BackgroundThe dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.ResultsWe first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).ConclusionsABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0036-2) contains supplementary material, which is available to authorized users.

Ovarian cancer is the fifth leading cause of cancer death and the most deadly gynecological malignancy in women. Epigenetic modifications play an important role in regulating gene transcription. Specifically, aberrant promoter hypermethylation has been implicated as a hallmark of cancer. In order to identify genes that are differentially methylated in ovarian cancer, we performed meDIP-chip in various ovarian cancer cell lines using Agilent 244K CpG island microarray. One of the targets, ARNTL which is a core component of the circadian clock is methylated in a sub-set of ovarian cancer cell lines. Combined bisulfite restriction analysis (COBRA) confirmed the results of the microarray. Additional analysis using ChIP-PCR revealed that promoter of ARNTL is enriched with the repressive histone mark H3K27me3 in CP70 and MCP2 ovarian cancer cells. Treatment with the EZH2 inhibitor (GSK126) significantly restored ARNTL expression in these cells (CP70 and MCP2). Further functional analysis demonstrated that overexpression of ARNTL inhibited cell growth and enhanced chemosensitivity of cisplatin in ovarian cancer cells. Finally, overexpression of ARNTL restored the rhythmic activity of c-MYC in ovarian cancer cells. These results suggested that ARNTL may be a tumor suppressor and is epigenetically silenced in ovarian cancer.

Previous studies have demonstrated that infection with human polyomavirus, such as JCPyV and BKPyV, might be associated with various human tumors. However, an association between human JCPyV and BKPyV infection and diffuse large B-cell lymphoma (DLBCL) has not been reported. The purpose of this study was to examine DLBCLs of the gastrointestinal tract for evidence of human polyomavirus infection. Nested PCR and DNA sequencing were employed for viral DNA detection and viral genotype identification. In addition, two viral proteins, the large tumor antigen (LT) and the major structural protein (VP1), were detected by immunohistochemistry (IHC). Human JCPyV and BKPyV DNA was detected in 14 out of 16 tissue samples (87.5%), whereby nine cases were infected with JCPyV and five cases were infected with BKPyV. Both archetypal and rearranged genotypes of JCPyV and BKPyV were detected in the tissues. LT was detected in 11 tissue samples (68.75%). However, VP1 was not detected in any of the tissue samples. The presence of human JCPyV and BKPyV DNA and protein in DLBCL tissues of gastrointestinal tract were first reported in this study. The current results provide evidence of a possible association between human JCPyV and BKPyV infection and DLBCL.

The TGF-β signaling regulates numerous cellular processes, including cell proliferation, cell differentiation, apoptosis, migration and cell fate. It was also demonstrated that TGF-β functions as a tumor suppressor in normal ovarian surface epithelium (OSE) cells but promotes tumor proliferation and epithelial-mesenchymal transition (EMT) during ovarian cancer progression. Nevertheless, the molecular mechanisms leading to this divert role of TGF-β signaling in ovarian cancer remains to be elucidated. Our previous studies demonstrated that several TGF-β/SMAD4 regulated targeted are epigenetically silenced by DNA methylation and histone modification including H3K27me3. We therefore hypothesized that the histone-lysine N-methyltransferase, EZH2 may act as an epigenetic switch to facilitate the TGF-β mediated EMT in ovarian cancer. In this study, we utilized our previously identified TGF-β responsive targets using combined ChIP-chip and expression arrays in an immortalized ovarian surface epithelial (IOSE) cell which showed TGF-β growth inhibition. Bioinformatics analysis using ENCODE ChiP-Seq data identified that several of these TGF-β targets are marked by EZH2. To investigate the effect of TGF-β signaling on the methylome of ovarian cancer, we performed MBDcap-Seq in SMAD4 knockdown CP70 cells. Our result showed that there are 99 and 73 TGF-β targets showing hyper- or hypo-methylation respectively after SMAD4 knockdown. Analysis of the gene functions on DAVID has uncovered that some of these hypermethylated genes are associated with EMT process and the TGF-β signaling pathway. On the other hand, the hypomethylated genes are associated with transcription repressor activity. We selected one of the hypermethylated target, LTBP2 which was previously found to be a tumor suppressor for further experiment. This gene was expressed in IOSE but down regulated in a panel of ovarian cancer cells showing overexpression of EZH2. Except for IOSE, promoter hypermethylation of LTBP2 was found in ovarian cancer cells as revealed by bisulphite pyrosequencing. In consistent with deep sequencing result, knock-down of SMAD4 further increased the promoter methylation in CP70 cells. Treatment of demethylation agent, 5azaDC partially restored its expression in these cancer cells. Interestingly, synergistic treatment of 5azaDC and EZH2 inhibitor, GSK343 resulted in a dramatic increase of LTBP2 expression in MCP3 and CP70 cells. Taken together, our result suggested that EZH2 may be involved in the epigenetic silencing of TGF-β/SMAD4-regulated tumor suppressors in ovarian cancer. The differential occupancy of EZH2 into these SMAD4 loci may act as an epigenetic switch to turn the function of TGF-β from a tumor suppressor into an EMT regulator. The therapeutic potential of targeting EZH2 in the inhibition of EMT in ovarian cancer deserves further investigation. Citation Format: Jora Meng-Ju Lin, Jacqueline Shay, Jian-Liang Chou, Pearlly S. Yan, Tim H.-M. Huang, Hung Cheng Lai, Michael W.y. Chan. The role of EZH2 as an epigenetic switch of the TGF-β/SMAD4 targets in regulating EMT in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2014-2318