Signaling Connections Controlling Cell Motility and Invasion

描述(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控的过程，它涉及细胞-基质接触部位的形成和更新，称为灶性粘连，基质-整合素附着部位，张力产生和细胞生存信号。一个由激酶和结构蛋白组成的复合体定位于粘连，本提案将重点研究Rho家族GTP酶的粘着斑激酶(FAK)和鸟核苷酸交换因子(Gef)的分子相互作用，称为ARHGEF28(另一种名称是p190Rhogef或Rgnef)。GEF是一种激活Rho家族GTP酶的蛋白质，在局部黏附形成和周转的调节中发挥作用。通常认为，GEF的作用远远低于基质整合素激活信号的下游。这个模型的一个例外是Rgnef，它直接与FAK结合，并定位于局部粘连。在之前的资助期间，我们表明通过显性-负性方法阻断Rgnef和FAK之间的相互作用可以抑制结肠癌的运动、基质降解和肿瘤进展。我们还创建了Rgnef基因敲除小鼠，对Rgnef-/-成纤维细胞的分析证实了Rgnef在RhoA调节、焦点黏附形成和整合素下游细胞迁移中的重要性。在这里，我们通过Rgnef-/-重构研究扩展了这些发现，并表明Rgnef磷脂酰肌醇结合作为一种接头蛋白(不依赖于环境基金)是FAK早期募集和局部粘连激活所必需的。我们假设，随后，在Rgnef-FAK复合体中，FAK介导的酪氨酸磷酸化导致Rgnef激活并促进RhoA/C GTP酶激活(依赖于环境基金)。这些事件将被测试为卵巢癌肿瘤进展的驱动因素，因为Rgnef和FAK的表达作为肿瘤分期的函数而升高，并且浆液性卵巢癌中高FAK水平与患者总体生存率下降相关。我们将测试Rgnef-FAK信号复合体的形成是否独立地促进FAK的激活，或者与下游RhoA/C GTP酶的激活一起推动卵巢癌细胞向上皮向间质转化和侵袭表型。我们提议的实验将结合细胞培养中的分子和机制信号研究与卵巢癌的小鼠肿瘤模型。AIM-1将通过使用Rgnef-/-成纤维细胞和卵巢肿瘤细胞进行功能获得细胞重建分析，确定有助于FAK激活、与Rho GTP酶连接、细胞运动和侵袭性细胞表型的Rgnef磷酸化位点和结构域。AIM-2将扩展Rgnef和FAK在人类肿瘤样本中的分析，并将在小鼠卵巢原位和遗传肿瘤模型中测试Rgnef的作用。这一多方面的方法将产生对成纤维细胞和卵巢癌细胞内Rgnef-FAK信号轴的全面了解，并为推动疾病进展的途径提供新的见解。

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.