Epigenetic Changes Induced by Estrogens and Xenoestrogens in Breast Cancer

描述(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)和邻苯二甲酸丁基苄酯(BBP)是否在人类乳腺癌的发生中发挥作用，如果是，这种影响是否由表观遗传机制介导。这项拟议的研究是基于越来越多的人担心，作为内分泌干扰物的雌激素环境化合物可能会对荷尔蒙敏感的器官(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，这进一步加剧了人们的担忧。因此，人们有理由担心，BPA和BBP具有雌激素性质，广泛分布在环境中，可能也会对人类乳房致癌。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在人乳腺上皮细胞MCF-10F中的致癌作用。这一强大而独特的模型的利用将为我们提供一个工具来探索BPA和BBP是否在乳腺癌的发生中具有相关性。此外，我们还发现，E2诱导的转化表型的表达与控制分支和导管形成的基因的高甲基化或低甲基化有关。这些都是我们假设异种雌激素BPA和BBP作为表观遗传调节器，通过高甲基化和/或组蛋白修饰导致关键基因沉默，从而导致乳腺癌的发生和发展，从而诱导肿瘤转化的理论基础。为此，我们提出了以下具体目标：确定异种雌激素BPA和BBP是否诱导人乳腺上皮细胞发生肿瘤转化，转化表型的表达是否与控制分支和导管形成的基因的表观遗传变化有关，如果是这样的话，确定改变它们的甲基化状态是否会逆转肿瘤过程。为了实现这一目标，我们将使用MCF-10F细胞和从缩乳术中获得的人乳腺上皮细胞的原代培养。这些细胞将被BPA和BBP处理，使用的方案类似于用E2处理这些细胞的方案，这将作为对照。甲基化研究将使用限制性地标基因组扫描(RLGS)进行，识别的基因将使用甲基化特异性聚合酶链式反应(MSP)进一步研究，然后确认它们的表达和功能作用。总之，这些研究将为外源性雌激素是否能够诱导HBEC肿瘤转化以及表观遗传学机制参与这一过程提供第一手证据。此外，操纵这些表观遗传修饰基因的甲基化状态和沉默不仅将提供对这些环境污染物如何参与乳腺癌发病的了解，还将为我们提供工具，以制定预防策略，以抵消其在普通人群中的影响。与公众健康相关：这些研究将提供BPA和BBP等异种雌激素类物质是否能够诱导HBEC肿瘤转化以及表观遗传机制参与这一过程的第一手证据。此外，操纵这些表观遗传修饰基因的甲基化状态和沉默，不仅将有助于理解这些广泛的环境污染物如何参与乳腺癌的启动，而且还将有助于制定预防策略，以抵消其在普通人群中的影响。

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.