DNA methylome changes by estradiol benzoate and bisphenol A links early-life environmental exposures to prostate cancer risk

Abstract

Developmental exposure to endocrine-disrupting chemicals (EDCs), 17β-estradiol-3-benzoate (EB) and bisphenol A (BPA), increases susceptibility to prostate cancer (PCa) in rodent models. Here, we used the methylated-CpG island recovery assay (MIRA)-assisted genomic tiling and CpG island arrays to identify treatment-associated methylome changes in the postnatal day (PND)90 dorsal prostate tissues of Sprague-Dawley rats neonatally (PND1, 3, and 5) treated with 25 µg/pup or 2,500 µg EB/kg body weight (BW) or 0.1 µg BPA/pup or 10 µg BPA/kg BW. We identified 111 EB-associated and 86 BPA-associated genes, with 20 in common, that have significant differentially methylated regions. Pathway analysis revealed cancer as the top common disease pathway. Bisulfite sequencing validated the differential methylation patterns observed by array analysis in 15 identified candidate genes. The methylation status of 7 (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4) of these 15 genes exhibited an inverse correlation with gene expression in tissue samples. Cell-based assays, using 5-aza-cytidine-treated normal (NbE-1) and cancerous (AIT) rat prostate cells, added evidence of DNA methylation-mediated gene expression of 6 genes (exception: Paqr4). Functional connectivity of these genes was linked to embryonic stem cell pluripotency. Furthermore, clustering analyses using the dataset from The Cancer Genome Atlas revealed that expression of this set of 7 genes was associated with recurrence-free survival of PCa patients. In conclusion, our study reveals that gene-specific promoter methylation changes, resulting from early-life EDC exposure in the rat, may serve as predictive epigenetic biomarkers of PCa recurrence, and raises the possibility that such exposure may impact human disease.

Keywords: Developmental origin of health and disease (DOHaD); Ingenuity®; NimbleGen rat DNA methylation promoter array; Pathway Analysis (IPA®); Sprague Dawley rats; The Cancer Genome Atlas (TCGA); early-life reprogramming; endocrine-disrupting chemicals (EDCs); epigenetics; methylated-CpG island recovery assay (MIRA); stem cell pluripotency.

Figure 1.

Schematic diagram of the experimental and analytical procedures. Sprague-Dawley (SD) rats were neonatally [postnatal day (PND) 1, 3, 5] treated with 17β-estradiol-3-benzoate (EB) at 25 μg/pup or 2,500 µg/kg body weight (BW), bisphenol A (BPA) at 0.1 µg/pup or 10 µg/kg BW, or corn oil as a control (Ctrl). DNA extracted from PND90 dorsal prostate tissue was subjected to promoter methylation array analysis, and genes with differentially methylated regions and inverse expression correlation were identified and validated.

Figure 2.

Representative results from genome-wide methylation study. A) Predicted CpG islands (light blue shaded areas) in the promoter region of differentially methylated genes (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, and Creb3l4) identified in this study. TSS stands for transcriptional start site whereas ATG stands for translational start codon. Individual CpG sites are represented by red vertical lines. Dark blue horizontal line marks the region selected for BS-sequencing; B) Position of the BS-sequenced region (blue line) relative to the NimbleGen probes covered regions (dirty yellow lines) of each gene; C) Significant methylation level of each gene in vehicle- (control), EB- and BPA-treated groups, which were measured by NimbleGen array probes (mBar values). The height of the mBar represents the probe intensity; red and green bars represent positive and negative methylation value, respectively, relative to their respective input control.

Figure 3.

Effect of neonatal exposure to EB or BPA on promoter methylation and gene expression in PND90 dorsal prostate. Promoter methylation status (left panel: Scatter Plot) and expression (right panel: Bar Graph) of candidate genes, in PND90 prostate tissues from SD rats treated with either corn oil (Ctrl; white), EB (green), or BPA (red), were analyzed using bisulfite sequencing and qPCR, respectively. Each circle in the scatter plot represents mean ±SEM of methylation percentage (averaged from 6 individual samples/animals) at a single CpG site in the gene promoter region. The % methylation of each CpG site in each sample was determined from bisulfite sequencing data derived from 8–12 clones. Gene expression data were expressed as mean ± SEM from 3 individual samples. Statistical significance was determined by one-way ANOVA and Tukey test when compared to Ctrl. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.

Signaling pathways associated with differentially methylated candidate genes were predicted by Ingenuity Pathway Analysis™. Genes with promoter hypermethylation are shown in red and genes with promoter hypomethylation are shown in green, with color intensity signifying the magnitude of differential methylation. Gray arrows indicate predicted association pathways; purple arrows indicate reported directional pathways.

Figure 5.

Effect of 5-aza-cytidine treatment on gene expression in NbE-1 and AIT cells. Gene expression was analyzed by qPCR in rat (A) normal prostate epithelial NbE-1 cells and (B) prostate cancer AIT cells treated with DMSO (Ctrl), or 0.5 µM or 1 µM 5-aza-2-deoxycytidine (5-aza), a DNA methylation inhibitor, for 8 d. Data (mean ± SEM ) is normalized to corresponding Rpl19 levels, and is expressed as fold change vs. Ctrl. *P < 0.05, and **P < 0.001 by one-way ANOVA and Tukey test when compared to Ctrl.

Figure 6.

Expression of the 7 candidate genes was associated with shorter recurrence-free survival of PCa patients. TCGA data consisting of 497 PCa patients were dichotomized into Groups 1 and 2 by K-means clustering analysis based on the 7 candidate genes. (A) Group 1 patients have longer time to recurrence than Group 2 patients; the two groups differ in recurrence-free survival. (B) Proportion of PCa patients having tumors with high (≥7) and low (<7) Gleason score.