[Background] Alcohol and diabetes are both suggested to damage the liver via toll-like receptor-4 (TLR4) and to enhance hepatitis C virus (HCV)-induced liver carcinogenesis. A connection between these two effects has recently been identified by our findings HCV NS5A protein induces TLR4 expression in hepatocytes that mediates synergistic liver tumor development in mice fed alcohol. It is yet to be determined if this synergism is also observed for diabetes. Stem cell-like gene profile is also linked to tumorigenesis, but its role in our liver tumor models has not been examined. [Objectives] We aimed to determine whether high-cholesterol, high saturated fat diet (HCFD) that causes obesity and insulin resistance, results in synergistic induction of liver tumor via TLR4 signaling in HCV NS5A transgenic (Tg) mice. We also determined if the stem cell markers are induced in NS5A Tg mice fed alcohol or HCFD in a manner dependent on TLR4. [Methods] Liver specific NS5A Tg and wild type (wt) mice with or without TLR4 deficiency (tlr4-/-) were fed HCFD supplemented with or without LPS (20 mg/kg diet) for 12 months. Liquid diet containing alcohol or isocaloric dextrin was also fed to NS5A Tg, tlr4-/-NS5A, wt, tlr4-/- mice. [Results] HCFD feeding activated TRAF6-TAK1 and induced steatohepatitis (NASH) and liver tumors in NS5A Tg mice but not in wt or tlr4-/-NS5A mice despite comparably elevated plasma concentrations of endotoxin. Further, LPS supplementation of HCFD resulted in aggravated NASH and an additional increment in liver tumor incidence (35%) in NS5A Tg mice as compared to those-fed HCFD without LPS (24%). In both alcohol and HCFD models, microarray analysis and qPCR demonstrated the stem cell marker Nanog is induced 7~19-fold in NS5A Tg mice but not in wt or NS5A/tlr4-/- mice. Nanog immunostaining was also co-localized with CD49f, a hepatoblast marker, in NS5A Tg mice. To determine if Nanog is downstream of TLR4, in vitro promoter and mRNA analysis were performed in Huh7 cells. LPS caused a 14-fold increase in Nanog promoter activity and a 9-fold increase in Nanog mRNA in NS5A-transfected cells, and these effects were largely abrogated by retroviral transduction of shRNA for TLR4. [Conclusions] These results demonstrate that HCFD like alcohol feeding induces hepatocellular tumors in NS5A Tg mice entirely dependent on TLR4. We also present the first evidence of Nanog transcriptional activation by NS5A/LPS-induced TLR4 signaling. These findings indicate TLR4 may promote liver tumorigenesis via both accentuated inflammation and direct induction of stem cell phenotype in liver diseases caused by HCV and diabetes/alcohol.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1866.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009