Characterization of growth factor control of Argonaute 2 and RAS interaction

Argonaute 2 和 RAS 相互作用的生长因子控制特征

• 批准号: 9394572
• 负责人: Ronald Francis Siebenaler
• 金额: $ 3.55万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9394572
• 关键词: Attenuated; Biology; Cancer Biology; Cell Line; Cell Proliferation; Cell Survival; Cells; Clinical; Colon; Complex; Data; Development; Dissociation; EGF gene; EGFR inhibition; Embryo; Epidermal Growth Factor Receptor; Event; Family; Fibroblasts; Future; Gene Expression Regulation; Genes; Global Change; Growth; Growth Factor; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; HRAS gene; Human; Hypoxia; KRAS2 gene; Knockout Mice; Lead; Lung; MAP Kinase Gene; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Monomeric GTP-Binding Proteins; Mutate; Mutation; N-terminal; Normal Cell; Oncogenes; Oncogenic; Outcome Study; Pancreas; Pathology; Pathway interactions; Patients; Phenotype; Phosphorylation; Physiology; Play; Prevention; Proteins; Quality of life; RAS Family Gene; RAS genes; RNA Interference; RNA-Induced Silencing Complex; Regulation; Research; Resistance; Role; Serum; Signal Transduction; Signal Transduction Pathway; Starvation; Testing; Therapeutic; base; cancer cell; cancer therapy; cell growth; endonuclease; improved; knock-down; member; mutant; novel; novel therapeutics; overexpression; prevent; protein expression; ras Proteins; targeted treatment; tumor

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.

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，占近 30% 含有 RAS 基因突变。三个 RAS 基因（KRAS、NRAS 和 HRAS）在细胞中发挥着核心作用 GTPase 开关的信号转导和 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，但在成功方面取得的进展甚微 临床上针对突变的 RAS，使新型 RAS 相互作用因子的鉴定成为一种具有潜在吸引力的策略 用于开发靶向治疗药物。最近的研究发现 N 之间存在相互作用 Argonaute 2 (AGO2) 的末端结构域是 RNA 诱导沉默复合物 (RISC) 的核心成分， 在野生型 (WT) 和突变型 KRAS 表达细胞系中切换 KRAS II 结构域。此外，这 相互作用对于 KRAS 介导的生长具有重要的功能。 KRAS 中 AGO2 的稳定敲低 依赖性细胞导致 KRAS 蛋白表达减少，随后细胞内 KRAS 蛋白表达减少 增殖。此外，与突变体 KRAS 的相互作用可通过阻止 AGO2 的 RNAi 功能来抑制 AGO2 的 RNAi 功能。 微小RNA解旋。 本研究将进一步探讨生长因子对 AGO2-RAS 的调节作用 生理学和癌症生物学。 EGFR 激活被证明可以减弱 AGO2 和 WT RAS在正常细胞中通过AGO2磷酸化，但突变KRAS对生长因子调节具有抵抗力。 尽管生长因子信号传导在 AGO2-RAS 生物学中具有潜在的调节作用，但其精确机制 该调节的功能作用尚不清楚。在这里我们建议通过以下方式研究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，从而 可能会导致 RAS 活性和细胞定位的变化。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。具体的 目标 2：鉴于已知突变型 KRAS 会抑制 AGO2 miRNA 解旋，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 并针对生长因子刺激后的目标。我们还将确定生长因子刺激的效果 全局 AGO2 miRNA 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 的缺失会导致 pEGFR 和 RAS 活性增加， 表明 AGO2、RAS 和 EGFR 之间存在重要的双向信号传导和串扰。我们将 描述 AGO2-RAS-EGFR 轴及其在细胞存活和生长中的作用。 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 在癌症生物学中的作用，可能为治疗突变 RAS 驱动的癌症提供临床靶点。