Proteolysis of many cellular regulators is controlled by ubiquitin ligases called SCFs since they are composed of three major subunits: Skp1, cull and one of many E-box proteins (Fbps). The substrate specificity of SCFs is determined by distinct Fbp subunits that act as substrate recognition factors. We have demonstrated that the cell cycle inhibitor p27 is degraded by the ubiquitin-pathway through the F-box protein Skp2. Importantly, destabilization of p27, which we and others have documented in human lymphomas and in epithelial cancers, correlates with tumor aggressivity. Our interest in the ubiquitin pathway lead us to the identification of a family of 26 human F-box proteins and the discovery that one member of this family, called 13-Trcp, regulates the stability of the proto-oncogene B-catenin. Because of their functions in regulating cell proliferation and given our preliminary results, we hypothesize that deregulation of the F-box proteins Skp2 and B-Trcp may participate in the genesis of human cancers.
In Aim 1 we will carry out immunohistochemical studies on Skp2 and B-Trcp expression in cancer samples obtained from two different tumor banks. We will concentrate our study on breast cancer, lymphoma and colorectal cancer. Our collection of tumor samples has concordant normal tissue and long-term follow-ups. In addition, these two tumor banks have extensive databases containing information on clinico-pathological features and standard molecular markers. Using univariate and multivariate analyses, we will determine the value of Skp2 and B-Trcp as novel prognostic markers for human cancers. Importantly, we will investigate possible causes for increased levels of these two F-box proteins and perform in vitro assays to measure their activities.
In Aim 2 we will then generate and characterize transgenic mice overexpressing Skp2, B-Trcp or corresponding dominant-negative mutants. Using this approach we will be able to investigate the effects of enforced expression of these Fbps in the mammary gland and lymphoid organs. Histopathological alterations expected in transgenic animal models will mimic those human tumors in which Skp2 and B-Trcp are overexpressed and will help us to define oncogenic pathways underlying the molecular abnormalities occurring in human tumors. Finally, in Aim 3 we will test whether the inhibition of Skp2 results in a cytostatic or cytotoxic effect in cancer cells. This will be achieved using specific membrane permeable peptides designed on the basis of our structural and biochemical information.
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