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Genome-Wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with Mir-26A Hypermethylation

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Genome-Wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with Mir-26A Hypermethylation Published OnlineFirst August 28, 2012; DOI: 10.1158/2159-8290.CD-12-0041 RESEARCH ARTICLE Genome-wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with miR-26a Hypermethylation Stefan T. Börno 1 , 3 , Axel Fischer 1 , Martin Kerick 1 , Maria Fälth 4 , 6 , Mark Laible 4 , Jan C. Brase 4 , 7 , Ruprecht Kuner 4 , Andreas Dahl 8 , Christina Grimm 1 , Behnam Sayanjali 1 , Melanie Isau 1 , 3 , Christina Röhr 1 , 3 , Andrea Wunderlich 1 , 3 , Bernd Timmermann 2 , Rainer Claus 5 , Christoph Plass5 , Markus Graefen 9 , Ronald Simon 10 , Francesca Demichelis 11 , 13 , Mark A. Rubin 12 , Guido Sauter 10 , Thorsten Schlomm 9 , Holger Sültmann 4 , Hans Lehrach 1 , and Michal R. Schweiger 1 Downloaded from cancerdiscovery.aacrjournals.org on October 2, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 28, 2012; DOI: 10.1158/2159-8290.CD-12-0041 ABSTRACT Prostate cancer is the second most common cancer among men worldwide. Altera- tions in the DNA methylation pattern can be one of the leading causes for prostate cancer formation. This study is the fi rst high-throughput sequencing study investigating genome-wide DNA methylation patterns in a large cohort of 51 tumor and 53 benign prostate samples using meth- ylated DNA immunoprecipitation sequencing. Comparative analyses identifi ed more than 147,000 cancer-associated epigenetic alterations. In addition, global methylation patterns show signifi cant differences based on the TMPRSS2–ERG rearrangement status. We propose the hypermethylation of miR-26a as an alternative pathway of ERG rearrangement-independent EZH2 activation. The observed increase in differential methylation events in fusion–negative tumors can explain the tumorigenic proc- ess in the absence of genomic rearrangements. SIGNIFICANCE: In contrast to TMPRSS2–ERG -rearranged tumors, the pathomechanism for gene fusion– negative tumors is completely unclear. Using a sequencing-based approach, our work uncovers signifi cant global epigenetic alterations in TMPRSS2–ERG gene fusion–negative tumors and provides a mechanistic explanation for the tumor formation process. Cancer Discov; 2(11); 1–12. ©2012 AACR. INTRODUCTION transmembrane protease serine 2 ( TMPRSS2 ) gene ( 4 ), most com- monly involving the v-ets erythroblastosis virus E26 oncogene homolog More than 900,000 men are diagnosed with prostate cancer (ERG) that is observed in approximately 50% of all prostate each year, making it the second most common cancer among cancer cases ( 5 ). The overexpression of ERG is thought to be men worldwide ( 1 ). The clinical course of prostate cancer is het- suffi cient for the initiation of prostate intraepithelial neoplasia erogeneous, ranging from indolent tumors requiring no therapy lesions, a precursor of prostate cancer ( 6 ). Other rearrangements during the patient’s lifetime to highly aggressive prostate cancer are less frequent and, interestingly, tend to be present in prostate developing into a metastatic disease. Despite its high prevalence, cancers harboring the TMPRSS2–ERG gene fusion (FUS +; refs. the clinical management of prostate cancer is limited by the low 7, 8 ). Recent work has uncovered frequent somatic deletions at specifi city of the existing diagnostic and prognostic tools and 5q21 and 6q21 including CHD1 and FOXO3 as well as recurrent the lack of effective systemic therapeutic strategies. SPOP mutations within ETS gene fusion–negative prostate can- Recent years have brought about a marked extension of cers, suggesting distinct subclasses within gene fusion–negative our understanding of the somatic basis of prostate cancer. tumors ( 9, 10 ). With 0.33 somatic protein altering mutations per megabase During prostate cancer progression, one of the most sig- (Mb), the mutation frequency in prostate cancer is signifi cantly nifi cantly upregulated genes is enhancer of zeste homolog 2 lower than in breast (∼1/Mb) or lung cancer (3.8/Mb) and lies ( EZH2) , which is also associated with invasiveness and high within the lowest range of cancer-associated mutations ( 2, 3 ). A malignancy of several other tumor entities ( 11, 12 ). The large proportion of prostate cancers harbor gene fusions involv- polycomb group protein EZH2 is responsible for silencing of ing members of the ETS family and the androgen-regulated homeobox (HOX) genes through H3K27 methylation during tissue development ( 13–15 ), and it links histone modifi ca- tions to DNA methylation as EZH2 target genes may consec- Authors’ Affi liations: 1 Department of Vertebrate Genomics and 2 Next Generation Sequencing Group, Max Planck Institute for Molecular Genet- utively