Oncogenic K-ras Stimulates Wnt Signaling in Colon Cancer Through Inhibition of GSK-3β

Background & Aims: Two key genetic events underlying the development of colon cancer are activation of the K-ras and Wnt signaling pathways. We have previously shown that these 2 pathways can cooperate to regulate vascular endothelial growth factor (VEGF) gene expression. The goal of this study was to define the molecular basis for this interaction. Methods: The effects of K-rasVal12 on VEGF and T-cell factor 4 (TCF-4) promoter activity, nuclear levels of β-catenin and β-catenin/TCF-4 complexes, glycogen synthase kinase 3β (GSK-3β) phosphorylation, and GSK-3β kinase activity were measured. LY294002 and PD98059 were used to define the role of specific ras effector pathways. Results: Oncogenic K-ras up-regulated the activity of the VEGF promoter, and selective mutagenesis of TCF-4 binding sites significantly blocked this induction. K-rasVal12 also induced the activity of a heterologous TCF-4 reporter construct in Caco-2 and HeLa cells. LY294002 and dominant negative phosphatidylinositol 3-kinase nearly completely blocked this induction. K-rasVal12 increased the stability of β-catenin, the levels of nuclear β-catenin, and the formation of nuclear β-catenin/TCF-4 complexes, and these effects were also blocked by LY294002. Finally, K-rasVal12 inhibited the kinase activity of total cellular GSK-3β and GSK-3β complexed with Axin. This effect was not mediated through phosphorylation at serine 9 but did depend on phosphatidylinositol 3-kinase. Conclusions: Our results suggest a unique cooperative interaction between 2 critical oncogenic pathways in colorectal tumorigenesis and highlight the pivotal role of GSK-3β.