[CANCER RESEARCH 59, 4973– 4983, October 1, 1999] Expression of OVCA1, a Candidate Tumor Suppressor, Is Reduced in Tumors and Inhibits Growth of Ovarian Cancer Cells 1 Wendy Bruening, 2 Amanda H. Prowse, 2 David C. Schultz, 2 Marina Holgado-Madruga, Albert Wong, and Andrew K. Godwin 3 Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [W. B., A. H. P., D. C. S., A. K. G.]; Wistar Institute, Philadelphia, Pennsylvania 18014 [D. C. S.]; and Department of Microbiology and Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [M. H-M., A. W.] ABSTRACT Loss of all or part of one copy of chromosome 17p is very common in ovarian and breast tumors. OVCA1 is a candidate tumor suppressor gene mapping to a highly conserved region on chromosome 17p13.3 that shows frequent loss of heterozygosity in breast and ovarian carcinomas. Western blot analysis of extracts prepared from breast and ovarian carcinomas revealed reduced expression of OVCA1 compared with extracts from normal epithelial cells from these tissues. Subcellular localization studies indicate that OVCA1 is localized to punctate bodies scattered throughout the cell but is primarily clustered around the nucleus. Attempts to create cell lines that stably expressed OVCA1 from the cytomegalovirus pro- moter were generally unsuccessful in a variety of different cell lines. This reduction of colony formation was quantified in the ovarian cancer cell line A2780, where it was demonstrated that cells transfected with plasmids expressing OVCA1 had a 50 – 60% reduction in colony number as com- pared with appropriate controls, and only a few of these clones expressed OVCA1, albeit at low levels. The clones that expressed exogenous OVCA1 were found to have dramatically reduced rates of proliferation. Reduced growth rates correlated with an increased proportion of the cells in the G 1 fraction of the cell cycle compared with the parental cell line and de- creased levels of cyclin D1. The low levels of cyclin D1 appeared to be caused by an accelerated rate of cyclin D1 degradation. Overexpression of cyclin D1 was able to override OVCA1’s suppression of clonal outgrowth. These results suggest that slight alterations in the level of OVCA1, such as would occur after reduction of chromosome 17p13.13 to hemizygosity, may result in cell cycle deregulation and promote tumorigenesis. INTRODUCTION Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about the molecular evolution of ovarian tumors. Only a few candidate tumor suppressor genes in sporadic ovarian cancer have thus far been identified. Although two familial breast/ovarian cancer genes, BRCA1 and BRCA2, have been identified, mutations in sporadic ovarian cancers are rare in these genes. Other recently identified tumor suppressor genes that have been analyzed for mutations in ovarian tumors include TSG101, PTEN, DPC4, and BARD1. However, there has been little evidence reported suggesting that these genes are important in the pathogenesis of sporadic ovarian cancers (1–7). In addition, several interesting candidate tumor suppressor genes, DOC2, NOEY2, and LOT1, have recently been identified, and their roles in the development of ovarian cancer are currently being investigated (8 –11). The TP53 tumor suppressor gene is, by far, the most frequently altered gene observed in ovarian cancer. In epithelial ovarian carcinomas, TP53 mutations are present in 50% of advanced-stage cancers. However, the low frequency of TP53 mutations in cancers confined to the ovary and the near absence of mutations in benign and borderline ovarian neoplasms suggest that TP53 alterations may be a relatively late event in the progression of ovarian cancer (12). LOH 4 for markers on the short arm of chromosome 17 is one of the most common genetic abnormalities in ovarian cancer. Two regions on 17p13, including TP53 at 17p13.1 and a more telomeric region at 17p13.3 defined by markers D17S5/30 (equivalent to YNZ22.1) and D17S28 (equivalent to YNH37.3), have received the most attention (13). It has been reported that YNZ22.1 had a rate of LOH as high as 80%, and YNH37.3 showed 65% LOH in ovarian carcinomas. Loss at either D17S5/S30 or D17S28 was observed in 43% of low malig- nant potential tumors, 80% of carcinomas without metastases, and 90% of advanced-stage carcinomas. Interestingly, in the low malig- nant potential tumors, allelic losses at YNZ22.1 and YNH37.3 were not accompanied by LOH at TP53, suggesting the loss of a more distal tumor suppressor gene in early tumorigenesis (14, 15). Alterations involving the NYH37.3/YNZ22.1 region on chromo- some 17p13.3 are not limited to ovarian cancer. A recent study by the European Breast Cancer Linkage Consortium of 1280 breast tumors found that the frequency of LOH observed on the p arm of chromo- some 17 was much higher than that observed on the q arm (16). Up to two-thirds of breast tumors show LOH at the YNZ22.1 locus (17–23), and this finding has been associated with markers of tumor aggression (16, 23–25). Breast tumors with LOH at YNZ22.1 have been associated with a higher risk of recurrence than those showing retention of this region (23, 25). This same region shows frequent LOH in small cell lung cancers (26 –28), colon cancers (29), primitive neuroectodermal tumors (30 –32), carcinoma of the cervix uteri (33– 36), medulloblastoma (37– 40), astrocytoma (41, 42), follicular thy- roid carcinoma (43), malignant melanoma (44), hepatocellular carci- noma (45), and leukemia and lymphoma (46). In many of these studies, changes on chromosome 17p13.3 occur in the absence of alterations involving TP53, suggesting that a tumor suppressor gene(s) residing in this region on chromosome 17p13.3 may be involved in the development of many types of cancers. We have previously reported the identification of a common region of allelic loss on 17p13.3 in ovarian cancer defined by the markers D17S28 and D17S5/S30 (47). These two loci span 20 kbp (47). By the use of positional cloning strategies, two candidate tumor suppres- sor genes, OVCA1 and OVCA2, have been identified that map to the region that is most commonly lost in ovarian and breast tumors, chromosome 17p13.3 (47, 48). OVCA1 demonstrates sequence sim- ilarity (20% identity) to one of the yeast enzymes in the diphthamide Received 9/15/98; accepted 8/5/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by NIH Grant RO1 CA70328, the Susan G. Komen Foundation, the United States Army Medical Research and Materiel Command (Contract DAMD17-96-1-6088), the Hoxie Harrison Smith Foundation, the Butler Family Fund, and an appropriation from the Commonwealth of Pennsylvania. W. B. was supported by a fellowship from the NIH. 2 The first three authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Room W-304, Philadel- phia, PA 19111. Phone: (215) 728-2756; Fax: (215) 728-2741; E-mail: a_godwin@ fccc.edu. 4 The abbreviations used are: LOH, loss of heterozygosity; SSCP, single-strand con- formation polymorphism; HA, hemagglutinin; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorting; TUNEL, terminal deoxynucleotidyl transferase-medi- ated nick end labeling; VNTR, variable number of tendem repeats; CMV, cytomegalo- virus; CD, Cowden disease; LDD, Lhermitte-Duclos disease; BZS, Bennayan-Zonena syndrome. 4973 Research. on October 4, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from