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Regulation of cyclooxygenase-2 expression by the Wnt and ras pathways
Yuzuru Araki1, Shu Okamura, S Perwez Hussain
1Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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.
Insights
Adenomatous polyposis coli (APC) down-regulates cyclooxygenase-2 (COX-2), while Wnt pathway activation, through beta-catenin, up-regulates COX-2. This implicates the Wnt pathway in colon and liver cancer development.
Area of Science:
- Molecular biology
- Cancer research
- Signal transduction
Background:
- Colorectal cancers frequently harbor mutations in adenomatous polyposis coli (APC) and K-ras.
- Loss of APC function activates the Wnt pathway, leading to beta-catenin accumulation and target gene transcription.
- The Wnt pathway's role in regulating cyclooxygenase-2 (COX-2) in cancer is not fully understood.
Purpose of the Study:
- To investigate the hypothesis that Wnt pathway activation regulates cyclooxygenase-2 (COX-2) expression.
- To elucidate the molecular mechanisms linking APC, beta-catenin, T-cell factor-4 (Tcf-4), and COX-2.
- To determine the role of the Wnt pathway in colon and liver carcinogenesis.
Main Methods:
- Utilized HT29-APC cell line to study COX-2 regulation by APC.
- Performed electrophoretic mobility shift assays (EMSA) to identify Tcf-4 binding to the COX-2 promoter.
- Employed luciferase reporter assays to assess COX-2 promoter activity.
- Investigated COX-2 mRNA and protein expression in HuH7 cells with mutant beta-catenin and K-ras.
Main Results:
- COX-2 was down-regulated upon induction of full-length APC in HT29-APC cells.
- A Tcf-4-binding element (TBE) was identified in the COX-2 promoter, which specifically bound Tcf-4.
- APC down-regulated COX-2 promoter activity via the TBE, while mutant beta-catenin up-regulated it.
- Coexpression of mutant beta-catenin and K-ras increased COX-2 protein levels in HuH7 cells.
Conclusions:
- COX-2 is negatively regulated by APC and positively regulated by nuclear beta-catenin accumulation.
- The Wnt signal transduction pathway plays a significant role in regulating COX-2.
- These findings implicate the Wnt pathway in the development of colon and liver cancers.