BACKGROUND Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor targeting estrogen receptors (ERs), has been implicated in the promotion of breast cancer. Perinatal exposure of BPA could induce longitudinal alteration of DNA hydroxymethylation in imprinted loci of mouse blood cells. To date, no data has been reported on the effects of BPA on DNA hydroxymethylation in breast cells. Therefore, we asked whether BPA can induce DNA hydroxymethylation change in human breast cells. Given that dysregulated epigenetic DNA hydroxymethylation is observed in various cancers, we wondered how DNA hydroxymethylation modulates cancer development, and specifically, whether and how BPA and its analogs promote breast cancer development via DNA hydroxymethylation. OBJECTIVES We aimed to explore the interplay of the estrogenic activity of bisphenols at environmental exposure dose levels with TET dioxygenase-catalyzed DNA hydroxymethylation and to elucidate their roles in the proliferation of ER+ breast cancer cells as stimulated by environmentally relevant bisphenols. METHODS Human MCF-7 and T47D cell lines were used as ER-dependent breast cell proliferation models, and the human MDA-MB-231 cell line was used as an ER-independent breast cell model. These cells were treated with BPA or bisphenol S (BPS) to examine BPA/BPS-related proliferation. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and enzyme-linked immunosorbent assays (ELISAs) were used to detect DNA hydroxymethylation. Crispr/Cas9 and RNA interference technologies, quantitative polymerase chain reaction (qPCR), and Western blot analyses were used to evaluate the expression and function of genes. Co-immunoprecipitation (Co-IP), bisulfite sequencing-PCR (BSP), and chromatin immunoprecipitation-qPCR (ChIP-qPCR) were used to identify the interactions of target proteins. RESULTS We measured higher proliferation in ER+ breast cancer cells treated with BPA or its replacement, BPS, accompanied by an ERα-dependent decrease in genomic DNA hydroxymethylation. The results of our overexpression, knockout, knockdown, and inhibition experiments suggested that TET2-catalyzed DNA hydroxymethylation played a suppressive role in BPA/BPS-stimulated cell proliferation. On the other hand, we observed that TET2 was negatively regulated by the activation of ERα (dimerized and phosphorylated), which was also induced by BPA/BPS binding. Instead of a direct interaction between TET2 and ERα, data of our Co-IP, BSP, and ChIP-qPCR experiments indicated that the activated ERα increased the DNA methyltransferase (DNMT)-mediated promoter methylation of TET2, leading to an inhibition of the TET2 expression and DNA hydroxymethylation. CONCLUSIONS We identified a new feedback circuit of ERα activation-DNMT-TET2-DNA hydroxymethylation in ER+ breast cancer cells and uncovered a pivotal role of TET2-mediated DNA hydroxymethylation in modulating BPA/BPS-stimulated proliferation. Moreover, to our knowledge, we for the first time established a linkage among chemical exposure, DNA hydroxymethylation, and tumor-associated proliferation. These findings further clarify the estrogenic activity of BPA/BPS and its profound implications for the regulation of epigenetic DNA hydroxymethylation and cell proliferation. https://doi.org/10.1289/EHP5862.

中文翻译：

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TET双加氧酶和DNA羟甲基化在双酚类刺激的乳腺癌细胞增殖中的作用。

背景技术双酚A（BPA）是一种普遍存在的靶向雌激素受体（ERs）的环境内分泌干扰物，已参与了乳腺癌的发展。围产期BPA暴露可能会引起小鼠血细胞印迹基因座中DNA羟甲基化的纵向改变。迄今为止，还没有关于双酚A对乳腺细胞DNA羟甲基化作用的报道。因此，我们询问BPA是否可以诱导人乳腺细胞中的DNA羟甲基化变化。鉴于在各种癌症中观察到表观遗传的DNA羟甲基化失调，我们想知道DNA羟甲基化如何调节癌症的发展，特别是BPA及其类似物是否以及如何通过DNA羟甲基化促进乳腺癌的发展。目的我们旨在探讨双酚在环境暴露剂量水平下的雌激素活性与TET双加氧酶催化的DNA羟甲基化的相互作用，并阐明它们在与环境有关的双酚刺激下在ER +乳腺癌细胞增殖中的作用。方法以人MCF-7和T47D细胞系为ER依赖性乳腺癌细胞增殖模型，以人MDA-MB-231细胞系为ER依赖性乳腺癌细胞模型。这些细胞用BPA或双酚S（BPS）处理，以检查BPA / BPS相关的增殖。超高效液相色谱-串联质谱（UHPLC-MS / MS）和酶联免疫吸附测定（ELISAs）用于检测DNA羟甲基化。Crispr / Cas9和RNA干扰技术，定量聚合酶链反应（qPCR）和Western印迹分析用于评估基因的表达和功能。共免疫沉淀（Co-IP），亚硫酸氢盐测序PCR（BSP）和染色质免疫沉淀qPCR（ChIP-qPCR）用于鉴定靶蛋白的相互作用。结果我们测量了用BPA或其替代物BPS处理的ER +乳腺癌细胞中较高的增殖，并伴随着ERα依赖性基因组DNA羟甲基化的降低。我们的过表达，敲除，敲除和抑制实验的结果表明，TET2催化的DNA羟甲基化在BPA / BPS刺激的细胞增殖中起抑制作用。另一方面，我们观察到TET2受ERα（二聚体和磷酸化）的激活负调控，这也是由BPA / BPS结合诱导的。我们的Co-IP，BSP和ChIP-qPCR实验数据表明，活化的ERα增强了DNA甲基转移酶（DNMT）介导的TET2的启动子甲基化，从而导致TET2的抑制，而不是TET2与ERα之间的直接相互作用。表达和DNA羟甲基化。结论我们确定了ER +乳腺癌细胞中ERα激活-DNMT-TET2-DNA羟甲基化的新反馈回路，并揭示了TET2介导的DNA羟甲基化在调节BPA / BPS刺激的增殖中的关键作用。而且，据我们所知，我们首次建立了化学暴露，DNA羟甲基化和肿瘤相关增殖之间的联系。这些发现进一步阐明了BPA / BPS的雌激素活性及其对表观遗传DNA羟甲基化和细胞增殖的调控的深远意义。https://doi.org/10.1289/EHP5862。