Protein Kinase C Substrates in Human Breast Cancer

描述（由申请人提供）：蛋白激酶C（PKC）是EGF刺激（erbB 2）和IL-8依赖性信号通路的上游组分，其活性与培养的人乳腺细胞的运动行为和转移性乳腺癌的发生有关。因此，除了PKC本身，PKC底物蛋白的鉴定将 为开发抗转移化疗提供了潜在的靶点。本实验室最近发现，微管蛋白和Cdc 42效应蛋白-4（CEP 4）是人类乳腺细胞中PKC的底物。微管蛋白Ser-165的磷酸化调节微管动力学，使生长期延长，这与细胞运动相关。的重要性将通过在非转化MCF-10A细胞和高转移性人癌细胞中测试β 6-微管蛋白的磷酸化位点突变体来研究β 6-微管蛋白磷酸化对微管动力学和细胞运动的影响（具体目标1）。 CEP 4磷酸化的机制意义将在人乳腺细胞表型中用磷酸化位点特异性突变体进行探索（特异性目的2）。由于CEP 4以高亲和力结合Cdc 42，因此将特别强调确定CEP 4磷酸化如何影响这种结合相互作用，从而影响Cdc 42信号传导。此外，IQGAP在PKC信号通路中的作用将被探索。IQGAP是一种已知的PKC底物，需要活化Cdc 42进行信号传导，也是细胞皮质MT捕获所需的组分。IQGAP重新定位于板状足响应于PKC活化而发生，并被用作由CEP 4磷酸化和活化的Cdc 42产生的促运动信号的读出。因此，IQGAP可能提供了一个连接点，在该连接点处，伸长的微管和CEP 4-Cdc 42复合物会聚以促进运动性。在体外三维培养试验中，将用MDA-MB-231细胞表达的磷酸化位点特异性突变体探索三种PKC底物对肿瘤生长和转移的意义（特异性目的3）。3-D测定将指导选择在原位裸鼠模型中分析哪种底物。对于体内研究，将在植入裸鼠脂肪垫中的磷酸化酶-MDA-MB-231细胞的转染子中评价磷酸化抗性PKC底物的影响。肿瘤形成、生长和转移将通过整个动物生物发光进行跟踪（特定目标3）。总之，这些研究的结果将建立一个连贯的机制模型，包括三个PKC底物（微管蛋白，CEP 4和IQGAP），并提供了一个基础，为进一步研究的运动途径。预期的发现可能为乳腺癌新型化疗药物的设计提供信息，并为预测转移潜力提供新的生物标志物。

DESCRIPTION (provided by applicant): Protein kinase C (PKC) is an upstream component of the EGF-stimulated (erbB2) and IL-8-dependent signaling pathways whose activity has been linked to motile behavior of cultured human breast cells and to the acquisition of metastatic breast cancer. Therefore, in addition to PKC itself, identification of PKC substrate proteins would offer potential targets for developing anti-metastasis chemotherapies. This laboratory recently discovered that ¿-tubulin and Cdc42 effector protein-4 (CEP4) are substrates of PKC in human breast cells. Phosphorylation of ¿-tubulin at Ser-165 modulates microtubule dynamics such that the growth phase is prolonged, which correlates with cell movement. The significance of ¿-tubulin phosphorylation to microtubule dynamics and cell movement will be investigated by testing phosphorylation site mutants of ¿6-tubulin both in non-transformed MCF-10A cells and in highly metastatic human cancer cells (Specific Aim 1). The mechanistic significance of CEP4 phosphorylation will be explored with phosphorylation site-specific mutants in phenotypes of human breast cells (Specific Aim 2). Since CEP4 binds Cdc42 with high affinity, special emphasis will be on determining how CEP4 phosphorylation affects this binding interaction and consequently how Cdc42 signaling is affected. In addition, a role for IQGAP will be explored in the PKC signaling pathway. IQGAP, a known PKC substrate that requires activated Cdc42 for signaling, is also a required component for MT capture at the cell cortex. Re-localization of IQGAP to lamellapodia occurs in response to PKC activation and be used as a read-out for pro-motility signals arising from CEP4 phosphorylation and activated Cdc42. Thus, IQGAP may provide a nexus at which elongating microtubules and the CEP4-Cdc42 complex converge to promote motility. The significance of the three PKC substrates to tumor growth and metastasis will be explored with phosphorylation site-specific mutants expressed by MDA-MB-231 cells in three dimensional culture assays in vitro (Specific Aim 3). The 3-D assays will guide the selection of which substrate to analyze in an orthotopic nude mouse model. For the in vivo studies, the impact of a phosphorylation-resistant PKC substrate will be evaluated in transfectants of luciferase-MDA-MB-231 cells implanted in the fat pads of nude mice. Tumor formation, growth, and metastasis will be tracked by whole animal bioluminescence (Specific Aim 3). Overall, the results of these studies will establish a coherent mechanistic model that incorporates three PKC substrates (¿-tubulin, CEP4, and IQGAP), and provides a foundation for further investigations of the motility pathway. The anticipated findings are likely to inform strategies for design of novel chemotherapeutics for breast cancer, and to provide new bio-markers for predicting metastatic potential.