Mechanisms of met-Induced Hepatocytes Survival

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ABSTRACT Hepatocellular carcinoma (HCC) is the one of most lethal forms of cancer (only second to pancreatic adenocarcinoma); it is anticipated that HCC will kill approximately 700,000 persons in 2007. No effective treatment for HCC exists as these tumors are notorious for being resistant to chemotherapeutic agents which kill cells mainly via induction of apoptosis. Indeed, escape from apoptosis is a cardinal feature of cancer cells. The molecular mechanisms involved in resistance of neoplastic hepatocytes to apoptosis are not well characterized. One pro-survival molecule that is a known promoter and maintenance factor for HCC is the Met oncogene, the tyrosine kinase receptor of Hepatocyte Growth Factor. Using structure-function studies during the past funding period we discovered that the intracellular cytoplasmic domain of human Met harbors a novel bona fide functional caspase-8/3 cleavage site at its c-terminal end with the following sequence DNAD?DEVD?TRPASFWETS (please note that [?] indicates the caspase cleavage site). Based on these and other observations, we have developed the novel hypothesis that the Met c-terminal tail acts as a novel ¿decoy substrate¿ to inhibit the apoptotic caspases such as caspase-8 (i.e. inhibits caspase-8 self activation). We further propose that the Met derived ?DEVD? peptide stays in the catalytic pocket of the caspase enzyme blocking its active site and preventing the natural course of the pro-apoptotic cascade. Accordingly we propose the following two specific aims: In Aim 1, we will investigate the functional role of the caspase ¿decoy¿ site (DNAD?DEVD?TRPASFWETS) present in the c-terminal end of human Met to caspase inhibition and promotion of hepatocyte survival and hepatocarcinogenesis. The approaches to be taken will be those of loss-of-function and gain-of-function models using in vitro hepatocyte cell culture models and in vivo transgenic mice to test this novel hypothesis. In Aim 2, we will assess the pathophysiological role of Met¿s caspase ¿decoy¿ site in human hepatocellular carcinoma (HCC). We will examine the status of Met¿s caspase ¿decoy¿ site cleavage, Metcaspase- 8 association and caspase-8 enzymatic activity in human HCC tissues and matched surrounding liver. We will also directly test our hypothesis that Met¿s caspase decoy site acts as a ¿kiss of death¿ for caspase-8 in HCC. To carry out these studies, we will employ immunoaffinity pull down experiments on human HCC cell lines (i.e. HepG2, Hep3B, HuH7) and tissues using caspase antibodies and MS/MS MALDITOF to achieve this goal.