Translational Epigenomics in Gastrointestinal Cancer

项目摘要 胃肠道（GI）癌症是美国一个重大的公共卫生负担。在胃肠道癌症中，结肠直肠癌 (CRC)它一直是人们关注的焦点，因为它是美国第三大常见癌症。CRC产生于 息肉到癌症进展序列，由分子改变（基因突变和表观遗传学）驱动 改变），以及其他因素，包括肿瘤微环境，肠 微生物组等。这些分子改变的鉴定和确定其在病因学中的作用 CRC的行为不完整，正在积极调查中。另一种胃肠道癌症， 近年来，食管腺癌（EAC）引起了相当大的关注，因为它的三倍增加， 近几十年来，由于其预后差（20%的5年生存率）。与CRC一样，EAC的出现是次要的， 基因突变和表观遗传改变，导致一种称为Barretts的癌前病变的形成 食管（BE），然后可以进展为EAC。虽然在预防和控制艾滋病毒/艾滋病方面取得了进展， 尽管我们已经认识到胃肠道癌的治疗，但仍然有大量的需要来促进我们对分子生物学的理解。 胃肠道癌症的病理学，并开发生物标志物，将转化为改善癌症筛查计划， 更好的患者管理和结果。为了满足这一需求，研究专家Ming Yu博士 制定了本研究计划，以实现以下目标：1）表征和功能 在启动期间询问表观遗传改变，特别是DNA甲基化变化， GI癌症的进展; 2）评估甲基化基因作为潜在的分子生物标志物。本研究 该计划是研究计划的一个组成部分，该单位主任，博士威廉格雷迪，已在开发的 Fred哈钦森癌症研究中心（FHCRC）（PQC 1 R 01 CA 194663; PQ 6 R 01 CA 220004）和几家 多学科NCI资助的研究网络（早期检测研究网络（EDRN; U 01 CA 086389- 08），巴雷特食管转化研究网络（BETRNet; 5 U 54 CA 163060），和一个桥接的 间隙UO 1（5 U 01 CA 182940））。此外，该计划代表了于博士独特的调查路线， 已启动，并将在未来五年内发挥主导作用。这些新的研究包括表征 增强子甲基化组，即基因增强子元件处的DNA甲基化变化，在GI癌模仿过程中 和进展;肿瘤增强子的鉴定和功能验证，以及 来源于正常和癌性胃肠道组织的类器官，以研究癌症期间的DNA甲基化 启动和进展。这些方法将为现有的研究计划提供令人兴奋的补充。 博士Yu是本提案中的研究专家，是成功领导这些研究的首席科学家。 研究计划在格雷迪实验室在FHCRC，并已成功的核心决定因素， 在格雷迪实验室工作了几年余博士作为EDRN的“整合节点”科学家发挥着核心作用， BETRNET和其他多研究者团队项目。在本研究计划中，余博士将进行全面的 使用全基因组亚硫酸氢盐评估癌症发生和进展期间的DNA甲基化变化 测序（WGBS），其在单个CpG上提供跨基因组的前所未有的CpG覆盖率 分辨率她将整合WGBS数据与RNA-seq，CHIP-seq和相同样本的临床数据，以获得 深入了解候选肿瘤增强子的特征，并测试异常DNA 癌症中增强子区域的甲基化是癌症中异常甲基化调节基因表达的主要方式。 表情为了阐明候选肿瘤增强子的DNA甲基化是否影响靶基因表达， 她将开发基于CRISPR-Cas9的DNA甲基化编辑工具，以靶向增强子区域，并评估 正常和癌组织细胞中DNA甲基化改变的生物学相关性。值得注意的是， 建立了从原代结肠上皮和CRC培养三维类器官的方法， 迄今为止最相关的离体培养系统。此外，她还将发现和验证有前途的 甲基化基因生物标志物，将作为BE和EAC以及CRC领域癌变的生物标志物 的位点余博士将利用她在EDRN和BTRNET的富有成效的合作， 这些生物标志物检测。突出的是，她将应用一种新的技术MethyLight Droplet Digital PCR， 开发用于推进一组创新的结肠癌风险甲基化DNA标记物的鉴定， 称为“现场癌变标记物”。成功完成拟议的研究将导致新的见解， 表观遗传学改变在胃肠道癌分子发病机制中的作用。此外，这些发现将 转化为DNA甲基化生物标志物的发现，可以改善胃肠道癌症的管理。

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.