Review

Endogenous Inhibitors of

Pia Nyberg, Liang Xie, and Raghu Kalluri

Center for Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Abstract factor, and hypoxic conditions that activate hypoxia-inducible Angiogenesis, the formation of new blood vessels, is required factor-1, which itself can up-regulate angiogenic proteins, as well as for many pathologic processes, including invasive tumor angiogenic oncogenes, such as Ras, and tumor suppressors, such as growth as well as physiologic organ/tissue maintenance. . Endogenous inhibitors of angiogenesis include various Angiogenesis during development and adulthood is likely antiangiogenic peptides, hormone metabolites, and regulated by a balance between endogenous proangiogenic modulators (Fig. 2; for reviews, see refs. 1, 4). To understand how and antiangiogenic factors. It is speculated that tumor growth systemic angiogenesis balance in the body is regulated by requires disruption of such balance; thus, the angiogenic endogenous inhibitors of angiogenesis requires an understanding switch must be turned ‘‘on’’ for progression. If the of how many such molecules exist and their role in inhibiting angiogenic switch needs to be turned on to facilitate the angiogenesis. This review will highlight the important features of tumor growth, the question remains as to what the physiologic each of these molecules and address their role in the regulation of status of this switch is in the adult human body; is it ‘‘off,’’ with angiogenesis balance. inhibitors outweighing the stimulators, or maintained at a Vascular components can modulate fine ‘‘balance,’’ keeping the proangiogenic properties of many endothelial cell behavior in addition to providing structural and factors at a delicate ‘‘activity’’ balance with endogenous functional support (5). A series of endogenous antiangiogenic inhibitors of angiogenesis. The physiologic status of this factors have been described, of which many are fragments of balance is important to understand as it might determine an naturally occurring (ECM) and basement individual’s predisposition to turn the switch on during membrane proteins (6). In this review, we divide the endogenous pathologic events dependent on angiogenesis. Conceivably, if inhibitors of angiogenesis into two major classes, matrix-derived the physiologic angiogenesis balance in human population inhibitors and non-matrix-derived inhibitors (Fig. 2). The reader will notice that most studies dealing with exists somewhere between off and even balance, an individ- ual’s capacity and rate to turn the switch on might reflect their endogenous inhibitors of angiogenesis address therapeutic impact normal physiologic angiogenic status. In this regard, although of these agents to control cancer progression when given at extensive knowledge has been gained in our understanding of pharmacologic doses. Nevertheless, interesting studies are emerg- endogenous growth factors that stimulate angiogenesis, the ing, which also highlight the role of these inhibitors in controlling activities associated with endogenous inhibitors are poorly cancer at physiologic concentrations. understood. In this review, we will present an overview of the knowledge gained in studies related to the identification and Matrix-Derived Inhibitors of Angiogenesis characterization of 27 different endogenous inhibitors of Arresten. The main component of vascular basement mem- angiogenesis. (Cancer Res 2005; 65(10): 3967-79) branes is type IV , forming a mesh-like structure with other macromolecules, such as laminin, heparan sulfate proteoglycans, Introduction fibronectin, and entactin (7). Type IV collagen is composed of six Angiogenesis, the formation of new capillaries, is among the key distinct gene products (i.e., a1-a6; for a review, see ref. 8). It has events in various tissue destructive pathologic processes, such as been observed earlier that a1 and a2 type IV collagen chains tumor growth, , arthritis, etc., as well as in physiologic isolated from the Engelbreth-Holm-Swarm sarcoma tumor are processes, like organ growth and development, , and inhibitory to capillary endothelial cell proliferation (9). reproduction (1). A hypothesis that tumor growth is angiogenesis Arresten is a recently identified endogenous inhibitor of dependent was first proposed by Folkman (2). The expansion of angiogenesis. It is a 26-kDa molecule derived from the non- tumors is strictly dependent on the neoangiogenesis, and the collagenous (NC1) domain of the a1 chain of type IV collagen (10). inhibition of vascular supply to tumors can suppress their growth In endothelial cell proliferation assays, a dose-dependent inhibition (3). Solid tumors cannot grow beyond 2 to 3 mm in diameter of basic FGF (bFGF)–stimulated endothelial cells proliferation is without being able to recruit their own blood supply. Angiogenesis detected with an ED50 value of as low as 10 nmol/L. Arresten is thought to depend on a delicate balance between endogenous selectively inhibits endothelial cell tube formation (10). Normally, stimulators and inhibitors (Fig. 1). Stimulators of angiogenesis when mouse aortic endothelial cells are cultured on Matrigel include growth factors, such as vascular endothelial (a solid gel of mouse basement membrane proteins), they rapidly (VEGF), (FGF), and platelet-derived growth align and form hollow tube-like structures (11), but arresten disrupts with this process. It also inhibits the formation of new blood vessels in Matrigel plug assay in mice (10). Arresten inhibits Requests for reprints: Raghu Kalluri, Center for Matrix Biology, Department of endothelial cell proliferation and migration (10). Arresten does not Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 have a significant effect on the proliferation of several cancer cell Brookline Avenue, Dana 514, Boston, MA 02215. Phone: 617-667-0445; Fax: 617-975- 5663; E-mail: [email protected]. lines even at very high doses. Arresten affects metastasis leading to I2005 American Association for Cancer Research. significant reduction of pulmonary nodules in arresten-treated www.aacrjournals.org 3967 Cancer Res 2005; 65: (10). May 15, 2005

