Characterization of growth factor control of Argonaute 2 and RAS interaction

项目摘要和摘要 RAS基因家族是人类癌症中最常见的突变基因之一，有近30%的突变 含有RAS基因突变的。三个RAS基因(KRAS、NRAS和HRAS)在细胞中起核心作用 作为GTP酶开关的信号转导，以及RAS蛋白的突变导致结构性活动 致癌转化。尽管进行了多年的密集研究，但成功的研究进展甚微。 临床靶向突变RAS，使鉴定新的RAS相互作用因子成为一个潜在的有吸引力的策略 用于靶向治疗的发展。最近的研究已经确定了N和N之间的相互作用 RNA诱导沉默复合体(RISC)的核心成分ArgAerte2(AGO2)的末端结构域，以及 在野生型(WT)和突变型KRAS表达细胞系之间切换KRAS的II结构域。此外，这一点 相互作用对KRAS介导的生长具有重要作用。在KRAS中稳定击倒AGO2 依赖细胞导致KRAS蛋白表达减少，随后细胞减少 扩散。此外，与突变体KRAS的相互作用通过阻止AGO2的RNAi功能来抑制 解开微核糖核酸。 本研究将进一步探讨生长因子调节AGO2-RAS在两组正常人群中的作用。 生理学和癌症生物学。EGFR的激活可以减弱AGO2和AGO2的相互作用 正常细胞中的WT RAS通过AGO2的磷酸化，而突变的KRAS对生长因子的调节是抵抗的。 尽管生长因子信号在AGO2-RAS生物学中具有潜在的调节作用，但其确切的机制 而这一规定的功能作用尚不清楚。在这里，我们建议通过以下途径研究AGO2-RAS-EGFR轴 具体目标1：EGFR介导的AGO2磷酸化改变RAS-AGO2相互作用 可能导致RAS活性和细胞定位的改变。在这里，我们将评估EGFR对AGO2的调节 正常生理学和癌症生物学中的磷酸化、RAS定位和RAS-GTP负载。特定的 目的2：鉴于突变的KRAS已知可以抑制AGO2 miRNA的解离，iCLIP-seq将在 AGO2-磷酸突变体和/或突变体KRAS的存在以确定AGO2 miRNA加工的变化 和生长因子刺激后的靶向。我们还将确定生长因子刺激的效果 关于全球AGO2 miRNA的加工、靶向和基因调控。具体目标3：初步研究 证明尽管血清饥饿，AGO2的丢失会导致pEGFR和RAS活性的增加， 提示AGO2、RAS和EGFR之间存在重要的双向信号和串扰。我们会 描述AGO2-RAS-EGFR轴及其在细胞生存和生长中的作用。 综上所述，该项目的结果有望提高对生长因子调控的理解 AGO2-RAS在癌症生物学中的研究，并可能为治疗突变的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.