Sucha Singh

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

Activation of β-Catenin and Yap1 in Human Hepatoblastoma and Induction of Hepatocarcinogenesis in Mice

BACKGROUND & AIMS Aberrant activation of β-catenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with the development of multiple tumor types. Yap functions as a transcriptional coactivator by interacting with TEA domain DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. METHODS We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the United States, consisting of 104 hepatocellular carcinoma, 62 intrahepatic cholangiocarcinoma, and 94 hepatoblastoma samples. We assessed β-catenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering RNAs, and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (ΔN90/β-catenin) and Yap1 (YapS127A) in livers of mice; tissues were collected, and histological and immunohistochemical analyses were performed. RESULTS We observed nuclear localization of β-catenin and Yap1 in 79% of hepatoblastoma samples but not in most hepatocellular carcinoma or intrahepatic cholangiocarcinoma samples. Yap1 and β-catenin coprecipitated in hepatoblastoma but not hepatocellular carcinoma cells. Small interfering RNA-mediated knockdown of Yap1 or β-catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to coactivate transcription with β-catenin; β-catenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and β-catenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumor cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEA domain factors was required for tumorigenesis in mice. CONCLUSIONS β-catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets.

Conditional β-catenin loss in mice promotes chemical hepatocarcinogenesis: Role of oxidative stress and platelet-derived growth factor receptor α/phosphoinositide 3-kinase signaling†

Activation of β‐catenin, the central effector of the canonical Wnt pathway and a recognized oncogene, has been implicated in hepatocellular carcinoma. We examined N‐nitrosodiethylamine (DEN)‐induced tumorigenesis in hepatic β‐catenin conditional knockout mice (β‐cat KO). Male β‐cat KO and age‐ and sex‐matched littermate controls were given a single intraperitoneal DEN injection and followed for 6‐12 months for hepatic tumors. Hepatic tumors were characterized for histology, proliferation, apoptosis, oxidative stress, and specific proteins by way of western blot, immunohistochemistry, and coprecipitation studies. For in vivo tumor intervention studies, specific inhibitors were administered intraperitoneally or through drinking water. Intriguingly, β‐cat KO mice showed a paradoxical increase in susceptibility to DEN‐induced tumorigenesis. This accelerated tumorigenesis is due to increased injury and inflammation, unrestricted oxidative stress, fibrosis, and compensatory increase in hepatocyte proliferation secondary to platelet‐derived growth factor receptor α (PDGFRα)/phosphoinositide 3‐kinase (PIK3CA)/Akt activation and c‐Myc overexpression. In vitro suppression of β‐catenin expression in hepatoma cells led to enhanced PDGFRα expression, which was abrogated in the presence of nuclear factor κB (NF‐κB) inhibitor. Daily treatment of 6‐month‐old DEN‐exposed β‐cat KO with PDGFRα inhibitor dramatically reduced tumor numbers and size. Inclusion of N‐acetyl‐L‐cysteine, a known antioxidant and NF‐κB inhibitor, in the drinking water led to complete abolition of tumorigenesis in DEN‐exposed β‐cat KO. Conclusion: Loss of β‐catenin impairs the livers ability to counteract DEN‐induced oxidative stress and enhances tumorigenesis through PDGFRα/PIK3CA/Akt signaling. Blockade of PDGFRα or oxidative stress dramatically affects β‐catenin–deficient tumorigenesis. Also, hepatoma cells use PDGFRα/PIK3CA signaling as an escape mechanism following β‐catenin suppression, and their sequential suppression profoundly impedes tumor proliferation. HEPATOLOGY 2010

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.