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Figure 1. The pathologic and physiologic angiogenic balance. It is speculated that, in normal adults, neoangiogenesis is tightly regulated, but the balance can be disrupted to favor increased blood vessel formation in many pathologic conditions (cancer, ocular disorders, psoriasis, etc.) and in physiologic repair settings (such as wound healing and reproduction). Here, we propose that angiogenic balance in the normal physiologic setting in a given individual may reflect the net capacity to launch an angiogenic response during pathologic setting. We propose the endogenous inhibitors of angiogenesis are important regulators of pathologic angiogenic switch. mice and inhibition of large and small renal cell carcinoma tumor proliferation or apoptosis of nonendothelial cells. The inhibition of growth (10). endothelial proliferation was not associated with a change in The existing evidence suggests that arresten might function via extracellular signal-regulated kinase (ERK) activation, as canstatin a1h1 and block the binding of a1h1 integrin to the type I did not alter the early phosphorylation of ERK after stimulation h collagen (10). a1 1 Integrin is a collagen receptor, but it also binds with serum and/or mitogens. This indicates that canstatin does not to other basement membrane constituents. In this regard, a1 in- primarily work by inhibiting proximal events activated by VEGF or tegrin neutralizing can suppress angiogenesis associated bFGF receptors. It is possible that canstatin acts by predisposing h with tumor growth (12). The 1 integrin is also involved in angio- cells to apoptosis. Canstatin also suppresses growth of tumors in genesis (13). Ablation or blocking of the interactions with integrin human xenograft mouse models, with histology revealing decreased a1h1 inhibits angiogenesis (12, 14), indicating that integrin a1h1 CD31-positive vasculature (18). It inhibits the phosphorylation of acts as a proangiogenic factor. Among the collagen , the Akt, focal adhesion kinase (FAK), mammalian target of rapamycin h a1 1 integrin activates the Ras-Shc-mitogen-activated protein (mTOR), eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), kinase (MAPK) pathway, thus promoting cell proliferation (15). In and ribosomal S6 kinase in cultured human umbilical vein addition, in the a1 integrin–deficient mice, tumors are less endothelial cells (HUVEC). It also induces Fas ligand (FasL) vascularized compared with wild-type mice (14, 16). Arresten expression, activates procaspase-8 and -9 cleavage, reduces might also function via binding to heparan sulfate proteoglycan mitochondrial membrane potential, and increases cell death. Thus, (10) and previous studies have shown that heparan sulfate canstatin-induced apoptosis seems to be associated with phospha- proteoglycan binds to the a1 NC1 domain of type IV collagen (17). tidylinositol 3-kinase (PI3K)/Akt inhibition and is dependent on Canstatin. Canstatin is a 24-kDa fragment of the a2 chain of type signaling events transduced through membrane death receptors IV collagen. Recombinant canstatin significantly inhibits endothe- (19). A functional receptor for canstatin is not yet identified, but lial and tube formation in a dose-dependent manner canstatin likely functions via cell surface integrins. (18). Canstatin inhibits serum-stimulated human endothelial cell In addition to the NC1 domains of collagen IV a1, a2, and a3 (see proliferation and induces apoptosis with no inhibitory effect on below) chains, the a6 chain possesses antiangiogenic activity and

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inhibits tumor growth. Soluble NC1 domain of the a6 chain suppresses primary tumor growth and metastasis in experimental regulates endothelial and migration (20). animal models without any apparent side effects, toxicity, or Endorepellin. is basement membrane heparan sulfate development of resistance (26, 30, 31). proteoglycan that plays key roles in vascular growth (21). The New insights into the molecular mechanisms associated with COOH-terminal end of perlecan, called endorepellin or perlecan for inhibition of tumor growth are emerging. Recent domain V, potently inhibits several aspects of angiogenesis: studies have reported that endostatin interferes with FGF-2- endothelial cell migration, collagen-induced endothelial tube induced signal transduction, blocking endothelial cell motility , and blood vessel growth in the chicken chorioal- (32), inducing apoptosis (33), causing G1 arrest of endothelial cells lantoic membrane assay and in mouse Matrigel plug assays. through inhibition of cyclin D1 (34), blocking VEGF-mediated Endorepellin is active at nanomolar concentrations in the in vitro signaling via direct interaction with the VEGF-R2/KDR/Flk-1 assays and blocks endothelial cell adhesion to fibronectin and type receptor tyrosine kinase in HUVECs (35), and blocking tumor I collagen without directly binding to these matrix proteins (22). necrosis factor–induced activation of c-Jun NH2-terminal kinase Endorepellin binds to endothelial cells as well as to squamous cell and c-Jun NH2-terminal kinase–dependent proangiogenic gene carcinoma cells and breast carcinoma cells via high-affinity expression (36). Endostatin rapidly down-regulates many genes in receptors. Interestingly, endorepellin binds endostatin, another growing endothelial cells, including immediate-early response matrix-derived inhibitor of angiogenesis, and counteracts its genes, cell cycle–related genes, and genes regulating apoptosis antiangiogenic effects (22). The exact mechanism of endorepellin inhibitors, MAPKs, FAKs, and G-protein-coupled receptors medi- is not yet known. In this regard, h1 integrin and a-dystroglycan ating endothelial , mitogenic factors, adhesion have been shown to interact with the COOH-terminal domain of molecules, and cell structure components (37). Recently, it is perlecan (a region containing endorepellin; refs. 23–25). shown that endostatin down-regulates many signaling pathways Endostatin. Endostatin is an endogenous collagen XVIII–derived in human microvascular associated with proangio- identified and purified from murine heman- genicactivityandatthesametimeup-regulatingmany gioendothelioma cell line (26) and later characterized in mice (27). antiangiogenic genes. Endostatin also affects signaling events that It corresponds to a 20-kDa fragment derived from the COOH- are not associated with angiogenesis, demonstrating the impor- terminal NC1 domain of type XVIII collagen (26, 28, 29). tance of interpathway communications in an intricate signaling Recombinant endostatin efficiently blocks angiogenesis and network (38).

Figure 2. The endogenous angiogenesis inhibitors. www.aacrjournals.org 3969 Cancer Res 2005; 65: (10). May 15, 2005

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Figure 3. Interactions of vascular endothelial cell–associated integrins with matrix-derived angiogenic inhibitors. Basement membrane collagen-derived endogenous inhibitors of angiogenesis are novel cryptic ligands for various integrins on proliferating endothelial cell surface. It is interesting to note that endostatin and , both basement membrane collagen-derived inhibitors, function via distinct integrins to negatively regulate proliferating endothelial cells through unique signaling pathways. The functional receptor/s for canstatin are still unknown, although it is known to bind avh3 and a3h1 integrins.

