Cellular Epigenetic Heterogeneity as a Predeterminant of Malignant Transformation Potential

项目概要/摘要 克隆扩增是癌症的一个关键特征，被认为是由基因改变启动的。 一个关键的看门人驱动基因。然而，并不是所有的正常细胞都容易受到恶性肿瘤的影响。 在这样的事件发生后，多种证据表明， 正常细胞可能会影响其引发癌症的潜力。这是一个特别具有挑战性的概念， 由于没有直接的方法来先验地鉴定哪些细胞可能对 在克隆扩增之前进行转化。我们提出了一系列创新的实验来测试 假设正常细胞中预先存在的表观遗传异质性影响恶性肿瘤的可能性， 在癌症驱动基因的遗传改变后的转化和进一步进展。 在具体目标1中，我们将使用单细胞全基因组亚硫酸氢盐测序的改进方案， 记录流式分选的小鼠结肠干细胞中DNA甲基化异质性的程度。为 这将首次提供了解在正常人中观察到的低水平DNA甲基化异常是否 组织是随机分布的，或者干细胞亚群是否具有多个协同的表观遗传 异常在特定目标2中，我们将首先产生大量的个体克隆扩增小鼠， 结肠类器官含有Apc、Trp 53、Kras和Braf的条件等位基因。每个类器官将 冷冻保存并同时进行全基因组亚硫酸氢盐测序以描绘每个甲基化组。 代表不同甲基化组的类器官将被解冻，癌症驱动因子被激活，然后进行测试。 通过结肠灌肠植入的致瘤性。这一创新实验旨在提供第一个直接 预先存在的表观遗传异质性对癌症易感性的贡献的证据。具体目标 3，我们将通过使用一种新的体内转录控制系统， 在激活条件键之前，瞬时上调或下调小鼠中的DNA甲基转移酶活性 癌症司机这一目标应该为先前存在的表观遗传变异的因果作用提供概念证明 影响癌症倾向。 这一建议涉及一个概念，有相当多的间接证据，但仍然是一个 由于前提所提出的技术和概念挑战，研究不足的领域。我们 拟议的实验是错综复杂的，但我们有相当多的专业知识，在所有领域的 该公司提出了一项建议，并为许多技术障碍开发了尖端的解决方案。我们可能无法 结论性地详细描述了所有易患恶性肿瘤的表观遗传变异，但这种探索性的 该项目应该为癌症中预先存在的表观遗传异质性的重要性提供原则证明 易感性这项研究的结果应该对我们对生命的理解产生重大影响。 癌症风险的累积，对癌症筛查和预防的影响。

PROJECT SUMMARY / ABSTRACT Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. This is a particularly challenging concept to investigate, since there is no straightforward way a priori to identify which cells may be susceptible or resistant to transformation prior to clonal expansion. We propose an innovative series of experiments to test the hypothesis that pre-existing epigenetic heterogeneity among normal cells affects the likelihood of malignant transformation and further progression upon genetic alteration of a cancer driver gene. In Specific Aim 1, we will use an improved protocol for single-cell whole genome bisulfite sequencing to document the degree of DNA methylation heterogeneity among flow-sorted mouse colon stem cells. For the first time, this will provide insight into whether low-level DNA methylation abnormalities observed in normal tissues are stochastically distributed, or whether subsets of stem cells bear multiple concerted epigenetic abnormalities. In Specific Aim 2, we will first generate large numbers of individual clonally expanded mouse colon organoids harboring conditional alleles for Apc, Trp53, Kras and Braf. Each organoid will be cryopreserved and concurrently subjected to whole genome bisulfite sequencing to delineate each methylome. Organoids representing diverse methylomes will be thawed, the cancer drivers activated, and then tested for tumorigenicity by colon enema engraftment. This innovative experiment aspires to provide the first direct evidence for the contribution of pre-existing epigenetic heterogeneity to cancer predisposition. In Specific Aim 3, we will provide empirical evidence for this concept by using a novel in vivo transcriptional control system to transiently up- or down-regulate DNA methyltransferase activity in mice, prior to activation of conditional key cancer drivers. This aim should provide a proof of concept for a causal role of pre-existing epigenetic variation affecting cancer propensity. This proposal addresses a concept for which there is considerable indirect evidence, but which remains a poorly studied area because of the technical and conceptual challenges presented by the premise. Our proposed experiments are intricate and complex, but we have considerable expertise in all areas of the proposal, and have developed cutting-edge solutions to many of the technical hurdles. We may not be able to conclusively delineate in detail all epigenetic variations that predispose to malignancy, but this exploratory project should provide a proof of principle for the importance of pre-existing epigenetic heterogeneity in cancer susceptibility. The outcome of this study should have a substantial impact on our understanding of lifetime accumulation of cancer risk, with implications for cancer screening and prevention.