Inhibition of β-catenin signaling by phenobarbital in hepatoma cells in vitro
Introduction
Phenobarbital (PB) is an antiepileptic drug due to its interference with γ-aminobutyric acid (GABA)-dependent neurotransmission (Macdonald and McLean, 1986). PB is capable of inducing tumors in rodents by a non-genotoxic mechanism via activation of the constitutive androstane receptor (CAR) and also involving cell–cell communication via the gap junction protein connexin 32 (Cx32) (Moennikes et al., 2000, Yamamoto et al., 2004). For a detailed overview of the toxicological properties of PB please refer to IARC (2001). Human relevance of tumorigenicity of PB through CAR activation is subject of an ongoing controversial debate (Braeuning, 2014, Braeuning et al., 2014, Braeuning et al., 2015, Braeuning and Schwarz, 2016, Elcombe et al., 2014, Yamada et al., 2014).
Despite decades of research on PB effects and CAR activation, the mechanism for the indirect activation of CAR by PB had not been elucidated until a few years ago (Mutoh et al., 2013): PB activates CAR via inhibition of the epidermal growth factor receptor (EGFR). This leads to diminished activity of the kinase Src, which in turn impacts on the phosphorylation of RACK1, thus enabling non-phosphorylated RACK1 to activate protein phosphatase 2A (PP2A) leading to dephosphorylation of CAR. Non-phosphorylated CAR then translocates to the nucleus and initiates pleiotropic transcriptional responses related to hepatocyte proliferation, hypertrophy and apoptosis, but also to xenobiotic metabolism and energy homeostasis. Cellular functions of CAR have e.g. been reviewed by Qatanani and Moore (2005) and by Whysner et al. (1996).
While chronic administration of PB to mice promotes the outgrowth of certain particular hepatoma sub-populations under appropriate experimental conditions, other tumor sub-populations, characterized by their individual tumorigenesis-driving genomic mutations and corresponding gene expression profiles, are significantly inhibited by PB in their growth, thus making the substance more a tumor selector than a tumor promoter (Braeuning and Schwarz, 2016): the formation of hepatocellular carcinoma (HCC) driven by pronounced activation of the canonical Wnt/β-catenin signaling pathway is inhibited by PB in a mouse model of hepatic APC (adenomatous polyposis coli protein) deficiency (Braeuning et al., 2016). Canonical Wnt/β-catenin signaling is one of the most prominent oncogenic signaling cascades and frequently activated in human as well as rodent tumors. Genomic alterations in different critical components of the cascade, for example in Ctnnb1 (encoding β-catenin) or Apc (encoding a negative regulator of β-catenin) lead to constitutive activation of the pathway. This contributes to tumor growth by triggering the expression of downstream target genes related to proliferation and tumorigenesis as reviewed by Lustig and Behrens (2003). Physiologically, the activity of the pathway is mainly regulated by post-translational modifications of β-catenin which impact on the degradation of the protein: phosphorylation of β-catenin at residues near the N-terminus by a cytosolic multi-protein complex which consists, amongst others, of APC and glycogen synthase kinase 3β (GSK3β), primes the protein for subsequent proteasomal degradation. This process is blocked by inhibition of GSK3β or the proteasome, or by Ctnnb1 mutations that eliminate the critical phosphorylation sites; inhibition of β-catenin degradation activates the pathway and leads to cytosolic accumulation and nuclear translocation of free, transcriptionally active β-catenin (Lustig and Behrens, 2003). Of note, β-catenin regulation might also occur via altered transcription of the Ctnnb1 gene (Gosens et al., 2010). Interestingly, the HCC from the abovementioned mouse model are defective in β-catenin degradation due to their lack of functional APC, but additionally exhibit strongly increased Ctnnb1 mRNA levels which also seem to contribute to β-catenin activation in this particular tumor type (Braeuning et al., 2016). Another mechanism of regulation is via enhanced translation of Ctnnb1 mRNA, a process which is mediated by Src kinase which phosphorylates the eukaryotic initiation factor 4E via extracellular signal-regulated kinase (ERK)-dependent signaling and thus regulates β-catenin protein levels by altering its synthesis, not its degradation (Karni et al., 2005). Moreover, mouse liver tumors with activated mitogen-activated protein kinase (MAPK)/ERK signaling due to activating mutations in the Ha-ras proto-oncogene upstream of ERK are also efficiently inhibited by PB (Aydinlik et al., 2001, Moennikes et al., 2000).
HCC from the APC-deficient mouse model described above are repressed by PB despite an almost complete lack of CAR expression (Braeuning et al., 2016). Moreover, MAPK-activated mouse liver tumors exhibit reduced expression of CAR, as compared to normal hepatocytes (Jaworski et al., 2007), and react less sensitive to PB stimulation with regard to the induction of CAR target genes related to xenobiotic metabolism (own unpublished data). It thus appears likely that CAR-independent effects mediate at least some of the tumor-inhibitory effects of PB. As tumor growth is driven by specific oncogenic signaling cascades, it appears plausible that PB might interfere with such cellular signal transduction pathways to inhibit the growth of certain tumor sub-populations. Interestingly, data from a recent publication on PB inhibition of calpains, a family of proteases, suggest an inhibition of β-catenin signaling by PB (Groll et al., 2016a).
Therefore, this study was designed to provide an in-depth analysis of the effects of PB on Wnt/β-catenin signaling in mouse hepatoma cells in vitro and to unravel the underlying molecular mechanisms.
Section snippets
Cell culture and treatment
Mouse hepatoma cells from line 70.4 (Groll et al., 2016a, Kress et al., 1992), stably transfected with the 8 x β-catenin/TCF-driven SuperTopFlash (STF) luciferase reporter vector (Braeuning et al., 2007), and a 70.4-derived sub-clone stably transfected with a doxycycline-inducible system for the expression of mutant, constitutively active human β-cateninS33Y (termed “70.4Mo”; Zeller et al., 2012) were cultivated in D-MEM/F12 medium supplemented with 10% fetal calf serum and antibiotics as
Results
In the course of a recent analysis of PB effects on calpain expression and activity it has been reported that PB inhibits β-catenin signaling in mouse hepatoma cells in vitro (Groll et al., 2016a). Neither a comprehensive description nor a mechanistic interpretation of this phenomenon has been published in the literature. It has also recently been shown that the outgrowth of β-catenin-driven HCC is reduced by PB in a mouse model of HCC (Braeuning et al., 2016). This was now followed up in order
Discussion
The present data show that PB inhibits signaling through the oncogenic β-catenin signaling pathway in hepatoma cells. While β-catenin inhibition by PB has been mentioned as a secondary finding in a single previous publication (Groll et al., 2016a), this is the first study which provides an in-depth characterization of the phenomenon and provides insight into the underlying molecular mechanisms.
The inhibitory effect of PB on β-catenin was shown to be independent of the nuclear receptor CAR and
Acknowledgments
The authors thank Johanna Mahr and Elke Zabinsky for expert technical assistance. The gift of Src kinase expression vectors by Dr. R. Lammers (Tübingen, Germany) and of erlotinib by Dr. M. Toulany (Tübingen, Germany) is greatly acknowledged. This work was funded by the German Federal Ministry of Education and Research (BMBF), grant FKZ 0315742 (Virtual Liver).
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