Oncogenic BARD1 Isoforms Expressed in Gynecological Cancers Lin Li, 1 Stephan Ryser, 1 Eva Dizin, 1 Dietmar Pils, 2 Michael Krainer, 2 Charles Edward Jefford, 1 Francesco Bertoni, 4 Robert Zeillinger, 3 and Irmgard Irminger-Finger 1 1 Laboratory of Molecular Gynecology and Obstetrics, Department of Gynecology and Obstetrics and Department of Genetics and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland; 2 Laboratory of Molecular Genetics, Department of Internal Medicine 1, Medical University of Vienna; 3 Molecular Oncology Group, Medical University of Vienna, Vienna, Austria; and 4 Institute of Experimental Oncology, Bellinzona, Switzerland Abstract BARD1 is required for protein stability and tumor suppressor functions of BRCA1, which depend on the ubiquitin ligase activity of the BRCA1-BARD1 heterodimer. The NH 2 -terminal RING domains of both proteins act as interaction modules and form a ubiquitin ligase, which has functions in DNA repair, cell cycle checkpoint regulation, and mitosis. Interest- ingly, up-regulated expression of truncated BARD1 isoforms was found to be associated with poor prognosis in breast and ovarian cancers and, in a hormonally regulated fashion, in the human cytotrophoblast, a cell type with properties reminis- cent of cancer cells. We therefore performed reverse tran- scription-PCR to determine the structure of BARD1 isoforms in cell lines derived from hormone-dependent and hormone- independent cancers. We found a specific combination of iso- forms, generated by differential splicing and alternative transcription initiation, mostly lacking the BRCA1 interaction domain, in gynecologic but not hematologic cancer cell lines. To investigate the prevalence of BARD1 isoforms in tumors, we applied immunohistochemistry to ovarian cancers, using antibodies distinguishing full-length BARD1 and isoforms. Expression of NH 2 terminally truncated BARD1 was correlated with advanced stage of cancer, and expression of spliced isoforms was typical for clear cell carcinoma, the ovarian cancer with worst prognosis, suggesting a role of BARD1 isoforms in cancer progression. To challenge this hypothesis, we silenced BARD1 isoforms in ovarian cancer cells that lacked wild-type BARD1 by siRNA interference, which led to a complete proliferation arrest. Thus, BARD1 isoform expres- sion is required for cancer cell proliferation, which is compatible with the notion that BARD1 isoforms act as cancer maintenance genes. [Cancer Res 2007;67(24):11876–85] Introduction BARD1 was discovered as a protein interacting with BRCA1 (1). BARD1 and BRCA1 form a stable heterodimer, which has functions in DNA repair, transcription regulation, RNA processing, and cell cycle regulation through the ubiquitin ligase function of the heterodimer (2). Depletion of BARD1 in vitro leads to genomic instability, loss of polarity, development of a premalignant phenotype (3), and embryonic lethality in knockout mice (4). As a tumor sup- pressor, BARD1 has also BRCA1-independent functions in mediat- ing p53-dependent apoptosis, which is inhibited by BRCA1 (5). It binds to p53, facilitating its phosphorylation and stabilization (6). Recently, a novel function of BARD1 in mitotic spindle assembly was described (7). The expression of BARD1 in most proliferative tissues, particularly in spleen and testis (3, 8), is consistent with its role in mitosis. Up-regulated expression of BARD1 was also observed in response to hypoxia, to genotoxic stress (5, 9), and to hormone signaling (10), suggesting that up-regulation of BARD1 might be associated with tumor suppressor pathways. In spite of this, BARD1 mutations in tumors are infrequent. Three missense mutations, Q564H, V695L, and S761N, and five other alterations were discovered in breast, ovarian, and endome- trial cancers (11). Loss-of-heterozygosity was found for Q564H and S671N mutations, substantiating the role of BARD1 as a tumor suppressor (11, 12). The V695L and S761N mutations were found in somatic breast tissue, whereas the Q564H mutation arose in the germline of a patient with clear cell ovarian carcinoma. At least six other alternations were subsequently identified (13–15), including the missense mutation C557S, which was linked to loss of p53 stability and apoptotic activity (16). BARD1y, an isoform presenting a deletion of exons 2 through 6, was found in a rat ovarian cancer cell line, which is resistant to apoptosis (6), and in HeLa cells (17). BARD1y lacks the RING finger domain, required for BRCA1-interaction, and the ankyrin repeats, which are involved in apoptosis and p53 binding (6, 9). Aberrantly elevated expression and cytoplasmic localization of truncated forms of BARD1 was found in breast and ovarian cancer and correlated with factors of poor prognosis (18). Interestingly, spliced isoforms of BARD1 are expressed, temporally and spatially controlled, in human cytotrophoblasts, which have cancer-like properties in the first trimester of pregnancy (19). Alternative splicing is a crucial mechanism for generating protein diversity and even proteins with novel antagonistic biological functions. Several cancer-associated alterations of splicing patterns have been reported for genes such as RON, RAC1, CD44, and MDM2 (20). Because differential splicing of tumor suppressor genes, such as BARD1, could have a profound effect on tumor development and progression, we characterized the expression pattern of BARD1 isoforms in ovarian, breast, endometrial, and cervical cancer cell lines, and we investigated their potential oncogenic properties in ovarian cancer. Materials and Methods Cancer cell lines. Cancer cell lines were a collection of the Universita ¨tsspital Wien. Ovarian cancer cell lines, composed of established ovarian cancer cell lines and newly generated ones from individual tumors (O1–O32), were as follows: A2780, Caov-3, ES-2, NIH:OVCAR-3, SK-OV-3, TOV-21G, TOV-112D, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for L. Li: Oncology Department, Beijing Hospital, Beijing, China. Requests for reprints: Irmgard Irminger-Finger, Laboratory of Molecular Gynecology and Obstetrics, Department of Gynecology and Obstetrics, Geneva University Hospitals, Maternite´ 30, Bld de la Cluse, CH-1211, Geneva, Switzerland. Phone: 41-22-382-43-27; Fax: 41-22-382-41-45; E-mail: Irmgard.irminger@hcuge.ch. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2370 Cancer Res 2007; 67: (24). December 15, 2007 11876 www.aacrjournals.org Research Article