Article

Discovery of Novel Hypermethylated Genes in Prostate Cancer Using Genomic CpG Island Microarrays

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
PLoS ONE (Impact Factor: 3.53). 02/2009; 4(3):e4830. DOI: 10.1371/journal.pone.0004830
Source: PubMed

ABSTRACT Promoter and 5' end methylation regulation of tumour suppressor genes is a common feature of many cancers. Such occurrences often lead to the silencing of these key genes and thus they may contribute to the development of cancer, including prostate cancer.
In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. Using computational and gene-specific validation approaches we have identified a large number of potential epigenetic biomarkers of prostate cancer. Further validation of candidate genes on a separate cohort of low and high grade prostate cancers by quantitative MethyLight analysis has allowed us to confirm DNA hypermethylation of HOXD3 and BMP7, two genes that may play a role in the development of high grade tumours. We also show that promoter hypermethylation is responsible for downregulated expression of these genes in the DU-145 PCa cell line.
This study identifies novel epigenetic biomarkers of prostate cancer and prostate cancer progression, and provides a global assessment of DNA methylation in prostate cancer.

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Available from: Vaijayanti V. Pethe, Jul 30, 2015
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    • "Perturbed DNA methylation patterns have been shown to arise during PCa tumorigenesis and have been implicated in PCa etiology and disease progression [6]. DNA methylation profiling studies using microarrays, MethylPlex–next-generation sequencing, and MeDIP-Seq have indeed identified a large number of DNA methylation changes in PCa [7] [8] [9] [10] [11]. However, few studies have investigated DNA methylation changes in CRPC, in part due to limited availability of tissues. "
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    ABSTRACT: Although prostate cancer (PCa) is the second leading cause of cancer death among men worldwide, not all men diagnosed with PCa will die from the disease. A critical challenge, therefore, is to distinguish indolent PCa from more advanced forms to guide appropriate treatment decisions. We used Enhanced Reduced Representation Bisulfite Sequencing, a genome-wide high-coverage single-base resolution DNA methylation method to profile seven localized PCa samples, seven matched benign prostate tissues, and six aggressive castration-resistant prostate cancer (CRPC) samples. We integrated these data with RNA-seq and whole-genome DNA-seq data to comprehensively characterize the PCa methylome, detect changes associated with disease progression, and identify novel candidate prognostic biomarkers. Our analyses revealed the correlation of cytosine guanine dinucleotide island (CGI)-specific hypermethylation with disease severity and association of certain breakpoints (deletion, tandem duplications, and interchromosomal translocations) with DNA methylation. Furthermore, integrative analysis of methylation and single-nucleotide polymorphisms (SNPs) uncovered widespread allele-specific methylation (ASM) for the first time in PCa. We found that most DNA methylation changes occurred in the context of ASM, suggesting that variations in tumor epigenetic landscape of individuals are partly mediated by genetic differences, which may affect PCa disease progression. We further selected a panel of 13 CGIs demonstrating increased DNA methylation with disease progression and validated this panel in an independent cohort of 20 benign prostate tissues, 16 PCa, and 8 aggressive CRPCs. These results warrant clinical evaluation in larger cohorts to help distinguish indolent PCa from advanced disease.
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    • "This left 2481 cancer-specific methylation events that may warrant further characterization (Fig. 3). Nearly all of the 56 previously reported prostate cancer methylated regions from pubmeth.org and a recent study (Kron et al. 2009) showed increased methylation in cancer tissues (Supplemental Table 10). "
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    • "Until recently, technical limitations have restricted these findings to either characterization of a handful of candidate loci or of overall abundance of 5-methylcytosine in the genome. Although a previously published study reported the DNA methylation profiles of prostate tumors at CpG islands across the genome (Kron et al. 2009), no study has examined the methylation profiles of normal prostate tissue necessary to determine the methylation changes that occur during or as a result of tumorigenesis. Here, we present quantitative DNA methylation levels at more than 26,000 loci across 14,000 gene promoters. "
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    ABSTRACT: Candidate gene-based studies have identified a handful of aberrant CpG DNA methylation events in prostate cancer. However, DNA methylation profiles have not been compared on a large scale between prostate tumor and normal prostate, and the mechanisms behind these alterations are unknown. In this study, we quantitatively profiled 95 primary prostate tumors and 86 benign adjacent prostate tissue samples for their DNA methylation levels at 26,333 CpGs representing 14,104 gene promoters by using the Illumina HumanMethylation27 platform. A 2-class Significance Analysis of this data set revealed 5912 CpG sites with increased DNA methylation and 2151 CpG sites with decreased DNA methylation in tumors (FDR < 0.8%). Prediction Analysis of this data set identified 87 CpGs that are the most predictive diagnostic methylation biomarkers of prostate cancer. By integrating available clinical follow-up data, we also identified 69 prognostic DNA methylation alterations that correlate with biochemical recurrence of the tumor. To identify the mechanisms responsible for these genome-wide DNA methylation alterations, we measured the gene expression levels of several DNA methyltransferases (DNMTs) and their interacting proteins by TaqMan qPCR and observed increased expression of DNMT3A2, DNMT3B, and EZH2 in tumors. Subsequent transient transfection assays in cultured primary prostate cells revealed that DNMT3B1 and DNMT3B2 overexpression resulted in increased methylation of a substantial subset of CpG sites that showed tumor-specific increased methylation.
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