The tumor suppressor TSG101, which is frequently mutated in sporadic breast cancer tumors, was identified in a yeast two-hybrid screen to interact with the PKN1 kinase, an effector protein for Rho. The carboxyl terminus of TSG101 containing the leucine zipper, which is frequently lost in breast cancer, was sufficient and required for its interaction with the PKN1 kinase. TSG101 also was found to interact with PKN1 in vivo, and preliminary experiments suggest that it functions to inhibit PKN1 kinase activation. This proposal seeks to expand on these initial observations to develop information on the mechanism by which disruption of the normal TSG101-PKN1 interaction might lead to cellular transformation and metastasis. Transient transfection experiments in fibroblasts will test whether TSG101 is a regulator and/or target of PKN1 kinase. Transfection experiments will be performed to determine whether a dominant negative and constitutively active PKN1 kinase can affect the cellular growth and cytoskeletal organization and whether TSG101 participates through PKN1 or independently to modulate these activities. A panel of different breast cancer cell lines with varying degrees of invasiveness will be evaluated for TSG101 mutations. These studies will determine whether the loss of TSG101 correlates with the invasive phenotype of these cell lines. Breast cancer cell lines which lack functional TSG101 such as MDA-435 cells will be used as recipients to study in detail the function of TSG101 and PKN1. Stable cell lines expressing PKN1 and TSG101 will be monitored for cellular morphology, anchorage dependent growth, and chemotaxis/chemoinvasion in order to see whether there is a change in the invasive phenotype. Together these studies may yield a mechanistic understanding of both the normal function of TSG101 and PKN1 and their role in cellular transformation and invasion.
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