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: 3.23). 02/2009; 4(3):e4830. DOI: 10.1371/journal.pone.0004830
Source: PubMed


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
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    • "However, to the best of our knowledge, this is the first time BMPER, BMP7, DUSP4 and LRP12 are reported to be methylated in lymphoma. From previous studies, DUSP4 and BMP7 are known to be methylated in gliomas [26] and gastric- and prostate cancer [27], [28], respectively. Even though large scale methylation studies have been performed on different lymphoma types increasing the knowledge about methylated genes, we were able to discover novel methylated genes using a cell line based approach and studying FL, DLBCL and BL at the same time. "
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    ABSTRACT: Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt's lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (BMP7, BMPER, CDH1, DUSP4 and LRP12), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes LRP12 and CDH1 showed the highest methylation frequencies (94% and 92%, respectively). BMPER (58%), DUSP4 (32%) and BMP7 (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of LRP12 and CDH1 methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "DNA methylation, occurring predominantly at CpG islands, is the most studied epigenetic modification and is frequently found within gene promoter regions. Genome-wide DNA methylation studies compared non-malignant prostate tissue and tumour tissue (review in Day and Bianco–Miotto, 2013) and found that the promoter hypermethylation of several genes, including FAM84A (NSE1) and SPOCK2 (Yang et al, 2013), EFEMP1 (Kim et al, 2011b), HOXD3 and BMP7 (Kron et al, 2009), GSTP1 (Goering et al, 2012), AOX1 and SPON2 (Kim et al, 2012), might serve as optimal biomarkers for differentiating PCa from non-malignant glands. Furthermore, there is substantial overlap in candidate methylated genes between different studies of genome-wide DNA methylation in prostate cancer (Day and Bianco–Miotto, 2013). "
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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.
    Full-text · Article · Jun 2014 · British Journal of Cancer
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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.
    Preview · Article · Apr 2013 · Neoplasia (New York, N.Y.)
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