Mutation of the retinoblastoma tumor suppressor gene (Rb1) causes the pediatric cancer retinoblastoma and contributes to most types of human cancer. Despite its central role in tumorigenesis, therapies designed to target this pathway have been slow to develop. A detailed understanding of the molecular mechanisms underlying Rb1 mediated tumor suppression will facilitate the development of such therapies. The long-term goal of this continuing project is to identify and characterize these molecular mechanisms. Rb1 protein (pRb) is a negative regulator of the cell cycle, and this cell cycle regulatory activity contributes to tumor suppression. Multiple mechanisms likely contribute to pRb mediated cell cycle control, including the well characterized mechanism involving binding of pRb to E2F transcription factors resulting in transcriptional repression of genes required for cell cycle progression. Another possible mechanism involves targeted degradation of Skp2 by pRb and consequent stabilization of p27Kip1. The ability of pRb to regulate E2F transcription factors is genetically separable from its ability to degrade Skp2. We hypothesize that pRb mediated degradation of Skp2 and stabilization of p27Kip1 contributes to prostate tumor suppression in vivo. Our working model is that post translational stabilization of p27Kip1 (p27) normally enforces a senescence like response to oncogenic stress, thereby curtailing tumor initiation and progression. In the absence of pRb, this tumor suppressive response is muted. The goal of this application is to test the hypothesis using in vitro and in vivo experimental model systems. We have created a mouse strain containing a mutant Rb1 allele (R654W) whose encoded protein is deficient for E2F binding and regulation, but may be competent to target Skp2 for degradation. We propose to use this mutant allele to assess the relative contribution of the pRb/E2F and pRb/p27 mechanisms to prostate tumor suppression in vivo. Two specific aims are proposed: 1) Test whether R654W pRb can suppress prostate tumorigenesis in the mouse;2) Characterize the contribution of the pRb/Skp2/p27 mechanism to R654W pRb tumor suppressor activity. Successful completion of these specific aims will identify a new mechanism contributing to Rb1 tumor suppression and suggest new therapeutic strategies to treat prostate cancer.