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.
This proposal will investigate newly discovered substrates of protein kinase C (PKC), an enzyme whose activity is critical to the ability of breast cells to spread throughout the body (metastasis). This research seeks to establish the mechanistic roles of these protein substrates that could lead to their use as chemotherapeutic targets and disease-related biomarkers for control and detection of metastatic human breast cancer.
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