Translational Epigenomics in Gastrointestinal Cancer

项目总结 在美国，胃肠道(GI)癌症是一个重大的公共卫生负担。在胃肠道癌症中，结直肠癌 结直肠癌一直是人们关注的焦点，因为它是美国第三常见的癌症。CRC通过以下方式产生 息肉到癌症的进展序列，由分子改变(基因突变和表观遗传学)驱动 改变)，以及包括肿瘤微环境、肠道在内的其他因素 微生物组等。这些分子改变的鉴定和它们在病因学中的作用 并且CRC的行为不完整，正在积极调查中。又一例胃肠道癌症患者 食管腺癌(EAC)最近备受关注，因为它的发病率增加了三倍 近几十年来，由于预后较差(20%的5年存活率)。与华侨银行一样，选管会的地位次于 基因突变和表观遗传学改变导致一种称为Barretts的癌前疾病的形成 食道(BE)，然后可以进展到EAC。尽管在预防和预防艾滋病方面取得了进展 在胃肠道癌症的治疗方面，仍然有很大的需要推动我们对分子的理解 胃肠道癌症的病理学和开发生物标记物，这些生物标记物将转化为改进的癌症筛查计划， 更好的患者管理和结果。为了满足这一需求，研究专家于明博士 我制定了这项研究计划，以实现以下目标：1)特征和功能 询问表观遗传变化，特别是DNA甲基化的变化，在启动和 2)评估甲基化基因作为潜在的分子生物标志物。这项研究 该计划是该股主任威廉·格雷迪博士在 弗雷德·哈钦森癌症研究中心(FHCRC)(PQC1R01CA194663；PQ6R01CA220004)，以及几个 NCI资助的多学科研究网络(早期发现研究网络(EDRN；U01 CA086389- 08)，巴雷特食道翻译研究网络(BETRNet；5U54CA163060)，以及连接 GAP UO1(5U01CA182940))。此外，这项计划代表了于博士独特的调查路线 发起并将在未来五年内发挥领导作用。这些新的调查包括对 增强子甲基组，基因增强子元件上的DNA甲基化变化，在胃肠道癌症模拟过程中 和进展；肿瘤增强剂的鉴定和功能验证，以及开发 从正常和癌胃肠道组织中提取的有机化合物用于研究癌症过程中的DNA甲基化 开始和进展。这些方法将为现有的研究计划提供一个令人兴奋的补充。 余博士，这项建议的研究专家，是成功领导这些研究的首席科学家 在FHCRC格雷迪实验室的研究计划，一直是成功的核心决定因素 格雷迪实验室在过去几年里。俞敏洪博士在EDRN中扮演着核心角色--“整合节点”科学家， BETRNet和其他多个调查员团队项目。在这项研究计划中，于博士将进行全面的 使用全基因组亚硫酸氢盐评估癌症发生和发展过程中DNA甲基化的变化 测序(WGBS)，它在单个CpG上提供了前所未有的跨基因组CPGS覆盖 决议。她将WGBS数据与RNA-SEQ、CHIP-SEQ和相同样本的临床数据进行整合，以获得 洞察候选肿瘤增强子的特征，并测试导致DNA异常的创新假说 肿瘤增强子区域甲基化是肿瘤异常甲基化调控基因的主要方式 表情。为了阐明候选肿瘤增强子上的DNA甲基化是否会影响靶基因的表达， 她将开发基于CRISPR-Cas9的DNA甲基化编辑工具，以针对增强子区域并评估 正常组织和癌组织细胞DNA甲基化改变的生物学相关性。值得注意的是，她有 建立了从原代结肠上皮和结直肠癌培养三维有机体的方法，这代表了 迄今为止最相关的体外培养系统。此外，她还将发现并验证有前途的 甲基化基因生物标记物将作为BE和EAC的生物标记物，以及结直肠癌的现场癌变 精神错乱。Yu博士将利用她在EDRN和BTRNET方面的富有成效的合作，成功地开发出 这些生物标记物分析。值得注意的是，她将应用一项新技术--甲基光微滴数字聚合酶链式反应 开发的目的是推进一组创新的结肠癌风险甲基化DNA标记物的识别， 称为“野外癌变标记物”。成功完成拟议的研究将带来对以下问题的新见解 表观遗传学改变在胃肠道肿瘤分子发病机制中的作用此外，这些调查结果将是 转化为DNA甲基化生物标记物的发现，可以改善胃肠道癌症的治疗。

PROJECT SUMMARY Gastro-intestinal (GI) cancer is a significant public health burden in the US. Among GI cancers, colorectal cancer (CRC) has been a focus of attention because it is the third most common cancer in the US. CRC arises through a polyp to cancer progression sequence, driven by molecular alterations (gene mutations and epigenetic alterations) in the normal colon cells, as well as by other factors including the tumor microenvironment, gut microbiome, etc. The identification of these molecular alterations and determination of their role in the etiology and behavior of CRC is incomplete and under active investigation. Another GI cancer that has received considerable attention recently is esophageal adenocarcinoma (EAC) because of its three-fold increase in the last few decades and because of its poor prognosis (20% 5-year survival). Like CRC, EAC arises secondary to gene mutations and epigenetic alterations that drive the formation of a pre-malignant condition called Barretts esophagus (BE) that can then progress to EAC. Although progress has been made in the prevention and management of GI cancers, there remains a substantial need to advance our understanding of the molecular pathology of GI cancers and to develop biomarkers that will translate into improved cancer screening programs, better patient management and outcomes. In order to address this need, the Research Specialist Dr. Ming Yu has developed this research plan to achieve the following objectives: 1) Characterize and functionally interrogate epigenetic alterations, in particular DNA methylation changes, during the initiation and progression of GI cancer; 2) Assess methylated genes as potential molecular