Cellular Epigenetic Heterogeneity as a Predeterminant of Malignant Transformation Potential

项目摘要/摘要 克隆性扩张是癌症的一个关键特征，被认为是由 关键的看门人驱动基因。然而，并不是所有的正常细胞似乎都对恶性肿瘤敏感。 在这样的事件之后发生的转变。多条证据表明，表观遗传异质性 正常细胞可能会影响它们的致癌潜力。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识别哪些细胞可能对 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 关于正常细胞中先前存在的表观遗传异质性影响恶性可能性的假说 癌症驱动基因的遗传改变的转化和进一步进展。 在具体目标1中，我们将使用一种改进的单细胞全基因组亚硫酸氢盐测序方法来 记录流式分选小鼠结肠干细胞间DNA甲基化异质性的程度。对于 这将首次深入了解在正常情况下观察到的低水平DNA甲基化异常 组织是随机分布的，或者干细胞亚群是否具有多个协调一致的表观遗传 异常现象。在特定目标2中，我们将首先生成大量个体克隆扩展小鼠 含有APC、TrP53、Kras和BRAF条件等位基因的结肠有机化合物。每一个有机化合物都将是 超低温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组。 代表不同甲基组的有机化合物将被解冻，癌症驱动因素被激活，然后测试 结肠灌肠植入法的致瘤性。这一创新的实验渴望提供第一个直接 先前存在的表观遗传异质性对癌症易感性的贡献的证据。以特定的目标 3，我们将通过使用一种新的体内转录控制系统来为这一概念提供经验证据 在激活条件键之前，瞬时上调或下调小鼠DNA甲基转移酶活性 癌症驱动因素。这一目的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。 这项提议涉及一个概念，有相当多的间接证据，但它仍然是一个 由于技术和概念方面的挑战，这一领域的研究很少。我们的 拟议的实验是错综复杂的，但我们在所有领域都有相当多的专业知识 此外，还提出了解决许多技术障碍的尖端解决方案。我们可能不能 最终详细描述了所有易患恶性肿瘤的表观遗传变异，但这一探索性的 项目应为癌症中先前存在的表观遗传异质性的重要性提供原则性证明。 敏感度。这项研究的结果应该会对我们对一生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.