Correlation between β-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma

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Abstract
Aberrant Wnt-signaling caused by mutants of β-catenin, a key regulator of the canonical Wnt-signaling pathway, is frequently detected in cancer. Only recently, it was suggested that in hepatocellular carcinoma (HCC) the expression of the target gene glutamine synthetase (GS) is a highly reliable marker for the identification of β-catenin mutations. In order to prove this hypothesis, 52 samples from human hepatocellular carcinomas were analysed for the activation of β-catenin and the expression of GS. In total, 45 samples stained positive for cytoplasmic/nuclear β-catenin. A strong correlation between expression of GS and activated β-catenin (100% of nuclear and 84% of cytosolic) was found. However, among 35 GS positive tumors that were analysed for β-catenin mutations no mutations were detected in 25 GS-positive carcinomas although 24 out of the 25 carcinomas exhibited at least abnormal expression of β-catenin. Since the mutational analysis identified 9 different point mutations of the β-catenin gene including the rare mutation H36P and the yet unknown mutation P44A it was asked whether these mutations may differently effect β-catenin target genes. Therefore, expression plasmids for different mutations were constructed and cotransfected with the TOP-flash luciferase reporter and a reporter carrying the GS-5'-enhancer. The experiments confirmed that there are differences between different β-catenin target sequences and different β-catenin mutations. In addition, the failure that the endogenous expression of GS in GS-negative cells was not induced by the transient transfection experiment indicated that the effect of β-catenin on the GS-5'-enhancer is only one aspect of gene activation induced by β-catenin.