Regulation of the Angiopoietin-2 Gene by hCG in Ovarian Cancer Cell Line OVCAR-3

Abstract

Angiogenesis is a crucial step in growing tissues including many tumors. It is regulated by pro- and antiangiogenic factors including the family of angiopoietins and their corresponding receptors. In previous work we have shown that in human ovarian cells the expression of angiopoietin 2 (ANG2) is regulated by human chorionic gonadotropin (hCG). To better understand the mechanisms of hCG-dependent regulation of the ANG2-gene we have now investigated upstream regulatory active elements of the ANG2-promoter in the ovarian carcinoma cell line OVCAR-3. We cloned several ANG2-promoter-fragments of different lengths into a luciferase reporter-gene-vector and analyzed the corresponding ANG2 expression before and after hCG stimulation. We identified regions of the ANG2-promoter between 1 048 bp and 613 bp upstream of the transcriptional start site where hCG-dependent pathways promote a significant downregulation of gene expression. By sequence analysis of this area we found several potential binding sites for transcription factors that are involved in regulation of ANG2-expression, vascular development and ovarian function. These encompass the forkhead family transcription factors FOXC2 and FOXO1 as well as the CCAAT/enhancer binding protein family (C/EBP). In conclusion, we have demonstrated that the regulation of ANG2-expression in ovarian cancer cells is hCG-dependent and we suggest that forkhead transcription factor and C/EBP-dependent pathways are involved in the regulation of ANG2-expression in ovarian cancer cells.

