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

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.