PKC ISOZYMES AND SUBSTRATES IN MAMMARY CARCINOGENESIS

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DESCRIPTION: Changes in cellular and molecular functions that contribute to the development and progression of breast cancer are largely undefined. Changes in activities and/or expression levels of components of growth regulatory signal transduction pathways that correlate with tumor progression have been noted. Understanding the functional significance of these biochemical correlates holds the promise for developing new and alternative strategies for prevention, detection, diagnosis and treatment of breast cancer. Protein kinase C (PKC) is a family of ubiquitously expressed enzymes known to be important in regulating basic mechanisms of cell growth and differentiation. PKC is also considered to be a significant signaling pathway in tumor promotion/progression since PKCs are the major cellular receptors for tumor promoting phorbol esters. The purpose of this proposal is to provide a clear understanding of the role of PKC isozymes and substrates in the transition of a normal breast cell into a cell with altered growth potential and subsequently into a cell with invasive, metastatic potential. PKC is actually a family of distinct isozymes that catalyze phospholipid-dependent protein phosphorylation. The role of individual isozymes in cellular processes has not yet been defined. Our preliminary data demonstrate increases in two PKC isozymes and decreases in two PKC substrates that correlate with conversion of preneoplasias to tumors and increased metastatic potential, (collaborations with Drs. D. Medina and D. Welch, respectively). Our goal is to test the functional significance of these biochemical correlates by manipulating the activities of individual isozymes through expression of PKCs, PKC dominant negative inhibitors and unique substrates. Ectopic genes will be introduced into cultured cells. Tumor incidence and metastatic potential of expressing clones will be determined in vivo with cells transplanted into the fat pad. Knowledge of the role of individual PKCs in regulation of mammary cell growth is essential for the development of new and alternate strategies for breast cancer treatment and prevention. Specific aims: 1. To compare levels of PKC isozymes and substrates in progressively transformed mammary cells and tumors. 2. To generate mammary cell lines with increased expression of individual PKCs or dominant negative inhibitor PKC constructs. 3. To determine the effects of expressing PKCs and PKC dominant negative inhibitors on tumor incidence and metastasis of transplanted mammary cells in vivo. 4. To generate cell lines with increased expression of two PKC substrates (clones 34 and 72) to assess the role of these proteins in growth and metastasis. 5. To determine the effects of expressing PKCs, PKC dominant negative inhibitors and substrates on 13762 rat mammary cells in culture.