Abstract B12: Genomic and epigenomic characterization of global DNA hypomethylation in human epithelial ovarian cancer
Authors: Zhang Wa , Klinkebiel David , Pandey Sanjit , Wang Dan , Liu Song , Guda Chittibabu , Odunsi Kunle , Karpf Adam R. 
Published: August 2014

Background: Epithelial ovarian cancer (EOC), and other solid tumors, contain a variable proportion of tumors characterized by global DNA hypomethylation. This phenotype is known to involve loss of DNA methylation at repetitive elements (RE) and at single copy gene promoters, including cancer-germline (a.k.a. cancer-testis) antigen genes. Global DNA hypomethylation in EOC coincides with tumor progression and reduced survival in EOC patients. Despite the clinical importance of global DNA hypomethylation, we currently have a limited understanding of the origin, the targets, and the consequences of this EOC phenotype.

Objectives: The purpose of this study was to: i) examine global gene expression in globally hypomethylated EOC, ii) to identify the molecular pathways deregulated in these tumors, iii) to discover and validate novel and clinically relevant targets of DNA hypomethylation in EOC, iv) to conduct DNA methylome analysis to more precisely define the genomic locations effected by global DNA hypomethylation in EOC, and v) to determine whether global DNA hypomethylation impact RE gene expression in EOC.

Methods: Global methylation status in EOC was determined using quantitative bisulfite pyrosequencing of the LINE1 RE. Affymetrix microarrays, RT-qPCR, and total RNAseq were used to measure gene expression in normal ovary (NO), LINE1 hypomethylated EOC, and LINE1 hypermethylated (i.e. normally methylated) EOC. Pathway analysis was conducted on differentially expressed genes (DEG) between NO and EOC and between the two EOC groups. Illumina Infinium 450K arrays and Agilent Sure Select methyl-seq were used to examine the DNA methylome of NO and EOC. Quantitative sodium bisulfite pyrosequencing was used to determine locus-specific DNA methylation in NO and EOC.

Results: Global mRNA expression was distinct in LINE1 hypomethylated EOC, with ~70% of DEG upregulated, implicating DNA hypomethylation in gene activation. The most significantly altered pathway in LINE1 hypomethylated EOC was cell cycle, implicating enhanced proliferation in this phenotype. Cancer germline genes not previously known to be regulated by DNA methylation in EOC, including CT45 and PRAME, were identified and characterized. DNA methylome analysis of LINE1 hypomethylated EOC indicated that hypomethylation is not evenly dispersed across the genome, but rather is regionally localized, including at nuclear lamina associated domains (LAD). Preliminary analysis of RNAseq data indicated that specific RE are overexpressed in LINE1 hypomethylated tumors.

Conclusions: Pathway analysis suggests that rapid cellular proliferation coupled with inherent inefficiencies of maintenance DNA methylation at specific genomic locations may contribute to global DNA hypomethylation in EOC. DNA hypomethylation is linked to gene activation in EOC, and important targets of this defect are cancer germline genes, which are likely to contribute to oncogenesis but may also be targeted by immunotherapy. Global DNA hypomethylation appears to be concentrated at specific genomic locations including LADs, and is connected not only to RE hypomethylation, but also to enhanced RE expression. Higher-order nuclear changes and enhanced RE expression are potential mechanisms driving genomic instability and/or poor prognosis in EOC patients displaying global DNA hypomethylation.

Citation Format: Wa Zhang, David Klinkebiel, Sanjit Pandey, Dan Wang, Song Liu, Chittibabu Guda, Kunle Odunsi, Adam R. Karpf. Genomic and epigenomic characterization of global DNA hypomethylation in human epithelial 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 B12.