, and VEGF 189 are the major forms produced, 95 some of these isoforms diffuse freely (VEGF 121 ), whereas others are sequestered tightly in the ECM (VEGF 165 and VEGF 189 ) because of binding to heparan sulphate proteoglycans. 96 The roles of the ECM-sequestered VEGF isoforms largely are unknown. However, the VEGF 189 and VEGF 165 isoforms, which are sequestered in the Table 2. The VEGF Family of Angiogenesis Stimulators Molecule Splice variants Receptor Target endothelial cell VEGF 121, 145, 165, VEGFR1 Vascular 183, 189, 206 VEGFR2 Vascular Neuropilin-1 Vascular Neuropilin-2 Vascular VEGF-B 167, 186 VEGFR1 Vascular Neuropilin-1 Vascular VEGF-C VEGFR3 Lymphatic VEGFR2 Vascular VEGF-D VEGFR3 Lymphatic VEGFR2 Vascular PIGF 131, 152, 219 VEGFR1 Vascular Neuropilin-1 Vascular VEGFR, VEGF receptor; PIGF, placental growth factor. ECM and the vicinity of cell membranes, have been shown to promote endothelial cell adherence, migration, and spreading in vitro. 97 This adhesion potentially is mediated by binding to 3 1 and v integrins, and not the VEGF receptors. Interestingly, tumstatin was shown to inhibit this binding. 97 Mice expressing only the ECMsequestered isoforms VEGF 164 and VEGF 188 (corresponding to VEGF 165 and VEGF 189 in human beings) have been generated. 98 The VEGF 188/188 mice show a cartilage defect leading to dwarfism and knee-joint dysplasia, which is caused by impaired vascularization of the epiphysis, leading to hypoxia and apoptosis within the tissue. 99 In vitro experiments have shown a defect in chondrocyte proliferation and survival in these mice. 99 The VEGF 188/188 mice also have defects in the development of the arterial vascularization of the retina. 98 It also has been shown by using the RIP1-Tag2 mouse model of pancreatic cancer that MMP-9 cleavage of the ECM leads to increased release of matrix-bound VEGF and thus enhanced angiogenesis and tumor growth. 100 Similarly, in the HPV 16 transgenic mouse model for epithelial carcinogenesis of the skin, altering MMP-9 levels affects tumor progression, possibly through the release of matrix-bound VEGF. 101,102 The absolute requirement of VEGF for physiologic angiogenesis and hematopoiesis during development was shown when the deletion of a single allele of the VEGF led to fetal death as a result of extensive vascular defects. 103,104 However, expression levels of VEGF rapidly decrease after birth when endothelial cells become quiescent, but can be found in areas of active physiologic angiogenesis such as in the skin, ovaries, and uterus, 105,106 and sites of pathologic angiogenesis such as tumors. 93,96 Circulating VEGF levels increase in most cancer types, including gastrointestinal cancers, because of enhanced

8 December 2005 GASTROINTESTINAL CANCER AND ANGIOGENESIS 2083 release from the matrix and high production by tumor cells, stromal cells, and inflammatory cells. 93,94,107 The secretion of VEGF by tumor cells is induced strongly by the hypoxic conditions within the growing tumor, caused by poor vessel function and high interstitial pressure. 27,108 It currently is believed that the increased VEGF and VEGF receptor expression, through the action of the hypoxia-inducible factor-1 in response to hypoxia, is a major mechanism involved in the regulation of tumor angiogenesis. 109,110 The von Hippel Lindau tumor-suppressor gene is another strong VEGF regulator. Mutations in this gene are found in von Hippel Lindau disease and sporadic kidney cancers. 111 von Hippel Lindau targets certain subunits of hypoxia-inducible factor for degradation, and the cells lacking von Hippel Lindau subsequently overproduce the products of hypoxia-inducible factor target genes, such as VEGF, thus promoting angiogenesis. 111 However, other factors including activation of oncogenes such as src, fos, v-ras, k-ras, v-raf, and v-yes, 112 expression of transforming growth factor- 113 and nitric oxide, 114 and inactivation of tumor-suppressor genes such as p lead to increased VEGF levels in tumors. VEGF first was discovered as a vascular permeability factor because of its potent capacity to induce vascular permeabilization. 116 VEGF induces the formation of fenestrations in the venular endothelium, leading to leakage of plasma and plasma proteins from the vessels. In tumor vessels, such extravasation leads to the formation of a fibrinous gel, which subsequently can function as a provisional matrix, supporting the proliferation of both EC and fibroblasts, leading to enhanced angiogenesis and synthesis of new ECM. 117 Because of its central role in tumor angiogenesis the inhibition of VEGF expression or its signaling is an attractive target for cancer treatment. With Food and Drug Administration approval of bevacizumab (Avastin; a VEGF-blocking antibody) for the treatment of metastatic colorectal cancer in combination with intravenous 5-fluorouracil (5-FU) based chemotherapy, this experimental hypothesis has received clinical validation. 