Abstract 3072: Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer

Abstract

Abstract Refractory to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in aberrant expression of downstream target genes in the disease. We hypothesized that aberrant expression of TGF-β/SMAD4 targets are mediated through epigenetic mechanism and also contribute to resistance to TGF-β meditated growth inhibition. Our previous report using chromatin immunoprecipitation microarray (ChIP-chip) identified FBXO32 as one of SMAD4 targets in immortalized ovarian surface epithelial cell (IOSE) (Qin et al., BMC Syst Biol, 17: 73, 2009). In the present study, we investigated the mechanism conferring FBXO32 down-regulation, its clinical significance, and its function in ovarian cancer. Our result showed that expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines (HeyC2, SKOV3, CP70, A2708, MCP2, MCP3) using real time RT-PCR. Promoter methylation of FBXO32 was seen in ovarian cancer cell lines, HeyC2 and SKOV3, that display constitutive TGF-β/SMAD4 signaling. Moreover, our finding that epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggested that epigenetic regulation of FBXO32 in ovarian cancer may be a common event. Re-expression of FBXO32 markedly impeded proliferation of a platinum-resistant ovarian cancer cell lines, HeyC2 and CP70 (colony number: HeyC2: 19.33 ± 3.06 vs 1 ± 0, P< 0.005; CP70: 46.5 ± 6.36 vs 1.5 ± 0.7, P< 0.001) by using colony formation assay, due to increased apoptosis of the cells (CP70; apoptotic cell%, control: 2.55 ± 0.17; FBXO32: 10.72 ± 1.07, P<0.05), and resensitized the cells to cisplatin (P < 0.05). Surprisingly, elevated apoptosis was not observed in HeyC2 cells which have much higher drug-resistance than CP70 cells (IC50, HeyC2: 5.6ug/ml, CP70: 2.2ug/ml). However, an increase in the G1 cell population was observed in FBXO32-transfected HeyC2 cells. In in vivo study, the FBXO32-transfecte CP70 showed smaller tumor size compared to vector control after day14 (P < 0.05) of tumor injection. In advanced stage ovarian cancer patients, significant methylation frequency (29.3%; P<0.05) of FBXO32 was observed by real-time qMSP and such an outcome further correlated with the protein expressed level of FBXO32 by IHC stain. Importantly, FBXO32 methylation was significantly associated with shorter progression-free survival, as determined by both Kaplan-Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio 1.003, P<0.05). In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-β/SMAD4 signaling, and FBXO32 methylation status predicts survival in ovarian cancer patients. Future regimen of treating ovarian cancer by ectopic expression of FBXO32 can be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3072.