The connection of β-catenin and phenobarbital in murine hepatocarcinogenesis: a critical discussion of Awuah et al., PLoS ONE 7(6):e39771, 2012

Abstract

Phenobarbital (PB) is frequently used as a promoter of rodent liver tumors. The mechanisms by which PB exerts its tumorpromoting activity are still not fully understood, but the constitutive androstane receptor (CAR) and b-catenin seem to be essentially involved: Experiments with Car knockout (KO) mice have proven that the presence of CAR is mandatory for PB-mediated tumor promotion (Yamamoto et al. 2004). In mice, PB selects for the outgrowth of hepatocellular tumors with activating mutations in the Ctnnb1 gene, which encodes the transcription factor b-catenin (Aydinlik et al. 2001). Similar observations were made with the PB-like tumor promoter PCB153 (Strathmann et al. 2006). Furthermore, CAR and b-catenin interact in the regulation of enzyme induction and hepatocyte proliferation triggered by PB (Braeuning et al. 2009; Braeuning et al. 2011). In the absence of PB, two groups have studied hepatocarcinogenesis induced by diethylnitrosamine (DEN), a potent tumor initiator, in b-catenin-deficient livers from transgenic mice with conditional hepatocyte-specific KO of Ctnnb1. The unexpected finding of both studies was an increased number of tumors in Ctnnb1 KO mice, which contradicts the classic view of b-catenin as an oncogenic driver of tumor formation (Rignall et al. 2011; Zhang et al. 2010). Possible explanations for this finding are increased inflammation and oxidative stress in the KO mice (Zhang et al. 2010), as well as the increased number of hepatocytes with high levels of O-ethylguanine after DEN injection (Rignall et al. 2011). Subsequent administration of PB promoted tumor formation in livers of wild-type (WT) C3H mice, whereas a lack of tumor promotion was observed in Ctnnb1 KO mice in the same experiment (Rignall et al. 2011). This can be attributed to the fact that the target cells of PB-mediated tumor promotion, namely Ctnnb1-mutated hepatoma cells, are largely absent from Ctnnb1 KO livers. The group of Dr. Monga has recently reported an increase in liver tumor burden of DEN/PB-treated Ctnnb1 KO mice, as compared to WT controls (Awuah et al. 2012). This seemingly contradicts previous findings (Rignall et al. 2011) and thus questions the established connection of PB and b-catenin, at least for readers who are not highly specialized in the field of tumor promotion. In their recent study, the authors treated 2-week-old C57BL/6 mice with a single dose of DEN, followed by chronic exposure to PB (Awuah et al. 2012). The age of mice at DEN injection is highly relevant since, in C3H mice, it has been shown that PB inhibits hepatocarcinogenesis when the initiator DEN is given to 2-week-old mice, whereas it promotes tumorigenesis after DEN injection at 6 weeks of age (Moennikes et al. 2000). Comparably, PB inhibits tumor development in DENinduced (2 weeks) mice from strain B6C3F1, a crossbreed of C3H and C57BL/6 (Diwan et al. 1984; Klaunig et al. 1987, 1988; Lee et al. 1998). The paradoxical phenomenon of tumor inhibition by PB has been reviewed by Lee (2000). Using CD1 mice, however, Tamano et al. (1994) have demonstrated tumor promotion by PB in animals treated with DEN at 2 weeks of age. Therefore, it cannot be excluded that PB has acted as a tumor promoter in the study by Awuah and colleagues. However, the majority of available studies argue against a tumor-promoting activity of PB when using the 2-week DEN treatment regimen, especially since the C57BL/6 strain is rather refractory to liver tumor formation and promotion (Maronpot 2009). A. Braeuning (&) Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tubingen, Wilhelmstr. 56, 72074 Tubingen, Germany e-mail: albert.braeuning@uni-tuebingen.de