Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of β-catenin-mutated mouse liver tumors

Abstract

Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Phenobarbital (PB) is an antiepileptic drug which promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like diethylnitrosamine (DEN). Here we have investigated the prevalence and patterns of mutations in two genes, Haras and b-catenin, both known mutational targets in mouse hepatocarcinogenesis. Liver tumors were generated by a single administration of DEN to 6 week old mice followed by feeding of PB (0.05%) containing or control diet for 39 weeks. Mutations at Ha-ras codon 61 were screened by allele-speci®c oligonucleotide hybridization; b-catenin mutations were detected by direct sequencing of PCR products spanning exon 2. In tumors from mice treated with DEN alone, the prevalence of Ha-ras mutations was *30% (6/20), while no b-catenin mutations (0/13) were detectable in tumors of this treatment group. By contrast, Ha-ras mutations were undetectable in tumors from mice treated with DEN/PB (0/32), while *80% (37/46) of tumors from this group showed b-catenin mutations. These results demonstrate that PB strongly aects the prevalence of mutations in the two cancer-related genes, presumably by positive and negative selection for cells harboring the respective mutation. Oncogene (2001) 20, 7812 ± 7816

Liver cell proliferation and tumor promotion by phenobarbital: relevance for humans?

“…The results of this experiment on the one hand demonstrate that tumor promotion by PB does in fact occur in hCAr mice, whereas, on the other hand, it becomes evident that tumor promotion in the hCAr group is significantly less pronounced than in WT mice treated according to the same experimental schedule. The particular phenotype of the tumors observed in DeN/PB-treated hCAr mice, i.e., eosinophilic, Ctnnb1-mutated, and glutamine synthetase-positive, exactly matches the observations in DeN/PB-treated WT animals , whereas this tumor phenotype is almost never seen in DeN-induced tumors not promoted by PB (Aydinlik et al 2001). This observation further substantiates that tumor promotion has occurred in the hCAr model.…”

“…The proliferative response of rodent hepatocytes following PB treatment in vitro is not observed in comparable cultures of their human counterparts (Parzefall et al 1991). An inseparable correlation of PB-induced proliferation and tumor growth, however, cannot be assumed with absolute certainty; in mice, PB exclusively promotes the outgrowth of a very distinct population of tumors, which carry activating mutations in the β-catenin gene Ctnnb1 (Aydinlik et al 2001). Accordingly, tumor promotion by PB is absent from mice with hepatocyte-specific knockout of Ctnnb1 (rignall et al 2011), which, however, still exhibit a proliferative response .…”

Deficiency in the gap junction protein Connexin32 alters p27Kip1 tumor suppression and MAPK activation in a tissue-specific manner

“…Increased activation of MAPK pathways in hepatocarcinogenesis is often a result of Ha-ras mutation (approximately 30% of DEN-induced mouse liver tumors (Schwarz et al, 2003) and 40% of radiationinduced mouse liver tumors (Lumniczky et al, 1998)). Phenobarbital treatment has been shown to decrease GJIC in liver with one study demonstrating decreased Ha-ras mutations and increased b-catenin mutations following Phenobarbital liver tumor promotion in wildtype DEN-treated mice (Aydinlik et al, 2001). Here, we did not detect any correlation between genotype and frequency of liver tumors harboring activating Ha-ras codon-61 mutations nor did we detect any b-catenin exon 2 or 3 mutations.…”

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

“…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).…”

“…Indirect evidence for the above assumption that PB did not act as a tumor promoter in their study comes from the author's notion that the ''utilized DEN/PB protocol in the WT C57BL/6 mice did not select for b-catenin gene mutations during hepatocarcinogenesis'' (Awuah et al 2012): PB and similarly acting compounds selectively promote the outgrowth of tumor cells with activated b-catenin in C3H (Aydinlik et al 2001), B6129SF2/J (Strathmann et al 2006), and C57BL/6 mice (MarxStoelting et al 2009; own unpublished analyses of tumors from the latter experiment). In addition, in Ctnnb1 KO livers, chronic PB treatment provides a selective advantage to the few remnant b-catenin-possessing hepatocytes, as compared to surrounding Ctnnb1 KO cells (Braeuning et al 2010).…”