AN ESTROGENICALLY REGULATED POTENTIAL TUMOR SUPPRESSOR GENE, PROTEIN TYROSINE PHOSPHATASE γ (PTPγ), IN HUMAN BREAST DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Suling Liu, B.S. * * * * * The Ohio State University 2003 Dissertation Committee: Approved by Dr. Young C. Lin, Adviser Dr. Robert W. Brueggemeier _______________________________ Dr. Yasuko Rikihisa Adviser Dr. Pui-kai Li Veterinary Biosciences Graduate Program ABSTRACT Except for skin cancer, breast cancer is the most common cancer among women and second only to lung cancer as the primary cause of cancer deaths in women. Among the endocrine factors associated with breast cancer, estrogens are considered to play a central role in human breast carcinogenesis. In 1988, Henderson et al. showed that breast cancer risks are increased by long-term exposure to estrogens, such as estradiol-17β (E2). Zeranol (Z) (Ralgro®) is a nonsteroidal agent with estrogenic activities and used as a growth promoter in the U.S. beef, veal and lamb industries. We showed E2 and Z induced human breast epithelial cell neoplastic transformation with the similar potency in the long-term exposure through the redox-pathway, in which estrogen metabolites undergo redox-cycling and produce free radicals which directly induce DNA damage leading to tumor initiation, and/or ERβ-mediated pathway and they have the similar potency in stimulating and inhibiting some target gene expressions in human breast cancer cells. Protein tyrosine phosphatase γ (PTPγ) is a member of the receptor-like family of tyrosine-specific phosphatases and has been implicated as a tumor suppressor gene in kidney and lung cancers. Little is known about PTPγ expression in human breast. In our study, we found that PTPγ was mainly immunolocalized to the epithelium and PTPγ mRNA expression was lower in cancerous than in normal breast tissues, both E2 and Z suppressed PTPγ levels to a much greater degree in cultured normal human breast tissues ii or epithelial cells co-cultured with stromal cells in comparison with the cultured epithelial cells alone. The results indicated that both E2 and Z downregulate PTPγ expression in human breast and that epithelial-stromal cell interaction is important in the regulation of PTPγ expression by estrogenically active agents. Furthermore, we showed that lower PTPγ was associated with higher ERα in cancerous human breast tissues, and the estrogenic downregulation of PTPγ expression by E2 and Z in human breast is associated with estrogen receptor α (ERα). Based on these findings, we hypothesize that PTPγ is a potential estrogen-regulated tumor suppressor gene in human breast cancer that may be involved in neoplastic processes of human breast epithelium, which lead us to examine the action of PTPγ on in vitro growth of MCF-7 human breast cancer cells and compare the estrogenic responses of human breast cancer cells with different expression levels of PTPγ. Our results showed that PTPγ overexpression decreased the doubling time of human breast cancer cell line MCF-7 and led to a decrease in the anchorage-independent growth of MCF-7 cells in soft agar. Furthermore, we described that PTPγ overexpression could reduce the estrogenic responses of MCF-7 cell proliferation to E2 and Z. Our data suggested that PTPγ is able to inhibit proliferation and anchorage-independent growth of human breast cancer cells in vitro and has anti-estrogenic activities in human breast cancer cells. The PTPγ may function as a potential tumor suppressor gene in regulating the process of tumorigenesis in human breast. Collectively, these studies provide new insight into the role of an endocrine disruptor, Zeranol, in the neoplastic transformation of human breast and the importance of PTPγ in the suppression of human breast carcinomas, and shed a light on the tight relationship among estrogen, PTPγ and breast cancer. The investigations not only further iii our understanding of human breast cancer biology but also provide rationales for the development of therapeutic approaches targeting on PTPγ as a breast cancer suppressor. iv Dedicated to my parents v ACKNOWLEDGMENTS I must first thank my advisor Dr. Young C. Lin, whose scientific guidance, support and mentorship has been invaluable. Dr. Lin, over the past four and a half years you have been a constant source of encouragement, advice and financial support for my study and research. I am especially grateful to my PhD committee members Drs. Robert W. Brueggemeier, Yasuko Rikihisa, and Pui-kai Li who provide me advice and direction throughout my graduate studies. I also show my appreciation to Drs. Yasuro Sugimoto and Samuel K. Kulp for their technical support and suggestions. Thanks also go to all members in Dr. Lin’s laboratory for their sincere friendship. To my parents, brother and sister, thank you for your continued support. Finally, to Xi, thank you for your encouragement, support, and love. vi