The broad goal of this application is to understand the mechanisms that regulate translation of the CDK inhibitor p27Kip1 (p27) in normal cells and in breast cancer cells, p27 is an essential component of normal cell cycle control and is expressed at elevated levels in quiescent cells and downregulated upon reentry into the cell cycle. There is a very strong correlation between low p27 expression and poor clinical prognosis for numerous types of cancer, including breast cancer, p27 levels are regulated by multiple mechanisms including translation initiation efficiency. Evidence is provided demonstrating that the ability to use the p27 5'-UTR for cap-independent translation strongly correlates with steady state p27 levels in a panel of breast cancer cell lines. A set of factors that bind to the p27 5'-UTR has been identified. Preliminary data show that differences in the binding of these factors to the 5'-UTR corresponds to differences in p27 expression. Based on these findings, a model is presented for the mechanism by which these factors mediate cap-independent translation of p27. The 1st specific aim is intended to test this model. This will involve defining the RNA sequences and structures required for translational initiation at the p27 internal ribosome entry site. The p27 5'-UTR-interacting proteins will be characterized and the mechanism by which they affect translation will be explored. The 2nd aim is to determine if changes in the p27 5'-UTR-interacting proteins play a role in decreased levels of p27 in breast cancer cells. This will involve examining the levels, binding activity, subcellular localization, and post-translational modifications of these proteins. These experiments will be performed using the same panel of breast cancer cells that have already been characterized for p27 levels and the ability to use the p27 5'-UTR for cap-independent translation. The same experiments will be performed using normal fibroblasts that are either quiescent or proliferating. These experiments are expected to lead to an understanding of how these proteins function in normal cells and whether alterations in this process contribute to the loss of p27 expression that is observed in some tumor cells.
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