Project 3 - Long Intergenic Non-Coding RNAs in the Malignant Progression of Barrett's Esophagus

项目总结/摘要 近年来，食管腺癌（EAC）的发病率以惊人的速度（>500%）增加， 几十年来，远远超过任何其他癌症类型，在美国。EAC患者的预后仍然是 穷人，治疗选择非常有限。虽然Barrett食管（BE），远端的柱状化生， 食管上皮是EAC唯一已知的前体，然而绝大多数BE患者不 发生发育不良或癌症;因此，驱动BE进展为EAC的因素仍然难以捉摸。我们 长期目标是阐明EAC进展的潜在机制， 可以开发出针对性的治疗方法来有效地控制这种致命的疾病。近日，利用 在BE相关病变的创新RNA测序中，我们鉴定了两个大的基因间非编码RNA 在~50%的EAC病变中显示出显著和选择性诱导的lincRNA。两种lincRNA均表现出细胞核 定位和初步的功能评估强烈表明这些lincRNA发挥促肿瘤发生作用， 在EAC进程中发挥作用。因此，我们的研究提供了这种疾病中lincRNA的第一个全球分析， 鉴定两种在食管癌发生中具有潜在致癌作用的新lincRNA。因此 目前建议的具体目标是：（目的1）阐明候选EAC相关的功能， lincRNA。我们将产生基于CRISPR/Cas9的诱导型lincRNA敲除EAC细胞系模型， 使用体外和体内两种方法全面表征候选lincRNA的表型效应 异种移植实验系统;（目的2）解剖上游的分子调控网络， EAC相关lincRNA的下游。我们将评估潜在的基因组/表观基因组机制 在EAC中驱动lincRNA表达;使用EAC细胞系模型，我们将进行全局基因表达 微阵列分析来描绘由lincRNA调节的基因/途径;并且我们将使用ChIRP-seq/- MS方法来绘制基因组占有率，并确定潜在的基因靶点和蛋白质伴侣， 候选lincRNA，从而建立候选lincRNA的调控路线图; EAC进展期间候选lincRNA中的时间和阶段相关失调。我们的初步 结果显示，两种lincRNA在高度异型增生（HGD）中被诱导，HGD是一种组织病理学替代物， EAC风险。因此，我们将验证和确定lincRNA失调的频率，在这些公开的研究中， 疾病进展的癌前阶段。此外，我们还将检测：非发育异常BE病变， 显示这些候选lincRNA的诱导;以及候选lincRNA是否 在非异型增生BE/低度异型增生（LGD）粘膜中显示早期诱导，来源于高风险患者 在随访中患上癌症的病人这些研究的成功将揭示分子机制 促进EAC进展;能够开发早期癌症的循证分子生物标志物 检测和监测;并为这种日益致命的癌症的靶向治疗开辟新的途径。

Project Summary/Abstract The incidence of esophageal adenocarcinoma (EAC) has increased at an alarming rate (>500%) in the last few decades, far exceeding any other cancer type, in the United States. The prognosis for EAC patients remains poor with very limited treatment options. Although Barrett’s esophagus (BE), a columnar metaplasia of the distal- esophagus epithelium, is the only known precursor of EAC, the vast majority of BE patients however do not develop dysplasia or cancer; consequently the factors driving progression from BE to EAC remain elusive. Our long-term objective is to elucidate the mechanisms underlying EAC progression, such that reliable biomarkers and targeted therapies can be developed for effective management of this deadly disease. Recently, using innovative RNA sequencing in BE-associated lesions, we identified two large intergenic non-coding RNAs (lincRNAs) showing marked and selective inductions in ~50% of EAC lesions. Both lincRNAs exhibited nuclear localization, and preliminary functional assessments strongly suggested these lincRNAs to play pro-tumorigenic roles during EAC progression. Our study thus provides the first global analysis of lincRNAs in this disease, identifying two novel lincRNAs with potential oncogenic roles in esophageal carcinogenesis. Accordingly, the specific goals of the current proposal are: (AIM 1) To elucidate the function of candidate EAC-associated lincRNAs. We will generate CRISPR/Cas9-based inducible lincRNA-knockout EAC cell line models to comprehensively characterize the phenotypic effects of candidate lincRNAs using both in vitro, and in vivo xenograft experimental systems; (AIM 2) To dissect the molecular regulatory networks upstream and downstream of EAC-associated lincRNAs. We will evaluate for potential genomic/epigenomic mechanisms driving lincRNA expression in EACs; using the EAC cell line models, we will perform global gene expression microarray profiling to delineate the genes/pathways modulated by the lincRNAs; and we will use ChIRP-seq/- MS approaches to map the genomic occupancy and to identify potential gene targets and protein partners of candidate lincRNAs, thus establishing a regulatory-roadmap of candidate lincRNAs; (AIM 3) To determine the timing and stage-associated deregulations in candidate lincRNAs during EAC progression. Our preliminary findings showed both lincRNAs being induced in high-grade dysplasia (HGD), a histopathologic surrogate for EAC risk. Accordingly, we will validate and ascertain the frequency of lincRNA deregulations in these overtly pre-malignant phases of disease progression. Additionally, we will test: whether non-dysplastic BE lesions, within close proximity to EAC, show induction of these candidate lincRNAs; and whether candidate lincRNAs show induction early-on in non-dysplastic BE/low-grade dysplasia (LGD) mucosa, derived from high-risk patients who developed cancer during follow-up. Success in these studies will uncover molecular mechanisms contributing to EAC progression; enable development of evidence-based molecular biomarkers for early cancer detection and surveillance; and open new avenues for targeted therapies in this increasingly fatal cancer.