Regulation of Genomic Instability in Early Breast Cancer

描述(申请人提供)：我们证明了在人乳腺上皮细胞(HMEC)中抑制p16/pRB通路，这是干细胞和许多肿瘤细胞常见的一种情况，会诱导几种对恶性转化至关重要的表型，包括动态表观遗传重塑，导致选定的CpG岛的靶向甲基化。在HMEC中抑制p16INK4a活性会增加E2F1转录因子的表达，从而增加其几个下游靶点的表达，如染色质重塑多梳组(PcG)蛋白、EZH2和SUZ12。这些PcG蛋白定位于特定的基因组序列，这些序列在转录时变得沉默，随后被DNA超甲基化修饰。鉴于间质-上皮相互作用在分化和肿瘤进展中的重要性，我们假设处于这种表观遗传可塑性状态的细胞可以通过微环境中的间质细胞信号来编程，以获得促进肿瘤发生的表观遗传变化。我们开发了一个体外模型系统，在这个系统中，表观遗传的癌细胞暴露在诱导上皮向间充质转化(EMT)的因素中，EMT是一种重要的恶性程序。与我们的假设一致，EMT伴随着表达变化、一组基因座的从头甲基化，包括E-钙粘素启动子，以及临床上相关的表型变化，如运动性增加。我们希望扩展这一强大的模型系统，以研究编程基因表达和肿瘤进展过程中获得的表观遗传学变化所必需的内在和外在调节因子。为了解决内在控制问题，我们假设，在HMEC中取消p16INK4a/PRB通路的不同成员将产生不同的表观遗传学变化，这取决于染色质重塑蛋白的表达水平和它们的靶点。为了解决外部控制，我们假设表观遗传可塑性的HMEC可以通过来自不同乳腺肿瘤亚型的周围成纤维细胞的信号进行差异编程。我们将通过特定的目标来检验这些假说：(1)确定调控(A)染色质重塑蛋白和(B)伴随着p16INK4a活性丧失的分化蛋白的表达程序是否改变了pRB途径其他成员的表达变化，即pRB、细胞周期蛋白D1和CDK4的表达变化；(2)确定伴随p16INK4a活性丧失的获得性DNA甲基化事件的程序是否受到pRB途径其他成员的表达变化的影响，(3)研究Lumina B和Basal样癌相关成纤维细胞对体外培养的表观可塑性HMEC的基因表达变化和获得性表观遗传学改变的影响，并(C)检验这些作用在体内的功能后果。公共卫生相关性：虽然获得性DNA甲基化在基因表达变化中的作用明显重要，但甲基化事件可以在肿瘤的临床评估中发挥额外的作用，为评估疾病的发生和发展、风险状态、监测预防策略、提供早期诊断和跟踪癌症的预后提供潜在的生物标志物。我们在表观遗传变化调控方面的新观察为评估个体恶性转化的易感性提供了潜在的标志物，也为预防和治疗提供了潜在的靶点。

DESCRIPTION (provided by applicant): We demonstrated that repression of the p16/pRb pathway in human mammary epithelial cells (HMEC), a condition common to stem cells and many tumor cells, induces several phenotypes critical to malignant transformation, including dynamic epigenetic remodeling resulting in the targeted methylation of selected CpG islands. Repression of p16INK4a activity in HMEC increases expression of the E2F1 transcription factor and, consequently, of several of its downstream targets such as the chromatin remodeling polycomb group (PcG) proteins, EZH2 and SUZ12. These PcG proteins localize to specific genomic sequences that become silenced for transcription and subsequently modified by DNA hypermethylation. Given the importance of stromal-epithelial interactions in differentiation and tumor progression, we hypothesized that cells in this epigenetically-plastic state could be programmed by signals from stromal cells within the microenvironment to acquire epigenetic changes that promote tumorigenesis. We developed an in vitro model system where epigenetically-plastic cells are exposed to factors that induce epithelial-to- mesenchymal transition (EMT), an important malignant program. Consistent with our hypothesis, the EMT was accompanied by expression changes, de novo methylation of a group of loci, including the E-cadherin promoter, and clinically relevant phenotypic changes such as increased motility. We wish to extend this powerful model system to investigate both intrinsic and extrinsic regulatory factors necessary for programming gene expression and epigenetic changes that are acquired during tumor progression. To address intrinsic control, we hypothesize that abrogation of different members of the p16INK4a/pRB pathway in HMEC will each generate a different spectrum of epigenetic changes, dependent on expression levels of chromatin remodeling proteins and sites to which they are targeted. To address extrinsic control, we hypothesize that epigenetically-plastic HMEC can be differentially programmed by signals from surrounding fibroblasts isolated from different breast tumor subtypes. We will test these hypotheses through Specific Aims that seek: (1) to determine if the program of expression changes that modulate levels of (a) chromatin remodeling proteins and (b) differentiation proteins that accompanies the loss of p16INK4a activity is phenocopied by expression changes for other members of the pRb pathway, i.e pRb, cyclin D1 and Cdk4, (2) to determine if the program of acquired DNA methylation events that accompanies the loss of p16INK4a activity is phenocopied by expression changes for other members of the pRb pathway, (3) to determine the effects of Luminal B and Basal-like carcinoma-associated fibroblasts on (a) gene expression changes and (b) acquired epigenetic alterations in epigenetically -plastic HMEC in vitro and to (c) test the functional consequences of these effects in vivo. PUBLIC HEALTH RELEVANCE: While the role of acquired DNA methylation in gene expression changes is of obvious importance, methylation events can play additional roles in the clinical assessment of tumors, providing potential biomarkers to assess the onset and progression of disease, risk states, monitor prevention strategies, provide early diagnosis and to track the prognosis of cancer. Our new observations in the regulation of epigenetic changes provide potential markers for assessing susceptibility to malignant transformation in individuals, as well as, potential targets for prevention and therapy.