Defining the Function of Age-Related Epimutation in Intestinal Tumorigenesis

项目摘要 表观突变--与DNA甲基化的表观遗传改变相关的有丝分裂稳定的基因沉默--是 现在被认为是人类癌症的共同特征。事实上，最近的表观基因组研究表明， 几乎所有的肿瘤类型都含有数百个异常高甲基化和沉默的启动子CpG岛， 相关（CGI相关）基因，强调需要确定驱动表型突变。在这方面我们 开发了一种新的肿瘤抑制基因p16（也称为细胞周期蛋白， 依赖性激酶抑制剂2A）。我们发现，基因工程启动子甲基化导致p16基因加速表达， 在衰老过程中小鼠体细胞组织中的表型突变。我们证明了p16表型突变使小鼠 到自发的肿瘤发展。此外，我们的初步工作表明，p16表位突变可以 与突变的Apc协同使用ApcMin/+小鼠加速肠肿瘤发生。重要的是我们 发现p16表位突变的功能超出了传统的细胞周期控制， 肠肿瘤的转化。因此，根据我们的初步数据，我们假设， p16表突变，通常在人散发性CRC中观察到，作为年龄相关表观遗传学改变的一部分， 与基因改变合作，推动肠癌的发生和发展。我们将通过1） 确定p16表型突变如何促进肠道肿瘤发生。我们将评估生物学 p16表型突变通过与两个关键信号通路的相互作用产生的后果：WNT-APC-β- catenin和MAPK-RAS-RAF; 2）确定逆转p16中的表观遗传缺陷是否抑制肿瘤 增长我们将通过应用基于CRISPR的逆转录聚合酶链反应来评估肿瘤对p16表型突变逆转的反应。 靶向p16启动子去甲基化;和3）确定转录因子是否作为p16启动子的阅读器。 甲基化DNA介导p16表型突变的功能。我们将研究分子事件， p16表观遗传沉默是DNA甲基化依赖的反式作用蛋白结合的结果， 随后募集染色质抑制复合物。这些研究将阐明异常的DNA 甲基化是调节肿瘤抑制基因表达的重要表观遗传机制， 结直肠肿瘤发生。从这一建议中得出的结果可能对设计产生独特的影响， 结肠癌表观遗传疗法。

PROJECT SUMMARY Epimutation – mitotically stable gene silencing associated with epigenetic alteration in DNA methylation – is now recognized as a common feature of human cancer. Indeed, recent epigenomic studies revealed that nearly all tumor types harbor hundreds of aberrantly hypermethylated and silenced promoter CpG island- associated (CGI-associated) genes, highlighting the need to identify driver epimutations. In this regard, we developed a novel mouse model of epimutation in a tumor suppressor gene p16 (also known as cyclin- dependent kinase inhibitor 2A). We found that engineered promoter methylation leads to accelerated p16 epimutation in mouse somatic tissues during aging. We demonstrated that p16 epimutation predisposes mice to spontaneous tumor development. In addition, our preliminary work shown that p16 epimutation can cooperate with mutational Apc to accelerate intestinal tumorigenesis using the ApcMin/+ mice. Importantly, we found that p16 epimutation functions beyond the classically known cell-cycle control to induce malignant transformation of intestinal neoplasms. Therefore, based on our strong preliminary data, we hypothesize that p16 epimutation, commonly observed in human sporadic CRCs as part of the age-related epigenetic alteration, cooperates with genetic alterations to drive intestinal cancer initiation and progression. We will test this by 1) Determine how p16 epimutation promotes intestinal tumorigenesis. We will assess the biological consequences of p16 epimutation through its interactions with two key signaling pathways: WNT-APC-β- catenin and MAPK-RAS-RAF; 2) Determine whether reversal of epigenetic defects in p16 suppresses tumor growth. We will evaluate tumor responses to the reversal of p16 epimutation by applying a CRISPR-based targeted p16 promoter demethylation; and 3) Determine whether transcription factors as a reader of methylated DNA mediate the function of p16 epimutation. We will investigate the molecular events by which p16 epigenetic silencing is a result from DNA methylation dependent trans-acting protein binding which subsequently recruits chromatin repressive complexes. These studies will elucidate how aberrant DNA methylation, an important epigenetic mechanism regulating the expression of tumor suppressor genes, drives colorectal tumorigenesis. The results derived from this proposal could have a unique impact in the design of colon cancer epigenetic therapies.