Kari Nejak-Bowen

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/β-catenin signaling, which is activated relatively early during regeneration mostly through post-translational modifications. Once activated, β-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/β-catenin signaling is now documented in rats, mice, zebrafish and patients. More recently, a regenerative advantage of the livers in β-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. β-Catenin is also pertinent in hepatic oval cell activation and differentiation. However, aberrant activation of the Wnt/β-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 β-catenin gene or in AXIN1/2, which encodes for a scaffolding protein critical for β-catenin degradation. Intriguingly, in experimental models hepatic overexpression of normal or mutant β-catenin is insufficient for tumorigenesis. In fact β-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 β-catenin signaling be one such pathway that may be redundant in regeneration and indispensible in HCC in a subset of cases?

ACCELERATED LIVER REGENERATION AND HEPATOCARCINOGENESIS IN MICE OVEREXPRESSING SERINE-45 MUTANT BETA-CATENIN

The Wnt/β‐catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45‐mutated‐β‐catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated β‐catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin‐D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2‐fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S‐phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of β‐catenin along with an increase in total and nuclear cyclin‐D1 protein at 40 hours in TG livers. To test if stimulation of β‐catenin induces regeneration, we used hydrodynamic delivery of Wnt‐1 naked DNA to control mice, which prompted an increase in Wnt‐1, β‐catenin, and known targets, glutamine synthetase (GS) and cyclin‐D1, along with a concomitant increase in cell proliferation. β‐Catenin‐overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed up‐regulation of β‐catenin, cyclin‐D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. Conclusion: β‐Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, β‐catenin overexpression makes TG mice susceptible to DEN‐induced HCC. HEPATOLOGY 2010

Cell Cycle Effects Resulting from Inhibition of Hepatocyte Growth Factor and Its Receptor c-Met in Regenerating Rat Livers by RNA Interference

Hepatocyte growth factor (HGF) and its receptor c‐Met are involved in liver regeneration. The role of HGF and c‐Met in liver regeneration in rat following two‐thirds partial hepatectomy (PHx) was investigated using RNA interference to silence HGF and c‐Met in separate experiments. A mixture of 2 c‐Met‐specific short hairpin RNA (ShRNA) sequences, ShM1 and ShM2, and 3 HGF‐specific ShRNA, ShH1, ShH3, and ShH4, were complexed with linear polyethylenimine. Rats were injected with the ShRNA/PEI complex 24 hours before and at the time of PHx. A mismatch and a scrambled ShRNA served as negative controls. ShRNA treatment resulted in suppression of c‐Met and HGF mRNA and protein compared with that in controls. The regenerative response was assessed by PCNA, mitotic index, and BrdU labeling. Treatment with the ShHGF mixture resulted in moderate suppression of hepatocyte proliferation. Immunohistochemical analysis revealed severe suppression of incorporation of BrdU and complete absence of mitosis in rats treated with ShMet 24 hours after PHx compared with that in controls. Gene array analyses indicated abnormal expression patterns in many cell‐cycle‐ and apoptosis‐related genes. The active form of caspase 3 was seen to increase in ShMet‐treated rats. The TUNEL assay indicated a slight increase in apoptosis in ShMet‐treated rats compared with that in controls. Conclusion: The data indicated that in vivo silencing of c‐Met and HGF mRNA by RNA interference in normal rats results in suppression of mRNA and protein, which had a measurable effect on proliferation kinetics associated with liver regeneration. (HEPATOLOGY 2007.)

Beta‐catenin signaling in murine liver zonation and regeneration: A Wnt‐Wnt situation!

Liver‐specific β‐catenin knockout (β‐Catenin‐LKO) mice have revealed an essential role of β‐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 β‐catenin activity in these events remains an enigma. Here we investigate to what extent β‐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 β‐catenin gene remained intact. Intriguingly, like β‐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 β‐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 β‐catenin‐TCF4 association and diminished Cyclin‐D1 expression. Conclusion: Wnt‐signaling is the major upstream effector of β‐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 β‐catenin activation during LR. (Hepatology 2014;60:964–976)

Wnt/β-catenin signaling in hepatic organogenesis

Wnt/β-catenin signaling has come to the forefront of liver biology in recent years. This pathway regulates key pathophysiological events inherent to the liver including development, regeneration, and cancer, by dictating several biological processes such as proliferation, apoptosis, differentiation, adhesion, zonation and metabolism in various cells of the liver. This review will examine the studies that have uncovered the relevant roles of Wnt/β-catenin signaling during the process of liver development. We will discuss the potential roles of Wnt/β-catenin signaling during the phases of development, including competence, hepatic induction, expansion, and morphogenesis. In addition, we will discuss the role of negative and positive regulation of this pathway and how the temporal expression of Wnt/β-catenin can direct key processes during hepatic development. We will also identify some of the major deficits in the current understanding of the role of Wnt/β-catenin signaling in liver development in order to provide a perspective for future studies. Thus, this review will provide a contextual overview of the role of Wnt/β-catenin signaling during hepatic organogenesis.

