A Novel RhoG Protein Interaction Network in Invadopodia

项目总结 在乳腺癌患者中，癌细胞从原发肿瘤及其周围的移行 随后转移到远处器官是导致死亡的主要原因。转移的细胞逃脱了 原发肿瘤并通过形成富含肌动蛋白的膜突起进入血流 一种降解细胞外基质(ECM)以允许入侵周围组织的侵袭足细胞。这个 Rho GTP酶是一类调节肌动蛋白的蛋白质家族，它对内侧足纲的组装进行调控 细胞骨架。Rho GTPase信号的失控与所有癌症阶段有关 进展，包括增殖、侵袭和转移。然而，人们对它们是如何 被激活，其激活的时间进程，或其上游调节器和 下游效应器。我们的长期目标是描述Rho的调节机制 促进癌细胞转移，特别是癌细胞迁移和侵袭的GTP酶。 我们的初步研究结果表明，RhoG基因在花序形成的调控中起关键作用。 乳腺癌细胞。根据我们的初步结果，我们的中心假设是RhoG的功能是 内生足纲形成的负性调节因子。确定RhoG信令组件的特征 在不动足纲的形成过程中，我们对RhoG相互作用组进行了蛋白质组学分析 并确定了几个潜在的RhoG结合伙伴。此应用程序侧重于以下子组 功能相关的蛋白质，包括可能涉及的上游和下游成分 在RhoG介导的信号传递中。本申请目的是表征这些新的蛋白质- 蛋白质相互作用及其在啮齿动物形成和细胞入侵中的作用。我们将测试我们的 通过追求两个具体目标来实现假设： 目的1.验证质谱学鉴定的RhoG相互作用伙伴。利用近似性- 基于标记实验，我们已经确定了一个潜在的RhoG相互作用蛋白网络。的目标是 这一目的是为了验证通过质谱学确定的相互作用。 目的2.鉴定出的蛋白质在啮齿动物形成过程中的作用。我们有 发现了一个可能在RhoG介导的调控中发挥作用的蛋白质相互作用网络 无足类动物的形成。这一目标的目标是表征不同RhoG相互作用的角色 在调节RhoG活性、内生孢子的形成和细胞入侵方面的合作伙伴。 预计这些研究产生的知识将促进我们的理解 了解Rho GTPase通路如何调控癌症进展中的关键步骤。未来的工作将建立 并扩展这些研究，以剖析该蛋白质网络在细胞中的功能 体内侵袭和转移。

PROJECT SUMMARY In breast cancer patients, the migration of cancer cells away from the primary tumor and their subsequent metastasis to distant organs is the leading cause of mortality. Metastatic cells escape the primary tumor and enter the bloodstream by developing actin-rich membrane protrusions called invadopodia that degrade the extracellular matrix (ECM) to allow invasion of surrounding tissues. The assembly of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton. Deregulation in Rho GTPase signaling has been associated with all stages of cancer progression, including proliferation, invasion and metastasis. However, little is known about how they are activated, the time course of their activation, or the identity of their upstream regulators and downstream effectors. Our long term goal is to characterize the mechanisms of regulation of Rho GTPases that contribute to cancer cell metastasis, in particular to cancer cell migration and invasion. Our preliminary results show that RhoG plays a key role in the regulation of invadopodia formation in breast cancer cells. Based on our preliminary results, our central hypothesis is that RhoG functions as a negative regulator of invadopodia formation. To characterize the RhoG signaling components involved in invadopodia formation, we have performed a proteomic analysis of the RhoG interactome and identified several potential RhoG binding partners. This application focuses on a subgroup of functionally related proteins, which includes potential upstream and downstream components involved in RhoG-mediated signaling. The objective of this application is to characterize these novel protein- protein interactions and their role during invadopodia formation and cell invasion. We will test our hypothesis by pursuing two specific aims: Aim 1. To validate the RhoG interaction partners identified by mass spectrometry. Using a proximity- based labeling assay we have identified a network of potential RhoG-interaction proteins. The goal of this aim is to validate the interactions identified by mass spectrometry. Aim 2. Characterization of the role of the identified proteins during invadopodia formation. We have uncovered a protein interaction network that may play a role in the regulation of RhoG-mediated invadopodia formation. The goal of this aim is to characterize the role of the different RhoG interaction partners on the regulation of RhoG activity, invadopodia formation, and cell invasion. It is expected that the knowledge generated from these studies will advance our understanding of how Rho GTPase pathways regulate critical steps during cancer progression. Future work will build up on this application and extend these studies to dissect the function of this protein network during cell invasion and metastasis in vivo.