Rehn et al. showed that recombinant ‘‘immobilized’’ human p44/42 MAPK, p38 MAPK, and p21-activated kinase activity (43). endostatin interacts with a5 and av integrins on the surface of Moreover, a recent study shows that endostatin action is human endothelial cells. Furthermore, this endostatin-integrin dependent on expression of E-selectin on the endothelial cells, interaction is of functional significance in vitro, as immobilized although a direct binding of endostatin to E-selectin was not endostatin promotes integrin-dependent endothelial cell functions observed (44). Therefore, more work needs to be done to sort out (39). Sudhakar et al. have further studied the receptors for soluble the exact mechanism of action associated with endostatin. endostatin and the downstream signaling events. Endostatin binds Endostatin binds to (45) and with low affinity to all to the a5h1 integrin and inhibits the migration of endothelial cells surface heparan sulfate proteoglycans that are involved in growth by blocking signaling pathways via Ras and Raf and further factor signaling (46–48). The antiangiogenic activity of endostatin downstream via ERK1 or p38 (Fig. 3; ref. 40). In the endothelial seems to depend on the interactions with heparan sulfate h cells, endostatin induces rapid clustering of a5 1 integrin proteoglycans possibly by an interaction between discontinuous associated with actin stress fibers and causes colocalization with sulfated domains in heparan sulfate proteoglycans and arginine the membrane anchor protein caveolin-1, which couples integrins clusters at the endostatin surface (49). Recent work has shown the to cytoplasmic signaling cascades (41). In these experiments, it was identity of a specific arginine-rich sequence motif of human shown that endostatin binds to both a5h1 and caveolin-1 and that endostatin that interacts with endothelial cell surface h1 integrin endostatin treatment induces phosphatase-dependent activation of and heparin and inhibits endothelial cell migration and tube caveolin-associated Src family kinases (42). The disassembly of formation. This Arg-Gly-Asp (RGD)–independent sequence is likely actin stress fibers/focal adhesions by endostatin effects cell-matrix to be the motif responsible for the antiangiogenic activity of interaction and cell motility via activation of Src and in a tyrosyl endostatin (50). phosphatase–dependent manner (42). On the other hand, Eriksson Endostatin inhibits the activation and activity of certain matrix et al. conclude that endostatin inhibits chemotaxis, without metalloproteinases (MMP; i.e., MMP-2, -9, and -13 and MT1-MMP) affecting intracellular pathways known to regulate endothelial cell and it binds directly to at least MMP-2 and -9 (51–53). However, migration and proliferation/survival, because they did not find any endostatin does not inhibit all MMPs, because it had no effect on effect of endostatin on phospholipase C-g, Akt/, the activation of MMP-8 (53). In addition to MMPs, endostatin has

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Endogenous Angiogenesis Inhibitors been shown to interfere with the actions of other , like the Anastellin—a fibronectin fragment. Incubation of soluble plasminogen activator system (54). Interestingly, certain MMPs can fibronectin with a small fibronectin-derived fragment, called generate endostatin-containing peptides differing in molecular size anastellin, results in a polymeric form of fibronectin that is (20-30 kDa) from human type XVIII collagen (55). These fragments strongly antimetastatic in tumor-bearing mice (64). Both anastellin inhibit the proliferation and migration of HUVECs in a similar and polymeric fibronectin reduce tumor growth in mice, and the fashion as native 20-kDa endostatin.1 tumors are less vascularized (65). Anastellin is unable to inhibit Endostatin has been shown to significantly reduce invasion of Matrigel plug angiogenesis in mice that lack plasma fibronectin, not only endothelial cells but also tumor cells into reconstituted but it is fully active in mice that are null for vitronectin, which like basement membrane, Matrigel (51). Although the main focus has fibronectin is a major endothelial cell adhesion protein. Interest- been on the antiangiogenic activity of endostatin, it should also be ingly, the activity of endostatin is impaired in both fibronectin- noted that endostatin inhibits tumor growth not only by acting via deficient and vitronectin-deficient mice. This suggests a shared endothelial cells but also by directly decreasing carcinoma cell mechanism of action for antiangiogenic factors derived from ECM migration. Furthermore, intravasation (the key step of carcinoma and plasma proteins (66). process leading to metastasis, where tumor cells invade through Fibulins. Proteolytic digestion of basement membrane prepara- blood vessel walls into the bloodstream) of oral carcinoma cells is tions by elastases and cathepsins releases fragments, which possess inhibited by endostatin (53). antiangiogenic activity (67). Two such fragments were COOH- Endostatin can exist in two forms: as a monomer or as a trimer terminal fragments corresponding to fibulin 1D and the domain III that exerts different or even opposite effects (e.g., trimeric of fibulin 5.2 Recently, fibulin 5 has been shown to antagonize VEGF endostatin in the NC1 domain is needed for endothelial cell signaling and inhibit endothelial angiogenic sprouting (68). Fibulin migration, but monomeric endostatin inhibits the migratory 5 also binds to extracellular superoxide dismutase (69). In contrast, activity; refs. 56, 57). In addition, it has been shown recently that another report recently shows that fibulin 5 promotes wound endostatin can exist as a soluble globular form or as an insoluble healing in vivo (70). Considering that wound healing is dependent form with abundant cross-h-sheets aggregating into amyloid on angiogenesis, this report opposes the notion that fibulin 5 is an deposits. Such different endostatin conformations have distinct inhibitor of angiogenesis. Future studies will hopefully shed more effects on plasminogen activation. Insoluble endostatin stimulates light on this opposing action of fibulin 5. plasminogen activation, whereas soluble endostatin has no effect . -1 (TSP-1) was the first on plasminogen activation (58). protein to be recognized as a naturally occurring inhibitor of The physiologic levels of circulating endostatin in the serum are angiogenesis (71). It is a large multifunctional ECM glycoprotein f40 to 100 ng/mL compared with the concentrations of that regulates various biological events, like cell adhesion, endostatin (0.2-20 mg/mL) that are effective in the inhibition of angiogenesis, cell proliferation and survival, transforming growth tumor growth