Signaling Connections Controlling Cell Motility and Invasion

控制细胞运动和侵袭的信号连接

• 批准号: 8692720
• 负责人: David D Schlaepfer
• 金额: $ 36.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8692720
• 关键词: Adaptor Signaling Protein; Adhesions; Antibodies; Automobile Driving; Binding; Biochemical; Biological Assay; Cancer Etiology; Cell Culture Techniques; Cell Survival; Cells; Cessation of life; Colon Carcinoma; Complex; Development; Diagnostic Neoplasm Staging; Disease Progression; Dominant-Negative Mutation; Epithelial; Evaluation; Event; Family; Female; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesions; Funding; Generations; Genetic; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Human; Image; Immunoblotting; In Situ; Injection of therapeutic agent; Institutional Review Boards; Integrins; Knockout Mice; Knowledge; Lipid Binding; Malignant Epithelial Cell; Malignant neoplasm of ovary; Mediating; Mesenchymal; Modeling; Molecular; Mus; Mutation; Names; Null Lymphocytes; Ovarian; Ovarian Carcinoma; PH Domain; Pathologic Processes; Pathway interactions; Patients; Peritoneal; Pharmaceutical Preparations; Phenotype; Phosphatidylinositols; Phosphorylation Site; Phosphotransferases; Process; Protein Tyrosine Kinase; Proteins; Recurrence; Regulation; Role; Sampling; Serous; Signal Pathway; Signal Transduction; Site; Staging; Staining method; Stains; Structural Protein; Survival Rate; Testing; Tumor Cell Invasion; Tumor stage; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; Viral Tumor Antigens; Woman; cancer cell; cell behavior; cell motility; chemotherapy; epithelial to mesenchymal transition; gain of function; insight; matrigel; mutant; neoplastic cell; ovarian neoplasm; public health relevance; reconstitution; research study; rho; rho GTP-Binding Proteins; tumor; tumor progression

DESCRIPTION (provided by applicant): The elucidation of mechanisms driving cell movement is critical to our understanding of normal development and pathological processes such as tumor invasion. Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions, sites of matrix-integrin attachment, tension generation, and cell survival signals. A complex of kinases and structural proteins are localized to adhesions and this proposal will focus on the molecular interactions of focal adhesion kinase (FAK) and a guanine nucleotide exchange factor (GEF) for Rho-family GTPases termed ARHGEF28 (alternative names are p190RhoGEF or Rgnef). GEFs are proteins that activate Rho-family GTPases and function in the regulation of focal adhesion formation and turnover. Canonically, GEFs are thought to function far downstream of matrix-integrin activating signals. An exception to this model is Rgnef, which binds directly to FAK and localizes to focal adhesions. In the previous funding period, we showed that blocking the interaction between Rgnef and FAK via a dominant-negative approach inhibited colon carcinoma motility, matrix degradation, and tumor progression. We also created an Rgnef knockout mouse and analyses of Rgnef-/- fibroblasts established the importance of Rgnef in RhoA regulation, focal adhesion formation, and cell migration downstream of integrins. Here we extend these findings by Rgnef-/- reconstitution studies and show that Rgnef phosphoinositide lipid binding as an adaptor protein (GEF independent) is required for early FAK recruitment and activation at focal adhesions. We hypothesize that subsequently, within an Rgnef-FAK complex, FAK-mediated tyrosine phosphorylation leads to Rgnef activation and promotes RhoA/C GTPase activation (GEF dependent). These events will be tested as drivers of ovarian carcinoma tumor progression as Rgnef and FAK expression are elevated as a function of tumor stage and high FAK levels in serous ovarian cancer are associated with decreased overall patient survival. We will test whether the formation of an Rgnef-FAK signaling complex promotes FAK activation independently, or in conjunction with downstream RhoA/C GTPase activation pushing ovarian carcinoma cells toward an epithelial to mesenchymal transition and invasive phenotype. Our proposed experiments will combine molecular and mechanistic signaling studies in cell culture with mouse tumor models of ovarian cancer. Aim-1 will identify phosphorylation sites and domains of Rgnef that contribute to FAK activation, connections to Rho GTPases, cell motility, and an invasive cell phenotype through gain-of-function cell reconstitution assays using Rgnef-/- fibroblasts and ovarian tumor cells. Aim-2 will expand the analysis of Rgnef and FAK in human tumor samples and will test the role of Rgnef in mouse ovarian orthotopic and genetic tumor models. This multi-faceted approach will yield a comprehensive understanding of Rgnef-FAK signaling axis within fibroblasts and ovarian cancer cells and provide new insights into pathways driving disease progression.

描述（由申请人提供）：阐明驱动细胞运动的机制对于我们理解正常发育和病理过程（如肿瘤侵袭）至关重要。细胞迁移是一个高度调节的过程，涉及称为粘着斑的细胞-基质接触位点、基质-整联蛋白附着位点、张力产生和细胞存活信号的形成和周转。一个复杂的激酶和结构蛋白定位于粘连，这个建议将集中在粘着斑激酶（FAK）和鸟嘌呤核苷酸交换因子（GEF）的Rho家族GTP酶称为ARHGEF 28（别名是p190 RhoGEF或Rgnef）的分子相互作用。GEF是激活Rho家族GTP酶的蛋白质，在粘着斑形成和转换的调节中起作用。典型地，GEF被认为在基质整合素激活信号的下游起作用。该模型的一个例外是Rgnef，其直接与FAK结合并定位于局灶性粘连。在之前的资助期间，我们表明通过显性阴性方法阻断Rgnef和FAK之间的相互作用可抑制结肠癌的运动性、基质降解和肿瘤进展。我们还创建了Rgnef敲除小鼠，并对Rgnef-/-成纤维细胞进行分析，确定了Rgnef在RhoA调节、粘着斑形成和整联蛋白下游细胞迁移中的重要性。在这里，我们通过Rgnef-/-重建研究扩展了这些发现，并表明Rgnef磷酸肌醇脂质结合作为衔接蛋白（GEF独立）是早期FAK募集和激活所需的局灶性粘连。 我们假设，随后，在Rgnef-FAK复合物中，FAK介导的酪氨酸磷酸化导致Rgnef活化并促进RhoA/C GT3活化（GEF依赖性）。这些事件将作为卵巢癌肿瘤进展的驱动因素进行测试，因为Rgnef和FAK表达作为肿瘤分期的函数升高，并且浆液性卵巢癌中的高FAK水平与患者总体生存期降低相关。我们将测试Rgnef-FAK信号传导复合物的形成是否独立地促进FAK活化，或与下游RhoA/C GT3活化一起将卵巢癌细胞推向上皮向间充质转化和侵袭性表型。我们提出的实验将结合联合收割机的分子和机制的信号转导研究，在细胞培养与小鼠肿瘤模型的卵巢癌。Aim-1将通过使用Rgnef-/-成纤维细胞和卵巢肿瘤细胞的功能获得性细胞重建测定来鉴定Rgnef的磷酸化位点和结构域，其有助于FAK活化、与Rho GTP酶的连接、细胞运动性和侵袭性细胞表型。Aim-2将扩大人肿瘤样品中Rgnef和FAK的分析，并将测试Rgnef在小鼠卵巢原位和遗传肿瘤模型中的作用。这种多方面的方法将全面了解成纤维细胞和卵巢癌细胞内的Rgnef-FAK信号传导轴，并为推动疾病进展的途径提供新的见解。