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

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 (<i>Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4</i>) 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: <i>Paqr4</i>). 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.

发育阶段暴露于内分泌干扰物（endocrine-disrupting chemicals, EDCs）17β-雌二醇-3-苯甲酸酯（17β-estradiol-3-benzoate, EB）与双酚A（bisphenol A, BPA），会增加啮齿类动物模型中前列腺癌（prostate cancer, PCa）的易感风险。本研究采用甲基化CpG岛富集检测（methylated-CpG island recovery assay, MIRA）辅助基因组平铺及CpG岛芯片技术，对新生期（出生后第1、3、5天，postnatal day 1,3,5, PND1、3、5）分别以25 μg/幼崽或2500 μg EB/kg体重（body weight, BW）、0.1 μg BPA/幼崽或10 μg BPA/kg体重处理的斯普拉格-道利大鼠，在出生后第90天（postnatal day 90, PND90）的背侧前列腺组织中，鉴定与处理相关的甲基化组变化。我们共鉴定得到111个与EB相关、86个与BPA相关的携带显著差异甲基化区域的基因，其中20个为两者共有。通路分析显示癌症为最显著的共同疾病通路。亚硫酸氢盐测序验证了芯片分析中15个候选基因的差异甲基化模式。在这15个基因中，7个基因（*Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4*）的甲基化状态与组织样本中的基因表达呈负相关。通过使用经5-氮杂胞苷（5-aza-cytidine）处理的正常大鼠前列腺细胞系NbE-1与癌性前列腺细胞系AIT的细胞实验，进一步证实了其中6个基因（Paqr4除外）的DNA甲基化介导的基因表达调控。这些基因的功能连通性与胚胎干细胞多能性相关。此外，利用癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据集进行聚类分析发现，这7个基因的表达与前列腺癌患者的无复发生存期显著相关。综上，本研究揭示，大鼠早期生命阶段暴露于内分泌干扰物所引发的基因特异性启动子甲基化改变，可作为前列腺癌复发的预测表观遗传生物标志物，同时提示此类暴露可能对人类疾病发生发展产生影响。