In their paper „Beta-Catenin Loss in Hepatocytes Promotes Hepatocellular Cancer after Diethylnitrosamine and Phenobarbital Administration to Mice”, Awuah et al. [1] report on an increase in liver tumor response in mice with hepatocyte-specific knockout (KO) of Ctnnb1 (encoding Beta-Catenin) as compared to respective wildtype mice. This finding was obtained using a regimen, where diethylnitrosamine (DEN) was used as initiator of hepatocarcinogenesis followed by continuous treatment with phenobarbital (PB), which is frequently used as a model tumor promoter. Readers not highly specialized in the field might therefore come to the conclusion that this finding is at variance with a recent report from our group [2] which demonstrated a complete lack of PB-mediated tumor promotion in mice with conditional hepatocyte-specific KO of Ctnnb1.
In their study, Awuah et al. have treated mice of strain C57BL/6 with DEN at 2 weeks of age before continuous PB exposure was started. In our study, C3H mice were treated with DEN at 5-6 weeks of age followed by PB exposure. The age of mice at the time of DEN treatment is of utmost importance since it has been shown that PB “paradoxically” inhibits rather than promotes hepatocarcinogenesis when initiation occurs in infant mice [3,4]. We are aware of a study by Tamano et al. [5] where promotional effects of PB in DEN (2 weeks) treated CD1 mice were reported and therefore do not exclude the possibility that strain differences may play some role in response to the DEN (2 weeks)/PB treatment regimen.
In any case, Awuah et al. have used only 2 groups in their study, Ctnnb1 KO and Ctnnb1 wildtype mice both treated with the DEN (2 weeks)/PB protocol, but show no data on respective DEN only controls. Thus, their claim that PB acted as a tumor promoter is not backed up by experimental data. Based on the above cited reports they cannot exclude the possibility that PB was either inactive as a tumor promoter or even inhibitory under the treatment regimen used. Supposed an ineffectiveness of PB, their finding of a higher tumor response in Ctnnb1 KO as compared to wildtype mice would be in agreement with previous results by our and the Monga group [2,6] that Ctnnb1 KO mice not treated with PB have a somewhat higher tumor response when compared to not PB-treated wildtype mice.
The assumption that PB was not active as a tumor promoter in their study is indirectly substantiated by a statement the authors made in the abstract of their paper, namely that the “utilized DEN/PB protocol in the WT C57BL/6 mice did not select for Beta-catenin gene mutations during hepatocarcinogenesis”. We and others have repeatedly shown that selection of Ctnnb1-mutated hepatocytes is a hallmark of PB-mediated tumor promotion in mouse liver [7,8]. This is not limited to hepatocarcinogenesis in C3H mice but is also seen in the C57BL strain, where PB promoted the outgrowth of glutamine synthetase-positive liver tumors [9], which were all mutated in Ctnnb1 (own unpublished observation). Moreover, PB has been shown in several studies to stimulate the outgrowth of eosinophilic lesions (e.g. see [3]) while the same study demonstrated that it inhibits the growth of basophilic lesions. Of note, the liver lesions shown in Figure 1C of the study of Awuah et al. appear to be basophilic rather than eosinophilic in nature, based on the impression taken from the low resolution photomicrograph shown.
In conclusion, the design of the study of Awuah et al. does not allow proving tumor promotional activity of PB in their experiment. We would therefore like to emphasize that this study does not disprove two central findings of our previous work, namely that (i) tumor promotion of hepatocarcinogenesis by PB is abolished in mice lacking Beta-catenin in their hepatocytes [2] and (ii) that PB selects for the outgrowth of Ctnnb1-mutated hepatocytes during mouse hepatocarcinogenesis [7].

Albert Braeuning, Michael Schwarz, Albrecht Buchmann

Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany

References
1. Awuah PK, Rhieu BH, Singh S, Misse A, Monga SP (2012) beta-Catenin Loss in Hepatocytes Promotes Hepatocellular Cancer after Diethylnitrosamine and Phenobarbital Administration to Mice. PLoS One 7: e39771.
2. Rignall B, Braeuning A, Buchmann A, Schwarz M (2011) Tumor formation in liver of conditional beta-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen. Carcinogenesis 32: 52-57.
3. Lee GH, Ooasa T, Osanai M (1998) Mechanism of the paradoxical, inhibitory effect of phenobarbital on hepatocarcinogenesis initiated in infant B6C3F1 mice with diethylnitrosamine. Cancer Res 58: 1665-1669.
4. Moennikes O, Buchmann A, Romualdi A, Ott T, Werringloer J, et al. (2000) Lack of phenobarbital-mediated promotion of hepatocarcinogenesis in connexin32-null mice. Cancer Res 60: 5087-5091.
5. Tamano S, Merlino GT, Ward JM (1994) Rapid development of hepatic tumors in transforming growth factor beta transgenic mice associated with increased cell proliferation in precancerous hepatocellular lesions initiated by N-nitrosodiethylamine and promoted by phenobarbital. Carcinogenesis 15: 1791-1798.
6. Zhang XF, Tan X, Zeng G, Misse A, Singh S, et al. (2010) Conditional beta-catenin loss in mice promotes chemical hepatocarcinogenesis: Role of oxidative stress and platelet-derived growth factor receptor alpha/phosphoinositide 3-kinase signaling. Hepatology 52: 954-965.
7. Aydinlik H, Nguyen TD, Moennikes O, Buchmann A, Schwarz M (2001) Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of beta-catenin-mutated mouse liver tumors. Oncogene 20: 7812-7816.
8. Calvisi DF, Ladu S, Factor VM, Thorgeirsson SS (2004) Activation of beta-catenin provides proliferative and invasive advantages in c-myc/TGF-alpha hepatocarcinogenesis promoted by phenobarbital. Carcinogenesis 25: 901-908.
9. Marx-Stoelting P, Borowiak M, Knorpp T, Birchmeier C, Buchmann A, et al. (2009) Hepatocarcinogenesis in mice with a conditional knockout of the hepatocyte growth factor receptor c-Met. Int J Cancer 124: 1767-1772.