Hepatitis B virus encoded X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). Aberrant activation of the Hedgehog (Hh) pathway has been linked to cancer. Thus, experiments were designed to test the hypothesis that HBx contributes to HCC via activation of Hh signaling. HBx expression correlated with up-regulation of Hh markers in human liver cancer cell lines, in HBx transgenic mice that developed HCC and in liver samples from HBV infected patients with HCC. The findings in human samples provide clinical validation of those in the HBx transgenic mice (HBxTg), and underscore the relevance of these transgenic mice to disease pathogenesis. Further, blockade of Hh signaling inhibited HBx stimulation of cell migration, anchorage independent growth, HCC tumorigenesis in HBx transgenic mice and tumor growth in xenograft model. These results suggest that the ability of HBx to promote cancer is at least partially dependent upon Hh activation and that activation of Hh signaling appears to be important for the development of HBx associated HCC. HBx also activates pathways that stimulate downstream Hh signaling, such as PI3K/AKT and Ras/Raf/MEK, also referred as non-canonical Hh signaling. Upon canonical Hh inhibition, compensatory activation of these pathways was seen in the presence of HBx in liver cancer cell lines and in HBxTg mice. Individual inhibition of these pathways also down-regulated Gli2 expression in HBx positive cell lines. These data suggests that in addition to canonical Hh signaling, activation of PI3K/AKT and ERK pathways by HBx leads to up-regulation of Gli2 expression in HBV-mediated HCC. This work identifies Hh pathway inhibition as a therapeutic strategy to slow tumor development and this work could lead to combination therapies that target Hh, AKT and ERK pathways, which may prevent or delay the appearance/progression of HCC. / Biology