Global DNA methylation profiling technologies and the ovarian cancer methylome.

Cytosine methylation in DNA constitutes an important epigenetic layer of transcriptional and regulatory control in many eukaryotes. Profiling DNA methylation across the genome is critical to understanding the influence of epigenetics in normal biology and disease, such as cancer. Genome-wide analyses such as arrays and next-generation sequencing (NGS) technologies have been used to assess large fractions of the methylome at a single-base-pair resolution. However, the range of DNA methylation profiling techniques can make selecting the appropriate protocol a challenge. This chapter discusses the advantages and disadvantages of various methylome detection approaches to assess which is appropriate for the question at hand. Here, we focus on four prominent genome-wide approaches: whole-genome bisulfite sequencing (WGBS); methyl-binding domain capture sequencing (MBDCap-Seq); reduced-representation-bisulfite-sequencing (RRBS); and Infinium Methylation450 BeadChips (450 K, Illumina). We discuss some of the requirements, merits, and challenges that should be considered when choosing a methylome technology to ensure that it will be informative. In addition, we show how genome-wide methylation detection arrays and high-throughput sequencing have provided immense insight into ovarian cancer-specific methylation signatures that may serve as diagnostic biomarkers or predict patient response to epigenetic therapy.

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.