The Role of DNA Methylation in Tumor Maintenance

描述(申请人提供)：在过去的十年里，人们对表观遗传学在癌症中的作用产生了极大的兴趣。启动子甲基化导致的抑癌基因沉默是肿瘤发生的重要表观遗传学机制之一。这项拟议应用的目的是通过减少已建立的结直肠肿瘤中的DNA甲基化来扩大我们对DNA甲基化在肿瘤维持中作用的理解，使用一种创新的小鼠模型系统，在该系统中，DNA甲基化过程可以通过转录抑制内源Dnmt1 DNA甲基转移酶基因而被可逆地和紧密地抑制。第一个具体目标是确定DNA甲基化在维持结直肠肿瘤中的作用。我们的转录抑制技术使我们能够避开常规和有条件敲除Dnmt1造成的细胞死亡，并使我们能够产生第一个具有有条件和可逆地转录抑制内源性Dnmt1的小鼠模型。我们的技术能够在肿瘤发生后抑制DNA甲基化过程，这将使肿瘤能够在正常的表观遗传影响下发展，从而能够在不影响肿瘤启动的情况下评估DNA甲基化在肿瘤维持中的作用。通过转录抑制而不是使用有毒化合物来抑制DNA甲基化将允许对DNA甲基化的作用进行更具体的评估。利用已经证实DNA甲基化作用的小鼠癌症模型，将增加观察到有希望的抗肿瘤反应的可能性。在严密的组织控制环境中对DNA甲基化水平进行时间控制的能力将提供独特的能力，以研究表观遗传学在肿瘤起始、进展、侵袭和转移的特定时间窗口中的作用。第二个具体目的是全面定位参与结直肠癌维持和发展的异常DNA甲基化变化，并通过与人类原发结直肠癌的比较来识别候选的表观遗传驱动事件。全基因组鸟枪亚硫酸盐测序(WGSBS)和基因表达微阵列分析相结合的基因组学方法将使我们能够有效地定位肿瘤特异性DNA甲基化变化与伴随的基因表达变化。在TCGA的背景下，实验室生产的500个人类原发结直肠癌样本的全面全基因组DNA甲基化数据将使我们能够对我们拟议的小鼠研究中获得的结果进行广泛的验证。这项拟议的应用可能会对表观遗传学对维持已建立的肿瘤的贡献产生有价值的见解，并将产生涉及结直肠肿瘤发展和维持的表观遗传学改变的全基因组视图。

DESCRIPTION (provided by applicant): The past decade has seen an explosion of interest in the role of epigenetics in cancer. Tumor suppressor gene silencing by promoter methylation is one of the key epigenetic mechanisms that contributes to tumorigenesis. The goal of the proposed application is to expand our understanding of the role of DNA methylation in tumor maintenance, by reducing DNA methylation in established colorectal tumors, using an innovative mouse model system in which the DNA methylation process can be reversibly and tightly inhibited through transcriptional repression of the endogenous Dnmt1 DNA methyltransferase gene. The first specific aim is to determine the role of DNA methylation in the maintenance of colorectal tumors. Our transcriptional repression technology enables us to circumvent cell lethality imposed by both conventional and conditional knock-out of Dnmt1, and allows us to produce the first mouse model with the capacity to conditionally and reversibly transcriptionally repress endogenous Dnmt1. The ability of our technology to suppress the DNA methylation process after the tumor has developed will allow tumors to develop under normal epigenetic influence, permitting an assessment of the role of DNA methylation in tumor maintenance without affecting tumor initiation. The inhibition of DNA methylation through transcriptional repression rather than the use of toxic compounds will allow a more specific assessment of the role of DNA methylation. The use of mouse cancer models for which the contribution of DNA methylation has been demonstrated will increase the likelihood of observing promising anti-neoplastic responses. The ability to temporally manipulate DNA methylation levels in tightly tissue-controlled context will provide unique abilities to investigate the role o epigenetics at defined time windows of tumor initiation, progression, invasion, and metastasis. The second specific aim is to comprehensively map aberrant DNA methylation changes involved in the colorectal tumor maintenance and development, and to identify candidate epigenetic driver events by comparison to human primary colorectal tumors. A combined genomics approach of whole genome shotgun bisulfite sequencing (WGSBS) and gene expression microarray analysis will allow us to efficiently map tumor- specific DNA methylation changes with an accompanying gene expression change. Comprehensive genome- wide DNA methylation data of 500 human primary colorectal cancer samples being produced in the lab in the context of TCGA will allow us to perform extensive validation of results obtained in our proposed mouse study. This proposed application will likely yield valuable insights into epigenetic contributions to the maintenance of established tumors, and will produce a genome-wide view of epigenetic alterations involved in the development and maintenance of colorectal tumors.