become hypermethylated ( 16, 17 ). In prostate cancer, ics; 3 Department of Biology, Chemistry, and Pharmacy, Free University, aberrant DNA methylation is found to be associated with Berlin; 4 Cancer Genome Research and 5 Division of Epigenomics and Can- altered EZH2 expression, correlates with tumor progression, cer Risk Factors, German Cancer Research Center (DKFZ) and National and is proposed to be one of the earliest events in oncogenesis Center for Tumor Diseases, Heidelberg; 6 Cellzome AG, Heidelberg; 7 Sividon Diagnostics GmbH, Cologne; 8 Biotechnology Center, Technical University ( 12, 13 , 15 , 18–20 ). Hypermethylation of the glutathione Dresden, Dresden; 9 Martini Clinic, Prostate Cancer Center and 10 Institute S-transferase pi 1 ( GSTP1 ) is considered to be a cardinal gate- of Pathology, University Medical Centre Hamburg-Eppendorf, Hamburg, keeper event in early prostate cancer development ( 21 ). Germany; 11 Institute for Computational Biomedicine and 12 Department Most epigenetic studies conducted so far are focused on of Pathology and Laboratory Medicine, Weill Cornell Medical College, specifi c gene regions and interrogate cancer-associated differen- New York, New York; and 13 Centre for Integrative Biology, University of Trento, Trento, Italy tial methylation events in general without asking if epigenetic Note: Supplementary data for this article are available at Cancer Discovery alterations might differ in specifi c subgroups of prostate can- Online (http://cancerdiscovery.aacrjournals.org/). cer. This might be particularly important for TMPRSS2–ERG - Corresponding Author: Michal-Ruth Schweiger, Max Planck Institute for negative tumors, in which the pathomechanism of oncogenesis Molecular Genetics, 14195 Berlin, Germany. Phone: 49-30-84131339; is so far unclear. Recent identifi cations of different patterns Fax: 49-30-84131380; E-mail: [email protected] of DNA methylation in promoters of benign, cancerous, and doi: 10.1158/2159-8290.CD-12-0041 metastatic prostate tissues as well as decreased levels of LINE-1 © 2012 American Association for Cancer Research. methylation in fusion-negative prostate cancer revealed a more NOVEMBER 2012CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on October 2, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 28, 2012; DOI: 10.1158/2159-8290.CD-12-0041 RESEARCH ARTICLE Börno et al. detailed picture of epigenetic alterations in prostate cancer samples and might be used as prostate cancer biomarkers ( 22 ). However, the epigenetic landscapes of prostate cancer (Supplementary Fig. S2C and S2D). subgroups are still not suffi ciently investigated to draw any con- Hypermethylated regions were more homogeneous among clusions. To understand alterations in the ERG fusion–negative the tumor samples (lower P values) than hypomethylated class of prostate cancer, we used a deep sequencing readout of regions. This might indicate a specifi c and site-directed meth- methylated DNA immunoprecipitation sequencing (MeDIP- ylation process as compared with a more unspecifi c global Seq) to screen 51 tumor and 53 benign prostate tissues ( 23–26 ). loss of methylation. We integrated the results with gene and microRNA (miRNA) Although most hypomethylated regions were detected in expression analyses and proposed a model for the development intergenic regions, only a quarter of the hypermethylated bins of aberrant DNA methylation patterns in ERG fusion–negative were located outside of genes and promoters (Supplementary (FUS −) prostate cancers. Fig. S2E). We found a peak of hypermethylation within ±2 kb windows around the transcription start sites (TSS). In addi- RESULTS tion, we detected that hypermethylation is enriched within conserved [OR = 1.47, confi dence interval (95% CI ) = 1.43–1.50, Catalogs of Tumor-Specifi c P = 1.08 × 10 −209 ] and miRNA (OR = 2.05, CI = 1.83–2.29, P = Epigenetic Alterations 8.79 × 10 −32 ) regions, whereas hypomethylation of these sites = = = For the analysis of genome-wide methylation patterns in is lower than expected (ORcons 0.42, CI 0.4–0.44, P 0 and = = = × −6 51 prostate cancers and 53 normal prostate tissues, we used ORmiRNA 0.63, CI 0.51–0.77, P 4.37 10 ; Supplementary MeDIP followed by high-throughput sequencing by oligo- Fig. S2F). Pathway analyses revealed the developmental gene nucleotide ligation and detection (SOLiD) ( 26 ). All tumors group to be the most signifi cantly differentially methylated. Of selected for this study were staged pT2–pT4 and had Gleason 231 homeobox genes ( 28 ), we identifi ed 175 with a signifi cant scores ranging from 6 to 9. The TMPRSS2–ERG fusion TSS-associated hypermethylation. The differential methylation was present in 17 tumors; 20 tumors were TMPRSS2–ERG - of homeobox genes also resulted in a more stringent impact negative as determined by PCR (Supplementary
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