118,119 The Role of VEGF and Endogenous Inhibitors of Angiogenesis in Human Gastrointestinal Cancer The Value of VEGF and Matrix-Derived Inhibitors of Angiogenesis as Prognostic Markers Numerous publications during recent years have focused on establishing a relationship between the expression of VEGF or endogenous inhibitors of angiogenesis (endostatin, TSP-1, and TSP-2) and progression of gastrointestinal cancers (Table 3). 120 In these studies a correlation between increased or decreased circulating levels of these factors, or expression of these molecules by the tumor tissue, to the outcome of the disease has been documented. These studies aimed to investigate the value and potential of developing these agents as prognostic markers for the disease. The results from the majority of these studies are summarized in Table 3. For the 5 most prevalent gastrointestinal cancers, namely esophageal, gastric, hepatocellular, pancreatic, and colorectal cancer, increased VEGF expression by the tumor and increased circulating levels of this growth factor correlate with advanced disease (Table 3). Although suggested as not having significant prognostic value for certain cancer types, 121,122 for others increased VEGF expression levels were shown to be more sensitive than the traditional staging of tumors in predicting the risk for relapses When analyzing the expression of the endogenous inhibitors in the context of tumor progression, it can be generalized for both endostatin and TSP-1 that high expression of these molecules by the tumor and high circulating levels indicates poor prognosis, likely reflecting a large tumor burden, although for some cancer forms the opposite has been found (Table 3). Nevertheless, most of these studies clearly indicate that these molecules can be of some value in the potential analysis of disease progression and prognosis. Clinical Trials to Establish Endogenous Inhibitors of Angiogenesis as Therapeutic Agents in Human Gastrointestinal Cancer Two matrix endogenous inhibitors of angiogenesis, endostatin and a small peptide mimetic of TSP-1, ABT-510, have entered clinical trials to establish their potential use as therapeutic agents for cancer treatment. The results from these clinical trials are summarized in Table 4. Recombinant human endostatin was shown in 3 separate phase I clinical trials to be well tolerated and safe without any serious adverse effects. 50,127,128 In these trials endostatin was given as a daily intravenous infusion with concentrations ranging from 15 to 600 mg/m 2 to patients with advanced metastasized cancers of various types (Table 4). 50,127,128 The observed antitumor effect was lower than expected, especially when compared with the effect of this molecule in preclinical studies. 46, However, the lack of effect partially may be a result of study design because patients with highly advanced disease potentially may not respond significantly to singleagent anti-angiogenic therapy. 50,129 Animal studies also have shown that administering endostatin continuously

9 2084 SUND ET AL GASTROENTEROLOGY Vol. 129, No. 6 Table 3. Expression and Prognostic Value of VEGF and Matrix-Derived Endogenous Inhibitors of Angiogenesis in Human Gastrointestinal Cancers Molecule Cancer type Patient number Correlation of expression with disease state a Correlation of circulating level and disease state Prognostic value of expression Reference VEGF Esophagus 45, Direct NA Mixed results 143, 144 Gastric 86, 156, 281 Direct NA Yes Hepatocellular 36, 46, 50, 21 Direct No correlation Mixed results 121, 122, Gallbladder and bile 50, 51, 24, 60, 68 Direct NA Yes duct Pancreas 142, 70, 30, 55, Direct NA Mixed results 123, Colorectal 152, 97, 81, 104, 65, 109, 150 Direct Direct Yesb 125, 126, Endostatin Hepatocellular 57, 108 Inverse No correlation Mixed results 163,164 Colorectal 30 NA Direct Yes b 165 TSP-1 Esophagus 43 Direct NA Yes 166 Gastric 30, 80 No correlation NA NA 167, 168 Hepatocellular 60 Direct NA Yes 169 Gallbladder and bile 67, 53 Direct NA Yes 170, 171 duct Pancreas 77 Direct NA Yes 172 Colorectal 61, 152, 100, 132 Inverse NA Yes 158, TSP-2 Gastric 32 Direct NA Yes 176 Colorectal 61 Inverse NA Yes 173 NA, not assessed. a Analysis of either mrna and/or protein levels has been used in the studies of the expression of VEGF and matrix-derived endogenous inhibitors of angiogenesis. b Prognostic value of measuring circulating values has been shown. 124, 148 yields better results than single daily injections/infusions, 46 a fact that potentially could have influenced the outcome of these clinical studies. Nevertheless, phase I clinical trials did show therapeutic benefit, in the form of partial regression and stabilization of disease for certain cancer types, such as pancreatic neuroendocrine tumors, colon cancer, soft-tissue sarcoma, and melanoma (Table 4). 