ARF6 function in cancers driven by RAS hyperactivation

项目摘要/摘要 RAS激活突变或RAS调控基因突变导致RAS过度激活是驱动因素 人类癌症的风险。1型神经纤维瘤病(NF1)是一种由NF1肿瘤缺失引起的发育性疾病 抑制基因，编码神经纤维素，一种通过其GTP酶使RAS失活的大蛋白 激活功能。NF1的缺失导致RAS的过度激活，增加了发生多发性硬化的风险 癌症，包括恶性周围神经鞘瘤(MPNSTs)。RAS激活促进信号转导 驱动肿瘤发生的几条下游通路，包括PI3K和MAPK信号通路。 虽然已经进行了许多直接或间接针对RAS的尝试，但还没有一次尝试 在临床上取得了成功，因此对控制RAS信号的精确机制有了新的见解 需要的。小分子GTPase蛋白ARF6与多种肿瘤的生长和转移密切相关。 在葡萄膜和皮肤黑色素瘤中，ARF6促进致癌GQ或-连环蛋白的转运到适当的 信号或转录增强的细胞内位置。我们的初步数据表明，ARF6可能 还通过控制RAS在细胞内的运输来调节NF1缺陷的MPNST中的RAS信号，以及 ARF6控制NF1缺陷肿瘤细胞的增殖。激活的ARF6似乎是RAS脂质所必需的 修饰，如棕榈酰化和法尼化，这是RAS膜定位和 用于与其效应器、RAF和p110(PI3K催化亚基)结合。根据这些初步数据，我们 假设ARF6通过控制RAS转运促进NF1缺陷组织的肿瘤发生 发信号。我们将通过追求以下目标来检验这一假设。在目标1中，我们将确定损失是否 NF1通过调节RAS亚细胞定位和信号转导激活ARF6，从而抑制肿瘤细胞的增殖。 这些研究将涉及多学科方法，以确定NF1如何调节ARF6以及ARF6如何 控制细胞增殖、RAS脂质修饰、细胞内定位和信号转导。在目标2中，我们将评估 ARF6在NF1缺陷人MPNST原位异种移植小鼠模型肿瘤生长中的作用这些 研究将使我们能够确定ARF6被敲除或药物抑制如何影响在体内的生长 在免疫功能低下的小鼠模型中，肿瘤来自人类癌细胞。在目标3中，我们将确定 在NF1基因工程小鼠模型中，ARF6是否对肿瘤的形成和生长是必需的 有缺陷的癌症。这些研究将涉及敲除Arf6或从药物上抑制Arf6在 免疫活性NF1/-；TrP53/-小鼠体内研究ARF6在NF1缺陷的小鼠肿瘤发生中的作用 有足够的免疫系统。这项提议将提供多学科的基本培训 方法和扩展候选人的癌症生物学知识(K99阶段)，并将建立 一个独立研究项目的基础，重点是ARF6在癌症中的作用，不仅是由 NF1缺乏症还表现为RAS过度激活(R00期)。

PROJECT SUMMARY/ABSTRACT Activating mutations in RAS or mutations in RAS regulatory genes that lead to hyperactivated RAS are drivers of human cancer. Neurofibromatosis type 1 (NF1) is a developmental disease caused by loss of the NF1 tumor suppressor gene, which encodes neurofibromin, a large protein that inactivates RAS through its GTPase activating function. Loss of NF1 leads to the hyperactivation of RAS and increases the risk of developing multiple cancers, including malignant peripheral nerve sheath tumors (MPNSTs). RAS activation promotes signaling of several downstream pathways that drive oncogenesis, including the PI3K and MAPK signaling pathways. Although many attempts have been made to target RAS either directly or indirectly, none of them have yet been successful in the clinic and therefore new insights into the precise mechanisms that govern RAS signaling are needed. The small GTPase protein ARF6 has been implicated in the growth and metastasis of many cancers. In uveal and cutaneous melanomas, ARF6 promotes the trafficking of oncogenic Gq or -catenin to appropriate intracellular locations where signaling or transcription is enhanced. Our preliminary data suggest that ARF6 may also be regulating RAS signaling in NF1-deficient MPNSTs by controlling RAS intracellular trafficking and that ARF6 controls proliferation of NF1-deficient tumor cells. Activated ARF6 appears to be necessary for RAS lipid modifications, such as palmitoylation and farnesylation, which are essential for RAS membrane localization and for binding with its effectors, RAF and p110 (a PI3K catalytic subunit). Based on these preliminary data, we hypothesize that ARF6 promotes tumorigenesis in NF1-deficient tissues by controlling RAS trafficking and signaling. We will test this hypothesis by pursuing the following aims. In Aim 1, we will determine whether loss of NF1 activates ARF6 to control tumor cell proliferation by regulating RAS subcellular localization and signaling. These studies will involve multidisciplinary approaches to determine how NF1 regulates ARF6 and how ARF6 controls cell proliferation, RAS lipid modifications, intracellular localization, and signaling. In Aim 2, we will assess ARF6 function in tumor growth in an orthotopic xenograft mouse model of NF1-deficient human MPNST. These studies will allow us to determine how ARF6 knockdown or pharmacologic inhibition affects in vivo growth of tumors derived from human cancer cells in an immunocompromised mouse model. In Aim 3, we will determine whether ARF6 is necessary for tumor formation and growth in a genetically engineered mouse model of NF1- deficient cancers. These studies will involve either knocking out Arf6 or pharmacologically inhibiting ARF6 in immunocompetent Nf1+/-; Trp53+/- mice to determine the in vivo role of ARF6 in NF1-deficient tumorigenesis in the presence of a competent immune system. This proposal will provide essential training in multidisciplinary approaches and expand the candidate’s knowledge of cancer biology (K99 phase) and will establish the foundations for an independent research program focused on the roles of ARF6 in cancers driven by not only NF1 deficiency but also hyperactivated RAS (R00 phase).