Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Abstract

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2'-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.

Keywords: genome-wide DNA methylation analysis; prostate cancer; pyrosequencing.

Figure 1.

Hierarchical cluster analysis of methylation findings from microarray data. DNA methylation hierarchical clustering shows high within-sample group similarity and between-sample heterogeneity. These analyses were done using Partek genome studio software with 450K methylation probes showing greatest variability across all samples. CpG methylation differences were considered significant above a cut-off P-value < 10⁻³ and 0.2-fold change in the β-value. The scale is based on β-value (M-value, as determined by Partek Genome Studio) for methylation score. AA: African-American; Cau: Caucasian; NI: Normal; Ca: Cancer.

Figure 2.

(A) Manhattan plot for differentially methylated gene analysis in cancer vs. normal prostate tissues in both African-American and Caucasian men. The Y-axis shows the –log₁₀ P-values of probe sets (P < 0.0005, and the X-axis shows their chromosomal position. Horizontal red line represented an arbitrary P-value of 0.001. The P-values were obtained using rank-sum Man-Whitney's U-test to compare cancer vs. normal for each group. (B) Venn diagram showing the number of differentially methylated genes probe sets of cancer vs. normal for AA and Cau groups and the number of overlapping probe sets between the 2 groups.

Figure 3.

Heat map hierarchical cluster analysis of gene validation of 25 novel selected promoter-associated DNA methylated genes in a panel of prostate cell lines: RWPE1 and pNT1A are primary immortalized prostatic epithelial cell lines derived from Caucasian (Cau) patients; Cau PCa cell lines are LNCaP, PC3, and DU145; African-American (AA) PCa cell lines are MDA-PCa-2b and E006AA. Percent methylated cytosines in the cell lines were obtained by pyrosequencing. Methylated DNA control (M-DNA) and unmethylated DNA control (UM-DNA) were purchased from Qiagen. Fold change calculation is based on a ratio of log transformed values (FDR adjusted P-value < 0.05; β-value > 0.2, except for 4 CpG loci included in our study). Unsupervised hierarchical clustering analysis of the most variable β-values was done with a false discovery rate (FDR) adjusted P-value < 0.05 as significant.

Figure 4.

Demethylation and gene expression. The androgen-dependent prostate cancer (PCa) cell line, LNCaP and the androgen independent PCa cell line DU145 from Caucasian patients were treated with 5′-aza-2′-dC (5 μmol⁻¹), TSA (250 noml⁻¹), or a combination of the 2 drugs. These cell lines had high methylation levels for the genes tested and all had low levels of expression at baseline. The fold change in gene expression level relative to mock-treated cells was determined by quantitative RT-PCR and expressed relative to GAPDH to correct for variation in the amounts of reverse-transcribed RNA. The data are representative of 3 independent experiments. The standard deviation of the mean is shown as error bar.

Figure 5.

(A) Quantitative DNA methylation analysis in human prostate tissues is shown in the box plot. The percent DNA methylation levels of promoter CpGs were analyzed in bisulfite-modified genomic DNA extracted from matched pair normal and prostate cancer (PCa) tissues (26 samples from AA; 30 samples from Cau) from men. Y-axis, percentage of methylated cytosines in the samples as obtained from pyrosequencing; X-axis, Nl (Normal) and Ca (Cancer) tissues. The box plot describes the median, interquartile range and maximum/minimum methylation. The P values were obtained using Mann-Whitney t-test. Pearson correlation and simple and multiple regression methods were used to compare methylation changes in cancer by race and cancer x race interactions. *Significant (P < 0.05) difference comparing normal and tumor. (B) Gene expression in prostate tissue samples. The relative mRNA transcript expression levels for SHANK2, MST1R, and SNRPN were analyzed in 32 matched pairs of Nl (normal) and PCa (prostate cancer) tissue samples by RT-PCR and expressed relative to GAPDH to correct for variation in amount of reverse-transcribed RNA. *Significantly different compared to cells treated with nonsense siRNA using an ANOVA with Holm post hoc test (P < 0.05).

Figure 6.

(A) Basal ABCG5 and SNRPN protein expression in Caucasian (Cau) prostate cancer (PCa) cell lines PC3 and LNCaP and African-American (AA) PCa cell lines E006AA and MDA PCa 2b. (B) Western blot validation of ABCG5 and SNRPN gene knockdowns in PCa cell lines. Cells were transfected with a nonsense sequence siRNA control (siNS) or one of 2 different gene-specific siRNAs (1 or 2). Knockdown of protein levels ranged from 60–90% compared to cells transfected with siNS, in agreement with qRT-PCR results (data not shown). Data shown are representative of 3–6 independent experiments. The amount of lysate loaded on the gel varied across the different knockdowns. If a protein was weakly expressed in 6A, we loaded more lysate in the knockdown experiments to get better signal to noise with the knockdowns. (C) and (D) Effects of gene knockdown on proliferation (C) and invasion (D) in Cau and AA PCa cell lines. siRNA-mediated knockdown of either ABCG5 or SNRPN led to a decrease in both invasion and proliferation by PC3 and LNCap cells. In contrast, loss of oncogenic function in AA cell lines was limited to a decrease in invasion in only the SNRPN knockdown. Data are represented as the mean ± SEM of 3–6 independent assays. *Significantly different compared to cells treated with siNS using an ANOVA with Holm post hoc test (P < 0.05).