50,127 Interestingly, in the phase I trials measurable effects on tumor blood flow, tumor metabolism, tumor cell apoptosis, and decreased levels of VEGF were observed. 128,132 Endostatin proceeded to phase II clinical trials for pancreatic neuroendocrine tumors. The prelim- Table 4. Results From Clinical Trials Using Endogenous Inhibitors of Angiogenesis Against Human Cancers Molecule Patient number RhEndostatin 26 Advanced cancer of various types RhEndostatin 15 Advanced cancer of various types RhEndostatin 21 Advanced cancer of various types ABT Advanced cancer of various types ABT Advanced cancer of various types Cancer type and state Dosage Results Reference mg/ m 2 /day IV infusion mg/ m 2 /day IV infusion mg/ m 2 /day IV infusion mg/ day SC injection mg/ day SC injection SD; 1 patient for 60 wk (melanoma), 1 50 patient for 20 wk (synovial cell sarcoma) PR; 1 patient for 44 wk (pancreatic 127 neuroendocrine tumor) SD; 1 patient for 16 wk (colon cancer), 1 patient for 12 weeks (soft-tissue sarcoma) No therapeutic effect observed 128 PR; 1 patient (soft-tissue sarcoma) 136 SD; 41% of patients at 8 wk of study, 15% at 16 wk SD; 27% of patients at 16 wk of study 177 RhEndostatin, recombinant human endostatin; IV, intravenous; SD, stabilization of disease; SC, subcutaneous; PR, partial regression of tumor.

10 December 2005 GASTROINTESTINAL CANCER AND ANGIOGENESIS 2085 inary analysis of the results from this study showed that 5% of patients had minor radiologic responses, and 62% of patients experienced stable disease in response to therapy. 133 The TSP-1 mimetic ABT-510 competes with TSP-1 for binding to CD36, thus blocking multiple pro-angiogenic signals and inducing apoptosis of endothelial cells. 134,135 In 2 phase I clinical trials of various cancer types, ABT-510 was shown to be well tolerated and no serious side effects were observed (Table 4). 136,137 Doses ranging from mg of ABT-510 were administered as a daily subcutaneous injection. Partial regression of tumor was observed in 1 patient with soft-tissue sarcoma and stabilization of disease was observed in 41% and 15% of patients after 8 and 16 weeks of therapy, respectively. 136 The stabilization of disease was prolonged for certain patients with effects lasting up to 24 weeks on therapy. 136 ABT-510 is currently in phase II clinical trials. 137 Bevacizumab (Avastin) as a Therapeutic Agent Against Human Gastrointestinal Cancer Bevacizumab (Avastin) is a humanized murine monoclonal antibody that binds to all VEGF isoforms, thus blocking the biological activity of this growth factor by inhibiting the interaction of VEGF with its receptor. 138 A phase III clinical study showed the efficacy of bevacizumab in the treatment of colorectal cancer. In this study, the efficacy of bevacizumab (measured as overall survival) was tested in combination with the standard treatment of irinotecan 125 mg/m 3, 5-FU 500 mg/m 3, and leucovorin 20 mg/m 3 (IFL) given once weekly for 4 weeks every 6 weeks in previously untreated metastatic colon or rectal carcinoma. Patients were randomized to receive standard bolus IFL plus placebo therapy (arm 1), bolus IFL plus bevacizumab (5 mg/kg every 2 weeks, arm 2), or 5-FU/ leucovorin plus bevacizumab (arm 3). Arm 3 subsequently was discontinued when the toxicity of bevacizumab in combination with bolus IFL met the prespecified criteria. 118 In the remaining 813 patients, randomized into arms 1 and 2, IFL plus bevacizumab treatment led to a significant improvement of the overall survival (20.3 vs 15.6 mo), progression-free survival (10.6 vs 6.4 mo), and an increased overall response rate (45% vs 35%). 118 Another study that tested the efficacy of bevacizumab in combination with 5-FU 500 mg/m 3 and leucovorin 500 mg/m 3 weekly for 6 weeks every 8 weeks as a third line of therapy, also showed a significant improvement of overall survival and response rate. Interestingly, in this study, bevacizumab was more effective at a concentration of 5 mg/kg than at the higher dose of 10 mg/kg. 139 Furthermore, in a separate study 6 patients with adenocarcinoma of the rectum received preoperative treatment with bevacizumab alone, before they were treated 2 weeks later with bevacizumab plus 5-FU, external beam radiation therapy, and surgery. 140 Analysis of these patients after the initial 2 weeks of bevacizumab therapy showed that tumor size decreased, with reduced tumor perfusion, vascular density, and a reduced number of circulating endothelial cells. This study showed that inhibition of tumor angiogenesis by bevacizumab accounted for the observed decrease of tumor size. 140 Bevacizumab is currently in clinical trials for treatment of a variety of human cancers as monotherapy or in combinations with chemotherapy and/or radiotherapy and/or other targeted therapies (for additional information see Ongoing trials for gastrointestinal cancers include bevacizumab combined with cetuximab, and bevacizumab in combination with oxaliplatin/5-fu/ leucovorin vs bevacizumab alone as a first line of therapy for colon cancer. 