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 TGFb/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer. Ovarian cancer is the fifth leading cause of death in women and the most deadly of gynecological malignancies. 1 The lifetime risk of ovarian cancer in women is B1.5%. 2 As ovarian cancer has few symptoms early in its course, the majority of patients are diagnosed with advanced-stage disease. Despite advances in chemotherapy, the poor prognosis for patients with ovarian cancer is reflected in the o20% 5-year survival rate after initial diagnosis for patients with stage III and IV disease, whereas survival of patients with stage I or II disease is 480% for the same period. 3 Current

Transforming growth factor-beta (TGF-beta)/SMAD signaling is a key growth regulatory pathway often dysregulated in ovarian cancer and other malignancies. Although loss of TGF-beta-mediated growth inhibition has been shown to contribute to aberrant cell behavior, the epigenetic consequence(s) of impaired TGF-beta/SMAD signaling on target genes is not well established. In this study, we show that TGF-beta1 causes growth inhibition of normal ovarian surface epithelial cells, induction of nuclear translocation SMAD4, and up-regulation of ADAM19 (a disintegrin and metalloprotease domain 19), a newly identified TGF-beta1 target gene. Conversely, induction and nuclear translocation of SMAD4 were negligible in ovarian cancer cells refractory to TGF-beta1 stimulation, and ADAM19 expression was greatly reduced. Furthermore, in the TGF-beta1 refractory cells, an inactive chromatin environment, marked by repressive histone modifications (trimethyl-H3K27 and dimethyl-H3K9) and histone deacetylase, was associated with the ADAM19 promoter region. However, the CpG island found within the promoter and first exon of ADAM19 remained generally unmethylated. Although disrupted growth factor signaling has been linked to epigenetic gene silencing in cancer, this is the first evidence demonstrating that impaired TGF-beta1 signaling can result in the formation of a repressive chromatin state and epigenetic suppression of ADAM19. Given the emerging role of ADAMs family proteins in growth factor regulation in normal cells, we suggest that epigenetic dysregulation of ADAM19 may contribute to the neoplastic process in ovarian cancer.

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.