[Mechanism of Wnt signaling pathway regulation by a truncated mutant of Axin2 in colorectal cancer]

Abstract

Background & objective: Axin2 gene which negatively regulates the Wnt signaling pathway was cloned recently. A truncated mutation of Axin2 (mtAxin2) is the most common mutation pattern in colorectal cancer (CRC) and could enhance the luciferase activity of T-cell factor (TCF). This study was to explore the mechanism of Wnt signaling pathway regulation by mtAxin2 in CRC.

Methods: The expression of beta-catenin in a mtAxin2-positive and a mtAxin2-negative CRC specimens was detected by immunohistochemistry. Plasmids containing wild-type or mutated Axin2 (pCMV-Flag-wtAxin2 and pCMV-Flag-mtAxin2) were constructed and co-transfected into 293 cells. The interactions of wtAxin2 and mtAxin2 with core components in Wnt signaling pathway was tested by co-immunoprecipitation (IP) and Western blot. TNT T3/T7 in vitro transcription and translation system were used to produce mtAxin2 and wtAxin2 proteins to verify the homodimerization of mtAxin2. Firefly and Renilla activities of mtAxin2 were detected with dual-luciferase report assay. Retinoid X receptor (RXR) and ecodysone receptor (ECR) plasmids were prepared for the experiment of restoration of TCF activity of mtAxin2.

Results: Immunohistochemistry showed that beta-catenin accumulated in the nuclei of mtAxin2-positive CRC cells and in cytoplasm of mtAxin2-negative CRC cells. Western blot showed that mtAxin2 bound to GSK-3beta, APC, beta-catenin, DVL-1 and PP2A as wtAxin2 did. After co-transfection of wtAxin2 and mtAxin2 in 293 cells, mtAxin2 competitively bound to beta-catenin. TNT T3/T7 experiment showed that wtAxin2 formed oligomers or dimers with Flag-wtAxin2, but not with Flag-mtAxin2. TCF activity in 293 cells was increased after transfection of pCMV-mtAxin2-RXR, but reduced after transfection of pCMV-mtAxin2-ECR or co-transfection of pCMV-mtAxin2-RXR and pCMV-mtAxin2-ECR, indicating the fusion of mtAxin2 and RXR formed dimers and restored its inhibitory effect on TCF activity.

Conclusion: Because of loss of DIX domain, mtAxin2 can not form dimmers, disturbs the degradation of beta-catenin, and leads to the nuclear accumulation of beta-catenin and activation of Wnt signaling pathway.