Frequent promoter hypermethylation of TGFBI in epithelial ovarian cancer

Abstract

Objectives. Using pharmacologic unmasking and genome-wide differential methylation analysis, we identified a novel methylated gene in ovarian cancers. Methods. Two ovarian cancer cells (OVCAR-3, ES-2) that showed synergistic growth inhibition by 5-aza-dC and cisplatin were selected. After treatment with 5-aza-dC, differential expression profiles were compared using microarray that contained 38,500 genes. Reactivation of candidate genes and their promoter methylation were validated by real-time RT-PCR, MS-PCR and bisulfite sequencing. Methylation status was tested by MS-PCR in 56 patients with epithelial ovarian cancer and compared to the 38 normal ovarian tissues. Results. We identified 103 candidate genes that were reactivated by 5-aza-dC treatment. Among those, SFN and TGFBI were commonly reactivated in both cells. Since SFN is a well known methylated marker, we selected TGFBI for further validation. Bisulfite sequencing revealed complete promoter methylation in ES-2 and partial methylation in OVCAR-3. In addition, silencing of TGFBI at the transcription level was reversed by 5-aza-dC treatment. TGFBI methylation was observed in 23 out of 38 (60.5%) cases of ovarian cancer, in no normal ovarian tissues (0 of 38, P = 0.001), and in 5 out of 18 (27.8%) borderline tumors (P = 0.044). In our cohort, we did not observe any association between methylation of TGFBI and clinicopathologic variables or clinical outcomes. Conclusion. Our results confirm that TGFBI is frequently methylated in ovarian cancer. Its methylation can be used as a novel epigenetic biomarker in discriminating ovarian cancer from non-cancer or borderline tumors. (C) 2010 Elsevier Inc. All rights reserved.