in various experiments. It has been shown that some factor-h (TGF-h) activation, and activation (ref. 63; for a of the antiangiogenic and antitumor effects of endostatin might be review, see ref. 72). Some studies suggest that TSP-1 may possess in fact represent pharmacologic effects at high doses and not dual activity (proangiogenic and antiangiogenic) depending on necessarily related to the physiologic function of endostatin (59). In proteases that generate fragments of TSP-1 (73, 74). It has been this regard, the physiologic levels of endostatin have no effect on shown to inhibit tumor growth and metastasis, thus making it a the growth of fibrosarcomas and melanomas in collagen XVIII/ potent inhibitor of in vivo neovascularization and tumorigenesis. endostatin knockout mice (60). Overexpression of TSP-1 in mice suppresses wound healing and Endostatin-like fragment from type XV collagen. Based on a tumorigenesis, whereas the lack of functional TSP-1 results in homology search with endostatin, a 22-kDa fragment of collagen XV increased vascularization of selected tissues (75–77). Expression of was found with 70% homology to endostatin. It inhibits the migration TSP-1 has been inversely correlated with malignant progression in of endothelial cells but has no effect on proliferation. Systemic breast and lung carcinomas and melanomas (78). To evaluate the administration of endostatin-like fragment from type XV collagen importance of TSP-1 for the progression of naturally arising tumors (EFC-XV) suppresses the growth of tumors in a xenograft renal in vivo, Lawler et al. have crossed TSP-1-deficient mice with p53- carcinoma model (61). Both NC1 domains of collagen XV and XVIII deficient mice. In the p53-null mice, the absence of TSP-1 contain a trimerization domain, a hinge region that is more sensitive decreased survival. They also determined more directly whether to proteolysis in collagen XVIII than collagen type XV. Unlike host TSP-1 inhibited tumor growth by implanting melanoma and endostatin, EFCXV does not bind to zinc or heparin. Both endostatin testicular teratocarcinoma cells into the TSP-1-null mice. The and EFC-XV inhibit FGF-2-induced or VEGF-induced angiogenesis tumors grew faster in the TSP-1-null background and exhibited an associated with chicken chorioallantoic membrane angiogenesis, but increase in vascular density, a decrease in the rate of tumor cell there are striking differences depending on which cytokine is used and apoptosis, and an increase in the rate of tumor cell proliferation whether free EFC-XV or XV-NC1 domains are used (62). Endostatin (79). The antiangiogenic activity of TSP-1 has been mapped to the and EFC-XV show a similar binding repertoire for ECM proteins. type 1 repeats and within the NH2-terminal portion of the molecule However, differences are detected using immunohistologic localiza- within the procollagen-like domain. TSP-1 and peptides from the tion in vessel walls and basement membrane zones (62). Although it type 1 repeat region (tryptophan-rich, heparin-binding sequences seems that the functions of endostatin and EFC-XVsomewhat overlap, and TGF-h1 activation sequences) were evaluated in two models of double knockout mice show no additional defects compared with the retinal angiogenesis. TSP-1 inhibited angiogenesis in both exper- single knockout mice (63). imental models, but peptides from the native TSP-1 sequence

1 Heljasvaara et al., unpublished observations. 2 Xie and Kalluri, unpublished data. www.aacrjournals.org 3971 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research containing both the tryptophan-rich repeat and the TGF-h1 with no effect on tumor cell proliferation. On the other hand, the activation sequence or containing only the tryptophan-rich, fragment of tumstatin containing amino acids 185 to 203 binds also heparin-binding sequence had distinct efficiencies in the two to both endothelial cells and melanoma cells but only inhibits the models. These results suggest that the type 1 repeats of TSP-1 proliferation of melanoma cells. The presence of cyclic RGD contain two subdomains that might independently influence the peptides do not compete for the avh3 integrin–mediated activity process of neovascularization (80). The existence of two sub- of tumstatin, although this peptide shows significant inhibition of domains also explains how TSP-1 may block FGF-2 and VEGF endothelial cell binding to vitronectin and fibronectin. These two h angiogenic signals by two independent pathways (81). TSP-1 is able distinct binding sites on tumstatin suggest unique av 3 integrin– to distinguish pathologic neovascularization from preexisting mediated mechanisms governing the two distinct antitumor vasculature due to the dependence of proliferating endothelial activities (91). A third proximal site, RGD site, capable of binding h cells on Fas/FasL-mediated apoptosis. TSP-1 up-regulates FasL to av 3 integrin, outside the NC1 domain sequence of tumstatin, expression on endothelial cells. Expression for the receptor of FasL, has also been proposed recently (94). How these different avh3 was low on quiescent endothelial cells but greatly enhanced by integrin sites regulate the antiangiogenic activity of tumstatin is not inducers of angiogenesis, thereby specifically sensitizing the understood completely. stimulated cells to apoptosis by inhibitor-generated FasL (82). The antiangiogenic domain of tumstatin within the 54– to 132– TSP-2 also shows antiangiogenic activity. Injection of TSP-2- region was named Tum-5. Recombinant Tum-5 inhibits transfected squamous cell carcinoma cells into the dermis of nude endothelial cell tube formation on Matrigel and induces G1 mice resulted in inhibition of tumor growth that was even stronger endothelial cell cycle arrest. Tum-5 possesses antiangiogenic than the inhibition observed with TSP-1-transfected cells. The activity and inhibits human growth in association combined overexpression of TSP-1 and TSP-2 completely pre- with a decrease in CD31-positive vasculature in nude mice (95). vented tumor formation. Extensive areas of necrosis were observed More specifically, the antiangiogenic activity of tumstatin is in TSP-2-expressing tumors, and both the density and the size of localized to a 25–amino acid region within the 69– to 98–amino tumor vessels were significantly reduced (83). Furthermore, tumor acid area as shown with overlapping synthetic peptides. Both of h angiogenesis was significantly enhanced in TSP-2-deficient mice. these peptides are antiangiogenic and function via av 3 integrin in Although TSP-2 deficiency did not affect tumor differentiation or a similar fashion as the full-length tumstatin (96). proliferation, tumor cell apoptosis was significantly reduced (84). The antiangiogenic and