383 Ablation of TGFbeta Receptor II Inhibits Liver Injury, Inflammation, Fibrosis and Carcinogenesis in Hepatocyte Specific TAK1 Deleted Mice

Background and Aims: Smad7 has been reported to inhibit inflammation. The role of Smad7 in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. We investigated the functional significance of Smad7 in HCC both In Vivo and In Vitro. Methods: Smad7 knockout (KO) and wildtype (WT) littermates were single injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days to induce HCC. The biological function of Smad7 was determined In Vitro in human HCC cell line (Hep3B) and primary cultures established from resected HCCs from KO and WT mice. Cell growth was evaluated by cell viability and colony formation, cell apoptosis by Annexin V/7-AAD double staining, cell cycle distribution by flow cytometry and cell invasion ability by Matrigel invasion assay. Signaling pathways and downstream effectors of Smad7 in HCC was evaluated by luciferase reporter activity assay and pathway-PCR-array. Results: Smad7 KO mice were more susceptible to DENinduced HCC than WT mice (78% vs 22%, P=0.018). HCCs from KO mice displayed a greater proliferative activity (P<0.01), and a reduced apoptotic index compared with the WT littermates (P<0.05). Smad7 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues (n=11, P<0.05). These In Vivo results indicated that Smad 7 may function as a potential tumor suppressor, which was further functionally tested In Vitro. Ectopic expression of Smad7 in Hep3B cells suppressed cell growth as evidenced by retarded cell growth curve (P<0.0001) and colony formation assay (P<0.01). Conversely, Smad7 increased cell apoptosis (P<0.05), consistent with enhanced cleaved caspase-9, caspase-3 and PARP, indicative of Smad7-induced apoptosis through the intrinsic caspase dependent pathway. Cell cycle analysis in Heb3B transfected with Smad7 revealed an increased proportion of cells arrested at G0/G1 phase (P<0.001), concomitant with a reduction in the proportion of cells in S-phase (P<0.001) as compared with control. Smad7 also repressed cell invasive ability (P<0.05). Moreover, primary culture cells form HCCs of KO mice showed a increased cell growth curve (P<0.01) and colony formation (P<0.01) compared with the cells generated from HCCs of WT mice. Moreover, pathway luciferase reporter assay indicated that Smad7 inhibited NF-κB and TGF-β signalings. Co-immunoprecipitation analysis showed that Smad7 directly interacts with TAB2, an upstream activator of NF-κB. The downstream targets of Smad7 was further revealed by TGF-β and NF-κB pathway PCR array as shown in Figure 1, which contribute to the tumor suppressive effect. Conclusion: Loss of Smad7 is sufficient to enhance susceptibility to HCC. Smad7 suppresses HCC growth through reducing cell proliferation and invasion, inducing G0/G1 phase arrest and apoptosis by inhibiting NF-κB and TGF-β signalings. Thus, Smad7 acts as a tumor-suppressor in the liver.