biomarkers. This research plan is an integral part of the research programs that the Unit Director, Dr. William Grady, has developed at the Fred Hutchinson Cancer Research Center (FHCRC) (PQC1R01CA194663; PQ6R01CA220004), and several multi-disciplinary NCI funded research networks (the Early Detection Research Network (EDRN; U01 CA086389- 08), the Barrett’s Esophagus Translational Research Network (BETRNet; 5U54CA163060), and a Bridging the Gap UO1 (5U01CA182940)). Furthermore, this plan represents unique lines of investigation that Dr. Yu has initiated and will be leading in the next five years. These new investigations include the characterization of enhancer methylomes, the DNA methylation changes at gene enhancer elements, during GI cancer imitation and progression; the identification and the functional validation of onco-enhancers, and development of organoids derived from normal and cancerous gastrointestinal tissues to study DNA methylation during cancer initiation and progression. These approaches will provide an exciting addition to the existing research programs. Dr. Yu, the Research Specialist in this proposal, is the principal scientist successfully leading the studies of these research programs in the Grady laboratory at the FHCRC and has been the central determinant of success of the Grady lab over the last few years. Dr. Yu has a central role as an “integrating node” scientist in the EDRN, BETRNET and other multi-investigator team projects. In this Research Plan, Dr. Yu will conduct a comprehensive assessment of DNA methylation changes during cancer initiation and progression, using whole genome bisulfite sequencing (WGBS), which provides unprecedented coverage of CpGs across genomes at single CpG resolution. She will integrate WGBS data with RNA-seq, CHIP-seq and clinical data of the same samples to gain insights into the features of a candidate onco-enhancer, and test the innovative hypothesis that aberrant DNA methylation at enhancer regions in cancer is the predominant way aberrant methylation in cancer regulates gene expression. To elucidate whether DNA methylation at candidate oncoenhancers affects target gene expression, she will develop CRISPR-Cas9 based DNA methylation editing tools to target enhancer regions and assess the biological relevance of DNA methylation alterations in cells from normal and cancerous tissues. Of note, she has established methods to culture 3-dimentional organoids from primary colon epithelium and CRC, which represent the most relevant ex vivo culture system to date. Moreover, she will also discover and validate promising methylated gene biomarkers that will function as biomarkers for BE and EAC, as well as CRC field cancerization loci. Dr. Yu will capitalize on her productive collaborations in the EDRN and BTRNET to successfully develop these biomarker assays. Prominently, she will apply a novel technology MethyLight Droplet Digital PCR that she developed to advance the identification of an innovative set of methylated DNA markers of colon cancer risk, termed ‘field cancerization markers’. Successful completion of the proposed studies will lead to novel insight into the roles of epigenetic alterations in molecular pathogenesis of GI cancer. Furthermore, these findings will be translated into the discovery of DNA methylation biomarkers that can improve the management of GI cancer.