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W2091337602 abstract "We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factorin vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosisin vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis. We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factorin vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosisin vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis. CEACAM1 1The abbreviations used are: CEACAM1, cell-cell adhesion molecule; CM, conditioned medium; HPAEC, human pulmonary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; MTT, 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium; DMEM, Dulbecco's modified Eagle's medium; m.o.i., multiplicity of infection; PARP, poly(ADP-ribose) polymerase; Ad, adenovirus; IFN, interferon; bFGF, basic fibroblast growth factor. 1The abbreviations used are: CEACAM1, cell-cell adhesion molecule; CM, conditioned medium; HPAEC, human pulmonary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; MTT, 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium; DMEM, Dulbecco's modified Eagle's medium; m.o.i., multiplicity of infection; PARP, poly(ADP-ribose) polymerase; Ad, adenovirus; IFN, interferon; bFGF, basic fibroblast growth factor. is a cell-adhesion molecule of the immunoglobulin supergene family (1Lin S.-H. Guidotti G. J. Biol. Chem. 1989; 264: 14408-14414Abstract Full Text PDF PubMed Google Scholar, 2Lin S.-H. Culic O. Flanagan D. Hixson D.C. Biochem. J. 1991; 278: 155-161Crossref PubMed Scopus (65) Google Scholar). Structurally, CEACAM1 is highly homologous to the carcinoembryonic antigen family of molecules (3Thompson J.A. Grunert F. Zimmermann W. J. Clin. Lab. Anal. 1991; 5: 344-366Crossref PubMed Scopus (580) Google Scholar, 4Beauchemin N. Draber P. Dveksler G. Gold P. Gray-Owen S. Grunert F. Hammarstrom S. Holmes K.V. Karlsson A. Kuroki M. Lin S.-H. Lucka L. Najjar S.M. Neumaier M. Obrink B. Shively J.E. Skubitz K.M. Stanners C.P. Thomas P. Thompson J.A. Virji M. von Kleist S. Wagener C. Watts S. Zimmermann W. Exp. Cell Res. 1999; 252: 243-249Crossref PubMed Scopus (323) Google Scholar). CEACAM1 has four extracellular Ig-like domains and a 71-amino acid cytoplasmic domain (2Lin S.-H. Culic O. Flanagan D. Hixson D.C. Biochem. J. 1991; 278: 155-161Crossref PubMed Scopus (65) Google Scholar). Structural and functional analyses have shown that CEACAM1 mediates homophilic cell adhesion through its first Ig domain (5Cheung P.H. Luo W. Qiu Y. Zhang X. Earley K. Millirons P. Lin S.-H. J. Biol. Chem. 1993; 268: 24303-24310Abstract Full Text PDF PubMed Google Scholar).The involvement of CEACAM1 in tumorigenesis was first noted by Hixsonet al. (6Hixson D.C. McEntire K.D. Obrink B. Cancer Res. 1985; 45: 3742-3749PubMed Google Scholar), who observed a general decrease in CEACAM1 protein expression in hepatomas. Down-regulation of CEACAM1 protein was subsequently demonstrated in colon (7Neumaier M. Paululat S. Chan A. Matthaes P. Wagener C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10744-10748Crossref PubMed Scopus (229) Google Scholar, 8Nollau P. Prall F. Helmchen U. Wagener C. Neumaier M. Am. J. Pathol. 1997; 151: 521-530PubMed Google Scholar, 9Nollau P. Scheller H. Kona-Horstmann M. Rohde S. Hagenmuller F. Wagener C. Neumaier M. Cancer Res. 1997; 57: 2354-2357PubMed Google Scholar), prostate (10Kleinerman D.I. Troncoso P. Lin S.H. Pisters L.L. Sherwood E.R. Brooks T. von Eschenbach A.C. Hsieh J.T. Cancer Res. 1995; 55: 1215-1220PubMed Google Scholar, 11Pu Y.S. Luo W., Lu, H.-H. Gingrich J. Greenberg N. Lin S.-H. J. Urol. 1999; 162: 892-896Crossref PubMed Scopus (23) Google Scholar), endometrium (12Bamberger A.M. Riethdorf L. Nollau P. Naumann M. Erdmann I. Gotze J. Brummer J. Schulte H.M. Wagener C. Loning T. Am. J. Pathol. 1998; 152: 1401-1406PubMed Google Scholar), and breast (13Riethdorf L. Lisboa B.W. Henkel U. Naumann M. Wagener C. Loning T. J. Histochem. Cytochem. 1997; 45: 957-963Crossref PubMed Scopus (94) Google Scholar, 14Huang J. Simpson J.F. Glackin C. Riethorf L. Wagener C. Shively J. Anticancer Res. 1998; 18: 3203-3212PubMed Google Scholar) carcinomas. These observations suggested that CEACAM1 may be a tumor suppressor. Our studies have shown that CEACAM1 can suppress the growth of prostate cancer in a mouse xenograft model (15Hsieh J.T. Luo W. Song W. Wang Y. Kleinerman D.I. Van N.T. Lin S.H. Cancer Res. 1995; 55: 190-197PubMed Google Scholar, 16Kleinerman D.I. Zhang W.W. Lin S.H. Nguyen T.V. von Eschenbach A.C. Hsieh J.T. Cancer Res. 1995; 55: 2831-2836PubMed Google Scholar, 17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar). CEACAM1 was subsequently shown to have anti-tumor activity in several different cell types, including colon (18Kunath T. Ordonez-Garcia C. Turbide C. Beauchemin N. Oncogene. 1995; 11: 2375-2382PubMed Google Scholar), bladder (19Kleinerman D.I. Dinney C.P.N. Zhang W.-W. Lin S.-H. Van N.T. Hsieh J.-T. Cancer Res. 1996; 56: 3431-3435PubMed Google Scholar), and breast carcinoma (20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar). In addition, the human (21Luo W. Talposky M. Earley K. Wood C. Wilson D. Logothetis C.J. Lin S.-H. Cancer Gene Ther. 1999; 6: 313-321Crossref PubMed Scopus (75) Google Scholar), rat (15Hsieh J.T. Luo W. Song W. Wang Y. Kleinerman D.I. Van N.T. Lin S.H. Cancer Res. 1995; 55: 190-197PubMed Google Scholar, 17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar), and mouse homologues of CEACAM1 (18Kunath T. Ordonez-Garcia C. Turbide C. Beauchemin N. Oncogene. 