The Role of DNA Methylation in Tumor Maintenance

描述（由申请人提供）：在过去的十年中，表观遗传学在癌症中的作用引起了人们的兴趣。启动子甲基化导致肿瘤抑制基因沉默是促进肿瘤发生的关键表观遗传机制之一。该申请的目标是扩大我们对DNA甲基化在肿瘤维持中的作用的理解，通过降低已建立的结直肠肿瘤中的DNA甲基化，使用一种创新的小鼠模型系统，其中DNA甲基化过程可以通过内源性Dnmt1 DNA甲基转移酶基因的转录抑制而被可逆和紧密地抑制。第一个具体目标是确定DNA甲基化在结直肠肿瘤维持中的作用。我们的转录抑制技术使我们能够规避常规和条件敲除Dnmt1所带来的细胞致命性，并使我们能够产生第一个具有条件和可逆转录抑制内源性Dnmt1能力的小鼠模型。我们的技术在肿瘤发生后抑制DNA甲基化过程的能力，将允许肿瘤在正常的表观遗传影响下发展，允许在不影响肿瘤起始的情况下评估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。使用DNA甲基化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。在严格的组织控制环境下暂时操纵DNA甲基化水平的能力将为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提供独特的能力。第二个具体目标是全面绘制参与结直肠肿瘤维持和发展的异常DNA甲基化变化，并通过与人类原发性结直肠肿瘤的比较来确定候选的表观遗传驱动事件。全基因组霰弹枪亚硫酸氢盐测序（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绘制肿瘤特异性DNA甲基化变化和伴随的基因表达变化。在TCGA的背景下，在实验室中产生的500个人类原发性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提出的小鼠研究中获得的结果进行广泛的验证。这一拟议的应用可能会对表观遗传对已建立肿瘤维持的贡献产生有价值的见解，并将产生涉及结肠直肠肿瘤发展和维持的表观遗传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.