Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer

Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Genome Research (Impact Factor: 14.63). 07/2011; 21(7):1028-41. DOI: 10.1101/gr.119347.110
Source: PubMed


Beginning with precursor lesions, aberrant DNA methylation marks the entire spectrum of prostate cancer progression. We mapped the global DNA methylation patterns in select prostate tissues and cell lines using MethylPlex-next-generation sequencing (M-NGS). Hidden Markov model-based next-generation sequence analysis identified ∼68,000 methylated regions per sample. While global CpG island (CGI) methylation was not differential between benign adjacent and cancer samples, overall promoter CGI methylation significantly increased from ~12.6% in benign samples to 19.3% and 21.8% in localized and metastatic cancer tissues, respectively (P-value < 2 × 10(-16)). We found distinct patterns of promoter methylation around transcription start sites, where methylation occurred not only on the CGIs, but also on flanking regions and CGI sparse promoters. Among the 6691 methylated promoters in prostate tissues, 2481 differentially methylated regions (DMRs) are cancer-specific, including numerous novel DMRs. A novel cancer-specific DMR in the WFDC2 promoter showed frequent methylation in cancer (17/22 tissues, 6/6 cell lines), but not in the benign tissues (0/10) and normal PrEC cells. Integration of LNCaP DNA methylation and H3K4me3 data suggested an epigenetic mechanism for alternate transcription start site utilization, and these modifications segregated into distinct regions when present on the same promoter. Finally, we observed differences in repeat element methylation, particularly LINE-1, between ERG gene fusion-positive and -negative cancers, and we confirmed this observation using pyrosequencing on a tissue panel. This comprehensive methylome map will further our understanding of epigenetic regulation in prostate cancer progression.