1995; 11: 2375-2382PubMed Google Scholar) were also shown to have tumor-suppressive activity.The domain responsible for the tumor-suppressive activity of CEACAM1 was determined by expressing various CEACAM1 mutants in prostate (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar,22Hsieh J.T. Earley K. Pong R.-C. Wang Y. Van N.T. Lin S.-H. Prostate. 1999; 41: 31-38Crossref PubMed Scopus (11) Google Scholar) and breast (20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar) cancer cells. Extensive structural and functional analyses suggested that the cytoplasmic domain is necessary for the anti-tumor activity of CEACAM1 (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar, 20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar, 22Hsieh J.T. Earley K. Pong R.-C. Wang Y. Van N.T. Lin S.-H. Prostate. 1999; 41: 31-38Crossref PubMed Scopus (11) Google Scholar). This in turn suggested that signal transduction mediated by the cytoplasmic domain of CEACAM1 may play an important role in its anti-tumor activity. These studies also revealed that the tumor-suppressive activity of CEACAM1 does not depend on the adhesion activity conferred by its first Ig domain (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar,20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar, 22Hsieh J.T. Earley K. Pong R.-C. Wang Y. Van N.T. Lin S.-H. Prostate. 1999; 41: 31-38Crossref PubMed Scopus (11) Google Scholar) and that the cytoplasmic domain of CEACAM1 is sufficient to elicit the tumor-suppressive activity (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar). Lastly, phosphorylation at serine 503 in the cytoplasmic domain of CEACAM1 was shown to be critical for the tumor-suppressive activity (23Estrera V.T. Chen D.-T. Luo W. Hixson D. Lin S.-H. J. Biol. Chem. 2001; 276: 15547-15553Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 24Fournes B. Sadekova S. Turbide C. Letourneau S. Beauchemin N. Oncogene. 2001; 20: 219-230Crossref PubMed Scopus (37) Google Scholar).Despite its strong anti-tumor activity in vivo, CEACAM1 does not significantly inhibit tumor cell proliferation in vitro, 2Y. S. Pu, K.-A. Do, W. Luo, C. J. Logothetis, and S.-H. Lin, unpublished data. 2Y. S. Pu, K.-A. Do, W. Luo, C. J. Logothetis, and S.-H. Lin, unpublished data. suggesting that CEACAM1 expression does not directly kill tumor cells in vitro. Thus, how CEACAM1 suppresses tumors in vivo is unknown. Because angiogenesis is essential for the growth and progression of solid tumors (25Folkman J. J. Natl. Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar), it is possible that the anti-tumor effect of CEACAM1 involves inhibition of tumor angiogenesis. In this study, we have shown that CEACAM1 expression in prostate cancer cells caused the release of a factor or factors that had a strong antiangiogenic effect in both in vitro and in vivo assays. In addition, we have shown that this antiangiogenic effect was due to induction of endothelial cell apoptosis. We have also found that the ability of various CEACAM1 mutants to induce endothelial cell apoptosis closely paralleled their tumor-suppressive activityin vivo. These observations suggest that the in vivo tumor suppression of CEACAM1 is due, at least in part, to its ability to inhibit tumor angiogenesis.DISCUSSIONAlthough the anti-tumor effect of CEACAM1 in vivo has been extensively documented, how CEACAM1 mediated tumor suppression was not clear. This study reports the antiangiogenic effect of CEACAM1. Several lines of evidence indicate that inhibition of tumor angiogenesis may be involved in CEACAM1-mediated tumor suppression. First, CEACAM1-expressing cells secreted a factor or factors that blocked endothelial cell migration in vitro and corneal angiogenesis in vivo. Second, CM from CEACAM1-expressing DU145 cells specifically inhibited in vitro proliferation of HUVECs and HPAECs but not primary epithelial cells or DU145 prostate cancer cells. Third, CEACAM1 CM inhibited endothelial cell growth by inducing apoptosis. Finally, the induction of endothelial cell apoptosis by CEACAM1 mutants correlated with their ability to suppress tumor growth in vivo.It is likely that expression of CEACAM1 in tumor cells induces the production of an inhibitory factor or factors that affect tumor angiogenesis, leading to suppression of the tumor growth in vivo. Normal growth is a balance between angiogenesis and antiangiogenesis activities in the tissues, and tumorigenesis is the result of an imbalance in the positive and negative regulators of angiogenesis. For example, studies by Bielenberg et al. (30Bielenberg D.R. Bucana C.D. Sanchez R. Mulliken J.B. Folkman J. Fidler I.J. Int. J. Oncol. 1999; 14: 401-408PubMed Google Scholar) showed that proliferating hemangiomas express high levels of bFGF and VEGF but are deficient in IFN-β, an endogenous inhibitor of angiogenesis. Dameron et al. (31Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Cold Spring Harbor Symp. Quant. Biol. 1994; LIX: 483-489Crossref Scopus (112) Google Scholar) showed that cells cultured from normal tissues secrete higher levels of inhibitors but lower levels of inducers than do cancer cells. Based on these observations, Dameron et al. (31Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Cold Spring Harbor Symp. Quant. Biol. 1994; LIX: 483-489Crossref Scopus (112) Google Scholar) postulated that normal adult tissues undergo little neovascularization because they often produce angiogenesis inhibitors. In contrast, tumor cells are angiogenic because they secrete angiogenic factors or have decreased levels of angiogenesis inhibitors. When a tumor suppressor is present, cells often secrete angiogenesis inhibitors and have an antiangiogenic phenotype. For instance, p53 stimulates the production of thrombospondin-1 (32Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Science. 