Wnt/β-Catenin Signaling Regulates Cytokine-Induced Human Inducible Nitric Oxide Synthase Expression by Inhibiting Nuclear Factor-κB Activation in Cancer Cells

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor kappa B (NF-kappa B) and has recently been shown to be a target of the Wnt/beta-catenin pathway. In this study, we tested the hypothesis that Wnt/beta-catenin signaling might regulate cytokine- or tumor necrosis factor a (TNF alpha)-induced hiNOS expression through interaction with NF-kappa B. A cytokine mixture of TNF alpha + interleukin (IL)-1 beta + IFN gamma 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 beta-catenin produced a dose-dependent decrease in NF-kappa B reporter activity and decreased cytokine mixture-induced hiNOS promoter activity. Gel shift for TNF alpha-induced hiNOS NF-kappa B activation showed decreased p50 binding and decreased NF-kappa B reporter activity in the beta-catenin-mutant HA beta 18 cells. Conversely, enhanced p50 binding and increased NF-kappa B reporter activity were seen in RA beta 85 cells, which lack beta-catenin signaling. Coimmunoprecipitation confirmed that beta-catenin complexed with both p65 and p50 NF-kappa B proteins. NF-kappa B-dependent Traf1 protein expression also inversely correlated with the level of beta-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased Pcatenin protein and increased TNF alpha-induced p65 NF-kappa B binding as well as iNOS and Traf1. expression. Finally, catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that beta-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-kappa B-dependent gene expression. These findings underscore the complex role of Wnt/beta-catenin, NF-kappa B, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis. [Cancer Res 2009;69(9):3764-71]