Signaling Connections Controlling Cell Motility and Invasion

描述（由申请人提供）：阐明驱动细胞运动的机制对我们理解正常发育和肿瘤侵袭等病理过程至关重要。细胞迁移是一个高度调控的过程，涉及细胞-基质接触位点（称为局灶黏附）、基质-整合素附着位点、张力产生和细胞存活信号的形成和周转。一种由激酶和结构蛋白组成的复合物定位于黏附，本提案将重点研究黏附激酶（FAK）和rho家族GTPases的鸟嘌呤核苷酸交换因子（GEF）的分子相互作用，称为ARHGEF28（可选名称为p190RhoGEF或Rgnef）。gef是一种激活rho家族gtpase的蛋白质，在调控局灶黏附形成和转换中起作用。通常，gef被认为在基质整合素激活信号的下游发挥作用。该模型的一个例外是Rgnef，它直接与FAK结合并定位于局灶粘连。在之前的资助期内，我们发现通过显性阴性途径阻断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敲除小鼠，并对Rgnef-/-成纤维细胞进行了分析，确定了Rgnef在RhoA调节、局灶粘连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磷酸肌苷脂结合作为一种适配器蛋白（不依赖于GEF）是早期FAK募集和局灶黏附激活所必需的。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 GTPase激活（GEF依赖）。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的表达随着肿瘤分期而升高，浆液性卵巢癌中高FAK水平与患者总体生存率降低相关。我们将测试Rgnef-FAK信号复合体的形成是单独促进FAK激活，还是与下游RhoA/C GTPase激活一起推动卵巢癌细胞向上皮间质过渡和侵袭性表型。我们提出的实验将结合细胞培养和卵巢癌小鼠肿瘤模型的分子和机制信号研究。目标-1将通过Rgnef-/-成纤维细胞和卵巢肿瘤细胞的功能获得性细胞重建分析，鉴定Rgnef的磷酸化位点和结构域，这些磷酸化位点和结构域有助于FAK激活、与Rho GTPases的连接、细胞运动和侵袭性细胞表型。Aim-2将扩大对人类肿瘤样本中Rgnef和FAK的分析，并将测试Rgnef在小鼠卵巢原位和遗传性肿瘤模型中的作用。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rgnnef - fak信号轴，并为驱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.