1994; 265: 1582-1584Crossref PubMed Scopus (1315) Google Scholar, 33Rastinejad F. Polverini P.J. Bouck N.P. Cell. 1989; 56: 345-355Abstract Full Text PDF PubMed Scopus (394) Google Scholar), which inhibits neovascularization in vivo and endothelial cell migration in vitro (34Good D.J. Polverini P.J. Rastinejad F., Le Beau M.M. Lemons R.S. Frazier W.A. Bouck N.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6624Crossref PubMed Scopus (886) Google Scholar). Similarly, Nishimori et al. (35Nishimori H. Shiratsuchi T. Urano T. Kimura Y. Kiyono K. Tatsumi K. Yoshida S. Ono M. Kuwano M. Nakamura Y. Tokino T. Oncogene. 1997; 15: 2145-2150Crossref PubMed Scopus (251) Google Scholar) reported that p53 induces the expression of the brain-specific angiogenesis inhibitor BAI1, which is absent or significantly reduced in glioblastoma cell lines. Van Meiret al. (36Van Meir E.G. Polverini P.J. Chazin V.R., Su Huang H.-J. de Tribolet N. Cavenee W.K. Nat. Genet. 1994; 8: 171-176Crossref PubMed Scopus (301) Google Scholar) also reported the release of an inhibitor of angiogenesis upon induction of wild-type p53 expression in glioblastoma cells. Therefore, we believe that the antiangiogenic effect of CEACAM1 most likely results from the production of an angiogenesis inhibitor or inhibitors. Using RNase protection and enzyme-linked immunosorbent assay analysis, we found that VEGF and bFGF did not participate in the antiangiogenic action of CEACAM1. Also, CEACAM1 CM induced endothelial cell apoptosis, probably by means of an apoptosis-inducing antiangiogenic factor rather than down-regulation of angiogenesis stimulators. Thus, down-regulation of CEACAM1 in several carcinomas may decrease angiogenesis inhibitors, leading to tumor progression.The identity of this inhibitory factor is not known. That the CEACAM1-induced factor inhibited the growth of endothelial cells but not epithelial cells suggests that it is not the apoptosis inducers Fas ligand, tumor necrosis factor, or transforming growth factor-β, which lack target cell specificity. We can also rule out angiopoietin-1, which is chemotactic for endothelial cells but has no proliferative effect on them (37Witzenbichler B. Maisonpierre P.C. Jones P. Yancopoulos G.D. Isner J.M. J. Biol. Chem. 1998; 273: 18514-18521Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar), and its inhibitor, angiopoietin-2. Consistent with this prediction, we found that angiopoietin-1 expression was not affected by CEACAM1 in an RNase protection assay (data not shown). We also believe the CEACAM1-induced factor is not a matrix metalloproteinase (MMP). Tumor-induced angiogenesis begins with dissolution of basement membrane surrounding pre-existing blood vessels, a process aided by MMPs that are produced by tumor cells and supporting cells. Increased MMP activity has been positively linked to increased metastatic and angiogenic potential of tumors (38Pluda J.M. Semin. Oncol. 1997; 24: 203-218PubMed Google Scholar), and up-regulation of MMP-2, -7, and -9, and stromelysin-3 mRNA has been detected during tumor invasion and metastasis (39Polette M. Clavel C. Cockett M. Girod de Bentzmann S. Murphy G. Birembaut P. Invasion Metastasis. 1993; 13: 31-37PubMed Google Scholar, 40Hahnel E. Harvey J.M. Joyce R. Robbins P.D. Sterrett G.F. Hahnel R. Int. J. Cancer. 1993; 55: 771-774Crossref PubMed Scopus (39) Google Scholar). Two observations suggest that the CEACAM1-induced factor is not related to MMPs or tissue inhibitor of metalloproteinase-1 or -2. First, CEACAM1 affects early tumor progression; there is no evidence that CEACAM1 affects tumor invasion or metastasis. Second, MMPs and their inhibitors do not cause endothelial cell apoptosis. Rather, they exert their effects indirectly by degradation of basement membrane. Therefore, the CEACAM1-induced factor is probably not an MMP or an MMP inhibitor.Other known angiogenesis inhibitors include angiostatin (41O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3150) Google Scholar), endostatin (42O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4211) Google Scholar), platelet factor 4 (43Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Science. 1990; 247: 77-79Crossref PubMed Scopus (622) Google Scholar, 44Gupta S.K. Hassel T. Singh J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7799-7803Crossref PubMed Scopus (145) Google Scholar, 45Gupta S.K. Singh J.P. J. Cell Biol. 1994; 127: 1121-1127Crossref PubMed Scopus (106) Google Scholar), 16-kDa prolactin fragment (46Clapp C. Martial J.A. Guzman R.C. Rentier-Delrue F. Weiner R.I. Endocrinology. 1993; 133: 1292-1299Crossref PubMed Scopus (255) Google Scholar), IFN-α (47Kaban L.B. Mulliken J.B. Ezekowitz R.A. Ebb D. Smith P.S. Folkman J. Pediatrics. 1999; 103: 1145-1149Crossref PubMed Scopus (206) Google Scholar), IFN-β (48Dong Z. Greene G. Pettaway C. Dinney C.P. Eue I., Lu, W. Bucana C.D. Balbay M.D. Bielenberg D. Fidler I.J. Cancer Res. 1999; 59: 872-879PubMed Google Scholar), IFN-induced protein 10 (49–51), antithrombin (52O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Science. 