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）和邻苯二甲酸丁基苄酯（BBP）是否在人类乳腺癌的发生中起作用，如果是，这种作用是否由表观遗传机制介导。这项拟议的研究是基于越来越多的关注，即雌激素环境化合物作为内分泌干扰化学品可能对乳房等乳房敏感器官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，进一步加剧了这种担忧。因此，BPA和BBP具有雌激素性质，广泛分布于环境中，对人类乳腺也可能致癌的担忧是有道理的。为了实现这些目标，我们将利用我们的体外体内模型，我们已经证明了E2在人乳腺上皮细胞MCF-10 F中的致癌性。利用这个强大而独特的模型将为我们提供一个工具，探索BPA和BBP是否在乳腺癌的发生相关。此外，我们发现E2诱导的转化表型的表达与控制分枝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可以通过表现为表观遗传调节剂诱导肿瘤转化的基础，表观遗传调节剂通过超甲基化和/或组蛋白修饰诱导关键基因沉默，导致乳腺癌的发生和进展。为此，我们提出了以下具体目标：以确定是否异种雌激素BPA和BBP诱导肿瘤转化在人类乳腺上皮细胞和转化表型的表达是否与表观遗传学变化的基因控制分支和ductulogenesis，如果是这种情况下，以确定是否修改其甲基化状态逆转肿瘤的过程。为了实现这一目标，我们将使用MCF-10 F细胞和从乳房缩小术中获得的人乳腺上皮细胞的原代培养。将使用与用E2处理这些细胞类似的方案用BPA和BBP处理细胞，其将用作对照。将使用限制性标志基因组扫描（RLGS）进行甲基化研究，并使用甲基化特异性PCR（MSP）进一步研究鉴定的基因，然后确认其表达和功能作用。总之，这些研究将提供第一手证据，异种雌激素物质是否能够诱导肿瘤转化HBEC和表观遗传机制参与这一过程。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可以了解这些环境污染物如何参与乳腺癌的发生，而且还可以为我们提供制定预防策略的工具，以抵消它们在普通人群中的影响。与公共卫生的关系：这些研究将提供第一手证据，证明BPA和BBP等外源性雌激素物质是否能够诱导HBEC的肿瘤转化，以及表观遗传机制是否参与了这一过程。此外，这些表观遗传修饰基因的甲基化状态和沉默的操纵不仅将提供这些广泛的环境污染物是如何参与乳腺癌的启动，而且还为制定预防策略，以抵消其在一般人群中的影响。

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.