Available from: John R Prensner, Jul 20, 2014
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    • "Furthermore, the false-negative rate of the 6-and 12-core biopsy techniques performed ex vivo on prostates removed due to biopsy-proven cancer was 425% (Svetec et al, 1998; Fink et al, 2001; Serefoglu et al, 2013). Thus, underdiagnosis of PCa is not uncommon and, therefore, standard hematoxylin and eosin (H&E) staining of core biopsies could be assisted by monitoring differentially expressed genes (Hessels et al, 2004; Bhavsar et al, 2013) and patterns of DNA methylation (Kim et al, 2011a; Day and Bianco–Miotto, 2013) as an initial biopsy strategy. Here, we analysed critically the potential usefulness of selected biomarkers for supporting conventional histological tests for PCa diagnosis. "
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    ABSTRACT: Background: We analysed critically the potential usefulness of RNA- and DNA-based biomarkers in supporting conventional histological diagnostic tests for prostate carcinoma (PCa) detection. Methods: Microarray profiling of gene expression and DNA methylation was performed on 16 benign prostatic hyperplasia (BPH) and 32 cancerous and non-cancerous prostate samples extracted by radical prostatectomy. The predictive value of the selected biomarkers was validated by qPCR-based methods using tissue samples extracted from the 58 prostates and, separately, using 227 prostate core biopsies. Results: HOXC6, AMACR and PCA3 expression showed the best discrimination between PCa and BPH. All three genes were previously reported as the most promising mRNA-based markers for distinguishing cancerous lesions from benign prostate lesions; however, none were sufficiently sensitive and specific to meet the criteria for a PCa diagnostic biomarker. By contrast, DNA methylation levels of the APC, TACC2, RARB, DGKZ and HES5 promoter regions achieved high discriminating sensitivity and specificity, with area under the curve (AUCs) reaching 0.95-1.0. Only a small overlap was detected between the DNA methylation levels of PCa-positive and PCa-negative needle biopsies, with AUCs ranging between 0.854 and 0.899. Conclusions: DNA methylation-based biomarkers reflect the prostate malignancy and might be useful in supporting clinical decisions for suspected PCa following an initial negative prostate biopsy.
    British Journal of Cancer 06/2014; 111(4). DOI:10.1038/bjc.2014.337 · 4.84 Impact Factor
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    • "Next generation sequencing can yield millions of single molecule reads and has been used to determine DNA methylation (Taylor et al., 2007; Bibikova and Fan, 2010; Laird, 2010; Feng et al., 2011; Kim et al., 2011; Komori et al., 2011; Ku et al., 2011; Nejman et al., 2014). Supplemented with DNA barcoding technology, which incorporates a unique index sequence into each PCR segment, this approach can provide a rapid way to simultaneously determine DNA methylation at the single-molecule level in large numbers of samples. "
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    ABSTRACT: Invasive cervix cancer (ICC) is the third most common malignant tumor in women and human papillomavirus 16 (HPV16) causes more than 50% of ICC. DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development. Next generation (Next Gen) sequencing has been proposed as a novel approach to determine DNA methylation. However, utilization of this method to survey CpG methylation in the HPV16 genome is not well described. Moreover, it provides additional information on methylation "haplotypes." In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing. Most of the CpG sites were highly consistent between the two approaches (overall correlation, r = 0.92), thus verifying that Next Gen sequencing is an accurate and convenient method to survey HPV16 methylation and thus can be used in clinical samples for risk assessment. Moreover, the CpG methylation patterns (methylation haplotypes) in single molecules identified an excess of complete-and non-methylated molecules and a substantial amount of partial-methylated ones, thus indicating a complex dynamic for the mechanisms of HPV16 CpG methylation. In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.
    Frontiers in Genetics 06/2014; 5:150. DOI:10.3389/fgene.2014.00150
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    • "data from ENCODE consortium available as user tracks in UCSC genome browser were utilized in this study[17]. The Methylplex NGS sequencing data custom tracks of benign cell line PrEC and PCa cell line LNCaP, prostate normal, benign adjacent, localized PCa, and metastatic PCa tissues from a recently published study by Kim et al. (Genome Res 2011) were also uploaded as tracks into UCSC genome browser and visualized[9]. This deep-sequencing data from a previously published dataset is deposited in NCBI GEO under accession number:GSE27619.The DSC3 genomic region ''Chr18:28,570,052-28,622,781'' (Hg19) (including both the promoter and coding region) was inspected for differential DNA methylation. "
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    ABSTRACT: Desmocollin 3 (DSC3), a member of the cadherin gene superfamily, is associated with pathogenesis of some cancers, but its role in prostate cancer (PCa) remains largely unknown. DSC3 gene expression level in available PCa microarray dataset was examined using the Oncomine database. DSC3 transcript expression in prostate cell line panel and an independent tissue cohort (n = 52) was estimated by quantitative PCR (Q-PCR). Epigenetic status of DSC3 gene promoter in PCa was investigated by uploading three dataset (ENCODE Infinium 450K array data and two methylation sequencing) in UCSC genome browser. While pyrosequencing analysis measured promoter DNA methylation, Q-PCR estimates were obtained for DSC3 transcript re-expression after 5-Aza-deoxycytidine (5-Aza) treatment. Clinical relevance of DSC3 expression was studied by Kaplan-Meier survival analysis. Finally, functional studies monitoring cell proliferation, migration and invasion were performed in prostate cell lines after siRNA mediated DSC3 knockdown or following 5-Aza induced re-expression. EMT markers Vimentin and E-cadherin expression was measured by Western Blot. Microarray data analyses revealed a significant decrease in DSC3 transcript expression in PCa, compared to benign samples. Q-PCR analysis of an independent cohort revealed DSC3 transcript down-regulation, both in PCa cell lines and tumor tissues but not in their benign counterpart. Examination of available NGS and Infinium data identified a role for epigenetic regulation DSC3 mRNA reduction in PCa. Pyrosequencing confirmed the increased DSC3 promoter methylation in cancer cell lines and restoration of transcript expression upon 5-Aza treatment further corroborated this epigenetic silencing mechanism. Importantly Kaplan-Meier analysis of an outcome cohort showed an association between loss of DSC3 expression and significantly increased risk of biochemical recurrence. Functional studies indicate a role for epithelial-mesenchymal transition in DSC3 regulated cell migration/invasion. Taken together, our data suggests that DNA methylation contributes to down-regulation of DSC3 in prostate cancer, and loss of DSC3 predicts poor clinical outcome.
    PLoS ONE 03/2014; 9(3):e92815. DOI:10.1371/journal.pone.0092815 · 3.23 Impact Factor
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