DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer

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.

Figure 1.

Hierarchical clustering of prostate tissues by DNA methylation. Unsupervised hierarchical clustering of 181 prostate tissues and 26,333 CpGs, by sample and by CpG. (Red branches) Tumor samples; (blue branches) benign adjacent samples; (red pixels) high DNA methylation; (green pixels) low DNA methylation.

Figure 2.

Differentially methylated CpGs of prostate tumors. Unsupervised hierarchical clustering of 181 prostate tissues based on the 5912 and 2151 CpG sites hypermethylated and hypomethylated in prostate tumors, respectively, as identified by 2-class SAM. (Red branches) Tumor samples; (blue branches) benign adjacent samples; (red pixels) high DNA methylation; (green pixels) low DNA methylation.

Figure 3.

GSTP1 CpG island hypermethylation in prostate tumors. (A) Diagram of the RefSeq annotation of the GSTP1 gene. (Green box) CpG island calculated by the UCSC Genome Browser. Circles are CpG sites assayed by HumanMethylation27. (Red circles) Probes that were identified to be hypermethylated in prostate tumors by 2-class SAM; (green circle) probe that was hypomethylated; (gray circle) probe that showed no significant change. The numbers below the circles indicate the relative distance in base pairs from the predicted TSS. (B) Heatmap depicts DNA methylation pattern of the seven probes near GSTP1. The dendrogram is based on the hierarchical clustering from Figure 2. (Red branches) Tumor samples; (blue branches) benign adjacent samples. Coordinates are based on the NCBI36/hg18 human genome assembly.

Figure 4.

Expression of DNMTs and EZH2 correlates with global hypermethylation in prostate tumors. Comparison of transcript levels of DNMTs and EZH2 measured by TaqMan qPCR with the average DNA methylation levels of CpG sites that are hypermethylated in prostate tumors. (Blue circles) Benign adjacent samples; (red circles) tumor samples. The P-value was calculated by linear regression analysis. y-axis: average DNA methylation levels (beta score); x-axis: relative gene expression levels [log₂(RQ)]; (black line) linear regression. (A) DNMT1 expression. (B) DNMT3A expression. (C) DNMT3A2 expression. (D) DNMT3B expression. (E) EZH2 expression. (F) Comparison of DNMTs and EZH2 transcript levels between benign adjacent tissues (blue) and tumors (red). Significant differences are indicated by asterisks; P-values were calculated by t-test. Standard errors are depicted by error bars. For part F, the y-axis is relative gene expression levels [log₂(RQ)].

Figure 5.

Overexpression of DNMTs and EZH2 results in increased methylation at a subset of prostate tumor hypermethylation sites. Ideal (black) and empirical (red) cumulative distribution functions of change in DNA methylation after DNMT or EZH2 transfection into cultured normal prostate cells. The empirical distribution functions are based on the 5912 CpGs that were hypermethylated in prostate tumors, while the ideal distribution functions are based on all 26,333 CpGs assayed on the array. Overexpression of (A) DNMT3A, (B) DNMT3A2, (C) DNMT3B1, (D) DNMT3B2, (E) DNMT3B3, (F) EZH2, (G) DNMT3A and EZH2, (H) DNMT3A2 and EZH2, (I) DNMT3B1 and EZH2, (J) DNMT3B2 and EZH2, and (K) DNMT3B3 and EZH2.