DNA Methylation In Ovarian Cancer Research Articles

Current data and future perspectives on DNA methylation in ovarian cancer (Review).

Ovarian cancer (OC) represents the most prevalent malignancy of the female reproductive system. Its distinguishing features include a high aggressiveness, substantial morbidity and mortality, and a lack of apparent symptoms, which collectively pose significant challenges for early detection. Given that aberrant DNA methylation events leading to altered gene expression are characteristic of numerous tumor types, there has been extensive research into epigenetic mechanisms, particularly DNA methylation, in human cancers. In the context of OC, DNA methylation is often associated with the regulation of critical genes, such as BRCA1/2 and Ras‑association domain family 1A. Methylation modifications within the promoter regions of these genes not only contribute to the pathogenesis of OC, but also induce medication resistance and influence the prognosis of patients with OC. As such, a more in‑depth understanding of DNA methylation underpinning carcinogenesis could potentially facilitate the development of more effective therapeutic approaches for this intricate disease. The present review focuses on classical tumor suppressor genes, oncogenes, signaling pathways and associated microRNAs in an aim to elucidate the influence of DNA methylation on the development and progression of OC. The advantages and limitations of employing DNA methylation in the diagnosis, treatment and prevention of OC are also discussed. On the whole, the present literature review indicates that the DNA methylation of specific genes could potentially serve as a prognostic biomarker for OC and a therapeutic target for personalized treatment strategies. Further investigations in this field may yield more efficacious diagnostic and therapeutic alternatives for patients with OC.

71P Single nucleotide polymorphisms in the folate metabolic pathway genes and global DNA methylation in ovarian cancer

71P Single nucleotide polymorphisms in the folate metabolic pathway genes and global DNA methylation in ovarian cancer

Sesquiterpene Lactones Modulated DNA Methylation through Inhibition of DNMTs in Ovarian Cancer Cells

Inappropriate DNA methylation of tumour suppressor genes can affect their expression and function, and as such, DNA methylation has been a promising target for anti-cancer therapy. In gynaecological malignancy, ovarian cancer has the highest associated rate of mortality. This study investigated the in vitro cytotoxicity of four sesquiterpene lactones (SLs), including dehydroleucodine (Deh), alantolactone (Ala), costunolide (Cos), and parthenolide (Pat), and their effect on DNA methylation in ovarian cancer. The binding of the SLs with DNA methyltransferases (DNMTs) were studied using molecular docking. The in vitro antiproliferative activity was evaluated in human ovarian cancer cell lines. SL-induced pro-apoptotic activity and effect on cell cycles was evaluated using enzymatic assays and flow cytometry. Global DNA methylation and DNMTs activity by SLs were measured using ELISA assays. DNA methylation and expression of tumour suppressor genes MHL1 and PTEN were quantified using pyrosequencing and RT-qPCR, respectively. SLs displayed strong binding to DNMT1, 3A and 3B in silico, with Deh exhibiting the strongest binding. SLs showed cytotoxicity in the order of Deh ≈ Ala > Pat > Cos, induced apoptosis, and arrested cell cycle at G2/M phase. Deh and Ala possessed DNMT inhibitory activity, reduced global and gene-specific DNA methylation, and increased expression of notable MHL1 and PTEN, which could contribute to the induction of apoptosis and cell death. In conclusion, Deh and Ala, possess strong cytotoxicity and hold the exciting potential as novel therapeutic agents in ovarian cancer.

DNA methylation changes in epithelial ovarian cancer histotypes

Survival after a diagnosis of ovarian cancer has not improved, and despite histological differences, treatment is similar for all cases. Understanding the molecular basis for ovarian cancer risk and prognosis is fundamental, and to this end much has been gleaned about genetic changes contributing to risk, and to a lesser extent, survival. There's considerable evidence for genetic differences between the four pathologically defined histological subtypes; however, the contribution of epigenetics is less well documented. In this report, we review alterations in DNA methylation in ovarian cancer, focusing on histological subtypes, and studies examining the roles of methylation in determining therapy response. As epigenetics is making its way into clinical care, we review the application of cell free DNA methylation to ovarian cancer diagnosis and care. Finally, we comment on recurrent limitations in the DNA methylation literature for ovarian cancer, which can and should be addressed to mature this field.

The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer.

To investigate SGI-110 as a "chemosensitizer" in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo, SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global (LINE1) and gene-specific hypomethylation in vivo, including TSGs (RASSF1A), proposed drivers of ovarian cancer cisplatin resistance (MLH1 and ZIC1), differentiation-associated genes (HOXA10 and HOXA11), and transcription factors (STAT5B). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting.

Abstract A65: Snail, a potent inducer of global DNA methylation in ovarian cancer