1999; 285: 1926-1928Crossref PubMed Scopus (422) Google Scholar), maspin (53Zhang M. Volpert O. Shi Y.H. Bouck N. Nat. Med. 2000; 6: 196-199Crossref PubMed Scopus (407) Google Scholar), and Gro-β (54Cao Y. Chen C. Weatherbee J.A. Tsang M. Folkman J. J. Exp. Med. 1995; 182: 2069-2077Crossref PubMed Scopus (123) Google Scholar). Angiostatin is a fragment of plasminogen (41O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3150) Google Scholar). Endostatin is a 20-kDa C-terminal fragment of collagen XVIII and specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth (42O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4211) Google Scholar). The 16-kDa N-terminal fragment of human prolactin was shown to inhibit VEGF-induced activation of Ras in capillary endothelial cells (46Clapp C. Martial J.A. Guzman R.C. Rentier-Delrue F. Weiner R.I. Endocrinology. 1993; 133: 1292-1299Crossref PubMed Scopus (255) Google Scholar). Thus, it seems that angiogenic inhibitors can be fragments of proteins whose functions are not related to angiogenesis. It will be interesting to see whether the CEACAM1-induced factor is a fragment of a known protein. In any event, it is very likely that the CEACAM1-induced factor is a previously unknown molecule. CEACAM1 1The abbreviations used are: CEACAM1, cell-cell adhesion molecule; CM, conditioned medium; HPAEC, human pulmonary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; MTT, 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium; DMEM, Dulbecco's modified Eagle's medium; m.o.i., multiplicity of infection; PARP, poly(ADP-ribose) polymerase; Ad, adenovirus; IFN, interferon; bFGF, basic fibroblast growth factor. 1The abbreviations used are: CEACAM1, cell-cell adhesion molecule; CM, conditioned medium; HPAEC, human pulmonary artery endothelial cells; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; MTT, 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyltetrazolium; DMEM, Dulbecco's modified Eagle's medium; m.o.i., multiplicity of infection; PARP, poly(ADP-ribose) polymerase; Ad, adenovirus; IFN, interferon; bFGF, basic fibroblast growth factor. is a cell-adhesion molecule of the immunoglobulin supergene family (1Lin S.-H. Guidotti G. J. Biol. Chem. 1989; 264: 14408-14414Abstract Full Text PDF PubMed Google Scholar, 2Lin S.-H. Culic O. Flanagan D. Hixson D.C. Biochem. J. 1991; 278: 155-161Crossref PubMed Scopus (65) Google Scholar). Structurally, CEACAM1 is highly homologous to the carcinoembryonic antigen family of molecules (3Thompson J.A. Grunert F. Zimmermann W. J. Clin. Lab. Anal. 1991; 5: 344-366Crossref PubMed Scopus (580) Google Scholar, 4Beauchemin N. Draber P. Dveksler G. Gold P. Gray-Owen S. Grunert F. Hammarstrom S. Holmes K.V. Karlsson A. Kuroki M. Lin S.-H. Lucka L. Najjar S.M. Neumaier M. Obrink B. Shively J.E. Skubitz K.M. Stanners C.P. Thomas P. Thompson J.A. Virji M. von Kleist S. Wagener C. Watts S. Zimmermann W. Exp. Cell Res. 1999; 252: 243-249Crossref PubMed Scopus (323) Google Scholar). CEACAM1 has four extracellular Ig-like domains and a 71-amino acid cytoplasmic domain (2Lin S.-H. Culic O. Flanagan D. Hixson D.C. Biochem. J. 1991; 278: 155-161Crossref PubMed Scopus (65) Google Scholar). Structural and functional analyses have shown that CEACAM1 mediates homophilic cell adhesion through its first Ig domain (5Cheung P.H. Luo W. Qiu Y. Zhang X. Earley K. Millirons P. Lin S.-H. J. Biol. Chem. 1993; 268: 24303-24310Abstract Full Text PDF PubMed Google Scholar). The involvement of CEACAM1 in tumorigenesis was first noted by Hixsonet al. (6Hixson D.C. McEntire K.D. Obrink B. Cancer Res. 1985; 45: 3742-3749PubMed Google Scholar), who observed a general decrease in CEACAM1 protein expression in hepatomas. Down-regulation of CEACAM1 protein was subsequently demonstrated in colon (7Neumaier M. Paululat S. Chan A. Matthaes P. Wagener C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10744-10748Crossref PubMed Scopus (229) Google Scholar, 8Nollau P. Prall F. Helmchen U. Wagener C. Neumaier M. Am. J. Pathol. 1997; 151: 521-530PubMed Google Scholar, 9Nollau P. Scheller H. Kona-Horstmann M. Rohde S. Hagenmuller F. Wagener C. Neumaier M. Cancer Res. 1997; 57: 2354-2357PubMed Google Scholar), prostate (10Kleinerman D.I. Troncoso P. Lin S.H. Pisters L.L. Sherwood E.R. Brooks T. von Eschenbach A.C. Hsieh J.T. Cancer Res. 1995; 55: 1215-1220PubMed Google Scholar, 11Pu Y.S. Luo W., Lu, H.-H. Gingrich J. Greenberg N. Lin S.-H. J. Urol. 1999; 162: 892-896Crossref PubMed Scopus (23) Google Scholar), endometrium (12Bamberger A.M. Riethdorf L. Nollau P. Naumann M. Erdmann I. Gotze J. Brummer J. Schulte H.M. Wagener C. Loning T. Am. J. Pathol. 1998; 152: 1401-1406PubMed Google Scholar), and breast (13Riethdorf L. Lisboa B.W. Henkel U. Naumann M. Wagener C. Loning T. J. Histochem. Cytochem. 1997; 45: 957-963Crossref PubMed Scopus (94) Google Scholar, 14Huang J. Simpson J.F. Glackin C. Riethorf L. Wagener C. Shively J. Anticancer Res. 1998; 18: 3203-3212PubMed Google Scholar) carcinomas. These observations suggested that CEACAM1 may be a tumor suppressor. Our studies have shown that CEACAM1 can suppress the growth of prostate cancer in a mouse xenograft model (15Hsieh J.T. Luo W. Song W. Wang Y. Kleinerman D.I. Van N.T. Lin S.H. Cancer Res. 1995; 55: 190-197PubMed Google Scholar, 16Kleinerman D.I. Zhang W.W. Lin S.H. Nguyen T.V. von Eschenbach A.C. Hsieh J.T. Cancer Res. 1995; 55: 2831-2836PubMed Google Scholar, 17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar). CEACAM1 was subsequently shown to have anti-tumor activity in several different cell types, including colon (18Kunath T. Ordonez-Garcia C. Turbide C. Beauchemin N. Oncogene. 1995; 11: 2375-2382PubMed Google Scholar), bladder (19Kleinerman D.I. Dinney C.P.N. Zhang W.