Laboratory Investigation (2010) 90, 414–425 & 2010 USCAP, Inc All rights reserved 0023-6837/10 $32.00 Promoter hypermethylation of FBXO32, a novel TGF-b/ SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer Jian-Liang Chou1,2, Her-Young Su3, Lin-Yu Chen1,2, Yu-Ping Liao4, Corinna Hartman-Frey5, Yi-Hui Lai1,2, Hui-Wen Yang1,2, Daniel E Deatherage6, Chieh-Ti Kuo6, Yi-Wen Huang6, Pearlly S Yan6, Shu-Huei Hsiao1,7, Chien-Kuo Tai1,2, Huey-Jen L Lin6,8, Ramana V Davuluri9, Tai-Kuang Chao10, Kenneth P Nephew5, Tim H-M Huang6, Hung-Cheng Lai*,3,11 and Michael W-Y Chan*,1,7 Resistance to TGF-b is frequently observed in ovarian cancer, and disrupted TGF-b/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-b/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-b/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; Po0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan–Meier analysis (Po0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, Po0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF- b/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer. Laboratory Investigation (2010) 90, 414–425; doi:10.1038/labinvest.2009.138; published online 11 January 2010 KEYWORDS: ovarian cancer; epigenetics; TGF-b; FBXO32 Ovarian cancer is the fifth leading cause of death in women ease. Despite advances in chemotherapy, the poor prognosis and the most deadly of gynecological malignancies.1 The for patients with ovarian cancer is reflected in the o20% lifetime risk of ovarian cancer in women is B1.5%.2 As 5-year survival rate after initial diagnosis for patients with ovarian cancer has few symptoms early in its course, the stage III and IV disease, whereas survival of patients with majority of patients are diagnosed with advanced-stage dis- stage I or II disease is 480% for the same period.3 Current 1Department of Life Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan, ROC; 2Institute of Molecular Biology, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan, ROC; 3Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC; 4Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC; 5Department of Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA; 6Department of Molecular Virology, Immunology, and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center, the Ohio State University, Columbus, OH, USA; 7Human Epigenomics Center, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan, ROC; 8Divison of Medical Technology, School of Allied Medical Professions, the Ohio State University, Columbus, OH, USA; 9Center for Systems and Computational Biology, Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA; 10Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC and 11Department of Obstetrics and Gynecology, National Defense Medical Center, Taipei, Taiwan, ROC *Correspondence: Dr MWY Chan, PhD, Room 452, Department of Life Science and Human Epigenomics Center, 168 University Road, National Chung Cheng University, Min-Hsiung, Chia-Yi, 621, Taiwan, ROC. E-mail: [email protected] or Dr H-C Lai, MD, PhD, Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, 5F, 325, Sec 2, Cheng-Gong Rd., Neihu District, Taipei 114, Taiwan, ROC. E-mail: [email protected] Received 30 April 2009; revised 3 November 2009; accepted 23 November 2009 414 Laboratory Investigation | Volume 90 March 2010 | www.laboratoryinvestigation.org Epigenetic dysregulation of FBXO32 J-L Chou et al prognostic indicators using clinicopathological variables, MATERIALS AND METHODS including stage and grade, neither accurately predict clinical Patient Samples outcomes nor provide biological insight into the disease. A total of 96 ovarian cancer samples were obtained from the Thus, a better understanding of the molecular carcinogenesis Tri-Service General Hospital (Taipei, Taiwan) (Supplemen- of ovarian cancer is required for developing more reliable tary Table S1). Five normal OSE (NOSE) cells were acquired prognostic markers. from patients during surgery for benign gynecological disease The