VITA October 17, 1974............................................Born – Liuyang, Hunan, China 1997................................................................B.S., Biology University of Science and Technology of China Hefei, Anhui, China 1997 – 1999 ...................................................Research Associate University of Science and Technology of China Hefei, Anhui, China 1999-present...................................................Graduate Research Associate, Department of Veterinary Biosciences The Ohio State University Columbus, Ohio PUBLICATIONS Research Publications S. Liu, Y. Sugimoto, S.K. Kulp, J. Jiang, H.L. Chang, K.Y. Park, Y. Kashida, and Y.C. Lin. Estrogenic down-regulation of Protein Tyrosine Phosphatase γ (PTPγ) in human breast is associated with Estrogen Receptor α. Anticancer Research 22: 3917-3924, 2002. S. Liu, S.K. Kulp, Y. Sugimoto, J. Jiang, H.L. Chang, and Y.C. Lin. Involvement of breast epithelial stromal interactions in the regulation of Protein Tyrosine Phosphatase γ (PTPγ) mRNA expression by estrogenically active agents. Breast Cancer Research and Treatment 71: 21-35, 2002. S. Liu, S.K. Kulp, Y. Sugimoto, J. Jiang, H.L. Chang, and Y.C. Lin. The (-)-enantiomer of gossypol possesses higher anticancer potency than racemic gossypol in human breast cancer. Anticancer Research 22: 33-38, 2002. vii S. Liu, Y. Sugimoto, C. Sorio, P. Melotti, and Y.C. Lin. Function analysis of estrogenically regulated Protein Tyrosine Phosphatase γ (PTPγ) in human breast cancer cell line MCF-7. Oncogene (submitted), 2003. S. Liu, and Y.C. Lin. Transformation of human breast epithelial cells MCF-10A by environmental disruptor, zeranol (Z), and estradiol-17β (E2). The Breast Journal (accepted, in press), 2003. S. Liu, Y. Sugimoto, H.L. Chang, J. Jiang, K.Y. Park, C. Sorio, P. Melotti, K. Huebner, and Y. C. Lin. Function analysis of estrogenically regulated Protein Tyrosine Phosphatase γ (PTPγ) in vitro in human breast cancer. American Association for Cancer Research 94th Annual Meeting: A4962, 2003. S. Liu, Y. Sugimoto, S.K. Kulp, J. Jiang, H.L. Chang, K.Y. Park, and Y.C. Lin. Estrogenically active agents downregulate Protein Tyrosine Phosphatase γ (PTPγ) expression in human breast through estrogen receptor α not estrogen receptor β. American Association for Cancer Research 93rd Annual Meeting: A5237, 2002. S. Liu, S.K. Kulp, J. Jiang, Y. Sugimoto, and Y.C. Lin. Importance of breast epithelial stromal interactions in the regulation of Protein Tyrosine Phosphatase γ (PTPγ) mRNA expression by estrogenically active agents. American Association for Cancer Research 92nd Annual Meeting Proceedings: A4737, 2001. Y.C. Lin, S. Liu, S.K. Kulp, J. Jiang, Y. Sugimoto, and M.K. Dowd, P. Wan. The (-) – enantiomer of gossypol (GP) is a potent inhibitor of normal and cancerous breast cell growth. American Association for Cancer Research 92nd Annual Meeting Proceedings 42: A387, 2001. S.K. Kulp, S. Liu, Y. Sugimoto, R.W. Brueggemeier, Y.C. Lin. Localization of Protein Tyrosine Phosphatase γ (PTPγ) to the mammary epithelium of ACI rats” Biol Reprod, 62(suppl 1): A191, 2000. H.L. Chang, Y. Sugimoto, S. Liu, J. Jiang, S.K. Kulp, K.Y. Park, Y. Kashida, and Y.C. Lin. Regulation of Protein Tyrosine Phosphatase γ (PTPγ) mRNA expression by estrogenically active agents in canine prostate. Biol Reprod, 64 (suppl 1), 2002. J. Jiang, S.K. Kulp, S. Liu, H.L. Chang, Y. Sugimoto, and Y.C. Lin. Estrogenic action elevates cyclin D1 mRNA expression in canine prostate. Biol Reprod, 63 (suppl 1), 2001. H.L. Chang, Y. Sugimoto, S. Liu, J. Jiang, S.K. Kulp, R.W. Brueggemeier, and Y.C. Lin. Development of an in vitro model for the screening of biologically active keratinocyte growth factor (KGF/FGF-7) receptor antagonists. Biol Reprod, 63 (suppl 1), 2001. M.P. Wick, Y. Sugimoto, S. Liu, and Y.C. Lin. Cellular and molecular functions of cultured myogenic cells derived from a day old calf. In vitro Cellular and Developmental viii Biology-Animal (Submitted), 2003. Y. Kashida, Y. Sugimoto, S. Liu, and Y.C. Lin. Protein Tyrosine Phosphatase γ (PTPγ) mRNA expression in the non-tumorous or tumorous uterus tissues. Biol Reprod, 68(suppl 1): A167, 2003. K.Y. Park, Y. Sugimoto, S. Liu, H.L. Chang, W. Ye, L.S. Wang, and Y.C. Lin. Involvement of adipocytes on local estrogen level. Biol Reprod, 68(suppl 1): A444, 2003. H.L. Chang, Y. Sugimoto, K.Y. Park, S. Liu, W. Ye, L.S. Wang, Y.W. Huang, and Y.C. Lin. Regulation of estrogen receptor-α mRNA expression by keratinocyte growth factor (KGF) in MCF-7 cells. Biol Reprod, 68(suppl 1): A659, 2003. J. Jiang, S.K. Kulp, Y. Sugimoto, S. Liu, H.L. Chang, and Y.C. Lin. Effects of estrogens and age on the growth of canine prostate cells. Biol Reprod, 68(suppl 1): A176, 2003. H.L Chang, Y. Sugimoto, K.Y. Park, S. Liu, W. Ye, L.S. Wang, Y.W. Huang, and Y.C. Lin. Regulation of estrogen receptor α mRNA by Keratinocyte growth factor (KGF) in MCF-7 cells.