-W. Lin S.-H. Van N.T. Hsieh J.-T. Cancer Res. 1996; 56: 3431-3435PubMed Google Scholar), and breast carcinoma (20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar). In addition, the human (21Luo W. Talposky M. Earley K. Wood C. Wilson D. Logothetis C.J. Lin S.-H. Cancer Gene Ther. 1999; 6: 313-321Crossref PubMed Scopus (75) Google Scholar), rat (15Hsieh J.T. Luo W. Song W. Wang Y. Kleinerman D.I. Van N.T. Lin S.H. Cancer Res. 1995; 55: 190-197PubMed Google Scholar, 17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar), and mouse homologues of CEACAM1 (18Kunath T. Ordonez-Garcia C. Turbide C. Beauchemin N. Oncogene. 1995; 11: 2375-2382PubMed Google Scholar) were also shown to have tumor-suppressive activity. The domain responsible for the tumor-suppressive activity of CEACAM1 was determined by expressing various CEACAM1 mutants in prostate (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar,22Hsieh J.T. Earley K. Pong R.-C. Wang Y. Van N.T. Lin S.-H. Prostate. 1999; 41: 31-38Crossref PubMed Scopus (11) Google Scholar) and breast (20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar) cancer cells. Extensive structural and functional analyses suggested that the cytoplasmic domain is necessary for the anti-tumor activity of CEACAM1 (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar, 20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar, 22Hsieh J.T. Earley K. Pong R.-C. Wang Y. Van N.T. Lin S.-H. Prostate. 1999; 41: 31-38Crossref PubMed Scopus (11) Google Scholar). This in turn suggested that signal transduction mediated by the cytoplasmic domain of CEACAM1 may play an important role in its anti-tumor activity. These studies also revealed that the tumor-suppressive activity of CEACAM1 does not depend on the adhesion activity conferred by its first Ig domain (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar,20Luo W. Wood C. Earley K. Hung M.-C. Lin S.-H. Oncogene. 1997; 14: 1697-1704Crossref PubMed Scopus (83) Google Scholar, 22Hsieh J.T. Earley K. Pong R.-C. Wang Y. Van N.T. Lin S.-H. Prostate. 1999; 41: 31-38Crossref PubMed Scopus (11) Google Scholar) and that the cytoplasmic domain of CEACAM1 is sufficient to elicit the tumor-suppressive activity (17Estrera V.T. Luo W. Phan D. Hixson D. Lin S.-H. Biochem. Biophys. Res. Commun. 1999; 263: 797-803Crossref PubMed Scopus (20) Google Scholar). Lastly, phosphorylation at serine 503 in the cytoplasmic domain of CEACAM1 was shown to be critical for the tumor-suppressive activity (23Estrera V.T. Chen D.-T. Luo W. Hixson D. Lin S.-H. J. Biol. Chem. 2001; 276: 15547-15553Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 24Fournes B. Sadekova S. Turbide C. Letourneau S. Beauchemin N. Oncogene. 2001; 20: 219-230Crossref PubMed Scopus (37) Google Scholar). Despite its strong anti-tumor activity in vivo, CEACAM1 does not significantly inhibit tumor cell proliferation in vitro, 2Y. S. Pu, K.-A. Do, W. Luo, C. J. Logothetis, and S.-H. Lin, unpublished data. 2Y. S. Pu, K.-A. Do, W. Luo, C. J. Logothetis, and S.-H. Lin, unpublished data. suggesting that CEACAM1 expression does not directly kill tumor cells in vitro. Thus, how CEACAM1 suppresses tumors in vivo is unknown. Because angiogenesis is essential for the growth and progression of solid tumors (25Folkman J. J. Natl. Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar), it is possible that the anti-tumor effect of CEACAM1 involves inhibition of tumor angiogenesis. In this study, we have shown that CEACAM1 expression in prostate cancer cells caused the release of a factor or factors that had a strong antiangiogenic effect in both in vitro and in vivo assays. In addition, we have shown that this antiangiogenic effect was due to induction of endothelial cell apoptosis. We have also found that the ability of various CEACAM1 mutants to induce endothelial cell apoptosis closely paralleled their tumor-suppressive activityin vivo. These observations suggest that the in vivo tumor suppression of CEACAM1 is due, at least in part, to its ability to inhibit tumor angiogenesis. DISCUSSIONAlthough the anti-tumor effect of CEACAM1 in vivo has been extensively documented, how CEACAM1 mediated tumor suppression was not clear. This study reports the antiangiogenic effect of CEACAM1. Several lines of evidence indicate that inhibition of tumor angiogenesis may be involved in CEACAM1-mediated tumor suppression. First, CEACAM1-expressing cells secreted a factor or factors that blocked endothelial cell migration in vitro and corneal angiogenesis in vivo. Second, CM from CEACAM1-expressing DU145 cells specifically inhibited in vitro proliferation of HUVECs and HPAECs but not primary epithelial cells or DU145 prostate cancer cells. Third, CEACAM1 CM inhibited endothelial cell growth by inducing apoptosis. Finally, the induction of endothelial cell apoptosis by CEACAM1 mutants correlated with their ability to suppress tumor growth in vivo.It is likely that expression of CEACAM1 in tumor cells induces the production of an inhibitory factor or factors that affect tumor angiogenesis, leading to suppression of the tumor growth in vivo. Normal growth is a balance between angiogenesis and antiangiogenesis activities in the tissues, and tumorigenesis is the result of an imbalance in the positive and negative regulators of angiogenesis. For example, studies by Bielenberg et al. (30Bielenberg D.R. Bucana C.D. Sanchez R. Mulliken J.B. Folkman J. Fidler I.J. Int. J. Oncol. 