Epigenetic Changes Induced by Estrogens and Xenoestrogens in Breast Cancer

描述（由申请人提供）：本申请的主要目的是确定异种雌激素物质双酚A （BPA）和邻苯二甲酸丁苯酯（BBP）是否在人类乳腺癌的发生中起作用，如果是，这种作用是否通过表观遗传机制介导。这项提议的研究是基于越来越多的关注，即作为内分泌干扰化学物质的雌激素环境化合物可能对激素敏感器官（如乳房）产生潜在的不利影响。有证据表明，天然雌激素，即17 -雌二醇（E2），是乳腺癌发生和发展的重要因素，这进一步加剧了这种担忧。因此，BPA和BBP这两种具有雌激素特性且广泛存在于环境中的物质，也可能对人类乳房具有致癌性的担忧是有充分理由的。为了实现这些目标，我们将利用我们的体外体内模型，在该模型中我们已经证明了E2对人类乳腺上皮细胞MCF-10F的致癌性。利用这一强大而独特的模型将为我们探索BPA和BBP是否与乳腺癌的发生有关提供工具。此外，我们发现e2诱导的转化表型的表达与控制分支和导管发生的基因的高甲基化或低甲基化有关。这些是我们假设异种雌激素BPA和BBP可以诱导肿瘤转化的基本原理，通过高甲基化和/或组蛋白修饰诱导关键基因沉默，从而导致乳腺癌的发生和发展。为此，我们提出以下具体目标：确定异种雌激素BPA和BBP是否诱导人乳腺上皮细胞的肿瘤转化，以及转化表型的表达是否与控制分支和导管发生的基因的表观遗传变化有关，如果是这样，确定修改它们的甲基化状态是否能逆转肿瘤过程。为了实现这一目标，我们将使用从缩乳成形术中获得的MCF-10F细胞和人乳腺上皮细胞的原代培养物。这些细胞将被BPA和BBP处理，使用类似于用E2处理这些细胞的方案，E2将作为对照。甲基化研究将使用限制性限制里程碑基因组扫描（RLGS）进行，鉴定的基因将使用甲基化特异性PCR （MSP）进一步研究，随后确认其表达和功能作用。总之，这些研究将为异种雌激素物质是否能够诱导HBEC的肿瘤转化以及这一过程中涉及的表观遗传机制提供第一手证据。此外，对这些表观遗传修饰基因的甲基化状态和沉默的操纵将不仅提供对这些环境污染物如何参与乳腺癌发病的理解，而且还将为我们提供制定预防策略的工具，以抵消它们对普通人群的影响。与公共卫生相关：这些研究将提供诸如BPA和BBP等异种雌激素物质是否能够诱导HBEC的肿瘤转化以及这一过程中涉及的表观遗传机制的第一手证据。此外，对这些表观遗传修饰基因的甲基化状态和沉默的操纵将不仅有助于了解这些广泛存在的环境污染物如何参与乳腺癌的发生，而且有助于制定预防策略来抵消它们对普通人群的影响。

DESCRIPTION (provided by applicant): The main objective of this application is to determine whether the xenoestrogenic substances bisphenol A (BPA) and butyl benzyl phthalate (BBP) play a role in the initiation of human breast cancer, and if so, whether this effect is mediated by epigenetic mechanisms. The proposed study is based on the growing concern that estrogenic environmental compounds that act as endocrine disrupting chemicals might have potential adverse effects on hormone-sensitive organs such as the breast. This concern is further fueled by evidence indicating that natural estrogens, namely 17 ¿-estradiol (E2), are important factors in the initiation and progression of breast cancer. Therefore, the concern that BPA and BBP, which have estrogenic properties and are widely distributed in the environment, might also be carcinogenic for the human breast is well justified. For accomplishing these goals we will utilize our in vitro in vivo model in which we have demonstrated the carcinogenicity of E2 in the human breast epithelial cells MCF-10F. The utilization of this powerful and unique model will provide us a tool for exploring whether BPA and BBP have relevance in the initiation of breast cancer. Furthermore, we have found that the expression of E2-induced transformation phenotypes is associated with hyper or hypomethylation of genes controlling branching and ductulogenesis. These are the basis for our rationale to postulate that the xenoestrogens BPA and BBP can induce neoplastic transformation by behaving as epigenetic modulators inducing silencing of critical genes by hypermethylation and/or histone modification that lead to the initiation and progression of breast cancer. For this purpose, we propose the following specific aim: To determine whether the xenoestrogens BPA and BBP induce neoplastic transformation in human breast epithelial cells and whether the expression of transformation phenotypes is associated with epigenetic changes in genes controlling branching and ductulogenesis, and if this is the case, to determine if modifying their methylation status reverts the neoplastic process. To accomplish this aim we will use MCF-10F cells and primary cultures of human breast epithelial cells obtained from reduction mammoplasty. The cells will be treated with BPA and BBP using a protocol similar to that used for the treatment of these cells with E2, which will serve as a control. Methylation studies will be performed using Restriction Landmark Genomic Scanning (RLGS) and the identified genes will be further studied using methylation specific PCR (MSP) followed by confirmation of their expression and functional role. Altogether, these studies will provide first hand evidence on whether xenoestrogenic substances are able to induce neoplastic transformation in HBEC and that epigenetic mechanisms are involved in this process. Furthermore the manipulation of the methylated status and silencing of those epigenetically modified genes will provide not only an understanding of how these environmental contaminants are involved in breast cancer initiation but also will give us tools for developing preventive strategies to counteract their effect in the general population. RELEVANCE TO PUBLIC HEALTH: These studies will provide first hand evidence on whether xenoestrogenic substances like BPA and BBP are able to induce neoplastic transformation in HBEC and that epigenetic mechanisms are involved in this process. Furthermore the manipulation of the methylated status and silencing of those epigenetically modified genes will provide not only an understanding how these widely environmental contaminants are involved in breast cancer initiation but also for developing preventive strategies to counteract their effect in the general population.