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.

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The Polycomb Group (PcG) Repressive Complexes 1 and 2 (PRC1 and PRC2) maintain gene expression patterns by epigenetically modifying chromatin. PRC2 suppresses transcription by catalyzing the trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of repressed chromatin. Although overexpression of PRC2 components has been implicated in cancer initiation, progression and metastasis how PRC2 is recruited to its target genes remains poorly understood. Recent work suggests the interaction of PRC2 with the long-non coding (lnc) RNA HOX transcript antisense intergenic RNA (HOTAIR) could serve as a potential guide to silence target genes, but the mechanism has yet to be defined. In this study, we performed whole transcriptome RNA-seq analysis of isogenic cisplatin- sensitive and -resistant A2780 ovarian cancer (OC) cell lines. Of the lncRNA examined, HOTAIR was markedly induced in the cisplatin resistant cells (5-fold upregulation compared to the cisplatin-sensitve A2780 parental line). shRNA depletion of HOTAIR resensitized the drug-resistant cells to cisplatin. Based on analysis of the HOTAIR promoter using luciferase assays, we identified two putative NF-κB binding sites and three E-box binding sites. To further investigate whether these sites contributed to HOTAIR expression, platinum-sensitive cells were treated with different growth factors and cytokines (0 to 48hrs) and analyzed for changes in HOTAIR expression. Treatment with TNF-α, a well-known inflammatory cytokine and established activator of NF-κB, resulted in 16 fold-induction of HOTAIR. In accord with these results, we observed significant enrichment of both NF-κB and cMYC on the HOTAIR promoter using ChIP assay. Pharmacological inhibition of cMYC and NF-κB reduced HOTAIR expression by 2-fold, and siRNA depletion of the NF-κB p65 subunit or cMYC similarly reduced (P<0.01) HOTAIR expression. Based on these results, we hypothesize a positive feedback loop for HOTAIR transcriptional regulation: HOTAIR inhibits Iκ-Bα (an inhibitor of NF-κB) allowing the lncRNA to enter the nucleus and activate not only its own expression but also cMYC expression to further drive HOTAIR expression as well as cMYC-inducible genes. We are currently testing this hypothesis. We believe this novel pathway, whose components have been implicated in numerous other cancers, may be involved in cisplatin-resistant OC. Citation Format: Ali R. Ozes, Dave Miller, Cong Guo, Anurag Bhattrai, Yunlong Liu, Kenneth P. Nephew. The transcriptional regulation of the long non-coding RNA HOTAIR in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5189. doi:10.1158/1538-7445.AM2013-5189

The Polycomb Repressive Complex 2 (PRC2) has been implicated in cancer and a role for PRC2 during DNA damage response (DDR) has recently been reported. To examine changes in gene expression with DDR in cisplatin (cddp)-resistant ovarian cancer (OC), we performed whole transcriptome RNA-seq analysis of isogenic cddp-sensitive and -resistant OC cell lines (A2780 and A2780-cp). Differential expression of PRC components (SUZ12, EZH1, SIRT1, PHC1 &2) (P<0.05), NF-κB pathway members (NCAM, ABCB9, RAGE, IL4R, IL6R, BCL2L11) (P<0.05), and long non-coding RNAs (lncRNAs) known to associate with PRC2 (P<0.05), including Hox transcript antisense intergenic RNA or HOTAIR, was observed. In tumors from patients with high grade serous OC at diagnosis, basal expression of HOTAIR was greater (P<0.01) compared to normal ovarian surface epithelium and marked overexpression of HOTAIR was observed in tumors obtained from patients who had developed platinum-resistant OC. Ablation of HOTAIR using dsiRNA resensitized A2780-cp to cddp, while ectopic over-expression of HOTAIR increased (P<0.05) A2780 cell survival after cddp treatment (3-fold vs. control). To further examine HOTAIR regulation, we conducted a promoter analysis using bioinformatics tools and luciferase assays. We identified a putative p65-NF-κB binding site (906-GGGACACCCC-915) 906 bp upstream of the HOTAIR transcription start site. Treatment of A2780 cells with the NF-κB activator TNF-α induced HOTAIR (16-fold vs. control) and NF-κB enrichment (3-fold assessed by ChIP assays) at the HOTAIR promoter. Furthermore, in A2780-cp compared to A2780, total Iκ-Bα levels were reduced (P<0.05) and nuclear p65 levels were increased, indicating that endogenous activation of NF-κB contributes to cddp resistance and DDR. Consistent with this observation, cddp treatment (20μM for 0-24hrs) of A2780 cells increased (P<0.05) HOTAIR expression by 5- and 16-fold at 8 and 24 hrs and decreased (P<0.05) Iκ-Bα protein levels at similar time points. Furthermore, inhibiting NF-κB by either gliotoxin (5μM) or Bay-11 (3μM) completely abolished cddp-induced HOTAIR expression in A2780 cells, demonstrating that NF-κB is a HOTAIR transcriptional activator during cddp-induced DDR. Importantly, EZH2 and histone H3 lysine-27 trimethylation (H3K27me3) levels were enriched (6- and 17-fold) in the Iκ-Bα promoter at 24 and 48 hours post cddp treatment, and HOTAIR depletion using dsiRNA reduced the observed EZH2-H3K27me3 enrichment at the Iκ-Bα promoter, demonstrating that HOTAIR recruits PRC2 complex to the Iκ-Bα promoter to prolong NF-κB activation during cddp-induced genotoxic stress. Mouse xenograft studies with A2780 cells overexpressing HOTAIR are ongoing. The results of this study support a role for HOTAIR as a positive regulator of the NF-κB pathway and PRC2 during cisplatin-induced DNA damage. We further suggest that HOTAIR may serve as a therapeutic target in cisplatin-resistant OC. Citation Format: Ali R. Ozes, Dave F. Miller, Yunlong Liu, Kenneth P. Nephew. Non-coding RNA HOTAIR connects DNA damage signaling to NF-κB activation in cisplatin resistant 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 3551. doi:10.1158/1538-7445.AM2014-3551

