Characterization of growth factor control of Argonaute 2 and RAS interaction

项目概要和摘要 RAS基因家族是人类癌症中最常见的突变基因之一， 含有RAS基因的突变三种RAS基因（KRAS、NRAS和HRAS）在细胞凋亡中起核心作用。 信号转导作为GT3开关，RAS蛋白的突变导致组成型活性， 致癌转化尽管经过多年的深入研究， 在临床上靶向突变型RAS，使新的RAS相互作用物的鉴定成为一种潜在的有吸引力的策略 用于靶向治疗的发展。最近的研究表明，N Argonaute 2末端结构域（AGO 2）是RNA诱导沉默复合物（RISC）的核心成分， 跨野生型（WT）和突变型KRAS表达细胞系的KRAS的开关II结构域。而且这 相互作用对KRAS介导的生长具有重要的功能。KRAS中AGO 2的稳定敲除 依赖性细胞导致KRAS蛋白表达减少，随后细胞凋亡减少。 增殖此外，与突变型KRAS的相互作用通过阻止AGO 2的表达来抑制AGO 2的RNAi功能。 microRNA解旋。 本研究将进一步探讨生长因子对AGO 2-RAS的调节作用， 生理学和癌症生物学EGFR激活被证明减弱AGO 2和 野生型RAS在正常细胞中通过AGO 2磷酸化，但突变型KRAS对生长因子调节具有抗性。 尽管生长因子信号在AGO 2-RAS生物学中具有潜在的调节作用， 这种调节的功能作用是未知的。在这里，我们建议研究AGO 2-RAS-EGFR轴，通过 以下目的：具体目的1：EGFR介导的AGO 2磷酸化改变RAS-AGO 2相互作用， 可能导致RAS活性和细胞定位的变化。在这里，我们将评估EGFR对AGO 2的调节作用。 在正常生理学和癌症生物学中的RAS磷酸化、RAS定位和RAS-GTP负载。具体 目的2：鉴于已知突变KRAS抑制AGO 2 miRNA解旋，iCLIP-seq将在 AGO 2磷酸突变体和/或突变KRAS的存在，以确定AGO 2 miRNA加工的变化 并在生长因子刺激后靶向。我们还将确定生长因子刺激的效果 全球AGO 2 miRNA加工，靶向和基因调控。具体目标3：初步研究 表明AGO 2的缺失导致pEGFR和RAS活性的增加，尽管血清饥饿， 提示AGO 2、RAS和EGFR之间存在重要的双向信号传导和串扰。我们将 表征AGO 2-RAS-EGFR轴及其在细胞存活和生长中的作用。 总之，本项目的结果有望提高对生长因子调控的理解 AGO 2-RAS在癌症生物学中的作用，并可能为治疗突变RAS驱动的癌症提供临床靶点。

Project Summary and Abstract The RAS gene family is among the most commonly mutated genes in human cancer with nearly 30% containing a mutation in a RAS gene. The three RAS genes (KRAS, NRAS, and HRAS) play a central role in cell signal transduction as GTPase switches, and mutations in RAS proteins lead to constitutive activity resulting in oncogenic transformation. Despite years of intensive research, little progress has been made in successfully targeting mutant RAS clinically, making the identification of novel RAS interactors a potentially attractive strategy for the development of targeted therapeutics. Recent studies have identified an interaction between the N terminal domain of Argonaute 2 (AGO2), a core component of RNA-induced silencing complex (RISC), and the Switch II domain of KRAS across both wild-type (WT) and mutant KRAS expressing cell lines. Furthermore, this interaction was functionally important to KRAS-mediated growth. Stable knockdown of AGO2 in KRAS dependent cells led to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. In addition, interaction with mutant KRAS inhibits the RNAi function of AGO2 by preventing microRNA unwinding. This study will further investigate the role of growth factor regulation of AGO2-RAS in both normal physiology and cancer biology. EGFR activation was demonstrated to attenuate the interaction of AGO2 and WT RAS in normal cells by AGO2 phosphorylation, but mutant KRAS was resistant to growth factor regulation. Despite the potential regulatory role for growth factor signaling in AGO2-RAS biology, the precise mechanism and functional role of this regulation is unknown. Here we propose to study the AGO2-RAS-EGFR axis through the following Aims: Specific Aim 1: EGFR-mediated phosphorylation of AGO2 alters RAS-AGO2 interaction that may lead to changes in RAS activity and cellular localization. Here, we will assess EGFR regulation of AGO2 phosphorylation, RAS localization, and RAS-GTP loading in normal physiology and cancer biology. Specific Aim 2: Given that mutant KRAS is known to inhibit AGO2 miRNA unwinding, iCLIP-seq will be performed in the presence of AGO2-phosphomutants and/or mutant KRAS to determine changes in AGO2 miRNA processing and targeting following growth factor stimulation. We will also determine the effect of growth factor stimulation on global AGO2 miRNA processing, targeting, and gene regulation. Specific Aim 3: Preliminary studies demonstrated that loss of AGO2 leads to an increase in pEGFR and RAS activity despite serum starvation, suggesting an important bidirectional signaling and crosstalk between AGO2, RAS and EGFR. We will characterize the AGO2-RAS-EGFR axis and its role in cell survival and growth. In summary, the results of this project are expected to improve understanding of growth factor regulation of AGO2-RAS in cancer biology and may provide clinical targets to treat mutant RAS-driven cancers.