RhoG Signaling in Invadopodia

侵袭伪足中的 RhoG 信号转导

• 批准号: 9096490
• 负责人: Rafael Garcia-Mata
• 金额: $ 16.53万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9096490
• 关键词: Actins; Adaptor Signaling Protein; Behavior; Biological Assay; Biosensor; Blood Circulation; Breast Cancer Cell; Breast Cancer Patient; Breast cancer metastasis; Cells; Coupled; Cytoskeleton; Distant; Extracellular Matrix; Extracellular Matrix Degradation; Fluorescence Resonance Energy Transfer; Fostering; Goals; Health; Invaded; Knowledge; Label; Life; Light; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Mission; Molecular; Neoplasm Metastasis; Organ; Pathway interactions; Phosphorylation; Play; Primary Neoplasm; Process; Protein Family; Proteomics; Regulation; Research; Role; Signal Pathway; Signal Transduction; Testing; Time; Tissues; Work; base; cancer cell; cell motility; cell type; improved; metastatic process; migration; mortality; neoplastic cell; novel; paxillin; prevent; public health relevance; rho GTP-Binding Proteins; spatiotemporal; therapeutic development; tumor progression

 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.

 描述（由申请人提供）：在乳腺癌患者中，癌细胞从原发肿瘤中迁移并随后转移到远处器官是死亡的主要原因。转移性细胞逃离原发性肿瘤并通过发展称为侵袭伪足的富含肌动蛋白的膜突起进入血流，所述侵袭伪足降解细胞外基质（ECM）以允许侵入周围组织。侵入伪足的组装受Rho GTP酶的调节，Rho GTP酶是调节肌动蛋白细胞骨架的蛋白家族。然而，很少有人知道它们是如何被激活的，它们激活的时间过程，或者它们的上游调节因子和下游效应因子的身份。我们的长期目标是表征促进癌症转移，特别是癌细胞迁移和侵袭的Rho GTPases的调节机制。本研究的目的是表征分子机制，调节RhoG介导的信号转导过程中invadopodia组装和拆卸。根据我们的初步结果，我们的中心假设是RhoG通过调节入侵伪足分解的动态来充当入侵伪足形成的负调节因子，该过程涉及衔接蛋白桩蛋白，但尚未表征信号成分。我们将通过追求两个具体目标来测试我们的假设：1）表征RhoG激活的时空动力学及其在侵袭伪足形成和细胞侵袭期间的作用。我们的假设是，RhoG激活在侵入伪足形成过程中在时间和空间上都受到严格调控，并在需要桩蛋白磷酸化的过程中在其分解中发挥作用。在这里，我们将使用一种新的RhoG FRET生物传感器，以确定时空激活的RhoG侵袭伪足形成过程中的活细胞。我们还将描述RhoG在侵袭伪足动力学、ECM降解和细胞侵袭调节中的作用。2)目的：鉴定侵袭伪足形成过程中RhoG特异性效应子。该目标的目的是表征直接RhoG下游效应子及其在侵袭伪足形成期间的作用。我们已经确定了几个潜在的RhoG效应器使用一种新的基于邻近的标记分析耦合到质谱。基于我们的初步结果，我们的工作假设是，这些RhoG特异性效应子中的一些对于侵袭伪足形成的调节至关重要。本文提出的工作有望揭示一种新的信号通路，该通路在侵袭伪足组装和功能的调节中发挥作用。这些结果预计将产生重要的积极影响，因为它们可以确定新的靶点，这些靶点可以推动治疗药物的开发，以预防或减缓乳腺癌转移的进展，此外还可以从根本上推进癌细胞迁移和侵袭领域。