The Role Of Pkc Epsilon In Breast Cancer Cell Surface Polyfibronectin Assembly And Pulmonary Metastasis

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Fibronectin polymerized and assembled into globules on the surface of breast cancer cells has been shown to mediate metastasis to the lungs. Disruption of the binding between these tumor-cell surface-associated fibronectin, or polymeric fibronectin (polyFn), and dipeptidyl peptidase (DPPIV) negatively impacts breast cancer lung colonization. This dissertation was designed to characterize the molecular mechanisms underlying assembly of surface coated polyFn to understand the regulation of breast cancer cell surface polyFn. In order to investigate the signaling pathways involved in polyFn assembly, we utilized pharmacological inhibitors for various signaling pathways that have been proposed to regulate cellular function. Mutant plasmid expression, siRNA knockdown of proteins and biochemical analyses were carried out to identify the particular kinase involved in regulating polyFn. The ability of rat breast cancer cells to assemble polyFn was impaired by disruption of PKC-epsilon function. Knockdown of fibronectin also abolished polyFn formation. Cells with knockdown of either PKC-epsilon or fibronectin both had diminished ability to colonize the lungs in rat tail vein injection metastasis model. To further substantiate the role of PKC-epsilon in lung metastasis-mediating polyFn, mass spectrometry analysis was performed on the PKC-epsilon complex. Two proteins were identified and tested with corresponding inhibitors to relate cellular function to polyFn assembly. Detailed biochemical analyses, immunofluorescent microscopy, mutant protein expression and pharmacological inhibition demonstrated that the subcellular localization of PKC-epsilon complex with actin cytoskeleton has a great impact on polyFn assembly. A focal adhesion kinase was also identified to be involved. Therefore, PKC-epsilon exhibits its effects on polyFn by controlling the activation of downstream signaling proteins. In summary, the presence of endogenous cellular fibronectin and catalytically competent PKC-epsilon is a prerequisite of successful polyFn assembly. Proper subcellular localization of PKCepsilon-actin complex facilitates PKCepsilon downstream signaling, most likely to Pyk2, promoting the formation of polyFn surface formation. These results present a novel potential signaling pathway for therapeutical intervention in breast cancer metastasis.

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