The goals of the previous funding period were to map, clone, and characterize a tumor suppressor gene at 13q21 in prostate cancer. In 9 papers including 7 published and 2 submitted, we identified the transcription factor KLF5 as a strong candidate for the 13q21 gene, and demonstrated three mechanisms that inactivate KLF5 in cancer cells, including genomic deletion, transcriptional downregulation, and excessive protein degradation. In addition, we found that KLF5 is an indispensable component of the TGFbeta signaling pathway;and that a ubiquitin E3 ligase that ubiquitinates and degrades KLF5, WWP1, is often overexpressed via copy number gain at 8q21 in prostate cancer, which is responsible for excessive degradation of KLF5 in cancer cells. Furthermore, it has been shown that VWVP1 negatively regulates the TGFbeta signaling pathway by inducing the degradation of Smad2, Smad4, and TGFbeta receptor type I. We therefore hypothesize that overexpression of the E3 ligase WWP1 in epithelial cells causes excessive protein degradation of KLF5 and other components of the TGFbeta signaling pathway, and thus makes cells resistant to the inhibitory effect of TGFbeta in cell proliferation. As a result, cells become more susceptible to other factor-induced carcinogenesis. We will further test and validate this hypothesis in three specific aims. 1) To assess molecular alterations of WWP1 and related molecules in human prostate cancer, clinical cancer specimens will be examined for copy number gain, overexpression, and mutations of WWP1 and for expression change in KLF5 and other components of the TGFbeta pathway. The alterations will be correlated with clinicopathological features of prostate cancer. 2) To examine the function of WWP1 in cell growth in the context of KLF5 and TGFbeta, cells expressing different levels of WWP1 will be examined for growth, cell cycle progression, tumorigenesis, and gene expression changes. 3) To test the role of WWP1 overexpression in carcinogenesis using genetically modified mice, WWP1 will be specifically overexpressed in the prostates of mice. Such mice will be crossed with NKX3.1 and PTEN knockout mice. Phenotypic and molecular alterations will be analyzed in these mice. Completion of these studies will likely show WWP1 to be a molecule useful for developing biomarkers and therapeutic targets as well as for understanding prostate cancer biology.
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