Wnt signaling regulates hepatobiliary repair following cholestatic liver injury in mice.

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).

Complete response of Ctnnb1-mutated tumours to ß-catenin suppression by locked nucleic acid antisense in a mouse hepatocarcinogenesis model.

BACKGROUND & AIMS: Hepatocellular cancer (HCC) remains a disease of poor prognosis, highlighting the relevance of elucidating key molecular aberrations that may be targeted for novel therapies. Wnt signalling activation, chiefly due to mutations in CTNNB1, have been identified in a major subset of HCC patients. While several in vitro proof of concept studies show the relevance of suppressing Wnt/ß-catenin signalling in HCC cells or tumour xenograft models, no study has addressed the impact of ß-catenin inhibition in a relevant murine HCC model driven by Ctnnb1 mutations. METHODS: We studied the in vivo impact of ß-catenin suppression by locked nucleic acid (LNA) antisense treatment, after establishing Ctnnb1 mutation-driven HCC by diethylnitrosamine and phenobarbital (DEN/PB) administration. RESULTS: The efficacy of LNA directed against ß-catenin vs. scrambled on Wnt signalling was demonstrated in vitro in HCC cells and in vivo in normal mice. The DEN/PB model leads to HCC with Ctnnb1 mutations. A complete therapeutic response in the form of abrogation of HCC was observed after ten treatments of tumour-bearing mice with ß-catenin LNA every 48h as compared to the scrambled control. A decrease in ß-catenin activity, cell proliferation and increased cell death was evident after ß-catenin suppression. No effect of ß-catenin suppression was evident in non-Ctnnb1 mutated HCC, observed after DEN-only administration. CONCLUSIONS: Thus, we provide the in vivo proof of concept that ß-catenin suppression in HCC will be of significant therapeutic benefit, provided the tumours display Wnt activation via mechanisms like CTNNB1 mutations.

ß-catenin signaling in murine liver zonation and regeneration: a Wnt-Wnt situation!

UNLABELLED: Liver-specific ß-catenin knockout (ß-Catenin-LKO) mice have revealed an essential role of ß-catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin-D1. However, what regulates ß-catenin activity in these events remains an enigma. Here we investigate to what extent ß-catenin activation is Wnt-signaling-dependent and the potential cell source of Wnts. We studied liver-specific Lrp5/6 KO (Lrp-LKO) mice where Wnt-signaling was abolished in hepatocytes while the ß-catenin gene remained intact. Intriguingly, like ß-catenin-LKO mice, Lrp-LKO exhibited a defect in metabolic zonation observed as a lack of glutamine synthetase (GS), Cyp1a2, and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to the absence of ß-catenin-TCF4 association and lack of Cyclin-D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked the ability to secrete Wnts from either liver epithelial cells (Wls-LKO), or macrophages including Kupffer cells (Wls-MKO), or endothelial cells (Wls-EKO). While Wls-EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls-LKO and Wls-MKO were viable but did not show any defect in hepatic zonation. Wls-LKO showed normal initiation of LR; however, Wls-MKO showed a significant but temporal deficit in LR that was associated with decreased ß-catenin-TCF4 association and diminished Cyclin-D1 expression. CONCLUSION: Wnt-signaling is the major upstream effector of ß-catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes, or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for ß-catenin activation during LR.

Beta-catenin signaling, liver regeneration and hepatocellular cancer: sorting the good from the bad.

Among the adult organs, liver is unique for its ability to regenerate. A concerted signaling cascade enables optimum initiation of the regeneration process following insults brought about by surgery or a toxicant. Additionally, there exists a cellular redundancy, whereby a transiently amplifying progenitor population appears and expands to ensure regeneration, when differentiated cells of the liver are unable to proliferate in both experimental and clinical scenarios. One such pathway of relevance in these phenomena is Wnt/ß-catenin signaling, which is activated relatively early during regeneration mostly through post-translational modifications. Once activated, ß-catenin signaling drives the expression of target genes that are critical for cell cycle progression and contribute to initiation of the regeneration process. The role and regulation of Wnt/ß-catenin signaling is now documented in rats, mice, zebrafish and patients. More recently, a regenerative advantage of the livers in ß-catenin overexpressing mice was reported, as was also the case after exogenous Wnt-1 delivery to the liver paving the way for assessing means to stimulate the pathway for therapeutics in liver failure. ß-Catenin is also pertinent in hepatic oval cell activation and differentiation. However, aberrant activation of the Wnt/ß-catenin signaling is reported in a significant subset of hepatocellular cancers (HCC). While many mechanisms of such activation have been reported, the most functional means of aberrant and sustained activation is through mutations in the ß-catenin gene or in AXIN1/2, which encodes for a scaffolding protein critical for ß-catenin degradation. Intriguingly, in experimental models hepatic overexpression of normal or mutant ß-catenin is insufficient for tumorigenesis. In fact ß-catenin loss promoted chemical carcinogenesis in the liver due to alternate mechanisms. Since most HCC occur in the backdrop of chronic hepatic injury, where hepatic regeneration is necessary for maintenance of liver function, but at the same time serves as the basis of dysplastic changes, this Promethean attribute exhibits a Jekyll and Hyde behavior that makes distinguishing good regeneration from bad regeneration essential for targeting selective molecular pathways as personalized medicine becomes a norm in clinical practice. Could ß-catenin signaling be one such pathway that may be redundant in regeneration and indispensible in HCC in a subset of cases?

ß-Catenin signaling in hepatocellular cancer: Implications in inflammation, fibrosis, and proliferation.

ß-Catenin signaling is implicated in hepatocellular carcinoma (HCC), although its role in inflammation, fibrosis, and proliferation is unclear. Commercially available HCC tissue microarray (TMA) of 89 cases was assessed for ß-catenin, one of its transcriptional targets glutamine synthetase (GS), proliferation (PCNA), inflammation (CD45), and fibrosis (Sirius Red). HCC cells transfected with wild-type (WT) or mutant-ß-catenin were evaluated for ß-catenin-T cell factor transactivation by TOPFlash reporter activity and expression of certain targets. Hepatocyte-specific-serine-45-mutated ß-catenin transgenic mice (TG) and controls (Con) were used to study thioacetamide (TAA)-induced hepatic fibrosis and tumorigenesis. Sustained ß-catenin activation was only observed in mutant-, not WT-ß-catenin transfected HCC cells. Aberrant intratumoral ß-catenin stabilization was evident in 33% cases with 9% showing predominant nuclear with some cytoplasmic (N/C) localization and 24% displaying predominant cytoplasmic with occasional nuclear (C/N) localization. N/C ß-catenin was associated with reduced fibrosis (p=0.017) and tumor-wide GS staining (p<0.001) while C/N correlated with increased intratumoral inflammation (p=0.064) and proliferation (p=0.029). A small subset of HCC patients (15.5%) lacked ß-catenin staining and exhibited low inflammation and fibrosis (p<0.05). TG and Con mice exposed to TAA showed comparable development of fibrosis and progression to cirrhosis and HCC. Taken together the data suggests a complex relationship of ß-catenin, inflammation, fibrosis and HCC. GS staining is highly sensitive in identifying HCC with nuclear ß-catenin, which may in turn represent ß-catenin mutations, and does so with high negative predictive value. Also, ß-catenin mutations and cirrhosis do not appear to cooperate in HCC pathogenesis in mice and men.