RhoG Signaling in Invadopodia

 描述(申请人提供)：在乳腺癌患者中，癌细胞离开原发肿瘤并随后转移到远处器官是导致死亡的主要原因。转移细胞通过形成富含肌动蛋白的膜突起(称为内向突起)逃离原发肿瘤进入血流，这种突起降解细胞外基质(ECM)，从而允许周围组织的侵袭。Rho GTP酶是一种调节肌动蛋白细胞骨架的蛋白质家族，它对内侧足纲的组装起调控作用。然而，关于它们是如何被激活的，它们被激活的时间进程，或者它们的上游调节因子和下游效应器的身份，人们知之甚少。我们的长期目标是确定Rho GTP酶调节肿瘤转移的机制，特别是癌细胞的迁移和侵袭。这项研究的目的是描述调控RhoG介导的信号转导的分子机制，这些信号在内侧足纲的组装和拆卸过程中发挥作用。根据我们的初步结果，我们的中心假设是，RhoG通过调节其分解的动力学，起到了负调控作用，在这个过程中涉及适配蛋白paxlin，但尚未对信号成分进行表征。我们将通过追求两个特定的目标来验证我们的假设：1)表征RhoG激活的时空动力学及其在隐足动物形成和细胞入侵中的作用。我们的假设是，RhoG的激活在失足动物形成过程中受到时间和空间上的严格调控，并在需要帕西林磷酸化的过程中在其分解过程中发挥作用。在这里，我们将使用一种新型的RhoG FRET生物传感器来确定活细胞中内含子形成过程中RhoG的时空激活。我们还将表征RhoG在调节介体动态、降解细胞外基质和细胞侵袭中的作用。2)确定RhoG特异的效应因子参与了不定足动物的形成。这一目的的目的是表征RhoG下游的即时效应器及其在内侧足形成过程中的作用。我们已经使用一种新的基于邻近标记的结合质谱学的分析方法确定了几种潜在的RhoG效应。根据我们的初步结果，我们的工作假设是，这些RhoG特异的效应器中的一些对于内陷形成的调节是关键的。这项工作有望揭示一种新的信号通路，它在Inadoodia组装和功能的调节中发挥作用。这些结果有望产生重要的积极影响，因为它们可能确定新的靶点，除了从根本上推进癌细胞迁移和侵袭的领域外，还可以推动治疗方法的发展，以防止或减缓乳腺癌的转移。

 DESCRIPTION (provided by applicant): 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. 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 metastasis, in particular to cancer cell migration and invasion. The objective of this study is to characterize the molecular mechanisms that regulate RhoG-mediated signaling during invadopodia assembly and disassembly. Based on our preliminary results, our central hypothesis is that RhoG functions as a negative regulator of invadopodia formation by regulating the dynamics of their disassembly, in a process that involves the adaptor protein paxillin and yet to be characterized signaling components. We will test our hypothesis by pursuing two specific aims: 1) To characterize the spatio-temporal dynamics of RhoG activation and its role during invadopodia formation and cell invasion. Our hypothesis is that RhoG activation is tightly regulated both temporally and spatially during invadopodia formation and plays a role in their disassembly in a process that requires paxillin phosphorylation. Here, we will use a novel RhoG FRET biosensor to determine the spatiotemporal activation of RhoG during invadopodia formation in live cells. We will also characterize the role of RhoG in the regulation of invadopodia dynamics, degradation of ECM and cell invasion. 2) To identify the RhoG-specific effectors involved in invadopodia formation. The objective for this Aim is to characterize the immediate RhoG downstream effectors and their role during invadopodia formation. We have identified several potential RhoG-effectors using a novel proximity-based labeling assay coupled to mass spectrometry. Based on our preliminary results, our working hypothesis is that some of these RhoG-specific effectors are critical for the regulation of invadopodia formation. The work proposed here is expected to shed light on a novel signaling pathway that plays a role in the regulation of invadopodia assembly and function. Such results are expected to have an important positive impact, because they may identify novel targets that could drive the development of therapeutics to prevent or slow the progression of breast cancer metastasis, in addition to fundamentally advance the fields of cancer cell migration and invasion.