proapoptotic activity of tumstatin is The antiangiogenic role of TSP-2 was further confirmed with an specific for endothelial cells. Maeshima et al. show that tumstatin implant system that continuously produces TSP-2. Fibroblasts, functions as an endothelial cell–specific inhibitor of protein which overexpress TSP-2 and transplanted into nude mice synthesis. Through an interaction with avh3 integrin, tumstatin resulting in increased levels of circulating TSP-2, inhibited tumor inhibits activation of FAK, PI3K, protein kinase B/Akt, and mTOR growth and angiogenesis of human squamous cell carcinomas, and prevents the dissociation of eukaryotic initiation factor 4E malignant melanomas, and Lewis lung carcinomas implanted at a protein from 4E-BP1 leading to the inhibition of Cap-dependent distant site (85). It has been shown recently that the antiangiogenic protein synthesis. Furthermore, these results establish a role for region of TSP-2 lies approximately within the 80-kDa fragment of integrins in mediating cell-specific inhibition of Cap-dependent the NH2-terminal globular region (86). Daily injections of TSP-2 protein synthesis and suggest a potential mechanism for the resulted in a significant inhibition of the growth of human selective effects of tumstatin on endothelial cells. Consequently, squamous cell carcinomas in vivo and reduced tumor vasculari- tumstatin causes endothelial cell apoptosis by inhibiting signaling zation. Possible mechanisms for this antiangiogenic activity are pathways involved in protein synthesis via interaction with avh3 inhibition of VEGF-induced endothelial cell migration, tube integrin (97). It is known that endothelial cells adhere to h h formation, and increased endothelial cell–specific apoptosis (86). immobilized VEGF mediated by av 3, a3 1, as well as other av Tumstatin. Synthetic peptides derived from the NC1 domain of integrins but not by VEGF receptors (VEGFR). This adhesion a3 chain of type IV collagen, residues 183 to 205, have been shown to almost totally abolishes endothelial cell apoptosis through the inhibit the proliferation of melanoma and other epithelial tumor cell interaction with integrins. Tumstatin can inhibit the adhesion h lines in vitro (87, 88) and bind to the CD47/av 3 integrin complex (89). between endothelial cells and immobilized VEGF via the interac- (89). The entire 28-kDa fragment of a3 chain of NC1 domain of type tion with avh3 integrin; thus, it induces apoptosis (98). IV collagen was named tumstatin (90, 91). In vivo overexpression of Although tumstatin and endostatin are both antiangiogenic tumstatin domains by tumor cells inhibits their invasive properties molecules derived from the NC1 domains of basement membrane in a mouse melanoma model (92). Tumstatin inhibits formation of , they share only 14% amino acid homology and their new blood vessels in Matrigel plug assays and suppresses tumor activities seem to be mediated via distinct signaling pathways. growth of human renal cell carcinoma and prostate carcinoma in Human tumstatin prevents angiogenesis via inhibition of endothe- mouse xenograft models. This is associated with in vivo endothelial lial cell proliferation and promotion of apoptosis with no effect on cell–specific apoptosis. The antiangiogenic activity is localized to a migration, whereas human endostatin prevents endothelial cell distinct region of the tumstatin molecule that is separate from the migration with no effect on VEGF-induced proliferation. The region responsible for the antitumor cell activity (90). Tumstatin has reason for these distinct functions of endostatin and tumstatin can two binding sites for avh3 integrin, one in the NH2-terminal end of be explained partly by different integrin-binding capabilities and the molecule (containing amino acids 54-132) that is associated induction of different signaling pathways. The activity of tumstatin h with the antiangiogenic properties and the other in the COOH- is mediated via binding to av 3 integrin and inhibition of protein terminal end (containing amino acids 185-203) that is associated synthesis via FAK/PI3K/Akt/mTOR/4E-BP1 pathway, whereas the with the antitumor cell activity (89, 91, 93). The tumstatin fragment activity of endostatin is mediated by a5h1 integrin and inhibition of containing amino acids 54 to 132 binds to both endothelial cells and FAK/c-Raf/MAPK-ERK kinase-1/2/p38/ERK1 MAPK pathway, with melanoma cells but only inhibits proliferation of endothelial cells, no effect on Cap-dependent protein synthesis (40).

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Does the physiologic level of endogenous circulating tumstatin There are several targets proposed for the action of (f300-360 ng/mL in mice) have an effect on angiogenesis and (for review, see ref. 113). It binds directly to the ATP synthase on tumor growth? Tumors in collagen IV a3 chain–deficient mice (that the surface of endothelial cells, which might play a role in are concomitantly deficient in tumstatin as well) grow faster than allowing the intracellular pH to drop, thus triggering apoptotic in wild-type mice. Furthermore, administration of exogenous events in endothelial cells (114). Both angiostatin (which contains tumstatin (missing physiologic levels) decreases the growth back the RGD motif) and plasmin specifically bind to avh3 integrin, to the wild-type level (99). However, physiologic angiogenesis and angiostatin significantly inhibits plasmin-induced cell h associated with tissue repair was not affected in these tumstatin- migration. This suggests that that binding of plasmin to av 3 deficient mice. As mentioned before, the antiangiogenic activity of integrin is required for its activity, and angiostatin may interfere tumstatin is mediated by integrin avh3 (40, 91, 96). This was further with such activity (115, 116). Angiomotin is also a potential confirmed by showing that tumstatin has no effect on the target for angiostatin, as it has been shown that cells that proliferation of mouse lung endothelial cells deficient in integrin contained angiomotin are able to bind and internalize angios- h3 or on neovascularization of Matrigel plugs in the integrin h3– tatin, leading to an induction of FAK activity (117). In addition, null mice (99). A clinical correlation study between levels of Claesson-Welsh et al. found that treatment with angiostatin has tumstatin and tumor progression of human bronchopulmonary no effect on growth factor–induced signal transduction but leads carcinoma was done recently, which highlights the potential to a RGD-independent induction of FAK activity. They also show significance of tumstatin as an endogenous angiogenesis inhibitor that angiostatin treatment of endothelial cells in the absence (100). A clear correlation between tumstatin