DNA methylome and the complexity of discovering prostate cancer biomarkers

Abstract

P rostate cancer (PCa) remains the most common malignancy and a leading cause of cancer-related deaths in men. Molecular discrimination at an early stage between indolent and aggressive primary tumors in pathologically confirmed PCa is required to develop personalized therapeutic interventions. Somatic epigenetic alterations include DNA methylation, histone modification, RNA interference, disordered micro-RNA gene expression and genomic imprinting among others. Epigenetic alterations in human PCa occur at the earliest phases of malignant transformation and remain detectable throughout the invasive and metastatic progression of the disease. Epigenetic alterations have created great promises and potentials as biomarkers for PCa screening, diagnosis, molecular staging, prognostication and risk stratification. Reversibility of epigenetic changes allows therapeutic modulation of the epigenome and can also provide preventive opportunities in PCa. DNA methylation is the addition of a methyl group at the 59-carbon of the cytosine ring adjacent to a guanine (CpG). This process is executed by a number of conserved enzymes known as DNA methyl transferases (DNMTs). Aberrant DNA methylation and specifically hypermethylation silencing appears to be the most frequent molecular event in several malignancies including PCa. It is not surprising that many groups are paying considerable attention to identifying the unique profile of hypermethylated CpG islands ‘methylotyping or methylation signature’ that could be used for screening or as predictive or prognostic biomarkers for PCa. Kobayashi et al. explored a small-scale DNA methylome by using the Illumina Human Methylation 27 platform. They quantitatively examined promoter DNA methylation levels at 26333 CpG sites encompassing 14104 genes in 95 primary PCa and 86 adjacent benign prostate tissues of flash-frozen specimens. These tissues were obtained from patients with organ-confined PCa who were subjected to radical retropubic prostatectomies. To determine the significance of observed differences for CpG site DNA methylation levels, they employed an unpaired twoclass significance analysis of microarray for the available tumors and the matched adjacent benign tissues. This analysis revealed 5912 CpG sites hypermethylated and 2151 CpGs hypomethylated in tumors compared to benign adjacent tissues. They also used a rational strategy for prediction analysis of the data by pre-sorting the CpGs with standard deviation for all samples and improving the statistical power by analyzing those CpGs with a standard deviation of equal or greater than 0.04. Using these criteria, they identified PCa methylation biomarkers for 87 CpGs (82 genes) with predictive diagnostic values and 69 CpGs of prognostic values which correlated with biochemical recurrence. Since one-third of the CpGs examined showed quantitatively significant changes in methylation, authors examined a very rational hypothesis to determine the correlation between the observed changes and the expression levels of maintenance and de novo DNMTs (DNMT1, DNMT3A, DNMT3A2 and DNMT3B) and DNMT-interacting proteins (DNMT3L and EZH2) which are believed to target methyltransferases to certain genomic locations. TaqMan qPCR gene expression assay using FAM/MGD-labeled probes from Applied Bioscience inventory revealed a significant correlation between overexpression of DNMT3A2 (r50.272, P50.0031), DNMT3B (r50.197, P50.0056), EZH2 (r50.211, P50.0037), and global hypermethylation in tumors suggesting a possible causal role. Kobayashi et al. extended the investigation to recapitulate the potential cause and effect relationship between increased levels of DNMT3A2, DNMT3B and EZH2 transcripts and global hypermethylation. They transiently transfected a primary culture of human prostate epithelial cells with various DNMTs plasmids (alone or with EZH2). After robust bioinformatics, they further discovered that DNMT3B1 and DNMT3B2 overexpression led to elevated methylation levels in a subset of CpG sites which demonstrated PCa-specific hypermethylation. Overall, the strengths of the study on promoter-DNA methylation profiling by Kobayashi et al. include: (i) the comprehensive nature of the methylation analysis involving hierarchical clustering of malignant and adjacent benign prostatic tissues, differential methylation assays, two-class significance analysis of microarray, permutations and associated bioinformatics; (ii) the inclusion of a reasonable population size; (iii) searching 26333 CpG representing 14104 genes; (iv) the prediction analysis of the methylation data to identify the most predictive diagnostic methylation signature; and (v) the identification of prognostic DNA methylation changes which correlate with biochemical tumor recurrence. Compared with other malignancies, genespecific and genome-wide methylation analyses in PCa were conducted with a limited number of samples or limited scopes. Previous studies have identified over 30 hypermethylated CpG loci in PCa. Among these, GSTP1 promoter methylation occurs in up to 90% of prostatic tumors and in 70% of prostatic intraepithelial neoplasia lesions. Other well-characterized PCahypermethylated genes include RASSF1A, CDH1 and CDKN2A. Departments of Cancer Genetics and Urology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA Correspondence: Dr S Koochekpour (Shahriar.koochekpour@Roswellpark.org) Asian Journal of Andrology (2011) 13, 661–662 2011 AJA, SIMM & SJTU. All rights reserved 1008-682X/11 $32.00