High-throughput DNA hypermethylation profiling in different ovarian epithelial cancer subtypes using universal bead array

Abstract

Abstract. DNA hypermethylation is common and plays a critical role in the regulation of gene expression. It is considered a major cause of carcinogenesis. High-throughput profiling method has been developed to analyze the methylation status of hundreds of pre-selected genes simultaneously. The aim of this study was to analyze promoter hypermethylation profiles of each subtype of ovarian epithelial cancer (OEC), to improve the understanding of the role of epigenetic silencing in carcinogenesis. DNA hypermethylation profiles on fresh frozen tissue samples of 5 serous, 3 mucinous, 5 endometrioid and 4 clear cell types of OEC, as well as 5 normal ovarian tissue samples as control. We used a highthroughput method for analyzing the hypermethylation status of 1,505 CpG loci selected from 871 genes simultaneously by GoldenGate Methylation Cancer Panel I (Illumina Human-6 v2 Expression BeadChip). Methylation status of seven genes was verified by methylation specific PCR (MSP). We identified 20, 37, 15 and 56 hypermethylated CpG locations in serous, mucinous, endometrioid and clear cell type OEC compared to control. Only 6 CpG loci were commonly hypermethylated across all subtypes of OEC. Hypermethylated loci of serous 17 (81.0%) and endometrioid type 10 (71.4%) were identical to that of clear cell type. However, mucinous type showed 17 peculiar loci (43.6%) out of 39 hypermethylated loci. The unique DNA hypermethylation patterns identified in different OEC subtypes suggest that their cause may involve different epigenetic mechanisms and the Bead chip used in this study is a useful tool to analyze DNA hypermethylation. IntroductionOvarian cancer is the fourth leading cause of cancer death in women (1). Patients (70%) have advanced disease (stage III or IV) upon presentation, with a 5-year survival between 15 and 20% at best with aggressive treatment. Ovarian epithelial cancer (OEC) accounts for over 90% of all cases and includes the following major histological subtypes: serous, mucinous, endometrioid and clear cell carcinomas. Cytogenetic and molecular analyses indicated that multiple genetic alterations were involved in the pathogenesis of ovarian cancer. BRCA1 and BRCA2 mutations are associated with increased ovarian cancer risks (2). Some studies have showed that p16 loss was associated with ovarian cancer prognosis (3). However, it remains unclear how genetic alterations lead to development and subsequent progression of ovarian cancer. Better understanding of the molecular mechanisms responsible for ovarian cancer development and progression will improve diagnosis and treatment of this disease.DNA methylation is an epigenetic alteration that plays an important role in carcinogenesis (4). The importance of aberrant CpG island methylation as an alternative mechanism to inactivate tumor suppressor genes has been recognized recently. Addition of a methyl group to the cytosine residues of CpG dinucleotide clusters in the 5' regulatory regions of genes occurs frequently in cancer cells, but seldom in nonmalignant cells. Ab...