P3: iNOS/NO upregulates Wnt/β-catenin signaling through dickkopf-1 (DKK1) inhibition

Abstract

Wnt signaling contributes to carcinogenesis and tumor progression but the detailed mechanism of constitutive activation or silencing of Wnt signaling is not well-established. We have shown that human iNOS (hiNOS) gene is a target of Wnt/ β -catenin signaling pathway, and Wnt/ β -catenin signaling regulates hiNOS by inhibiting NF- κ B activity in cancer cells. DKK1 is a potent secreted Wnt antagonist that plays a key role in the negative regulation of the canonical Wnt system, by interacting with Wnt co-receptor proteins in the LRP family to inhibit Wnt signaling. However, the relationship between induced NO synthesis and DKK1-mediated inhibition of Wnt signaling has not been examined. The purpose of this study was to determine if iNOS expression and NO influence Wnt/ β -catenin activity, and if so whether this was mediated by an effect on DKK1 expression. Stable cell lines transfected with human iNOS (hiNOS) or DKK-1genes were established in the SW480 colon cancer cell line. Stable cell lines transfected with shRNA-iNOS were established in SW480 colon and MCF7 breast cancer cell lines. A Wnt signaling pathway microarray analysis (SuperArray) was employed to study the global transcriptional response to hiNOS gene expression. The Greiss reaction was used for the detection of NO production. RT-PCR and Western blotting were used for gene expression analysis. Banked cancer tissues were examined for hiNOS and DKK1 expression. Human iNOS (hiNOS) gene expression was inversely correlated with DKK1 expression in human colon and breast cancer tumor samples and intestinal adenoma tumors from Min ( APC min /+ ) mice. HiNOS gene expression promoted canonical Wnt signaling in cancer cells. HiNOS expression transcriptionally downregulated DKK1 gene expression in the Wnt-related gene superarray, and in cancer cell lines stably expressing hiNOS. Cytokines that induced iNOS expression in cancer cells and primary human hepatocytes decreased constitutive DKK1 protein levels. Knocking down hiNOS gene by shRNA restored DKK1 expression and inhibitory effect on Wnt signaling. These findings demonstrate that iNOS/NO down-regulate DKK1 gene expression at the transcriptional level in vitro, and are consistent with the in vivo findings in cancer tissues where iNOS and DKK1 expression are inversely correlated. These data identify a previously unrecognized mechanism by which the iNOS/NO pathway promotes cancer by inhibiting DKK1 which unleashes Wnt/ β -catenin signaling leading to enhanced β -catenin target gene activation. The findings provide new insight into the complex control mechanisms of iNOS/NO, DKK1, and Wnt/ β -catenin during carcinogenesis. Nothing to disclose.