Oncogenic K-ras stimulates Wnt signaling in colon cancer through inhibition of GSK-3beta.
OBJECTIVE
Two key genetic events underlying the development of colon cancer are activation of the K-ras and Wnt signaling pathways. We have previously shown that these 2 pathways can cooperate to regulate vascular endothelial growth factor (VEGF) gene expression. The goal of this study was to define the molecular basis for this interaction.


METHODS
The effects of K-ras(Val12) on VEGF and T-cell factor 4 (TCF-4) promoter activity, nuclear levels of beta-catenin and beta-catenin/TCF-4 complexes, glycogen synthase kinase 3beta (GSK-3beta) phosphorylation, and GSK-3beta kinase activity were measured. LY294002 and PD98059 were used to define the role of specific ras effector pathways.


RESULTS
Oncogenic K-ras up-regulated the activity of the VEGF promoter, and selective mutagenesis of TCF-4 binding sites significantly blocked this induction. K-ras(Val12) also induced the activity of a heterologous TCF-4 reporter construct in Caco-2 and HeLa cells. LY294002 and dominant negative phosphatidylinositol 3-kinase nearly completely blocked this induction. K-ras(Val12) increased the stability of beta-catenin, the levels of nuclear beta-catenin, and the formation of nuclear beta-catenin/TCF-4 complexes, and these effects were also blocked by LY294002. Finally, K-ras(Val12) inhibited the kinase activity of total cellular GSK-3beta and GSK-3beta complexed with Axin. This effect was not mediated through phosphorylation at serine 9 but did depend on phosphatidylinositol 3-kinase.


CONCLUSIONS
Our results suggest a unique cooperative interaction between 2 critical oncogenic pathways in colorectal tumorigenesis and highlight the pivotal role of GSK-3beta.
Li
Jingnan
J
Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.
Mizukami
Yusuke
Y
Zhang
Xiaobo
X
Jo
Won-Seok
WS
Chung
Daniel C
DC
D018938:Caco-2 Cells; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003110:Colonic Neoplasms / Q000235:genetics* / Q000503:physiopathology; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D011905:Genes, ras; D038362:Glycogen Synthase Kinase 3 / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000096:biosynthesis; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000502:physiology; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics; D051153:Wnt Proteins; D051176:beta Catenin
C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D051153:Wnt Proteins; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3