1999; 14: 401-408PubMed Google Scholar) showed that proliferating hemangiomas express high levels of bFGF and VEGF but are deficient in IFN-β, an endogenous inhibitor of angiogenesis. Dameron et al. (31Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Cold Spring Harbor Symp. Quant. Biol. 1994; LIX: 483-489Crossref Scopus (112) Google Scholar) showed that cells cultured from normal tissues secrete higher levels of inhibitors but lower levels of inducers than do cancer cells. Based on these observations, Dameron et al. (31Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Cold Spring Harbor Symp. Quant. Biol. 1994; LIX: 483-489Crossref Scopus (112) Google Scholar) postulated that normal adult tissues undergo little neovascularization because they often produce angiogenesis inhibitors. In contrast, tumor cells are angiogenic because they secrete angiogenic factors or have decreased levels of angiogenesis inhibitors. When a tumor suppressor is present, cells often secrete angiogenesis inhibitors and have an antiangiogenic phenotype. For instance, p53 stimulates the production of thrombospondin-1 (32Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Science. 1994; 265: 1582-1584Crossref PubMed Scopus (1315) Google Scholar, 33Rastinejad F. Polverini P.J. Bouck N.P. Cell. 1989; 56: 345-355Abstract Full Text PDF PubMed Scopus (394) Google Scholar), which inhibits neovascularization in vivo and endothelial cell migration in vitro (34Good D.J. Polverini P.J. Rastinejad F., Le Beau M.M. Lemons R.S. Frazier W.A. Bouck N.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6624Crossref PubMed Scopus (886) Google Scholar). Similarly, Nishimori et al. (35Nishimori H. Shiratsuchi T. Urano T. Kimura Y. Kiyono K. Tatsumi K. Yoshida S. Ono M. Kuwano M. Nakamura Y. Tokino T. Oncogene. 1997; 15: 2145-2150Crossref PubMed Scopus (251) Google Scholar) reported that p53 induces the expression of the brain-specific angiogenesis inhibitor BAI1, which is absent or significantly reduced in glioblastoma cell lines. Van Meiret al. (36Van Meir E.G. Polverini P.J. Chazin V.R., Su Huang H.-J. de Tribolet N. Cavenee W.K. Nat. Genet. 1994; 8: 171-176Crossref PubMed Scopus (301) Google Scholar) also reported the release of an inhibitor of angiogenesis upon induction of wild-type p53 expression in glioblastoma cells. Therefore, we believe that the antiangiogenic effect of CEACAM1 most likely results from the production of an angiogenesis inhibitor or inhibitors. Using RNase protection and enzyme-linked immunosorbent assay analysis, we found that VEGF and bFGF did not participate in the antiangiogenic action of CEACAM1. Also, CEACAM1 CM induced endothelial cell apoptosis, probably by means of an apoptosis-inducing antiangiogenic factor rather than down-regulation of angiogenesis stimulators. Thus, down-regulation of CEACAM1 in several carcinomas may decrease angiogenesis inhibitors, leading to tumor progression.The identity of this inhibitory factor is not known. That the CEACAM1-induced factor inhibited the growth of endothelial cells but not epithelial cells suggests that it is not the apoptosis inducers Fas ligand, tumor necrosis factor, or transforming growth factor-β, which lack target cell specificity. We can also rule out angiopoietin-1, which is chemotactic for endothelial cells but has no proliferative effect on them (37Witzenbichler B. Maisonpierre P.C. Jones P. Yancopoulos G.D. Isner J.M. J. Biol. Chem. 1998; 273: 18514-18521Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar), and its inhibitor, angiopoietin-2. Consistent with this prediction, we found that angiopoietin-1 expression was not affected by CEACAM1 in an RNase protection assay (data not shown). We also believe the CEACAM1-induced factor is not a matrix metalloproteinase (MMP). Tumor-induced angiogenesis begins with dissolution of basement membrane surrounding pre-existing blood vessels, a process aided by MMPs that are produced by tumor cells and supporting cells. Increased MMP activity has been positively linked to increased metastatic and angiogenic potential of tumors (38Pluda J.M. Semin. Oncol. 1997; 24: 203-218PubMed Google Scholar), and up-regulation of MMP-2, -7, and -9, and stromelysin-3 mRNA has been detected during tumor invasion and metastasis (39Polette M. Clavel C. Cockett M. Girod de Bentzmann S. Murphy G. Birembaut P. Invasion Metastasis. 1993; 13: 31-37PubMed Google Scholar, 40Hahnel E. Harvey J.M. Joyce R. Robbins P.D. Sterrett G.F. Hahnel R. Int. J. Cancer. 1993; 55: 771-774Crossref PubMed Scopus (39) Google Scholar). Two observations suggest that the CEACAM1-induced factor is not related to MMPs or tissue inhibitor of metalloproteinase-1 or -2. First, CEACAM1 affects early tumor progression; there is no evidence that CEACAM1 affects tumor invasion or metastasis. Second, MMPs and their inhibitors do not cause endothelial cell apoptosis. Rather, they exert their effects indirectly by degradation of basement membrane. Therefore, the CEACAM1-induced factor is probably not an MMP or an MMP inhibitor.Other known angiogenesis inhibitors include angiostatin (41O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3150) Google Scholar), endostatin (42O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4211) Google Scholar), platelet factor 4 (43Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Science. 1990; 247: 77-79Crossref PubMed Scopus (622) Google Scholar, 44Gupta S.K. Hassel T. Singh J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7799-7803Crossref PubMed Scopus (145) Google Scholar, 45Gupta S.K. Singh J.P. J. Cell Biol. 1994; 127: 1121-1127Crossref PubMed Scopus (106) Google Scholar), 16-kDa prolactin fragment (46Clapp C. Martial J.A. Guzman R.C. Rentier-Delrue F. Weiner R.I. Endocrinology. 