transforming growth factor-b signaling pathway has at the Indiana University as described previously.17,25 All an important role in controlling proliferation, differentiation, studies involving human ovarian epithelial samples were and other cellular processes, including the growth of ovarian approved by the Institutional Review Boards of the Tri-Ser- surface epithelial (OSE) cells.4 With each ovulation, the OSE vice General Hospital (Taiwan) and the Indiana University. covering the ovary undergoes rupture, followed by pro- 5 liferation-mediated repair. The growth inhibitory effect of Cell Culture and Epigenetic Treatment TGF-b on the OSE may have a key role in preventing over- 5 Immortalized OSE (IOSE) cells were derived by transducing proliferation of OSE, and dysregulation of TGF-b signaling the catalytic subunit of human telomerase and the papilloma may be crucial to the development of epithelial ovarian virus subunit E7 into primary ovarian epithelial cells, as cancer. In addition, resistance to TGF-b signaling is com- 26 6,7 described previously. Cells were maintained in a 1:1 mix- monly observed in ovarian cancer, suggesting that dimin- ture of medium 199 (Sigma, St Louis, MO, USA) and 105 ished responsiveness to TGF-b is a key event. However, few (Sigma) supplemented with 10% fetal bovine serum (FBS) studies have examined the consequences of disrupted TGF-b (Invitrogen, Carlsbad, CA, USA), 400 ng/ml hydrocortisone signaling in ovarian cancer. (Sigma), 10 ng/ml EGF, and 50 Units/ml of penicillin/strep- Ovarian tumorigenesis is a multistep process that includes tomycin (Invitrogen).26 Ovarian cancer cell lines, namely the accumulation of acquired (somatic) epigenetic changes in 8 A2780, CP70, MCP2, MCP3, were propagated with RPMI critical genes. Epigenetic alterations, including DNA methyl- 1640 (Invitrogen) containing 10% FBS. The HeyC2 cell was ation and histone modifications, have important roles in 9–11 12–16 cultured with DMEM containing 5% FBS, 1% NEAA, 1% gene regulation, and we along with others have Gln, and 1% HEPES. The SKOV3 cell was cultured previously shown that tumor-suppressor genes can be tran- with McCoy’s 5A containing 10% FBS, 1% NEAA, 1% Gln, scriptionally silenced by epigenetic modifications in ovarian and 1% HEPES. For epigenetic treatment, 1 Â 106 cells were cancer. We have also shown that epigenetically modified seeded onto 90-mm plates and treated with 5 mM50-aza-20- genes can be used for both ovarian cancer diagnosis and 17,18 deoxycytidine (5-azaDC; Sigma) for 72 h or with trichostatin prognosis. A (TSA, 0.5 mM; Sigma) for 12 h. For 5-azaDC treatment, Our previous study using chromatin immunoprecipitation media were changed and a new drug was added every 24 h. microarray (ChIP–Chip) identified FBXO32 as a TGF-b/SMAD target gene in OSE.19 FBXO32 (also known as atrogin-1) is a member of the F-box protein family and constitutes one of the DNA Extraction four subunits of the ubiquitin protein ligase complex.20,21 The DNA was extracted using the Tissue and Cell Genomic DNA FBXO32 protein has been reported to have a role in muscle Purification Kit (Genemark, Taiwan). DNA was eluted in 1 atrophy,22 and recent findings suggest that FBXO32 is a novel 50 ml distilled water and stored at À20 C until use. apoptosis regulator23 and is negatively regulated by a pro- survival signal.24 Interestingly, Tan et al23 also showed that Bisulfite Conversion and Combined Bisulfite Restriction FBXO32 was transcriptionally silenced by epigenetic mechan- Analysis isms in MCF-7 breast cancer cells. In this study, we found that Genomic DNA (0.5 mg) was bisulfite modified using the EZ FBXO32 was downregulated in a panel of ovarian cancer cell DNA Methylation Kit (Zymo Research, Orange, CA, USA) lines. Promoter hypermethylation of FBXO32 was observed in according to the manufacturer’s protocol. For combined bi- ovarian cancer cells, showing constitutive SMAD4 nuclear sulfite restriction analysis (COBRA), bisulfite-modified DNA translocation.15