gonadotropin - angiopoietin - cancer - angiogenesis

References

• 1 Jones PF. Not just angiogenesis – wider roles for the angiopoietins.  J Pathol. 2003;  201 515-527
• 2 Augustin HG, Young Koh G, Thurston G, Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system.  Nat Rev Mol Cell Biol. 2009;  10 165-177
• 3 Thurston G. Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis.  Cell Tissue Res. 2003;  314 61-68
• 4 Geva E, Jaffe RB. Role of angiopoietins in reproductive tract angiogenesis.  Obstet Gynecol Surv. 2000;  55 511-519
• 5 Hazzard TM, Christenson LK, Stouffer RL. Changes in expression of vascular endothelial growth factor and angiopoietin-1 and -2 in the macaque corpus luteum during the menstrual cycle.  Mol Hum Reprod. 2000;  6 993-998
• 6 Etoh T, Inoue H, Tanaka S, Barnard GF, Kitano S, Mori M. Angiopoietin- 2 is related to tumor angiogenesis in gastric carcinoma: possible in vivo regulation via induction of proteases.  Cancer Res. 2001;  61 2145-2153
• 7 Helfrich I, Edler L, Sucker A, Thomas M, Christian S, Schadendorf D, Augustin HG. Angiopoietin-2 levels are associated with disease progression in metastatic malignant melanoma.  Clin Cancer Res. 2009;  15 1384-1392
• 8 Choi JH, Choi KC, Auersperg N, Leung PC. Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells.  Endocr Relat Cancer. 2005;  12 407-421
• 9 Konishi I. Gonadotropins and ovarian carcinogenesis: a new era of basic research and its clinical implications.  Int J Gynecol Cancer. 2006;  16 16-22
• 10 Steinmeyer C, Berkholz A, Gebauer G, Jäger W. The expression of hCG receptor mRNA in four human ovarian cancer cell lines varies considerably under different experimental conditions.  Tumour Biol. 2003;  24 13-22
• 11 Gawronska B, Leuschner C, Enright FM, Hansel W. Effects of a lytic peptide conjugated to beta HCG on ovarian cancer: studies in vitro and in vivo.  Gynecol Oncol. 2002;  85 45-52
• 12 Runnebaum IB, Brüning A. Glucocorticoids inhibit cell death in ovarian cancer and up-regulate caspase inhibitor cIAP2.  Clin Cancer Res. 2005;  11 6325-6332
• 13 Rainey WH, Sawetawan C, Shay JW, Michael MD, Mathis JM, Kutteh W, Byrd W, Carr BR. Transformation of human granulosa cells with the E6 and E7 regions of human papillomavirus.  J Clin Endocrinol Metab. 1994;  78 705-710
• 14 Pietrowski D, Keck C. Differential regulation of ANG2 and VEGF-A in human granulosa lutein cells by choriogonadotropin.  Exp Clin Endocrinol Diabetes. 2004;  112 208-214
• 15 Potente M, Urbich C, Sasaki K, Hofmann WK, Heeschen C, Aicher A, Kollipara R, DePinho RA, Zeiher AM, Dimmeler S. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization.  J Clin Invest. 2005;  115 2382-2392
• 16 Furuyama T, Kitayama K, Shimoda Y, Ogawa M, Sone K, Yoshida-Araki K, Hisatsune H, Nishikawa S, Nakayama K, Nakayama K, Ikeda K, Motoyama N, Mori N. Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice.  J Biol Chem. 2004;  279 34741-34749
• 17 Daly C, Wong V, Burova E, Wei Y, Zabski S, Griffiths J, Lai KM, Lin HC, Ioffe E, Yancopoulos GD, Rudge JS. Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1).  Genes Dev. 2004;  18 1060-1071
• 18 Goettsch W, Gryczka C, Korff T, Ernst E, Goettsch C, Seebach J, Schnittler HJ, Augustin HG, Morawietz H. Flow-dependent regulation of angiopoietin-2.  J Cell Physiol. 2008;  214 491-503
• 19 Shi F, LaPolt PS. Relationship between FoxO1 protein levels and follicular development, atresia, and luteinization in the rat ovary.  J Endocrinol. 2003;  179 195-203
• 20 Xue Y, Cao R, Nilsson D, Chen S, Westergren R, Hedlund EM, Martijn C, Rondahl L, Krauli P, Walum E, Enerbäck S, Cao Y. FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue.  Proc Natl Acad Sci USA. 2008;  105 10167-10172
• 21 Sterneck E, Tessarollo L, Johnson PF. An essential role for C/EBPbeta in female reproduction.  Genes Dev. 1997;  11 2153-2162
• 22 Huang AM, Rudelius M, Sharan S, McAllister JM, Raffeld M, Christenson LK, Sterneck E. The Cebpd (C/EBPdelta) gene is induced by luteinizing hormones in ovarian theca and interstitial cells but is not essential for mouse ovary function.  PLoS ONE. 2007;  19, 2 e1334
• 23 Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.  Science. 1997;  277 ((5322)) 55-60
• 24 Goede V, Schmidt T, Kimmina S, Kozian D, Augustin HG. Analysis of blood vessel maturation processes during cyclic ovarian angiogenesis.  Lab Invest. 1998;  78 1385-1394
• 25 Zygmunt M, Herr F, Keller-Schoenwetter S, Kunzi-Rapp K, Münstedt K, Rao CV, Lang U, Preissner KT. Characterization of human chorionic gonadotropin as a novel angiogenic factor.  J Clin Endocrinol Metab. 2002;  87 5290-5296
• 26 Fisher RA, Savage PM, MacDermott C, Hook J, Sebire NJ, Lindsay I, Seckl MJ. The impact of molecular genetic diagnosis on the management of women with hCG-producing malignancies.  Gynecol Oncol. 2007;  107 413-419
• 27 Yu Q, Stamenkovic I. Angiopoietin-2 is implicated in the regulation of tumor angiogenesis.  Am J Pathol. 2001;  158 563-570
• 28 Ahmad SA, Liu W, Jung YD, Fan F, Wilson M, Reinmuth N, Shaheen RM, Bucana CD, Ellis LM. The effects of angiopoietin-1 and -2 on tumor growth and angiogenesis in human colon cancer.  Cancer Res. 2001;  61 1255-1259
• 29 Geva E, Ginzinger DG, Moore 2nd DH, Ursell PC, Jaffe RB. In utero angiopoietin-2 gene delivery remodels placental blood vessel phenotype: a murine model for studying placental angiogenesis.  Mol Hum Reprod. 2005;  11 253-260
• 30 Arrieta O, Michel Ortega RM, Angeles-Sánchez J, Villarreal-Garza C, Avilés-Salas A, Chanona-Vilchis JG, Aréchaga-Ocampo E, Luévano-González A, Jiménez MA, Aguilar JL. Serum human chorionic gonadotropin is associated with angiogenesis in germ cell testicular tumors.  J Exp Clin Cancer Res. 2009;  28 120
• 31 Crawford RA, Iles RK, Carter PG, Caldwell CJ, Shepherd JH, Chard T. The prognostic significance of beta human chorionic gonadotropin and its metabolites in women with cervical carcinoma.  J Clin Pathol. 1998;  51 685-688
• 32 Lopez D, Sekharam M, Coppola D, Carter WB. Purified human chorionic gonadotropin induces apoptosis in breast cancer.  Mol Cancer Ther. 2008;  7 2837-2844
• 33 Wulff C, Wilson H, Largue P, Duncan WC, Armstrong DG, Fraser HM. Angiogenesis in the human corpus luteum: localization and changes in angiopoietins, tie-2, and vascular endothelial growth factor messenger ribonucleic acid.  J Clin Endocrinol Metab. 2000;  85 4302-4309
• 34 Miyabayashi K, Shimizu T, Kawauchi C, Sasada H, Sato E. Changes of mRNA expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in eCG/hCG-treated immature female rats.  J Exp Zool A Comp Exp Biol. 2005;  303 590-597
• 35 Molskness TA, Stouffer RL, Burry KA, Gorrill MJ, Lee DM, Patton PE. Circulating levels of total angiopoietin-2 and the soluble Tie-2 receptor in women during ovarian stimulation and early gestation.  Fertil Steril. 2006;  86 1531-1533

1 These authors contributed equally to this work.

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