Tri‐iodothyronine induces hepatocyte proliferation by protein kinase a‐dependent β‐catenin activation in rodents

Thyroid hormone (T3), like many other ligands of the steroid/thyroid hormone nuclear receptor superfamily, is a strong inducer of liver cell proliferation in rats and mice. However, the molecular basis of its mitogenic activity, which is currently unknown, must be elucidated if its use in hepatic regenerative medicine is to be considered. F‐344 rats or C57BL/6 mice were fed a diet containing T3 for 2‐7 days. In rats, administration of T3 led to an increased cytoplasmic stabilization and nuclear translocation of β‐catenin in pericentral hepatocytes with a concomitant increase in cyclin‐D1 expression. T3 administration to wild‐type (WT) mice resulted in increased hepatocyte proliferation; however, no mitogenic response in hepatocytes to T3 was evident in the hepatocyte‐specific β‐catenin knockout mice (KO). In fact, T3 induced β‐catenin‐TCF4 reporter activity both in vitro and in vivo. Livers from T3‐treated mice demonstrated no changes in Ctnnb1 expression, activity of glycogen synthase kinase‐3β, known to phosphorylate and eventually promote β‐catenin degradation, or E‐cadherin‐β‐catenin association. However, T3 treatment increased β‐catenin phosphorylation at Ser675, an event downstream of protein kinase A (PKA). Administration of PKA inhibitor during T3 treatment of mice and rats as well as in cell culture abrogated Ser675‐β‐catenin and simultaneously decreased cyclin‐D1 expression to block hepatocyte proliferation. Conclusion: We have identified T3‐induced hepatocyte mitogenic response to be mediated by PKA‐dependent β‐catenin activation. Thus, T3 may be of therapeutic relevance to stimulate β‐catenin signaling to in turn induce regeneration in selected cases of hepatic insufficiency. (Hepatology 2014;59:2309–2320)

Spontaneous repopulation of β‐catenin null livers with β‐catenin‐positive hepatocytes after chronic murine liver injury

Prolonged exposure of mice to diet containing 0.1% 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) results in hepatobiliary injury, atypical ductular proliferation, oval cell appearance, and limited fibrosis. Previously, we reported that short‐term ingestion of DDC diet by hepatocyte‐specific β‐catenin conditional knockout (KO) mice led to fewer A6‐positive oval cells than wildtype (WT) littermates. To examine the role of β‐catenin in chronic hepatic injury and repair, we exposed WT and KO mice to DDC for 80 and 150 days. Paradoxically, long‐term DDC exposure led to significantly more A6‐positive cells, indicating greater atypical ductular proliferation in KO, which coincided with increased fibrosis and cholestasis. Surprisingly, at 80 and 150 days in KO we observed a significant amelioration of hepatocyte injury. This coincided with extensive repopulation of β‐catenin null livers with β‐catenin‐positive hepatocytes at 150 days, which was preceded by appearance of β‐catenin‐positive hepatocyte clusters at 80 days and a few β‐catenin‐positive hepatocytes at earlier times. Intriguingly, occasional β‐catenin‐positive hepatocytes that were negative for progenitor markers were also observed at baseline in the KO livers, suggesting spontaneous escape from cre‐mediated recombination. These cells with hepatocyte morphology expressed mature hepatocyte markers but lacked markers of hepatic progenitors. The gradual repopulation of KO livers with β‐catenin‐positive hepatocytes occurred only following DDC injury and coincided with a progressive loss of hepatic cre‐recombinase expression. A few β‐catenin‐positive cholangiocytes were observed albeit only after long‐term DDC exposure and trailed the appearance of β‐catenin‐positive hepatocytes. Conclusion: In a chronic liver injury model, β‐catenin‐positive hepatocytes exhibit growth and survival advantages and repopulate KO livers, eventually limiting hepatic injury and dysfunction despite increased fibrosis and intrahepatic cholestasis. (HEPATOLOGY 2011;)

Modeling a human hepatocellular carcinoma subset in mice through coexpression of met and point‐mutant β‐catenin

Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to its poorly understood molecular basis. In the current study, we investigated two independent cohorts of 249 and 194 HCC cases for any combinatorial molecular aberrations. Specifically we assessed for simultaneous HMET expression or hMet activation and catenin β1 gene (CTNNB1) mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we coexpressed hMet and β‐catenin point mutants (S33Y or S45Y) in hepatocytes using sleeping beauty transposon/transposase and hydrodynamic tail vein injection and characterized tumors for growth, signaling, gene signatures, and similarity to human HCC. Missense mutations in exon 3 of CTNNB1 were identified in subsets of HCC patients. Irrespective of amino acid affected, all exon 3 mutations induced similar changes in gene expression. Concomitant HMET overexpression or hMet activation and CTNNB1 mutations were evident in 9%‐12.5% of HCCs. Coexpression of hMet and mutant‐β‐catenin led to notable HCC in mice. Tumors showed active Wnt and hMet signaling with evidence of glutamine synthetase and cyclin D1 positivity and mitogen‐activated protein kinase/extracellular signal‐regulated kinase, AKT/Ras/mammalian target of rapamycin activation. Introduction of dominant‐negative T‐cell factor 4 prevented tumorigenesis. The gene expression of mouse tumors in hMet‐mutant β‐catenin showed high correlation, with subsets of human HCC displaying concomitant hMet activation signature and CTNNB1 mutations. Conclusion: We have identified cooperation of hMet and β‐catenin activation in a subset of HCC patients and modeled this human disease in mice with a significant transcriptomic intersection; this model will provide novel insight into the biology of this tumor and allow us to evaluate novel therapies as a step toward precision medicine. (Hepatology 2016;64:1587‐1605)

β-Catenin Loss in Hepatocytes Promotes Hepatocellular Cancer after Diethylnitrosamine and Phenobarbital Administration to Mice

Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 µg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p<0.05). Tumors in KO were β-catenin and glutamine synthetase negative and HGF/Met, EGFR & IGFR signaling was unremarkable. A significant increase in PDGFRα and its ligand PDGF-CC leading to increased phosphotyrosine-720-PDGFRα was observed in tumor-bearing KO mice (p<0.05). Simultaneously, these livers displayed increased cell death, stellate cell activation, hepatic fibrosis and cell proliferation. Further, PDGF-CC significantly induced hepatoma cell proliferation especially following β-catenin suppression. Our studies also demonstrate that the utilized DEN/PB protocol in the WT C57BL/6 mice did not select for β-catenin gene mutations during hepatocarcinogenesis. Thus, DEN/PB enhanced HCC in mice lacking β-catenin in the liver may be due to their ineptness at regulating cell survival, leading to enhanced fibrosis and regeneration through PDGFRα activation. β-Catenin downregulation also made hepatoma cells more sensitive to receptor tyrosine kinases and thus may be exploited for therapeutics.

Muc1 is protective during kidney ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

ADAR1 Prevents Liver Injury from Inflammation and Suppresses Interferon Production in Hepatocytes

Adenosine deaminase acting on RNA 1 (ADAR1) is an essential protein for embryonic liver development. ADAR1 loss is embryonically lethal because of severe liver damage. Although ADAR1 is required in adult livers to prevent liver cell death, as demonstrated by liver-specific conditional knockout (Alb-ADAR1(KO)) mice, the mechanism remains elusive. We systematically analyzed Alb-ADAR1(KO) mice for liver damage. Differentiation genes and inflammatory pathways were examined in hepatic tissues from Alb-ADAR1(KO) and littermate controls. Inducible ADAR1 KO mice were used to validate regulatory effects of ADAR1 on inflammatory cytokines. We found that Alb-ADAR1(KO) mice showed dramatic growth retardation and high mortality because of severe structural and functional damage to the liver, which showed overwhelming inflammation, cell death, fibrosis, fatty change, and compensatory regeneration. Simultaneously, Alb-ADAR1(KO) showed altered expression of key differentiation genes and significantly higher levels of hepatic inflammatory cytokines, especially type I interferons, which was also verified by inducible ADAR1 knockdown in primary hepatocyte cultures. We conclude that ADAR1 is an essential molecule for maintaining adult liver homeostasis and, in turn, morphological and functional integrity. It inhibits the production of type I interferons and other inflammatory cytokines. Our findings may provide novel insight in the pathogenesis of liver diseases caused by excessive inflammatory responses, including autoimmune hepatitis.