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

Abstract

Aberrant Wnt-signaling caused by mutants of beta-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 beta-catenin mutations. In order to prove this hypothesis, 52 samples from human hepatocellular carcinomas were analysed for the activation of beta-catenin and the expression of GS. In total, 45 samples stained positive for cytoplasmic/nuclear beta-catenin. A strong correlation between expression of GS and activated beta-catenin (100% of nuclear and 84% of cytosolic) was found. However, among 35 GS positive tumors that were analysed for beta-catenin mutations no mutations were detected in 25 GS-positive carcinomas although 24 out of the 25 carcinomas exhibited at least abnormal expression of beta-catenin. Since the mutational analysis identified 9 different point mutations of the beta-catenin gene including the rare mutation H36P and the yet unknown mutation P44A it was asked whether these mutations may differently effect beta-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 beta-catenin target sequences and different beta-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 beta-catenin on the GS-5'-enhancer is only one aspect of gene activation induced by beta-catenin.

Figure 1

Examples of β-catenin phenotypes detected in human hepatocellular carcinomas. A: Magnification of the squared area seen in 'D' with β-catenin located in the cytoplasm and also in several nuclei (arrowheads). B: Tumor with normal confinement of β-catenin to the cell membrane and C: cytoplasmic staining of β-catenin without detectable expression in the nuclei. D and E: serial sections of an HCC expressing glutamine synthetase (E) and nuclear β-catenin (D). Original magnification: 400× (A, B, C) and 100× (D, E). The mutation of β-catenin in the serial sections was S33C.

Figure 2

Correlation between expression of GS and the phenotype of β-catenin expression. The %age of GS-expressing tumors with nuclear (N), cytosolic (C) and normal membranous (M) β-catenin expression is indicated. Numbers on top indicate the total amount of HCCs in each group.

Figure 3

Effect of different mutations of β-catenin on the expression of the reporter gene 'TOP-flash'. Cells from the cell-line HuH7 were transfected with the reporter gene 'TOP-flash' harbouring LEF-1/TCF binding sites for β-catenin and the corresponding 'FOP-flash' without these sites. Each of the two reporters was cotransfected with several expression plasmids for different β-catenin forms: The construct 'bCATa' as a control not expressing any functional protein, the vector 'pBatem' expressing a truncated β-catenin, 'pCS2', expressing a form with a mutation of all N-terminal phosphorylation sites to alanine and in addition plasmids with mutations found in human HCCs: H36P, P44A, S45F and S45P. The expression from each co-transfection with 'TOP-flash' was compared to cotransfection with 'FOP-flash' in order to determine the factor of enhancement mediated by the mutants.

Figure 4

Effect of different mutations of β-catenin on the expression of a reporter gene with the main 5'-enhancer of GS. Cells from the cell-line HuH7 were transfected with the reporter gene 'HIII/EV_pT81' containing the main GS 5'-enhancer together with different expression plasmids for β-catenin forms (compare legend to Fig. 3). Expression was compared to expression from pT81 in order to determine the factor of enhancement mediated by the mutants.