Multiple roles of the candidate oncogene ZNF217 in ovarian epithelial neoplastic progression Peixiang Li 1 , Sarah Maines-Bandiera 1 , Wen-Lin Kuo 2 , Yinghui Guan 2 , Yu Sun 1 , Mark Hills 3 , Guiqing Huang 2 , Collin C. Collins 2 , Peter C.K. Leung 1 , Joe W. Gray 2,4 and Nelly Auersperg 1 * 1 Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada 2 University of California San Francisco Cancer Center, San Francisco, CA 3 The Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada 4 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA The transcription factor ZNF217 is often amplified in ovarian can- cer, but its role in neoplastic progression is unknown. We intro- duced ZNF217-HA by adenoviral and retroviral infection into normal human ovarian surface epithelial cells (OSE), i.e., the source of ovarian cancer, and into SV40 Tag/tag expressing, p53/ pRB-deficient OSE with extended but finite life spans (IOSE). In OSE, ZNF217-HA reduced cell–substratum adhesion and acceler- ated loss of senescent cells, but caused no obvious proneoplastic changes. In contrast, ZNF217-HA transduction into IOSE yielded two permanent lines, I-80RZ and I-144RZ, which exhibited telo- merase activity, stable telomere lengths, anchorage independence and reduced serum dependence, but were not tumorigenic in SCID mice. This immortalization required short-term EGF treat- ment near the time of crisis. The permanent lines were EGF-inde- pendent, but ZNF217-dependent since siRNA to ZNF217 inhibited anchorage independence and arrested growth. Array CGH revealed genomic changes resembling those of ovarian carcino- mas, such as amplicons at 3q and 20q, and deletions at 4q and 18, associated with underexpressed annexin A10, N-cadherin, desmo- collin 3 and PAI-2, which have been reported as tumor suppres- sors. The lines overexpressed EEF1A2, SMARA3 and STAT1 and underexpressed other oncogenes, tumor suppressors and extracel- lular matrix/adhesion genes. The results implicate ZNF217 as an ovarian oncogene, which is detrimental to senescing normal OSE cells but contributes to neoplastic progression in OSE with inacti- vated p53/RB. The resemblance of the genomic changes in the ZNF217-overexpressing lines to ovarian carcinomas provides a unique model to investigate interrelationships between these changes and ovarian neoplastic phenotypes. ' 2007 Wiley-Liss, Inc. Key words: ZNF217; oncogene; ovarian cancer; immortalization The candidate oncogene ZNF217, predicted to encode alterna- tively spliced Kru ´ppel-like transcription factors, was originally identified because of its location in an amplicon on chromosome 20q13.2 in breast cancer cell lines and tumors and by its recurrent expression in other malignancies. 1 ZNF217 is expressed in normal tissues, and is overexpressed in all cell lines and tumors where the gene locus is amplified. 2 Amplification of 20q, which involves a large number of potentially relevant genes, has since been described in many types of human cancers. 3–10 The 20q region, and ZNF217 specifically, is amplified in a high proportion of ovar- ian carcinomas, and expression levels of ZNF217 correlate with their copy numbers in primary ovarian cancers. 11,12 However, because of the coexistence of other amplified genes in the same region, it has been difficult to define the specific influence of ZNF217 on malignant progression. An increased copy number of ZNF217 is associated with reduced survival of ovarian cancer patients. 13,14 In experimental systems, 20q13 amplification has been associated with genome instability and immortalization in cultured human uroepithelial cells. 15,16 Furthermore, overexpres- sion of ZNF217 in 2 human mammary epithelial cell lines with fi- nite lifespans resulted in the establishment of permanent cell lines with increased telomerase activity, stabilized telomere length and resistance to TGF-b-mediated growth inhibition. 17 Recently, ZNF217 overexpression was found to be associated with resist- ance to chemotherapy and telomere dysfunction. 18 We investigated the functional consequences of ZNF217 over- expression by transducing the gene into human ovarian surface epithelial (OSE) cells, which are thought to be the source of ovar- ian epithelial carcinomas. 19 Normal OSE cells were investigated soon after explantation into culture and after finite extension of their life span with SV40 Tag/tag (IOSE). 20 Transient adenoviral infection of ZNF217-HA into OSE caused selective loss of senes- cent cells, but no other obvious changes. In 2 separate IOSE lines, retroviral infection with ZNF217-HA in conjunction with brief epi- dermal growth factor/hydrocortisone treatment during crisis gave rise to permanent cell lines with activated telomerase, stable telo- mere lengths, increased anchorage- and serum-independence and genomic changes resembling those of ovarian cancers. The results support the hypothesis that ZNF217 contributes to the neoplastic progression of epithelial ovarian carcinomas. Material and methods Cell culture Normal ovarian surface epithelial cells (OSE) were obtained at surgery for nonmalignant gynecological disorders. Ethical permits were obtained as required by the University of BC. The origin of each culture was identified by a hyphenated number. OSE cells in low passage were transfected with SV40 large T and small t antigen (Tag/tag) to give rise to IOSE (‘‘immortalized OSE’’), which have an extended but finite lifespan. 20 OSE and IOSE cells were trans- fected or infected with HA-tagged ZNF217 constructs and control vectors as described below. The cultures were maintained in me- dium 199/MDCB105 (Sigma, Mississauga, ON, Canada) with 10% (for OSE) and 5% (for IOSE) FBS (Hyclone, Logan, UT) in humidified 5% CO 2 /air. OSE and IOSE cells senesced after approx- imately 4–5 and 16–22 passages, respectively. In some experiments, 10 ng/ml epidermal growth factor/1.0 lg/ml hydrocortisone (EGF/ HC) (Sigma, Mississauga, ON, Canada) was added. To evaluate the influence of EGF on signal transduction path- ways in ZNF217-infected IOSE cells that were approaching crisis, cells were grown with and without EGF/HC. Cells grown without EGF/HC senesced by passage 15 and could not be subcultured fur- ther. Cells grown in the presence of EGF/HC from passage 14 on could be subcultured twice more to passage 17. Inhibitors of the This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat. Grant sponsor: US Department of Energy, Office of Science, Office of Biological and Environmental Research; Grant number: DE-AC03- 76SF00098; Grant sponsor: US National Institutes of Health, NCI P50; Grant numbers: CA 58207; P01 CA 64602; Grant sponsor: National Cancer Institute of Canada. *Correspondence to: Department of Obstetrics and Gynaecology, Uni- versity of British Columbia, BC Women’s Hospital, Rm. 2H30, 4490 Oak Street, Vancouver BC, V6H 3V5, Canada. Fax: 1604-875-2725. E-mail: auersper@interchange.ubc.ca Received 9 May 2006; Accepted after revision 27 July 2006 DOI 10.1002/ijc.22300 Published online 31 January 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 120, 1863–1873 (2007) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer