Cancer Letters

Cancer Letters

Volume 433, 1 October 2018, Pages 242-251
Cancer Letters

Original Articles
Integrated transcriptomic and epigenomic analysis of ovarian cancer reveals epigenetically silenced GULP1

https://doi.org/10.1016/j.canlet.2018.06.030Get rights and content

Highlights

  • GULP1 is specifically methylated in ovarian cancer.

  • GULP1 is associated with clinicopathological parameters in ovarian cancer patients.

  • GULP1 is a potential tumor suppressor gene.

Abstract

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFβ2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p < 0.0003), worse overall (p = 0.02) and disease specific survival (p = 0.01). Depletion of GULP1 in OC cells led to increased pro-survival signaling, inducing survival and colony formation, whereas reconstitution of GULP1 negated these effects, suggesting that GULP1 is required for maintaining cellular growth control.

Introduction

Ovarian cancer (OC) was estimated to represent 22,240 new cases in 2018 in the United States, leading to 14,070 deaths [1]. Advances in molecular biology have revealed that genetics alone cannot explain the pervasive changes in gene expression profile of sporadic cancers. Other molecular changes, such as epigenetic abnormalities, are also recognized to have an impact on gene expression and tumorigenesis. The potential reversibility of epigenetic mechanisms make them attractive targets for the prevention, diagnosis and treatment of cancer. Numerous approaches have been undertaken to assess the OC methylome [2,3] and to provide new targets for further studies. Moreover, although aberrant promoter methylation patterns of specific genes in ovarian tumor cells were previously demonstrated in several studies [[4], [5], [6], [7]], to date, no reliable markers with clinical utility have been identified. Therefore, a better understanding of the epigenetic mechanisms responsible for OC initiation and progression is imperative for an improved management of this disease.

DNA methylation, the most studied epigenetic change in cancer, refers to the addition of a methyl group to the cytosine ring to form a methyl cytosine, but only on cytosines that precede a guanosine in the DNA sequence (CpG dinucleotide). CpG islands frequently span the promoter/regulatory region of genes with tumor suppressor activity and are usually unmethylated in normal cells [8]. The human genome in normal cells is not methylated uniformly, containing unmethylated segments interspersed with methylated regions, whereas in cancer cells, methylation patterns are altered, undergoing global DNA hypomethylation, as well as hypermethylation of CpG islands in the selected promoters/regulatory regions [9]. Aberrant promoter methylation induces gene silencing, a common phenomenon in human cancer cells and likely one of the earliest events in carcinogenesis [[9], [10], [11], [12], [13]]. The discovery of OC-specific methylated genes that are correlated with down-regulation of expression could lead to the identification of potential biomarker candidates for risk assessment, prognosis and early detection of OC. Moreover, deciphering molecular pathways that may be deregulated due to the inactivation of a given gene by methylation may reveal novel targets for potential therapeutic interventions.

To comprehensively understand the methylation alterations in ovarian cancer, we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using cell lines. This comprehensive approach coupled with an innovative computational analysis led to the discovery of novel OC-specific epigenetically silenced genes. Here, we focused on the engulfment gene GULP1 and report its aberrant silencing by methylation in multiple cohorts of primary OC samples, and its tumor suppressor activity. Our current data hypothesize that silencing of GULP1 by methylation plays an important role in ovarian carcinogenesis, playing a role in pro-survival and anti-apoptotic pathways.

Section snippets

Cell lines

Normal ovarian cell lines OSE2A, OSE2B and OSE7 (kindly provided by Dr. Ie-Ming Shih - Johns Hopkins University), were maintained in the DMEM F12 medium containing 10% Fetal Bovine Serum (FBS), penicillin (100 units/mL) and streptomycin (100 μg/mL). OC cell lines IGROV, A2780 and 2008 (kindly provided by Dr. Stephen B Howell - University of California, San Diego, CA) were maintained in RPMI1640 medium containing either 10% FBS (IGROV, A2780) or 5% FBS (2008), penicillin (100 units/mL) and

Genome-wide methylation profiles of OC by the integration of pharmacologic unmasking data in cell lines and expression array data of primary OC and normal ovarian epithelium samples

We conducted an Affymetrix microarray analysis on 15 late stage primary OC samples, 10 normal ovarian surface epithelium brushing of tumor-free female patients, 3 immortalized normal ovarian cell lines (OSE2A, OSE2B and OSE7) and 3 OC cell-lines (IGROV, A2780 and 2008). All cell lines were treated with 5-aza-dC.

A linear regression model (false discovery rate = 0.05) using all the data set identified 5718 probes differentially downregulated in cancer samples compared to healthy controls and 4870

Discussion

Numerous strategies using different platforms have been undertaken to identify cancer-specific methylated genes. We employed a genome-wide microarray platform using a carefully selected cohort of normal and OC tissues/cell lines, coupled with functional pharmacologic unmasking of epigenetically silenced genes by treating cancer cell lines with a demethylating agent. We identified 43 novel potentially OC specific methylated genes by computational approach and additional filtering criteria, and 5

Acknowledgments

We thank Dr. Richard Roden/Dr. T.C. Wu –Johns Hopkins University for kindly providing us with 15 ovarian cancer frozen samples. We thank Dr. Birrer (and his team - NCI) for the microarray data on 10 normal ovarian brushing samples from patients without ovarian cancer (normal Epithelium Brushings). We thank Jacqueline C. Junn, Juna Lee, Luciane T. Kagohara and Christina Michailidi for technical assistance.

Funding

This work was supported by a career development award to M. O. Hoque from Specialized Program of Research Excellence P50 CA098252 (T-C Wu) National Institutes of Health/National Cancer Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of interest

None.

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    All named authors have agreed to the submission and have participated in the study to a sufficient extent to be named as authors. The authors declare no potential conflicts of interest.

    1

    These authors contributed equally to this study (LM and MB).

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