1993; 133: 1292-1299Crossref PubMed Scopus (255) Google Scholar), IFN-α (47Kaban L.B. Mulliken J.B. Ezekowitz R.A. Ebb D. Smith P.S. Folkman J. Pediatrics. 1999; 103: 1145-1149Crossref PubMed Scopus (206) Google Scholar), IFN-β (48Dong Z. Greene G. Pettaway C. Dinney C.P. Eue I., Lu, W. Bucana C.D. Balbay M.D. Bielenberg D. Fidler I.J. Cancer Res. 1999; 59: 872-879PubMed Google Scholar), IFN-induced protein 10 (49–51), antithrombin (52O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Science. 1999; 285: 1926-1928Crossref PubMed Scopus (422) Google Scholar), maspin (53Zhang M. Volpert O. Shi Y.H. Bouck N. Nat. Med. 2000; 6: 196-199Crossref PubMed Scopus (407) Google Scholar), and Gro-β (54Cao Y. Chen C. Weatherbee J.A. Tsang M. Folkman J. J. Exp. Med. 1995; 182: 2069-2077Crossref PubMed Scopus (123) Google Scholar). Angiostatin is a fragment of plasminogen (41O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3150) Google Scholar). Endostatin is a 20-kDa C-terminal fragment of collagen XVIII and specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth (42O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4211) Google Scholar). The 16-kDa N-terminal fragment of human prolactin was shown to inhibit VEGF-induced activation of Ras in capillary endothelial cells (46Clapp C. Martial J.A. Guzman R.C. Rentier-Delrue F. Weiner R.I. Endocrinology. 1993; 133: 1292-1299Crossref PubMed Scopus (255) Google Scholar). Thus, it seems that angiogenic inhibitors can be fragments of proteins whose functions are not related to angiogenesis. It will be interesting to see whether the CEACAM1-induced factor is a fragment of a known protein. In any event, it is very likely that the CEACAM1-induced factor is a previously unknown molecule. Although the anti-tumor effect of CEACAM1 in vivo has been extensively documented, how CEACAM1 mediated tumor suppression was not clear. This study reports the antiangiogenic effect of CEACAM1. Several lines of evidence indicate that inhibition of tumor angiogenesis may be involved in CEACAM1-mediated tumor suppression. First, CEACAM1-expressing cells secreted a factor or factors that blocked endothelial cell migration in vitro and corneal angiogenesis in vivo. Second, CM from CEACAM1-expressing DU145 cells specifically inhibited in vitro proliferation of HUVECs and HPAECs but not primary epithelial cells or DU145 prostate cancer cells. Third, CEACAM1 CM inhibited endothelial cell growth by inducing apoptosis. Finally, the induction of endothelial cell apoptosis by CEACAM1 mutants correlated with their ability to suppress tumor growth in vivo. It is likely that expression of CEACAM1 in tumor cells induces the production of an inhibitory factor or factors that affect tumor angiogenesis, leading to suppression of the tumor growth in vivo. Normal growth is a balance between angiogenesis and antiangiogenesis activities in the tissues, and tumorigenesis is the result of an imbalance in the positive and negative regulators of angiogenesis. For example, studies by Bielenberg et al. (30Bielenberg D.R. Bucana C.D. Sanchez R. Mulliken J.B. Folkman J. Fidler I.J. Int. J. Oncol. 1999; 14: 401-408PubMed Google Scholar) showed that proliferating hemangiomas express high levels of bFGF and VEGF but are deficient in IFN-β, an endogenous inhibitor of angiogenesis. Dameron et al. (31Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Cold Spring Harbor Symp. Quant. Biol. 1994; LIX: 483-489Crossref Scopus (112) Google Scholar) showed that cells cultured from normal tissues secrete higher levels of inhibitors but lower levels of inducers than do cancer cells. Based on these observations, Dameron et al. (31Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Cold Spring Harbor Symp. Quant. Biol. 1994; LIX: 483-489Crossref Scopus (112) Google Scholar) postulated that normal adult tissues undergo little neovascularization because they often produce angiogenesis inhibitors. In contrast, tumor cells are angiogenic because they secrete angiogenic factors or have decreased levels of angiogenesis inhibitors. When a tumor suppressor is present, cells often secrete angiogenesis inhibitors and have an antiangiogenic phenotype. For instance, p53 stimulates the production of thrombospondin-1 (32Dameron K.M. Volpert O.V. Tainsky M.A. Bouck N. Science. 1994; 265: 1582-1584Crossref PubMed Scopus (1315) Google Scholar, 33Rastinejad F. Polverini P.J. Bouck N.P. Cell. 1989; 56: 345-355Abstract Full Text PDF PubMed Scopus (394) Google Scholar), which inhibits neovascularization in vivo and endothelial cell migration in vitro (34Good D.J. Polverini P.J. Rastinejad F., Le Beau M.M. Lemons R.S. Frazier W.A. Bouck N.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6624Crossref PubMed Scopus (886) Google Scholar). Similarly, Nishimori et al. (35Nishimori H. Shiratsuchi T. Urano T. Kimura Y. Kiyono K. Tatsumi K. Yoshida S. Ono M. Kuwano M. Nakamura Y. Tokino T. Oncogene. 1997; 15: 2145-2150Crossref PubMed Scopus (251) Google Scholar) reported that p53 induces the expression of the brain-specific angiogenesis inhibitor BAI1, which is absent or significantly reduced in glioblastoma cell lines. Van Meiret al. (36Van Meir E.G. Polverini P.J. Chazin V.R., Su Huang H.-J. de Tribolet N. Cavenee W.K. Nat. Genet. 