expression and mildly of growth factors results in increased apoptosis, whereas developed vascular networks was observed (100). proliferation does not change. Angiostatin also inhibits migration MMPs are capable of degrading type IV collagen and liberating and tube formation associated with proliferating endothelial fragments containing tumstatin (101). MMP-9 is the most effective cells (118). in cleaving tumstatin-containing fragments from type IV collagen, Cleaved III and prothrombin kringle-2. but MMP-2, -3, and -13 also can release tumstatin. MMP-9-deficient Circulating clotting factors in the blood seem to play an important mice have decreased levels of tumstatin in their blood, and the role in angiogenesis. In addition to angiostatin (see above), TSPs tumors in MMP-9-null mice grow faster than the tumors in wild- (see above), and platelet factor-4 (PF-4; see below), the antiangio- type mice (99). genic forms of antithrombin III (119) and prothrombin kringle-2 Recent studies also show that tumstatin peptide can prevent (120) have been shown to posses antiangiogenic properties. glomerular hypertrophy in the early stages of diabetic nephropathy Cleavage of the COOH-terminal loop of antithrombin induces a (102). Increase in the renal expression of VEGF, Flk-1, and Ang-2 conformational change in the molecule, and the cleaved confor- was inhibited by tumstatin in mice with diabetic nephropathy mation has potent antiangiogenic and antitumor activity in mouse (102). models. In addition, the latent form of intact antithrombin, which is similar in conformation to the cleaved molecule, also inhibits angiogenesis and tumor growth (119). In this regard, prothrombin Non-Matrix-Derived Inhibitors of Angiogenesis kringle-2 domain also exhibits anti–endothelial cell proliferative Angiostatin. Cleavage of plasminogen (contains five kringles) activity (120). by proteases results in the formation of 38- to 45-kDa Chondromodulin-I. Although cartilage contains many angio- antiangiogenic peptides that contain homologous triple-disulfide genic factors during ossification, in adults it is an avascular tissue. bridged kringle domains: kringle-1 to -4 or kringle-1 to -3. They The 25-kDa cartilage-specific NC1 matrix protein chondromodulin-I are collectively called angiostatin (103–105). Subsequent studies is a strong inhibitor of angiogenesis (121). The expression pattern have also shown that plasminogen kringle-5 by itself also of the gene suggests a role for chondromodulin-I in the exhibits antiangiogenesis activity (106). Angiostatin is a cryptic morphogenesis during embryonic development (122). The level fragment of plasminogen that possesses antiangiogenic proper- of chondromodulin-I transcripts is substantially reduced in ties, a property not shared by the parent molecule (plasmino- chondrosarcomas or in other benign cartilage tumors. Human gen). Angiostatin was originally purified from serum and urine of chondrosarcoma cells in nude mice produce tumors with mice bearing s.c. Lewis lung carcinoma, where the growth of cartilaginous matrix. Local administration of recombinant human metastases was inhibited by tumor-generated angiostatin (107). chondromodulin-I almost completely blocks vascular invasion Several members of the human MMP family, including matrilysin and tumor growth in vivo. Furthermore, it inhibits the growth of (MMP-7) and gelatinase B and A (MMP-9 and -2), metalloelastase colon adenocarcinoma in vivo, implying therapeutic potential for (MMP-12), and stromelysin-1 (MMP-3), hydrolyze human plas- other solid tumors (123). minogen to generate angiostatin fragments (104, 108–110) after Soluble Fms-like tyrosine kinase 1. The soluble version of plasminogen is converted to plasmin by plasminogen activator, VEGFR-1 [soluble Fms-like tyrosine kinase 1 (sFlt-1)] was identified which is followed by reduction by phosphoglycerate kinase (111). by Kendall et al. (124, 125). In addition to the full-length receptor, Angiostatineen inhibits endothelial cell proliferation and migra- VEGFR-1 gene encodes for a small soluble form (sFlt-1) carrying tion (107). It is suggested that the different kringle domains may only six Ig domain via an attenuate splicing (124, 126, 127). sFlt-1 contribute to the overall antiangiogenic function of angiostatin has a strong affinity for VEGF and placental growth factor (124, by their distinct antimigratory activities (e.g., kringle-4, the 128, 129). In human, VEGF circulates at 75 pg/mL and sFlt-1 potent fragment in inhibiting endothelial cell migration, which circulates at a concentration of 23 ng/mL (130). Therefore, VEGF has only marginal antiproliferative activity). In contrast, kringle-1 can circulate in a bound state (bound to sFlt-1) as well as in free to -3, which is equivalent to angiostatin in inhibiting endothelial state depending on the amount of sFlt-1 in the circulation. In this cell proliferation, manifests only a modest antimigratory effect regard, several reports have suggested that sFlt-1 can serve as an (112). antitumor agent by inhibiting VEGF (130–132). The relevance for www.aacrjournals.org 3973 Cancer Res 2005; 65: (10). May 15, 2005

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research sFlt-1 at the normal physiologic concentration in the regulation of diabetic retinopathy (82, 163–167). It also has neurotrophic activity cancer progression is not understood. both in retina and in the central nervous system. PEDF expression Interferons. IFNs, pleiotropic cytokines that regulate antiviral, is suppressed by hypoxia, and changes in PEDF correlate with the antitumor, apoptotic, and cellular immune responses, were the first development of retinal neovascularization in animal models of endogenous antiangiogenic regulators identified. IFNs inhibit hypoxic eye disease. angiogenesis induced by tumor cells in mice (133). IFN-a or IFN- PEDF possesses several physiologic properties that make it a h has biological activity against squamous cell carcinomas and potentially important protein in the regulation of angiogenesis, in inhibits angiogenesis in tumor-bearing nude mice. When tumor the neuronal cell survival, and in the protection of neurons from cells are treated in vitro with low-dose IFN-a, there is a significant neurotoxic agents. Its antiangiogenic activity is selective, in that drop in their secretion of interleukin (IL)-8, the major angiogenic PEDF targets only new vessel growth but spares existing ones, and factor produced by tumors (134). IFN-a reduces urokinase-type it is reversible. It is a protein that is highly up-regulated in the Go plasminogen activator and plasminogen activator inhibitor-1 phase of early-passage cells compared with rapidly proliferating or activity (135) and significantly inhibits MMP-9 enzymatic activity senescent cells