SignificanceCD24+CD133+ liver cancer stem cells (LCSCs) express higher levels of the inducible nitric oxide synthase (iNOS) and possess self-renewal and tumor growth properties. iNOS is associated with more aggressive hepatocellular carcinoma (HCC), leading to the upregulation of Notch1 signaling. The activation of Notch1 by iNOS/NO is dependent on cGMP/PKG-mediated activation of TACE and upregulation of iRhom-2. The expression of iNOS, CD24, and CD133 correlates with the expression of activated TACE and Notch signaling in more aggressive human HCC. These findings have implications for understanding how LCSCs are regulated in the setting of chronic inflammation, where signals to upregulate iNOS are often present. Targeting iNOS could have therapeutic benefit in HCC.

Human hepatocellular carcinoma (HCC) is driven and maintained by liver cancer stem cells (LCSCs) that display stem cell properties. These LCSCs are promoted by the intersecting of Notch and Wnt/β-Catenin signaling pathways. In this study, we demonstrate that LCSCs with markers CD90, CD24, CD13, and CD133 possess stem properties of self-renewal and tumorigenicity in NOD/SCID mice. The increased expression of these markers was correlated with advanced disease stage, larger tumors, and worse overall survival in 61 HCC cases. We also found that both Notch and Wnt/β-catenin signaling pathways played important roles in increasing the stem-ness characteristics of LCSCs. Our data suggested that Notch1 was downstream of Wnt/β-catenin. The active form of Notch1 intracellular domain (NICD) expression depended on Wnt/β-catenin pathway activation. Moreover, Notch1 negatively contributed to Wnt/β-catenin signaling modulation. Knock down of Notch1 with lentivirus N1ShRNA up-regulated the active form of β-catenin. Ectopic expression of NICD with LV-Notch1 in LCSCs attenuated β-catenin/TCF dependent luciferase activity significantly. In addition, there was a non-proteasome mediated feedback loop between Notch1 and Wnt/β-catenin signaling in LCSCs. The central role of Notch and the Wnt/β-catenin signaling pathway in LCSCs may provide an attractive therapeutic strategy against HCC.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, anti-viral, and anti-proliferative effects. In this study, we examined the effects of IFN-γ on autophagy and cell growth in human hepatocellular carcinoma (HCC) cells. IFN-γ inhibited cell growth of Huh7 cells with non-apoptotic cell death. IFN-γ induced autophagosome formation and conversion/turnover of microtubule associated protein 1 light chain 3 (LC3) protein. Furthermore, overexpression of IRF-1 also induced autophagy in Huh7 cells. Silencing IRF-1 expression with target small hairpin RNA blocked autophagy induced by IFN-γ. Silencing of the autophagy signals Beclin-1 or Atg5 attenuated the inhibitory effect of IFN-γ on Huh7 cells with decreased cell death. Additionally, IFN-γ activated autophagy in freshly cultured human HCC cells. Together, these findings show that IFN-γ induces autophagy through IRF-1 signaling pathway and the induction of autophagy contributes to the growth-inhibitory effect of IFN-γ with cell death in human liver cancer cells.

Nitric oxide (NOÁ), an important mediator of inflammation, and B-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that B-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-B-catenin or anti-Tcf-4 antibody. Overexpression of B-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of B-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3B, increased cytosolic and nuclear B-catenin level, NOS2 expression, and NOÁ production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased B-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic B-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that B-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/ B-catenin signaling pathway may contribute to cancer by increasing NOÁ production.

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor KB (NF-KB) and has recently been shown to be a target of the Wnt/B-catenin pathway. In this study, we tested the hypothesis that Wnt/B-catenin signaling might regulate cytokine-or tumor necrosis factor A (TNFA)-induced hiNOS expression through interaction with NF-KB. A cytokine mixture of TNFA + interleukin (IL)-1B + IFN; induced a 2-to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of B-catenin produced a dose-dependent decrease in NF-KB reporter activity and decreased cytokine mixture-induced hiNOS promoter activity. Gel shift for TNFAinduced hiNOS NF-KB activation showed decreased p50 binding and decreased NF-KB reporter activity in the Bcatenin-mutant HAB18 cells. Conversely, enhanced p50 binding and increased NF-KB reporter activity were seen in HAB85 cells, which lack B-catenin signaling. Coimmunoprecipitation confirmed that B-catenin complexed with both p65 and p50 NF-KB proteins. NF-KB-dependent Traf1 protein expression also inversely correlated with the level of Bcatenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased Bcatenin protein and increased TNFA-induced p65 NF-KB binding as well as iNOS and Traf1 expression. Finally, Bcatenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that B-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-KB-dependent gene expression. These findings underscore the complex role of Wnt/B-catenin, NF-KB, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis. [Cancer Res 2009;69(9):3764-71]