Purpose: Epigenetic changes, particularly in DNA methylation, have been implicated in acquired resistance to platinum in ovarian cancer (OC). The goal of the current study was to analyze and integrate global RNA expression and DNA methylation profiles of platinum resistant tumors compared to untreated, platinum-sensitive ovarian tumors, as well as to measure genomic and epigenomic changes induced by guadecitabine (SGI-110) in tumors. Methods: An ongoing phase I/II multi-institutional clinical trial uses the novel DNA methyltransferase (DNMT) inhibitor guadecitabine to re-sensitize recurrent platinum resistant OC to carboplatin. Patients enrolled in this trial had recurrent platinum resistant OC. Tumor biopsies were collected at baseline and after two cycles of guadecitabine administered daily for 5 days at a low (30mg/m2) dose (28 days per cycle). RNA and DNA were extracted from 48 and 57 baseline tumors and analyzed for next generation sequencing approaches to interrogate transcriptomes (RNA-seq) and methylomes (Infinium Human Methylation450 (HM450) arrays), respectively. Differential gene expression and DNA methylation profiles were generated and used for Ingenuity Pathway Analysis (IPA) to identify the top altered pathways in response to guadecitabine. Expression of DNMTs was examined by real-time RT-PCR and immunohistochemistry. LINE1 methylation and promoter methylation of selected genes (MAGE-A2, MAGE-A3, MAGE-A11, NY-ESO, RASSF1, MLH1, and HOXA11) were quantified by pyrosequencing before and after guadecitabine treatment (n=12 paired samples). Results: Analysis of a limited number of paired samples before and after treatment (n=8) revealed significant changes in global gene expression profiles induced by guadecitabine, with 960 altered genes representing immunopathway enrichment including cytokine production in macrophages and T helper cells by IL-17A and IL-17F, granulocyte /agranulocyte adhesion and inflammation, IL-8 signaling, p38 MAPK signaling, cAMP-mediated signaling, and innate immunity. Epigenetic profiling using HM450 revealed extensive methylation changes when comparing recurrent platinum resistant ovarian tumors (n=42) to primary, untreated ovarian cancer specimens analyzed as part of the TCGA project (n=10). Six hundred and four promoters were significantly differentially methylated (adjusted p<0.05, absolute methylation changes β>0.2), among which, 498 and 106 were hypermethylated or hypomethylated respectively in recurrent platinum resistant ovarian tumors. IPA analysis of baseline tumor transcriptome and methylome demonstrated significant enrichment in a wide range of pathways associated with cancer, stem cells, inflammation and the immune system. DNMT1, 3A, and 3B mRNA levels in the tumors were highly variable (n=19). Analysis of a limited number of paired samples (n=7) revealed no significant changes in global methylation or in DNMT expression levels induced by treatment with guadecitabine (adjusted p>0.05). However, the DNMT inhibitor induced significant methylome alterations in selected patients. Significant hypomethylation of MAGE-A3 and MAGE–A11 promoters (p<0.05) was detected. Correlations between methylation changes and clinical outcomes are being explored. Conclusions: These data suggest that treatment with the DNMT inhibitor guadecitabine induces a reactivation of immune responses in human OC. Correlations between methylation changes and expression profiles are being explored. Citation Format: Fang Fang, Horacio Cardenas, Dave Miller, Aaron Buechlein, Qing Yu, Yunlong Liu, Guanglong Jiang, Pietro Taverna, Harold Keer, Doug Rusch, Daniela Matei, Kenneth P. Nephew. Epigenome and genome alterations in platinum resistant ovarian tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A70.