The overall goal of this proposal is to understand the mechanisms involved in maintaining the survival of breast carcinoma cells. In marked contrast to normal mammary epithelial cells, breast carcinoma cells are exposed to the hypoxic, potentially lethal environment found within many tumors. Hypoxia actually provides a strong selective pressure for the survival of the most aggressive and metastatic cells. Based on recent findings, the hypothesis that hypoxia selects for the survival of breast carcinoma cells that are able to activate the AKT/PKB kinase and exhibit AKT-dependent survival will be examined in the first specific aim. A key component of this aim will be to assess the involvement of specific AKT isoforms, which have been linked to breast cancer, in hypoxic survival. Both genetic and biochemical approaches will be used to address these issues. In the second aim, a novel paradigm for breast carcinoma survival will be investigated that involves autocrine simulation of AKT activation and AKT-mediated survival by VEGF, a hypoxia-induced cytokine. A well-established antisense oligonucleotide approach will be used to reduce VEGF expression in metastatic breast carcinoma cells, and the effects of reduced VEGF expression on AKT activation and survival in hypoxia metastasis of breast carcinoma cells will be studied in the third aim. The pattern of neuropilin expression as a metastasis of breast carcinoma progression will be determined by in situ hybridization. In addition, the impact of modulating neuropilin expression and function on the survival and metastasis of breast carcinoma cells will be established using genetic approaches. Finally, in the fourth aim, the hypothesis will be examined that AKT-mediated survival is enhanced by the alpha6 integrins, a class of receptors that have been linked to breast cancer progression, and that one mechanism by which the alpha6 integrins contribute to breast carcinoma survival is to cooperate with VEGF signaling to activate AKT. The ability of alpha6beta1 and alpha6beta4 integrins to protect breast carcinoma cells from hypoxia-induced apoptosis, as well as the contribution of these integrins to metastasis, will be evaluated using a genetic approach to alter their expression. Collectively, the results obtained from this study will establish a novel mechanism that regulates the survival of metastatic breast carcinoma cells.
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