Functional consequences of WNT3/Frizzled7-mediated signaling in non-transformed hepatic cells

We have previously demonstrated that WNT3 and Frizzled7 (FZD7) expression levelswere upregulated in hepatocellular carcinoma (HCC) and that they directly interact to activate the canonical Wnt/β–catenin pathway in HCC cell lines. In this study, we investigated the functional consequences of WNT3 and FZD7 expression levels in non-transformed hepatic cells to address the question of whether WNT3/FZD7-mediated signal transduction could be involved in cellular transformation. After stable transfection of WNT3 and FZD7, the activation of the Wnt/β–catenin pathway was confirmed by western blot, immunostaining and quantitative real-time reverse transcriptase–PCR (qRT–PCR) analysis in two non-transformed hepatocyte-derived cell lines. In vitro characteristics of the malignant phenotype were measured, including cell proliferation, migration, invasion and anchorage-independent growth in soft agar. Stable expression of WNT3 and FZD7 in the two cell lines led to cellular accumulation of β-catenin and expression of downstream target genes activated by this pathway. In the stable WNT3/FZD7-expressing clones, hepatic cell proliferation, migration, invasion as well as soft agar colony formation were enhanced compared with the non-transformed control cells. The epithelial–mesenchymal transition (EMT) factors, Twist, Snail and Vimentin, were increased in cells expressing WNT3 and FZD7. However, the WNT3/FZD7-expressing cells did not form tumors in vivo. We conclude that activation of the WNT3/FZD7 canonical pathway has a role in the early stages of hepatocarcinogenesis by promoting the acquisition of a malignant phenotype with features of EMT.