and thus is also linked to both cell cycle and cell and protein expression (136). IFN-a and IFN-h treatment senescence (168, 169). also inhibits angiogenesis by down-regulation of bFGF expression Recent study highlights two beneficial effects of PEDF treatment (137–139), but other studies suggest that the action of IFN-a is not on tumor growth and expansion. One is the suppression of tumor mediated by bFGF or VEGF (140–142). angiogenesis. Overexpression of PEDF was found to significantly Interleukins. ILs are a family of leukocyte-derived proteins with inhibit melanoma growth and vessel formation in G361 nude mice broad-ranging effects on multiple physiologic properties, including xenografts (170). PEDF is also expressed strongly in normal murine angiogenesis. ILs bearing a NH2-terminal Glu-Leu-Arg (ERL) motif, kidney, and the loss of angioinhibitory activity may contribute to such as IL-8, tend to display proangiogenic properties, whereas pathologic angiogenesis in Wilms’ tumor (171). In addition, PEDF those lacking this motif have been found to inhibit angiogenesis may serve as a multifunctional antitumor agent in neuroblastomas, (143). IL-1, mainly secretable IL-1h, is involved in inflammation, inhibiting angiogenesis while promoting the numbers of Schwann tumor growth, and metastasis. It is shown that IL-1h inhibits FGF- cells and differentiated tumor cells that in turn produce PEDF stimulated angiogenesis by an autocrine pathway (144). However, (172). The loss of PEDF expression was also detected in glioma recent study found that the IL-1 receptor antagonist reduced progression (173). In PEDF-deficient mice, stromal vessels were inflammatory responses and inhibited tumor development in increased and associated with epithelial cell hyperplasia (174). It is mice (145). IL-4 inhibits bFGF-induced angiogenesis (146). IL-12 a key inhibitor of stromal vasculature and epithelial tissue growth and IL-18 are IFN-g-inducing cytokines. IL-12 can inhibit in mouse prostate and pancreas. The other activity of PEDF is angiogenesis through the downstream , such as IFN- induction of FasL-dependent apoptosis in tumor cells. PEDF up- inducible protein-10 and monokine induced by IFN-g (147–149). regulates FasL on endothelial cells. Expression of the essential Tumors in mice treated with IL-12 are significantly smaller and partner of FasL, Fas/CD95 receptor, was low on quiescent have extensive necrosis compared with untreated tumors (150). endothelial cells but greatly enhanced by inducers of angiogenesis, The combination of IL-12 and IL-2 mediated synergistic antitumor thereby specifically sensitizing the stimulated cells to apoptosis by activity in preclinical tumor models (151). The IL-12 plasmid DNA inhibitor-generated FasL. The antiangiogenic activity of PEDF, both gene transfer significantly prevented the growth and vasculariza- in vitro and in vivo, was dependent on this dual induction of Fas tion of highly angiogenic sarcoma and TS/A murine mammary and FasL and the resulting apoptosis (82). carcinoma tumors in nude and/or syngeneic mice (152). IL-18 PEDF can be given therapeutically as a soluble protein or by inhibits FGF-stimulated endothelial cell proliferation in vitro and viral-mediated gene transfer (175, 176). It is stable and nontoxic suppresses the FGF-induced corneal neovascularization by sys- when injected systemically. Gene transfer of PEDF suppresses temic administration in mice (153). IL-18 can also inhibit tumor vascularization and growth while prolonging survival in embryonic angiogenesis. syngeneic murine models of thoracic malignancies. Gene transfer 2-Methoxyestradiol. 2-Methoxyestradiol (2-ME), an endogenous of PEDF using adeno-associated viral vectors also inhibited estradiol metabolite, is an inhibitor of angiogenesis, with direct ischemia-induced neovascularization (177). effect on cancer cells (154–157). 2-ME is orally active molecule with A recent study suggests that, in physiologic conditions, a critical low affinity for estrogen receptors (158). Its mechanism of action is balance between PEDF and VEGF exists, and PEDF may counteract attributed to its ability to bind to the colchicine binding site of the angiogenic potential of VEGF. Under oxidative stress, PEDF tubulin and the inhibition of superoxide dismutase enzymatic decreases, disrupting the angiogenic balance (178). This critical activity (154, 159–161). Recently, another novel mechanism for balance between PEDF and VEGF is important to prevent the 2-ME was proposed (162). 2-ME-mediated destabilization of development of choroidal neovascularization (163, 179). In addition, microtubules was associated with a block in nuclear accumulation bone angiogenesis and matrix modeling may also be mediated both and activity of hypoxia-inducible factor-1a (oxygen and protea- by dynamic intraplay between PEDF and by VEGF (180). some–independent pathway), leading to significant reduction in PEX. It has been shown that PEX, a noncatalytic COOH- the VEGF levels (162). The endogenous physiologic role for 2-ME as terminal hemopexin-like domain of MMP-2, prevents binding of an inhibitor of angiogenesis is not yet understood. MMP-2 to integrin avh3, thus inhibiting proteolytic activity on the Pigment epithelium-derived factor. Because pigment epithe- cell surface and disrupting angiogenesis (181). Delivery of PEX by lium-derived factor (PEDF), a noninhibitory member of the serpin viral vectors resulted in suppressed endothelial cell invasion and superfamily, was identified to be responsible for the avascularity of formation of capillary-like structures, blocking of bFGF-induced ocular compartments in 1999, it is the most potent inhibitor of MMP-2 activation and angiogenesis, and inhibition of tumor- angiogenesis in the mammalian eye and is involved in the induced angiogenesis and tumor growth in nude mice (182). PEX pathogenesis of angiogenic eye diseases, such as proliferative formation seems to depend on the stage of angiogenesis, reaching

