KRAS-regulated Ago2-mediated sorting of extracellular RNAs into exosomes

项目概要/摘要 大约30 - 50%的结直肠肿瘤在KRAS中携带激活的错义突变。晚期核内体- 衍生的外泌体通常从癌细胞中分泌并充当细胞-细胞通讯的介质。 已报道外泌体与肿瘤侵袭性和转移相关。我们有 先前报道，结肠直肠癌细胞的突变KRAS状态极大地影响细胞外的 外泌体的蛋白质和RNA组成。我们最近还报道了Argonaute 2（Ago2）和 在结肠癌细胞中，几种Ago2相关的miRNA与外泌体的结合受KRAS-MEK-Erk信号传导的控制。 具体地，丝氨酸387上Ago2的磷酸化阻止Ago2与多泡内体结合 (MVE)并分类成外泌体。这一发现确定了一种分子机制， 控制多种miRNA、miRNA相关蛋白和潜在的其他类型的RNA的分泌 转化为外泌体在这个提议的项目中，我将确定Argonaute 2在RNA分选中的确切作用， 外泌体，包括使用这种Ago2- 控制机制以及miRNA分选中是否存在选择性。我还将决定 其它RNA依赖于Ago2将它们分选到外来体中。最后，我将研究功能性后果 Ago2介导的KRAS依赖性结肠癌细胞RNA分泌对受体细胞基因表达的影响 和表型。 !

Project Summary/Abstract Approximately 30-50% of colorectal tumors carry activating missense mutations in KRAS. Late endosome- derived exosomes are often secreted from cancer cells and serve as mediators of cell-cell communication. Exosomes have been reported to be associated with tumor aggressiveness and metastasis. We have previously reported that the mutant KRAS status of colorectal cancer cells greatly affects the extracellular protein and RNA composition of exosomes. We also recently reported that sorting of Argonaute 2 (Ago2) and several Ago2-associated miRNAs to exosomes is controlled by KRAS-MEK-Erk signaling in colon cancer cells. Specifically, phosphorylation of Ago2 on serine 387 prevents Ago2 association with multivesicular endosomes (MVE) and sorting into exosomes. This finding identifies a molecular mechanism that has the potential to control the secretion of multiple miRNAs, miRNA-associated proteins, and potentially additional types of RNAs into exosomes. In this proposed project, I will identify the exact role of Argonaute 2 in sorting RNAs to exosomes, including determining what proportion of miRNAs are secreted in exosomes using this Ago2- controlled mechanism as well as whether there is selectivity in miRNA sorting. I will also determine whether other RNAs rely on Ago2 for their sorting into exosomes. Finally, I will investigate the functional consequence of Ago2-mediated RNA secretion from KRAS-dependent colon cancer cells on recipient cell gene expression and phenotypes both in vitro and in vivo. !