Mechanisms of Met Induced Hepatocytes Survival

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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, 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-3 and caspase-8 (i.e. inhibits caspase-8 self activation). We further propose that the Met DNAD?DEVD?T decoy site 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 three specific aims: In Aim 1, we will investigate the functional role of the caspase ¿decoy¿ site (- EDNAD?DEVD?TRPASFWETS) present in the c-terminal end of human Met to caspase inhibition and promotion of hepatocyte survival and hepatocarcinogenesis by using loss-of-function and gain-of-function approaches. In Aim 2, we will test if Met inhibits the caspase by a novel serpin-like mechanism hence covalently trapping the caspase active site site by making a thioester linkage with the carbonyl carbon of the scissile peptide bond (the bond between D and T residues) and the Cys catalytic residue in caspase. In Aim 3, we will determine the status of Met caspase ¿decoy¿ site cleavage, Met-caspase-3/8 association and caspase- 3/8 enzymatic activity in human HCC cell lines and HCC tissues and matched surrounding liver. Collectively these studies will establish novel insights into the molecular mechanisms of HCC growth and cell survival. They may open avenues for rational drug design (i.e. peptidomimetic agonists and antagonists corresponding to the Met caspase decoy site) to treat liver diseases ranging from hepatitis to HCC.