Abstract Snail plays a critical role in the epithelial to mesenchymal transition (EMT). To investigate the role of Snail in this event, we established inducible Snail expression in ovarian cancer cell lines. Reverse Phase Protein Assay (RPPA) revealed that Dnmt1 was upregulated after snail expression in SKOv3-Snail inducible cells. Dnmt1, a major enzyme responsible for transferring a methyl group to the cytosine in a CpG dinucleotide that commonly occurs in the promoter region of genes, is required for heterochromatin maintenance and transcriptional silencing in somatic cells. To evaluate the function of Snail in mediating DNA methylation in ovarian cancer, we first validated the correlated expression of Snail and Dnmt1 by Western blotting in SKOv3-Snail and HO8910PM-Snail inducible cells. Under non-inducible condition, Dnmt1 was mainly localized in the nucleus. However, Dnmt1 was increased in the cytoplasm and had little change in the nucleus when Snail was induced. In a parallel experiment, two other DNA methyltransferases Dnmt3a and Dnmt3b, attribute to de novo DNA methylation during embryonic development, remained localized in the nucleus and had no altered expression when Snail was induced. We also examined 5-Methylcytosine (5-MC) expression in SKOv3-Snail and HO8910PM-Snail inducible cells using immunofluorescent staining. Nuclear 5-MC, which infers global DNA methylation, became apparent when Snail was induced. To further extend this observation in ovarian cancer, we analyzed the expression of 5-MC in an ovarian cancer progression tissue array. We found that 5-MC was expressed in nucleus in all ovarian cancer and only a small portion of positive stain in normal ovarian epithelial and benign neoplasm. We further discovered that expression of 5-MC not only correlated with the stage of ovarian cancer but also significantly elevated in metastatic lesions. In our previous study, we demonstrated that Snail expression correlated with stages of ovarian cancer and its expression was elevated in metastatic leisions, which was also in accordance with the pattern of 5-MC pattern in ovarian cancer cells. To identify the underlying molecular mechanism, we performed NimbleGen Multiplex CpG Island Plus RefSeq Promoter Array using Snail inducible ovarian cancer cell lines. After normalization, a 1.25 fold of increase in total DNA methylation was found in Snail inducible cells in comparison with the un-inducible cells. The increased DNA methylation is consistent with the Snail-5-MC pattern in ovarian cancer cells. In summary, our data show that Snail can alter the global DNA methylation in ovarian cancer, through the regulation of Dnmt1. Further study will shed a new light into this intricate epigenetic regulation and may provide novel therapeutic intervention for targeting ovarian cancer. Citation Format: Jin Hongyan, Zhou Jiayi, Zhao Xiaoyan, Jia Luoqi, Lu Yilin, Yu Yinhua. Snail, a potent inducer of global DNA methylation in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A65.

Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers

Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating “hit” during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan–Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.

Epigenetic suppression of the TGF-beta pathway revealed by transcriptome profiling in ovarian cancer

Epithelial ovarian cancer is the leading cause of death among gynecologic malignancies. Diagnosis usually occurs after metastatic spread, largely reflecting vague symptoms of early disease combined with lack of an effective screening strategy. Epigenetic mechanisms of gene regulation, including DNA methylation, are fundamental to normal cellular function and also play a major role in carcinogenesis. To elucidate the biological and clinical relevance of DNA methylation in ovarian cancer, we conducted expression microarray analysis of 39 cell lines and 17 primary culture specimens grown in the presence or absence of DNA methyltransferase (DNMT) inhibitors. Two parameters, induction of expression and standard deviation among untreated samples, identified 378 candidate methylated genes, many relevant to TGF-beta signaling. We analyzed 43 of these genes and they all exhibited methylation. Treatment with DNMT inhibitors increased TGF-beta pathway activity. Hierarchical clustering of ovarian cancers using the 378 genes reproducibly generated a distinct gene cluster strongly correlated with TGF-beta pathway activity that discriminates patients based on age. These data suggest that accumulation of age-related epigenetic modifications leads to suppression of TGF-beta signaling and contributes to ovarian carcinogenesis.

Aberrant DNA methylation in ovarian cancer: is there an epigenetic predisposition to drug response?

Epigenetic regulation of gene expression has been observed in a variety of tumor types. We have used microarray technology to evaluate the predisposition of drug response by aberrant methylation in ovarian cancer. Results indicate that loss of gene activity due to hypermethylation potentially confers a predisposition in certain cancer types and is an early event in disease progression. Methylation profiles of ovarian cancer might be useful for early cancer detection and prediction of chemotherapy outcome in a clinical context.

DNA Methylation in Ovarian Cancer: II. Expression of DNA Methyltransferases in Ovarian Cancer Cell Lines and Normal Ovarian Epithelial Cells

Objective.The aim of this study was to investigate whether expression of the enzymes that catalyze cytosine CpG island methylation, DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b is altered in human ovarian cancer. Aberrations in DNA methylation are common in cancer and have important roles in tumor initiation and progression. Tumors that display frequent and concurrent inactivation of multiple genes by methylation are designated as having a CpG Island methylator phenotype, or CIMP. To date, colon, gastric, and most recently ovarian cancers meet the CIMP criteria for cancer. We hypothesized that altered expression of DNA methyltransferases can result in hypermethylation events seen in CIMP cancers.Methods. DNMT1, DNMT3a, and DNMT3b mRNA levels in eight ovarian cancer cells lines (Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5) were compared to DNMT expression in normal ovarian surface epithelial cells using semi-quantitative reverse transcription-polymerase chain reaction.Results. In HeyA8 and HeyC2 ovarian cancer cells, DNMT1 expression levels were up to threefold higher (P < 0.05) than in normal ovarian surface epithelial cells. SK-OV-3 and PA-1 displayed increased DNMT3b expression (P < 0.05) compared to normal ovarian surface epithelial cells. Transcript levels for DNMT3a, however, were similar in cancer and normal ovarian cells.Conclusions. We observed differential expression of the DNMT genes in some ovarian cancer cell lines and conclude that alterations in DNMT expression might contribute to the CIMP phenotype in ovarian cancer. However, based on the lack of aberrant DNMT expression in some of the cancer cell lines examined, we further suggest that another mechanism(s), in addition to DNMT overexpression, accounts for methylation anomalies commonly observed in ovarian cancer.