Regulation of Invadopodia Formation by RhoG Specific GEFs and GAPs

RhoG 特异性 GEF 和 GAP 对侵袭伪足形成的调节

• 批准号: 9099158
• 负责人: Rafael Garcia-Mata
• 金额: $ 40.87万
• 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: 2016-04-12 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099158
• 关键词: Actins; Affect; Behavior; Binding; Biological Assay; Biosensor; Blood Circulation; Breast Cancer Cell; Breast Cancer Patient; Breast cancer metastasis; Cell physiology; Cells; Coupled; Cytoskeleton; Development; Distant; Extracellular Matrix; Fluorescence Resonance Energy Transfer; Fostering; Future; GTP Binding; GTPase-Activating Proteins; Goals; Guanine Nucleotide Exchange Factors; Health; Invaded; Label; Life; Light; Mass Spectrum Analysis; Mediating; Membrane; Mission; Molecular; Neoplasm Metastasis; Organ; Pathway interactions; Pattern; Primary Neoplasm; Process; Protein Family; Proteins; Regulation; Research; Role; Signal Pathway; Signaling Molecule; Specificity; Staging; Techniques; Testing; Time; Tissues; Work; base; cancer cell; cancer type; cell motility; improved; innovation; malignant breast neoplasm; migration; mortality; novel; novel therapeutics; prevent; public health relevance; rho; rho GTP-Binding Proteins; therapeutic development; tool; 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 Rho GTPases signaling pathways that contribute to cancer cell metastasis, in particular to cancer cell migration and invasion. The objective of this study is to characterize the molecular mechanisms that regulate RhoG in the formation of invadopodia in breast cancer. Based on our preliminary results, our central hypothesis is that RhoG functions as a negative regulator of invadopodia formation, and functions during their disassembly, in a process that involves the exchange factor SGEF and a yet to be identified RhoGAP/s. We will test our hypothesis by pursuing three specific aims: Aim 1. To identify the RhoG-specific RhoGAPs involved in invadopodia formation. We have used a novel proximity-based labeling assay coupled to mass spectrometry to identify several potential RhoG GAPs. Here, we will characterize their role in regulating RhoG activity, and their function during invadopodia formation and cell invasion. Aim 2. To characterize the role of SGEF in the regulation of RhoG during invadopodia formation. Here, we will test our working hypothesis that SGEF, a RhoG-specific GEF, is activated by Src, maintains the active pool of RhoG that prevents invadopodia formation, and needs to be inactivated for invadopodia to form. Aim 3. To characterize the spatio-temporal dynamics of RhoG activation during invadopodia formation. Based on preliminary results, our working hypothesis is that RhoG activation is tightly regulated both temporally and spatially during invadopodia formation. Here, we will use a novel RhoG FRET biosensor to determine the spatial and temporal activation pattern of RhoG during invadopodia formation in live cells, and the role of RhoGAPs and RhoGEFs in regulating its activity. The approach proposed here is innovative because it applies cutting edge techniques to examine aspects of invadopodia formation that have either not been examined before and/or have not been accessible with previously available tools. The proposed research is significant because by characterizing this pathway, it has the potential to identify novel targets that could drive the development of new therapeutics to prevent or slow down metastasis in breast cancer and other cancer types that utilize invadopodia to invade other tissues.

 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有助于癌细胞转移，特别是癌细胞迁移和侵袭的Rho GTPases信号通路。本研究的目的是表征在乳腺癌侵袭伪足形成中调节RhoG的分子机制。基于我们的初步结果，我们的中心假设是，RhoG作为侵入伪足形成的负调节因子发挥作用，并在其分解过程中发挥作用，该过程涉及交换因子SGEF和尚未确定的RhoGAP/s。我们将通过追求三个具体目标来测试我们的假设：目标1。鉴定侵袭伪足形成过程中RhoG特异性RhoGAP。我们已经使用了一种新的基于邻近的标记分析耦合到质谱法，以确定几个潜在的RhoG GAP。在这里，我们将描述其在调节RhoG活性的作用，以及它们在侵袭伪足形成和细胞侵袭过程中的功能。目标2.研究SGEF在侵袭伪足形成过程中对RhoG的调节作用。在这里，我们将测试我们的工作假设，即SGEF，一个RhoG特异性GEF，被Src激活，维持RhoG的活性池，防止侵袭伪足形成，并且需要被灭活以形成侵袭伪足。目标3.描述侵入伪足形成过程中RhoG激活的时空动态。基于初步的结果，我们的工作假设是，RhoG的激活受到严格的时间和空间的调控过程中invadopodia的形成。在这里，我们将使用一种新的RhoG FRET生物传感器来确定在活细胞中侵袭伪足形成过程中RhoG的空间和时间激活模式，以及RhoGAP和RhoGEFs在调节其活性中的作用。 这里提出的方法是创新的，因为它采用了尖端技术来检查invadopodia形成的方面，以前没有被检查和/或以前可用的工具无法访问。这项拟议中的研究意义重大，因为通过表征这一途径，它有可能确定新的靶点， 开发新的治疗方法，以预防或减缓乳腺癌和利用侵袭伪足侵入其他组织的其他癌症类型的转移。

项目成果

Regulation of circular dorsal ruffles, macropinocytosis, and cell migration by RhoG and its exchange factor, Trio.

• DOI: 10.1091/mbc.e16-06-0412
• 发表时间: 2017-07-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Valdivia A;Goicoechea SM;Awadia S;Zinn A;Garcia-Mata R
• 通讯作者: Garcia-Mata R

The small GTPase RhoG regulates microtubule-mediated focal adhesion disassembly.

小 GTP 酶 RhoG 调节微管介导的粘着斑分解。

• DOI: 10.1038/s41598-019-41558-7
• 发表时间: 2019
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Zinn,Ashtyn;Goicoechea,SilviaM;Kreider-Letterman,Gabriel;Maity,Debonil;Awadia,Sahezeel;Cedeno-Rosario,Luis;Chen,Yun;Garcia-Mata,Rafael
• 通讯作者: Garcia-Mata,Rafael

ARHGAP17 regulates the spatiotemporal activity of Cdc42 at invadopodia.

ARHGAP17调节Cdc42在Invadopodia上的时空活性。

• DOI: 10.1083/jcb.202207020
• 发表时间: 2023-02-06
• 期刊: The Journal of cell biology

The RhoA dependent anti-metastatic function of RKIP in breast cancer.

• DOI: 10.1038/s41598-021-96709-6
• 发表时间: 2021-08-31
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Kalpana G;Figy C;Feng J;Tipton C;De Castro JN;Bach VN;Borile C;LaSalla A;Odeh HN;Yeung M;Garcia-Mata R;Yeung KC
• 通讯作者: Yeung KC