Trafficking of eRNAs in K-Ras Mutant Colon Cancer

K-Ras 突变结肠癌中 eRNA 的贩运

• 批准号: 8588220
• 负责人: Robert J. Coffey
• 金额: $ 39.57万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588220
• 关键词: Affect; Behavior; Biogenesis; Cancer Model; Cells; Ceramides; Cholesterol; Clinical Markers; Coculture Techniques; Colon Carcinoma; Complex; Data; Deposition; Flow Cytometry; Gene Activation; Gene Silencing; Generations; Immune; KRAS2 gene; Lead; Link; Lipoproteins; Luciferases; Mediating; Membrane Microdomains; Messenger RNA; MicroRNAs; Microscopy; Modeling; Modification; Multivesicular Body; Neoplasm Metastasis; Oncogenic; Pathway interactions; Pattern; Population; Process; Proteins; RNA; RNA Processing; RNA Sequences; RNA Transport; RNA-Binding Proteins; Regulation; Reporter; Role; Secretory Vesicles; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Sphingomyelinase; Staging; Technology; Testing; Tumor Biology; Ubiquitination; Vesicle; base; cancer cell; cholesterol trafficking; colon cancer cell line; extracellular; intercellular communication; late endosome; mutant; new technology; next generation sequencing; novel; particle; receptor; scaffold; trafficking; tumor; tumor microenvironment; tumorigenesis

(Project Leader: Alissa Weaver) Exosomes are late endosomal-derived secretory vesicles that are frequently secreted by cancer cells and have been linked to tumor aggressiveness and metastasis. In addition to carrying proteins, exosomes are enriched in a variety of extracellular RNAs (eRNAs), including miRNAs and mRNAs. A major unanswered question is whether and how cellular signaling affects the export of eRNAs into exosomes and other described RNA transport vehicles. Recent advances in sequencing technologies combined with advanced intracellular microscopy and flow cytometry vesicle sorting approaches make it possible to perform in-depth analysis of the specificity and regulation of eRNA export. These studies should have a profound impact on our understanding of eRNA secretion mechanisms and tumor biology. Using an isogenically matched colon cancer cell line model of KRAS signaling, we recently found that an oncogenic mutant of KRAS selectively affects both the protein and RNA content of colon cancer cell exosomes. In addition, we find that mutant KRAS-expressing cells have differential import of a model miRNA into late endosomes. Finally, we find that KRAS mutant cell exosomes have increased content of lipid raft-associated proteins, suggesting differential control of exosome biogenesis and cholesterol trafficking. Based on these data, we hypothesize that KRAS signaling selectively controls the sorting of specific RNAs and proteins into exosomes. To test this hypothesis, we propose to comprehensively analyze the eRNA content from the isogenically matched KRAS colon cancer cell lines and determine whether specific sorting mechanisms exist to control eRNA export. We will also identify how K-RAS signaling controls two critical regulatory points in RNA secretion: subcellular localization of RNA machineries to late endosomes and exosome biogenesis pathways.

(项目负责人：Alissa Weaver)Exosome是一种晚期内体衍生的分泌小泡，通常由癌细胞分泌，与肿瘤的侵袭性和转移有关。除了携带蛋白质外，外切体还 富含多种胞外RNA，包括miRNAs和mRNAs。一个未获答复的重大问题 问题是细胞信号是否以及如何影响eRNA输出到外体和其他 描述了RNA运输工具。与先进测序技术相结合的最新进展 细胞内显微镜和流式细胞术囊泡分选方法使深入进行 二是分析额尔纳出口的特殊性及其调控。这些研究应该会对 我们对Erna分泌机制和肿瘤生物学的理解。使用同源匹配的冒号 KRAS信号转导的癌细胞模型，我们最近发现KRAS的一个癌基因突变体选择性地 影响结肠癌细胞外切体的蛋白质和RNA含量。另外，我们发现那个突变体 表达KRAS的细胞有差异地将模型miRNA输入到晚期内吞体内。最后，我们发现， KRAS突变细胞外切体增加了脂筏相关蛋白的含量，提示存在差异 控制外切体的生物发生和胆固醇的运输。根据这些数据，我们假设KRAS 信号选择性地控制特定的RNA和蛋白质进入外切体的分选。为了测试这一点 假设，我们建议从同源匹配的KRAS中综合分析Erna含量 并确定是否存在控制Erna输出的特定分选机制。我们 还将确定K-RAS信号如何控制RNA分泌的两个关键调节点：亚细胞 RNA机制在晚期内体和外体生物发生途径中的定位。