1994; 8: 171-176Crossref PubMed Scopus (301) Google Scholar) also reported the release of an inhibitor of angiogenesis upon induction of wild-type p53 expression in glioblastoma cells. Therefore, we believe that the antiangiogenic effect of CEACAM1 most likely results from the production of an angiogenesis inhibitor or inhibitors. Using RNase protection and enzyme-linked immunosorbent assay analysis, we found that VEGF and bFGF did not participate in the antiangiogenic action of CEACAM1. Also, CEACAM1 CM induced endothelial cell apoptosis, probably by means of an apoptosis-inducing antiangiogenic factor rather than down-regulation of angiogenesis stimulators. Thus, down-regulation of CEACAM1 in several carcinomas may decrease angiogenesis inhibitors, leading to tumor progression. The identity of this inhibitory factor is not known. That the CEACAM1-induced factor inhibited the growth of endothelial cells but not epithelial cells suggests that it is not the apoptosis inducers Fas ligand, tumor necrosis factor, or transforming growth factor-β, which lack target cell specificity. We can also rule out angiopoietin-1, which is chemotactic for endothelial cells but has no proliferative effect on them (37Witzenbichler B. Maisonpierre P.C. Jones P. Yancopoulos G.D. Isner J.M. J. Biol. Chem. 1998; 273: 18514-18521Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar), and its inhibitor, angiopoietin-2. Consistent with this prediction, we found that angiopoietin-1 expression was not affected by CEACAM1 in an RNase protection assay (data not shown). We also believe the CEACAM1-induced factor is not a matrix metalloproteinase (MMP). Tumor-induced angiogenesis begins with dissolution of basement membrane surrounding pre-existing blood vessels, a process aided by MMPs that are produced by tumor cells and supporting cells. Increased MMP activity has been positively linked to increased metastatic and angiogenic potential of tumors (38Pluda J.M. Semin. Oncol. 1997; 24: 203-218PubMed Google Scholar), and up-regulation of MMP-2, -7, and -9, and stromelysin-3 mRNA has been detected during tumor invasion and metastasis (39Polette M. Clavel C. Cockett M. Girod de Bentzmann S. Murphy G. Birembaut P. Invasion Metastasis. 1993; 13: 31-37PubMed Google Scholar, 40Hahnel E. Harvey J.M. Joyce R. Robbins P.D. Sterrett G.F. Hahnel R. Int. J. Cancer. 1993; 55: 771-774Crossref PubMed Scopus (39) Google Scholar). Two observations suggest that the CEACAM1-induced factor is not related to MMPs or tissue inhibitor of metalloproteinase-1 or -2. First, CEACAM1 affects early tumor progression; there is no evidence that CEACAM1 affects tumor invasion or metastasis. Second, MMPs and their inhibitors do not cause endothelial cell apoptosis. Rather, they exert their effects indirectly by degradation of basement membrane. Therefore, the CEACAM1-induced factor is probably not an MMP or an MMP inhibitor. Other known angiogenesis inhibitors include angiostatin (41O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3150) Google Scholar), endostatin (42O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4211) Google Scholar), platelet factor 4 (43Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Science. 1990; 247: 77-79Crossref PubMed Scopus (622) Google Scholar, 44Gupta S.K. Hassel T. Singh J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7799-7803Crossref PubMed Scopus (145) Google Scholar, 45Gupta S.K. Singh J.P. J. Cell Biol. 1994; 127: 1121-1127Crossref PubMed Scopus (106) Google Scholar), 16-kDa prolactin fragment (46Clapp C. Martial J.A. Guzman R.C. Rentier-Delrue F. Weiner R.I. Endocrinology. 1993; 133: 1292-1299Crossref PubMed Scopus (255) Google Scholar), IFN-α (47Kaban L.B. Mulliken J.B. Ezekowitz R.A. Ebb D. Smith P.S. Folkman J. Pediatrics. 1999; 103: 1145-1149Crossref PubMed Scopus (206) Google Scholar), IFN-β (48Dong Z. Greene G. Pettaway C. Dinney C.P. Eue I., Lu, W. Bucana C.D. Balbay M.D. Bielenberg D. Fidler I.J. Cancer Res. 1999; 59: 872-879PubMed Google Scholar), IFN-induced protein 10 (49–51), antithrombin (52O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Science. 1999; 285: 1926-1928Crossref PubMed Scopus (422) Google Scholar), maspin (53Zhang M. Volpert O. Shi Y.H. Bouck N. Nat. Med. 2000; 6: 196-199Crossref PubMed Scopus (407) Google Scholar), and Gro-β (54Cao Y. Chen C. Weatherbee J.A. Tsang M. Folkman J. J. Exp. Med. 1995; 182: 2069-2077Crossref PubMed Scopus (123) Google Scholar). Angiostatin is a fragment of plasminogen (41O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3150) Google Scholar). Endostatin is a 20-kDa C-terminal fragment of collagen XVIII and specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth (42O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4211) Google Scholar). The 16-kDa N-terminal fragment of human prolactin was shown to inhibit VEGF-induced activation of Ras in capillary endothelial cells (46Clapp C. Martial J.A. Guzman R.C. Rentier-Delrue F. Weiner R.I. Endocrinology. 1993; 133: 1292-1299Crossref PubMed Scopus (255) Google Scholar). Thus, it seems that angiogenic inhibitors can be fragments of proteins whose functions are not related to angiogenesis. It will be interesting to see whether the CEACAM1-induced factor is a fragment of a known protein. In any event, it is very likely that the CEACAM1-induced factor is a previously unknown molecule. We thank Dr. Nguyen T. Van for flow cytometry analysis and Dr. Kenneth Wu for endothelial cells." @default.
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W2091337602 title "Inhibition of Prostate Tumor Angiogenesis by the Tumor Suppressor CEACAM1" @default.
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