FRLSVDGRSC EDINECSSSP CSQECANVYG SYQCYCRRGY QLSDVDGVTC 451 EDIDECALPT GGHICSYRCI NIPGSFQCSC PSSGYRLAPN GRNCQDIDEC 501 VTGIHNCSIN ETCFNIQGAF RCLAFECPEN YRRSAATRCE RLPCHENREC 551 SKLPLRITYY HLSFPTNIQA PAVVFRMGPS SAVPGDSMQL AITGGNEEGF 601 FTTRKVSPHS GVVALTKPVP EPRDLLLTVK MDLSRHGTVS SFVAKLFIFV

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F. , Capony, F. , Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-l in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases. Epithelial ovarian cancer is the leading cause of death from gynecological cancer in western countries. Contributing to the high mortality is the propensity for local spread of cancer cells within the peritoneal cavity and in lymphatic vessels and their hidden location, which often accounts for late diagnosis (1). Therefore, there is an urgent need to define the components involved in invasion of the peritoneal cavity by ovarian cancer cells in order to develop new therapies and new marker(s) allowing earlier detection of the disease (2). One approach proven to be effective in defining markers involved in metastasis and invasion of breast cancer has been to identify estrogen-regulated proteins in estrogen-responsive breast cancer cell lines (3, 4). Assays in the primary tumors of some of these proteins (progesterone receptor, cathepsin D, pS2) have proved to be useful as prognostic indicators (4, 5). A similar approach can be used for defining ovarian cancer markers by using the available estrogen receptor-positive epithelial ovarian cancer cell lines (6). Several factors have been proposed to facilitate the emergence of epithelial ovarian cancers, including the number of ovulations (1). The role of estrogens in ovarian cancers is more controversial than for endometrial and breast cancers. However, several studies point to estrogens having a facilitative role in ovarian cancer. The estrogen receptor, which is mandatory for estrogen action (7), is expressed in >60% of ovarian cancers (8). The growth of human ovarian cancer cell lines containing estrogen receptors is stimulated in culture by estrogen, as is that of breast cancer cell lines (6, 9, 10). Estrogens also increase expression of genes associated with cell proliferation such as c-myc (11) or with metastasis such as the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. gene for cathepsin D, a lysosomal protease (10). Recently, a prospective epidemiological study on 240,000 U.S. postmenopausal women has shown that estrogen replacement therapy increased the risk of ovarian cancer by 40% after 4 years and 70% after 11 years of treatment (12). Since estrogen replacement therapy of menopause is increasingly used in western countries, it is critical to specify the role of estrogen in carcinogenesis. Our approach is to identify the genes that are under estrogen control and to define the function of the corresponding proteins. A major secreted protein induced by estrogen, but not by other classes of steroids, in the BG1 ovarian cancer cells has been previously described (10). In this manuscript, we report the identification of this protein as fibulin-1, an extracellular matrix and plasma protein (13, 14). MATERIALS AND METHODS Cell Culture. The three following estrogen receptor-positive human ovarian adenocarcinoma cell lines used in this study displayed characteristics of epithelial cells. The BG1 ovarian carcinoma cell line was derived from the primary tumor tissue of a stage II, poorly differentiated ovarian adenocarcinoma. Tumor cells were stained with cytokeratin antibody but not with vimentin-specific antibody (9). This cell line obtained from C. E. Welander (Emory University, Atlanta) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum (FCS). The PEO4 cell line, derived from ascites of a stage III serous ovarian adenocarcinoma, displayed ultrastructural characteristics of epithelial cells (15). This cell line, obtained from Thomas Hamilton (Fox Chase Cancer Center, Philadelphia), was cultivated in RPMI 1640 medium supplemented with 10% FCS and 10 ,tg of insulin per ml (6). The SKOV3 adenocarcinoma cell line, which also contains estrogen receptors (16), was obtained from the American Type Culture Collection and was cultivated in DMEM containing 10% FCS. All culture media contained 0.5% gentamycin. Metabolic Labeling of Estrogen-Regulated Proteins. The effects of estrogen on metabolically labeled proteins were examined as described (4, 10). Cells were deprived of steroids in phenol red-free medium supplementedwith5% dextran-coated charcoal (DCC)-stripped FCS for 5 days and then treated with 10 nM 17,B-estradiol (E2) or the control ethanol vehicle for 2 days. Proteins synthesized by ovarian carcinoma cells were labeled as Abbreviations: E2, 173-estradiol; FCS, fetal calf serum; mAb, monoclonal antibody. tPresent address: Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098. :Present address: Centre National de la Recherche Scientifique, Unite Propre de Recherche 9008, 1919 route de Mende, B.P. 5051, 34033 Montpellier Cedex 1, France. 1To whom reprint requests should be addressed.