Mechanisms of Met Induced Hepatocytes Survival

肝细胞癌是仅次于胰腺的最致命的癌症之一 腺癌)。预计2007年肝细胞癌将导致大约70万人死亡。没有效果 肝癌的治疗是因为这些肿瘤对化疗药物具有耐药性而臭名昭著 主要通过诱导细胞凋亡来杀伤细胞。事实上，逃脱凋亡是癌细胞的一个基本特征。 肿瘤肝细胞抗凋亡的分子机制尚不清楚 特色化的。一种促生存分子是已知的肝细胞癌的启动子和维持因子，那就是蛋氨酸 癌基因，肝细胞生长因子的酪氨酸激酶受体。利用结构-功能研究，我们 发现人Met的胞内结构域含有一种新的真正的功能 Caspase-8/3裂解位点位于其c-末端，序列如下：DNAD？DEVD？TRPASFWETS (请注意[？]指示caspase切割位点)。基于这些和其他观察，我们有 提出了新的假设，即Met c-末端尾部作为一种新的诱饵底物来抑制 凋亡的caspase，如caspase-3和caspase-8(即抑制caspase-8的自我激活)。我们进一步 建议Met DNAD？DEVD？T诱骗位点留在caspase酶阻断的催化口袋中 其活性部位和防止促凋亡级联反应的自然过程。因此，我们建议 以下三个具体目标：在目标1中，我们将研究caspase诱骗位点(-)的功能作用 EDNAD、DEVD、TRPASFWETS)存在于人Met的c-末端，对caspase抑制和 应用功能丧失和功能获得促进肝细胞存活和肝癌的发生 接近了。在目标2中，我们将测试Met是否通过一种新的丝氨酸样机制抑制caspase 通过与Caspase活性中心的羰基碳进行硫酯连接，共价捕获Caspase活性中心 半胱氨酸酶中的半胱氨酸催化残基与D和T残基之间的剪切性多肽键。在《目标3》中， 我们将确定Met caspase诱骗位点切割、Met-caspase-3/8结合和caspase-3/8结合状态。 3/8人肝癌细胞系、肝细胞癌组织及配对的癌旁肝组织中的酶活性。集体地 这些研究将建立对肝癌生长和细胞存活的分子机制的新见解。 它们可能为合理的药物设计开辟道路(即拟肽类激动剂和相应的拮抗剂 到Met caspase诱骗位点)，用于治疗从肝炎到肝细胞癌的各种肝病。

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.