141 In addition, preoperative bevacizumab/5- FU/radiation therapy is being tested further for rectal cancer. 141 Bevacizumab monotherapy is currently in phase II clinical trials for hepatocellular cancer, and phase II and I trials are ongoing for pancreatic cancer with bevacizumab/ gemcitabine and bevacizumab/capecitabine/radiation therapy, respectively. 141 Bevacizumab has been combined with other forms of treatment such as chemotherapy, radiotherapy, and other targeted therapies with good results, whereas clinical trials with this molecule as a monotherapy have been less successful in terms of increased survival. 142 One proposed explanation is that the anti-angiogenic effect of bevacizumab, besides inhibiting recruitment of tumor vasculature, also normalizes a portion of it, which subsequently leads to efficient delivery of other drugs and/or an enhanced effect of radiotherapy. 27,140 This hypothesis clearly indicates that the balance of anti-angiogenic and antitumor treatment of cancer is very intricate and requires further optimization and testing. It also appears from the failure of bevacizumab to increase survival in certain cancer settings that the potential therapeutic effect of this molecule might be dictated by the VEGF dependency of a given tumor at the time of therapy (Figure 3). Over the entire course of cancer progression, tumors can use multiple pro-angiogenic proteins and thus there might be a need to design anti-angiogenic therapies that affect many of these simultaneously (Figure 3). The side effects seen in the patients treated with bevacizumab mostly were mild and related to the vascular system, including thrombosis, bleeding, hypertension, and proteinuria. Severe effects including gastrointestinal perforations and central nervous system hemorrhages also have been reported. 118,139 Sur-

11 2086 SUND ET AL GASTROENTEROLOGY Vol. 129, No. 6 Figure 3. The hypothesis that cancer progression likely is associated with emerging dependence on different pro-angiogenic factors. Fastgrowing cancers such as colorectal cancer likely depend on 1 angiogenic factor such as VEGF, whereas slow-growing cancers such as breast cancer might undergo more genetic instability, favoring the use of several angiogenic factors for tumor growth, 178 thus making it less likely for an agent that neutralizes 1 angiogenic factor to show clinical efficacy. prisingly, when several of the bevacizumab trials were designed, there was no diagnostic eligibility testing, such as measurement of VEGF expression or tumor vascular density, which potentially could select for a patient population with potentially higher clinical benefit with the use of bevacizumab. 119 Summary Endogenous stimulators and matrix-derived inhibitors of angiogenesis are molecules that are produced naturally and circulate in the body. These molecules are important in maintaining the angiogenic balance that influences the rate of new blood vessel formation. Many of the matrix-derived endogenous angiogenesis inhibitors are products from the ECM degradome and can be released by tumor-associated proteolytic enzymes. Others are molecules sequestered pericellularly in the ECM. The VBM is a specialized ECM that surrounds all blood vessels, providing a structural scaffold for endothelial cell and pericyte interactions and provides these cells with signals that affect cell growth, differentiation, and apoptosis. The VBM is a source of many matrix-derived endogenous angiogenesis inhibitors such as tumstatin, canstatin, arresten, and endostatin. The matrix-derived endogenous inhibitors of angiogenesis are attractive candidates for potential cancer therapy due to low toxicity and very promising efficacy results in animal studies. Endostatin and a TSP-1 mimetic, ABT-510, are currently in phase II clinical studies for certain cancer types. VEGF is a growth factor that strongly induces new vessel formation by aiding tumor angiogenesis, and this molecule is overexpressed in most human cancers. Treatment with bevacizumab (Avastin), an anti-vegf antibody, significantly increases the survival of patients with metastatic colorectal cancer. Currently, several other clinical studies are ongoing to evaluate the potential role of bevacizumab therapy for other forms of cancer. However, it is becoming clear that anti-angiogenic therapy most likely will be used in combination with radiation and chemotherapy as an additional method to maximize the total antitumor effect in patients with advanced disease. In addition to their value as therapeutic agents, VEGF and matrix-derived endogenous angiogenesis inhibitors potentially also can be used as prognostic markers to assess for tumor progression. 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