Regulation of cyclooxygenase-2 expression by the Wnt and ras pathways

Abstract

Mutations in the adenomatous polyposis coli (APC) gene and K-ras occur in the majority of human colorectal cancers. Loss of functional APC protein activates the Wnt signal transduction pathway, allowing the nuclear accumulation of beta-catenin, which then binds to T-cell factor-4 (Tcf-4), causing increased transcriptional activation of downstream target genes. We investigated the hypothesis that the activation of the WNT pathway regulates cyclooxygenase-2 (COX-2). COX-2 was down-regulated after the induction of full-length APC in the HT29-APC cell line. We identified a Tcf-4-binding element (TBE) in the COX-2 promoter that specifically bound to Tcf-4 in an electrophoretic mobility shift assay. COX-2 promoter luciferase activity is down-regulated by APC in a promoter reporter construct containing the, TBE but not with mutant TBE. Mutant beta-catenin expression up-regulated the COX-2 promoter activity and the endogenous COX-2 mRNA expression in HuH7, hepatocellular carcinoma cell line, which is partially abrogated by cotransfection with a dominant-negative Tcf-4 expression vector. Although beta-catenin alone did not increase COX-2 protein to detectable levels in HuH7 cells, coexpression of both mutant beta-catenin and mutant K-ras increased COX-2 protein expression, which is consistent with the previous reports that K-ras can stabilize COX-2 mRNA. Taken together, our data support the hypothesis that COX-2 is down-regulated by APC and up-regulated by nuclear beta-catenin accumulation, and additionally implicate the Wnt signal transduction pathway in colon and liver carcinogenesis.