High frequency of β-catenin mutations in mouse HCCs induced by a non-genotoxic CAR agonist

Activation of Wnt/β-catenin signaling is frequent in human and rodent hepatocarcinogenesis. Although in mice, the tumor promoting activity of agonists of constitutive androstane receptor (CAR) occurs via selection of carcinogen-initiated cells harbouring β-catenin mutations, the molecular alterations leading to hepatocellular carcinoma (HCC) development by The CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), in the absence of genotoxic injury are unknown. Here we show that CAR activation per se induced HCC in mice and that 91% of them carried β-catenin point mutations or large in-frame deletions/exon skipping targeting Ctnnb1 exon 3. Point mutations in HCCs induced by TCPOBOP alone displayed different nucleotide substitutions compared to those found in HCCs from mice pre-treated with diethylnitrosamine (DENA). Moreover, unlike those occurring in HCCs from DENA+TCPOBOP mice, they did not result in increased expression of β-catenin target genes, such as Glul, Lgr5, Rgn, Lect2 and Ccnd1, or nuclear translocation of β-catenin when compared to the control liver. Remarkably, in the non-tumoral liver tissue, chronic CAR activation led to down-regulation of these genes and to a partial loss of glutamine synthetase (GS)-positive hepatocytes. These results thus show that while chronic CAR activation per se induces HCCs carrying β-catenin mutations, it concurrently down-regulates the Wnt/β-catenin pathway in non-tumoral liver. They also indicate that the relationship between CAR and β-catenin may be profoundly different between normal and neoplastic hepatocytes. 3 Controls, 7 TCP 42 w, 9 Dena + TCP, 3 TCP 28 w.