Dual-function of beta-catenin in human tumor hepatocytes

ß-catenin is part of the cell-cell adhesion complex, where it plays a structural role but is also the key effector of the Wnt pathway, where it is endowed with a transcriptional regulatory activity. Oncogenic mutations of ß-catenin are present in about one third of hepatocellular carcinoma. In most tumors, ß-catenin mutations are heterozygous meaning that mutated and wild-type proteins co-exist in tumor cells. We address here the interplay between wild-type and mutated ß-catenins in liver tumor cells. We have designed a RNA interference strategy in HepG2 cells that allows uncoupling of the two functions of ß-catenin in the same cellular background: nuclear/transcriptional activity, a function almost exclusively mediated by the mutated ß-catenin, and membrane/structural activity, which is mediated by the degradable WT ß-catenin. The N-terminus part of ß-catenin contains the GSK-3ß phosphorylation sites that are crucial for the regulation of its turnover. This region encoded by the exon3 of ß-catenin is deleted on one allele of the CTNNB1 gene in HepG2 cells. Thus, a high amount of a truncated form of 76kDa is co-expressed with a 92-kDa full-length ß-catenin in HepG2 cells. We designed small interfering RNAs (siRNAs), named “sißcat-WT”, “sißcat-mut” and “sißcat-both” to target, respectively and specifically, either the wild-type (WT), the mutated form of ß-catenin, or both. We performed a transcriptional analysis of HepG2 cells treated with each siRNA. This analysis was performed in triplicates.