REGULATION OF PAR 4 BY ONCOGENIC RAS

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Prostate apoptosis response-4 (Par-4) is a leucine zipper domain- containing protein that is required for stimulus-dependent apoptosis in both prostatic and non-prostatic cells. This widely expressed protein is not sufficient on its own to induce apoptosis, but can sensitize the cells to the action of apoptotic stimuli. We examined various tumors and corresponding normal tissues for Par-4 protein expression, and found that Par-4 levels are quantitatively decreased in renal cell carcinomas (RCCs) and pancreatic cancer but not in prostate cancer relative to the corresponding normal cells. This suggested that Par-4 down-regulation may play a role in the process of malignant transformation in certain cancers. Immortalized fibroblasts expressing the ras oncogene serve as an excellent model to study signaling pathways essential for transformation and tumorigenesis. Our preliminary studies in immortalized fibroblasts suggested that endogenous Par-4 protein expression is diminished by oncogenic Ras, and that restoration of Par-4 inhibits oncogenic Ras- inducible transformation without altering cell viability. These findings led to the hypothesis that down-regulation of Par-4 by oncogenic Ras is necessary for transformation because Par-4 may inhibit key oncogenic Ras-inducible pathway(s) that are required for transformation. Three specific aims proposed to test this hypothesis are: (1) To determine the molecular mechanism(s) by which oncogenic ras down-regulates Par-4; (2) To determine the molecular mechanism(s) by which replenishment of Par- 4 prevents the process of transformation; and (3) To identify the structural domains of Par-4 that regulate suppression of transformation. The signaling mechanisms invoked by oncogenic Ras and Par-4 will be elucidated in fibroblasts in vitro, and the findings will be extended to examine whether the structural domains of Par-4 that inhibit transformation, also prevent oncogenic ras-inducible tumor growth in fibroblast and epithelial cell backgrounds in vivo. The proposed studies will help dissect out the functional domains of Par-4 that regulate the anti- transformation and apoptosis-sensitization properties of the protein. Thus, these studies are expected to uncover the signaling pathways used by oncogenic Ras to overcome the mechanisms that interfere with transformation and identify novel mechanisms that are utilized by Par-4 to block cellular transformation or tumor growth. The findings may help future studies to explore the feasibility of using Par-4 over-expression for tumor growth control.