Epigenetic Regulation of Medulloblastoma

描述(申请人提供)：本项目的主要目标是了解髓母细胞瘤形成过程中表观遗传学控制的两个重要方面，即癌症干细胞和实体瘤之间的差异，以及DNA甲基化依赖的基因沉默在肿瘤进展中的作用。我们将这两个问题联系在一起的实验原理是，我们观察到正常的小鼠胚胎干细胞不需要唯一的维持DNA甲基转移酶Dnmt1，而所有已知的体细胞都需要这种基因才能存活。人们越来越意识到多能基因对胚胎干细胞自我更新至关重要，这些基因可能在肿瘤干细胞中发挥重要的功能作用，这促使我们测试这一途径在髓母细胞瘤来源的脑瘤干细胞中的重要性。为了解决这个问题，我们利用了一个含有两个肿瘤抑制基因Ptch和P53突变的小鼠模型，当突变时，会导致高度穿透性和致命性的髓母细胞瘤。为了检验表观遗传控制在癌症干细胞中的作用，我们将首先对Ptch-P53髓母细胞瘤进行鉴定，以确定它们是否包含自我更新的癌症干细胞群体。然后，我们将测试这些髓母细胞瘤中的自我更新细胞是像其他体细胞一样依赖Dnmt1，还是像胚胎干细胞一样依赖Dnmt1。为了解决表观遗传沉默的作用，我们将利用Dnmt1的一个活性亚型等位基因，并询问基因组DNA低甲基化是诱导还是抑制髓母细胞瘤的形成。根据肿瘤的情况，Dnmt1亚型小鼠已经揭示了DNA低甲基化在促进染色体不稳定和防止异常基因沉默方面的重要作用。我们将使用Dnmt1亚型小鼠的遗传学方法，确定在髓母细胞瘤的进展中，是染色体机制还是表观遗传机制占优势。最后，我们最近证明，胚胎干细胞中的瞬时去甲基化导致印迹的全局丢失，并通过一种未知的机制在来源的小鼠成纤维细胞中丢失P53的表达。我们从易患髓母细胞瘤的Ptch小鼠中提取ES细胞，以便在干细胞中进行一过性去甲基化，并测试表观遗传学机制是否会导致P53基因缺失，并促进髓母细胞瘤体内肿瘤的发展。这些结果将扩展先前的结果，即全球印记的丢失可以促进肿瘤的发生，并为剖析暂时性表观遗传变化对细胞生长调节和体内肿瘤易感性产生长期影响的机制提供了一个模型。公共卫生相关性：这项建议测试了四个关于表观遗传学改变对髓母细胞瘤发生和发展的贡献的假说。我们问，癌症干细胞是否与胚胎干细胞相似，因为它们不依赖DNA甲基转移酶Dnmt1，以及全球瞬时去甲基化是否通过促进癌症干细胞的扩张而促进癌症？我们通过询问基因沉默是否有助于肿瘤的启动或进展来测试表观遗传改变的贡献，以及印记基因是否是这些儿科脑瘤中放松调控的表观遗传靶点的关键功能类别？对这些问题的回答应该有助于洞察最常见的儿童脑瘤的起源，并可能影响对癌症表观遗传学的更广泛理解。

DESCRIPTION (provided by applicant): The overarching goal of this project is to understand two important aspects of epigenetic control during medulloblastoma formation, namely the difference between cancer stem cells and bulk tumor, and the role of DNA methylation dependent gene silencing in tumor progression. Our experimental rationale that connects these two questions is an observation we made that normal mouse embryonic stem cells do not require the sole maintenance DNA methyltransferase Dnmt1, whereas all known somatic cells tested require this gene for survival. The increasing awareness that pluripotency genes, those genes important for embryonic stem cell self-renewal, may play an important functional role in cancer stem cells encourages us to test the importance of this pathway in brain tumor stem cells derived from medulloblastoma. To approach this question, we are utilizing a mouse model harboring mutations in two tumor suppressor genes, Ptch and p53, that when mutated lead to a highly penetrant and lethal medulloblastoma. To examine the role for epigenetic control in cancer stem cells we will first characterize Ptch-P53 medulloblastomas to determine whether they contain a self- renewing cancer stem cell population. We will then test whether the self-renewing cells within these medulloblastomas are Dnmt1 dependent like other somatic cells or are Dnmt1 independent like embryonic stem cells. To address the role of epigenetic silencing, we will utilize a viable hypomorphic allele of Dnmt1 and ask whether genomic DNA hypomethylation induces or suppresses medulloblastoma formation. Depending on tumor context, Dnmt1 hypomorphic mice have revealed important roles for DNA hypomethylation in promoting chromosomal instability and alternatively in preventing aberrant gene silencing. We will determine whether chromosomal or epigenetic mechanisms prevail in medulloblastoma progression using a genetic approach with Dnmt1 hypomorphic mice. Finally, we have recently demonstrated that transient demethylation in embryonic stem cells leads to global loss of imprinting, and loss of p53 expression in derived mouse fibroblasts through an unknown mechanism. We have derived ES cells from medulloblastoma prone Ptch mice in order to carry out transient demethylation in stem cells and test whether epigenetic mechanisms lead to loss of p53 and promote medulloblastoma tumor progression in vivo. These results will extend prior results that global loss of imprinting can promote tumorigenesis, and provide a model to dissect the mechanism through which transient epigenetic changes exert long term effects on cell growth regulation, and tumor predisposition in vivo. PUBLIC HEALTH RELEVANCE: This proposal tests four hypotheses regarding the contribution of epigenetic alterations to medulloblastoma initiation and progression. We ask are cancer stem cells similar to embryonic stem cells in their lack of dependence on the DNA methyltransferase Dnmt1, and does global transient demethylation promote cancer by potentiating cancer stem cell expansion? We test the contribution of epigenetic alterations by asking does gene silencing contribute to tumor initiation or progression, and are imprinted genes a key functional class of epigenetic targets that become deregulated in these pediatric brain tumors? Answers to these questions should yield insight into the origins of the most common pediatric brain tumor, and will likely impact a broader understanding of cancer epigenetics.