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the maximum level during the late stage when the vessels are Vasostatin. Vasostatin, a NH2-terminal domain of human maturating (181). inclusive of amino acids 1,180, is a potent angiogenesis Platelet factor-4. PF-4 is a protein released from platelet inhibitor. It selectively inhibits endothelial cell proliferation and a-granules during platelet aggregation that has been shown to have angiogenesis in response to stimulation from growth factors and antiangiogenic properties both in vitro and in vivo (183). suppresses tumor growth (198, 199). Burkitt, colon, and ovarian Recombinant human PF-4 inhibits blood vessel proliferation in tumors in mice treated with vasostatin were smaller than tumors in the chicken chorioallantoic membrane assay in a dose-dependent control mice (150). Combination of vasostatin and IP-10 reduced manner. PF-4 inhibits angiogenesis by associating directly with tumor growth more effectively than each agent alone, but complete FGF-2, inhibiting its dimerization and blocking FGF-2 binding to regression was not observed (200). Vasostatin specifically inhibits endothelial cells. The inhibitory activities are associated with the endothelial cell attachment to laminin and reduces subsequent COOH-terminal, heparin-binding region of the molecule (183) and endothelial cell growth induced by bFGF (201). Furthermore, gene more specifically in the short COOH-terminal segment (amino acids therapy experiments with intramuscular delivery of vasostatin DNA 47-70). A minor modification of this active segment of PF-4, an is effective in the inhibition of angiogenesis and tumor growth in addition of a ERL, a critical domain present in proangiogenic murine tumor models (202). chemokines, surprisingly elicits several times greater antiangiogenic potential than the original peptide. Thus, tailored PF-4 peptides Summary and Future Perspective represent a new class of antiangiogenic agents with a defined mode Relative systemic levels of proangiogenic and antiangiogenic of action and a strong in vivo activity (184). Protein assays show that factors likely govern tumor progression by regulating the PF-4 and FGF-2 bind to adjacent or overlapping sites together ‘‘angiogenic balance.’’ Conversion of dormant carcinomas to an covering a 12-kDa stretch of heparan sulfate, suggesting that these invasive malignant carcinomas is considered to involve a shift in three components may form a ternary complex. This enables PF-4 favor of enhanced angiogenesis potential. Influenced by oncogenes to inhibit FGF-2-stimulated endothelial cell proliferation through and tumor suppressor genes, disruption of the ‘‘angiogenic heparan sulfate–dependent mechanism (185). checkpoint’’ via increase in angiogenic factors, such as VEGF, or fragment. The intact prolactin (23 kDa) is enzymat- decrease in the physiologic levels of endogenous inhibitors of ically cleaved in several different tissues to generate a 16-kDa (16K angiogenesis, like TSP-1, tumstatin, and endostatin, could represent PRL) and a 8-kDa fragment (186). Although the intact prolactin has an important step in the progression of cancer. Currently, at least activities consistent with proangiogenesis, the generation of the 27 different protein and small molecules are known to exist in the 16K PRL fragment exposes a cryptic antiangiogenic activity (187, body that function as inhibitors of angiogenesis. Therefore, it is 188). The opposing angiogenesis activities of intact prolactin and quite possible that genetic control of the physiologic levels of NH -terminal 16K PRL offers an insight into the body’s mechanism 2 endogenous inhibitors of angiogenesis might constitute a critical to regulate angiogenesis (189). The mechanism by which such line of defense against the conversion of dormant neoplastic events opposing action is regulated can in part be explained by differential into a malignant phenotype of cancer. regulation of MAPK signaling pathway (190). Additionally, the 16K How these endogenous inhibitors orchestrate the formation of a PRL inhibits VEGF-induced activation of Ras in capillary barrier to counteract the effects of physiologic levels of proangio- endothelial cells (191). genic growth factors and cytokines is not yet understood. For Tissue inhibitors of matrix metalloproteinases. Tissue endogenous inhibitors that need to be generated via proteolysis of inhibitors of matrix metalloproteinases (TIMP) suppress MMP larger precursor proteins, regulation of enzymatic activity also activity and ECM turnover. In addition to their MMP inhibitory likely plays a key role in the formation of the angiogenic barrier. activity, TIMPs have pluripotent effects on cell growth, apoptosis, Further studies are needed to address the function of physiologic and differentiation (192–194). TIMP-2 inhibits angiogenic factor– levels of these 27 inhibitors in controlling cancer progression. It is induced endothelial cell proliferation in vitro and angiogenesis conceivable that genetic defects reflected by loss-of-function single in vivo, independent of MMP inhibition. These effects require nucleotide polymorphisms in the sequence of the protein inhibitors a h integrin–mediated binding of TIMP-2 to endothelial cells. 3 1 or enzymes that generate angiogenesis inhibitors could determine Further, TIMP-2 induces a decrease in total protein tyrosine how fast a set of neoplastic events switch from a nonlethal lesion to phosphatase activity associated with h integrin subunit as well 1 a malignant and angiogenic tumor. as dissociation of the phosphatase SHP-1 from h integrin. TIMP- 1 Lastly, the potential therapeutic application for these endoge- 2 treatment also results in an increase in protein tyrosine nous inhibitors of angiogenesis is being considered in the clinic phosphatase activity associated with tyrosine kinase receptors and early results from clinical trials with angiostatin, endostatin, FGF receptor-1 and KDR (195). TSP-1 (ABT-510), and 2-ME (Panzam) suggest that more laboratory Troponin I. Troponin I (Tn I) is a novel cartilage-derived studies are required to better understand the mechanism of action angiogenesis inhibitor, which inhibits endothelial cell proliferation associated with each of these drug candidates (203–209). Such and angiogenesis in both in vivo and in vitro model systems. Tn I continuing efforts will enable us to design more relevant and also inhibits metastasis of a wide variety of tumors in vivo. Tn I is a targeted clinical trial guided by the inherent antiangiogenic subunit of the troponin complex, which along with tropomyosin is mechanism of each particular inhibitor. responsible for the calcium-dependent regulation of striated muscle contraction (196). Interestingly, Tn I is capable of inhibiting actomyosin ATPase, which might constitute for its angiogenesis, Acknowledgments tumor growth, and metastasis inhibitory activity (196). Tn I inhibits Received 7/13/2004; revised 12/1/2004; accepted 2/24/2005. both bFGF-stimulated and basal levels of endothelial cell Grant support: NIH grants DK 55001 and DK 62987, Center for Matrix Biology at the Beth Israel Deaconess Medical Center research funds, Emil Aaltonen Foundation, proliferation probably via an interaction of Tn I with the cell Ann L. and Herbert J. Siegel Philantropic Jewish Communal Fund, and Joseph Lubrano surface bFGF receptor on capillary endothelial cells (197). Memorial Goldfield Family Charitable trust fund. www.aacrjournals.org 3975 Cancer Res 2005; 65: (10). May 15, 2005

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