β-Catenin Regulates Vitamin C Biosynthesis and Cell Survival in Murine Liver

Because the Wnt/β-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of β-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and l-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in β-catenin-null livers and greater expression in β-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active β-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit β-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of β-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between −163 and −157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by β-catenin. Significantly lower serum ascorbate levels were observed in β-catenin knock-out mice secondary to decreased expression of regucalcin and also of l-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to β-catenin loss and regucalcin decrease was contributing to apoptosis, β-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and l-gulonolactone oxidase expression, β-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of β-catenin in cell survival.

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

β-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 β-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-β-catenin were evaluated for β-catenin-T cell factor transactivation by TOPFlash reporter activity and expression of certain targets. Hepatocyte-specific-serine-45-mutated β-catenin transgenic mice (TG) and controls (Con) were used to study thioacetamide (TAA)-induced hepatic fibrosis and tumorigenesis. Sustained β-catenin activation was only observed in mutant-, not WT-β-catenin transfected HCC cells. Aberrant intratumoral β-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 β-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 β-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 β-catenin, inflammation, fibrosis and HCC. GS staining is highly sensitive in identifying HCC with nuclear β-catenin, which may in turn represent β-catenin mutations, and does so with high negative predictive value. Also, β-catenin mutations and cirrhosis do not appear to cooperate in HCC pathogenesis in mice and men.

Conditional genetic elimination of hepatocyte growth factor in mice compromises liver regeneration after partial hepatectomy.

Hepatocyte growth factor (HGF) has been shown to be indispensable for liver regeneration because it serves as a main mitogenic stimulus driving hepatocytes toward proliferation. We hypothesized that ablating HGF in adult mice would have a negative effect on the ability of hepatocytes to regenerate. Deletion of the HGF gene was achieved by inducing systemic recombination in mice lacking exon 5 of HGF and carrying the Mx1-cre or Cre-ERT transgene. Analysis of liver genomic DNA from animals 10 days after treatment showed that a majority (70–80%) of alleles underwent cre-induced genetic recombination. Intriguingly, however, analysis by RT-PCR showed the continued presence of both unrecombined and recombined forms of HGF mRNA after treatment. Separation of liver cell populations into hepatocytes and non-parenchymal cells showed equal recombination of genomic HGF in both cell types. The presence of the unrecombined form of HGF mRNA persisted in the liver in significant amounts even after partial hepatectomy (PH), which correlated with insignificant changes in HGF protein and hepatocyte proliferation. The amount of HGF produced by stellate cells in culture was indirectly proportional to the concentration of HGF, suggesting that a decrease in HGF may induce de novo synthesis of HGF from cells with residual unrecombined alleles. Carbon tetrachloride (CCl4)-induced regeneration resulted in a substantial decrease in preexisting HGF mRNA and protein, and subsequent PH led to a delayed regenerative response. Thus, HGF mRNA persists in the liver even after genetic recombination affecting most cells; however, PH subsequent to CCl4 treatment is associated with a decrease in both HGF mRNA and protein and results in compromised liver regeneration, validating an important role of this mitogen in hepatic growth.

Complete response of Ctnnb1-mutated tumours to β-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/β-catenin signalling in HCC cells or tumour xenograft models, no study has addressed the impact of β-catenin inhibition in a relevant murine HCC model driven by Ctnnb1 mutations. METHODS We studied the in vivo impact of β-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 β-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 β-catenin LNA every 48h as compared to the scrambled control. A decrease in β-catenin activity, cell proliferation and increased cell death was evident after β-catenin suppression. No effect of β-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 β-catenin suppression in HCC will be of significant therapeutic benefit, provided the tumours display Wnt activation via mechanisms like CTNNB1 mutations.

Beta-catenin-NF-κB interactions in murine hepatocytes: a complex to die for.

Wnt/β‐catenin signaling plays an important role in hepatic homeostasis, especially in liver development, regeneration, and cancer, and loss of β‐catenin signaling is often associated with increased apoptosis. To elucidate how β‐catenin may be regulating hepatocyte survival, we investigated the susceptibility of β‐catenin conditional knockout (KO) mice and their wild‐type (WT) littermates to Fas and tumor necrosis factor‐α (TNF‐α), two common pathways of hepatocyte apoptosis. While comparable detrimental effects from Fas activation were observed in WT and KO, a paradoxical survival benefit was observed in KO mice challenged with D‐galactosamine/lipopolysaccharide. KO mice showed significantly lower morbidity and liver injury due to early, robust, and protracted activation of NF‐κB in the absence of β‐catenin. Enhanced NF‐κB activation in KO mice was associated with increased basal inflammation and Toll‐like receptor 4 expression and lack of the p65/β‐catenin complex in hepatocytes. The p65/β‐catenin complex in WT livers underwent temporal dissociation allowing for NF‐κB activation to regulate hepatocyte survival following TNF‐α‐induced hepatic injury. Decrease of total β‐catenin protein but not its inactivation induced p65 activity, whereas β‐catenin stabilization either chemically or due to mutations repressed it in hepatomas in a dose‐dependent manner, whereas β‐catenin stabilization repressed it either chemically or due to mutations. Conclusion: The p65/β‐catenin complex in hepatocytes undergoes dynamic changes during TNF‐α–induced hepatic injury and plays a critical role in NF‐κB activation and cell survival. Modulation of β‐catenin levels is a unique mode of regulating NF‐κB activity and thus may present novel opportunities in devising therapeutics in specific hepatic injuries. (HEPATOLOGY 2013)