The overall goal of this project is to elucidate the role of KLF6 tumor suppressor in hepatocellular cancer (HCC) a highly prevalent tumor in patients with liver disease and cirrhosis. In HCC, KLF6 is deleted and/or mutated in a number of tumors and accumulates in the cytoplasm of both preneoplastic and HCC cells, suggesting that dysregulation of KLF6 may also occur in a premalignant milieu. The hypotheses are that a) KLF6 is a growth suppressor in normal hepatocytes, and its inactivation through loss, mutation or alternative splicing contributes to the development of hepatocellular cancer and; b) Alternative splicing of KLF6 is an early event in malignant transformation of hepatocytes, leading to loss of wild type function and gain-of-function of new transcriptional targets that contribute to carcinogenesis. We will test these hypotheses by following three Specific Aims: 1) To correlate the relative expression of KLF6 alternative splice forms by real-time PCR with histologic progression from human to cirrhosis HCC, and examine the possible release of KLF6 splice form mRNA or protein into the circulation in patients with HCC; 2) To replicate KLF6 dysregulation associated with human HCC in mouse models by: a. Generating a liver transgenic mouse overexpressing the predominant splice form found in human HCC downstream of the transthyretin promoter; b. Determining the response of KLF6 mice, which are phenotypically normal, to carcinogenic stress induced by either chemical or genetic means (KLF6 -/- are embryonic lethal) c. Abrogating wild type KLF6 function in liver in vivo through administration of siRNA that specifically downregulates wild type but not splice forms of KLF6; 3. To generate animals with conditional deletion of KLF6 in adult mouse liver using a tamoxifen-inducible cre-recombinase vector. The findings from these studies could lead to important new insights into the pathogenesis of HCC, ultimately yielding novel diagnostic and therapeutic strategies for this often-fatal neoplasm in millions of patients at risk worldwide.
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