SHP-1/STAT3訊息傳遞路徑於肝癌標靶治療之分子機制研究

Abstract

Abstract Human hepatocellular carcinoma (HCC) is the fifth most prevalent solid tumor in the world and the fourth main inducer of cancer-related death. Surgical resection, the only curative treatment for HCC, is feasible in only 20-30% of the patients at diagnosis. Unfortunately, systemic chemotherapy or liver transplantation is limited for patients with HCC. In 2007, sorafenib (NexavarR), a multiple kinase inhibitor, has shown survival benefits in patients with advanced HCC and become the first clinically approved drug for HCC. First, we have showed that sorafenib sensitizes HCC cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) through the inhibition of signal transducer and activator of transcription 3 (STAT3) (Chapter 1). In this study, we found that sorafenib inhibits STAT3 through up-regulating the activity of Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1), a protein tyrosine phosphatase and a key inhibitory regulator of STAT3, in HCC cells. To examine whether sorafenib’s effect on STAT3 is related to its kinase inhibitory activity, we further generated a series of sorafenib derivatives which lack activities on kinases. Interestingly, we have observed SC-1, a sorafenib derivative which is close to sorafenib structurally but no kinase activity, showed even better in vivo activity than sorafenib in HCC tumors. These results suggest that SHP-1-dependent STAT3 inhibition is a major kinase-independent target of sorafenib (Chapter 2). In addition to sorafenib, we also found that dovitinib, another multiple kinase inhibitor, shows the anti-HCC effect via a SHP-1/STAT-dependent signaling pathway and overcomes the resistance of sorafenib (Chapter 3). Finally, to elucidate the molecular mechanism by which sorafenib increases SHP-1 phosphatase activity, we demonstrated the conformation-based changes in SHP-1 to promote catalytic activity after sorafenib treatment (Chapter 4). Sorafenib increases SHP-1 activity in vitro and in vivo, indicating that sorafenib affects phosphatase activity directly. Based on a series of deletion mutants of SHP-1, we observed that N-terminal SH2 domains (N1) strongly involved in sorafenib-induced STAT3 inhibition and apoptotic effect. Moreover, D61, a critical residue in N1 responsible to form inhibitory salt bridge with catalytic domain, also abrogates the biological effect of sorafenib. In co-IP experiments, sorafenib impairs the interaction between N1 and PTP directly. Furthermore, the role of elevated SHP-1 served as tumor suppressor of HCC was confirmed in cells expressed constitutively activate mutant (dN1 and D61A). Together, we propose a conformational change model for sorafenib-induced SHP-1 activity. Sorafenib potentially opened the inhibited structure of SHP-1 through impairing the linkage between N-SH2 and PTP domains to increase phosphatase activity.