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


CONCLUSIONS
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.
Wang
Feng-Wei
FW
Department of Radiation Oncology, Tianjin People's Hospital, Tianjin, 300130, P. R. China. wfengwei2004@tom.com
Wen
Ling
L
Zhu
Si-Wei
SW
Yao
Qiang
Q
Cai
Yu-Mei
YM
Ma
Gang
G
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D025601:Adenomatous Polyposis Coli Protein / Q000378:metabolism; D060466:Axin Protein; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism*; D000072261:Dishevelled Proteins; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids; D015398:Signal Transduction; D051820:TCF Transcription Factors / Q000378:metabolism; D014162:Transfection; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism*
C491761:AXIN2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D025601:Adenomatous Polyposis Coli Protein; D060466:Axin Protein; D003598:Cytoskeletal Proteins; C000606578:DVL1 protein, human; D000072261:Dishevelled Proteins; D010750:Phosphoproteins; D051820:TCF Transcription Factors; D051153:Wnt Proteins; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3