Background & Aims Aberrant activation of βcatenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with development of multiple tumor types. Yap functions as a transcriptional co-activator by interacting with TEAD 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 US, consisting of 104 hepatocellular carcinoma (HCC), 62 intrahepatic cholangiocarcinoma (ICC), and 94 hepatoblastoma samples. We assessed βcatenin 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 (si)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 ( N90-βcatenin) and Yap1 (YapS127A) in livers of mice; tissues were collected and histologic and immunohistochemical analyses were performed. Results We observed nuclear localization of βcatenin and Yap1 in 79% of hepatoblastoma samples, but not in most HCC or ICC tissues. Yap1 and β catenin co-precipitated in hepatoblastoma but not HCC cells. siRNA-mediated knockdown of Yap1 or β catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to co-activate transcription with βcatenin; βcatenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and βcatenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumors cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEAD 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.

The Wnt/b-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-b-catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated b-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 b-catenin along with an increase in total and nuclear cyclin-D1 protein at 40 hours in TG livers. To test if stimulation of b-catenin induces regeneration, we used hydrodynamic delivery of Wnt-1 naked DNA to control mice, which prompted an increase in Wnt-1, b-catenin, and known targets, glutamine synthetase (GS) and cyclin-D1, along with a concomitant increase in cell proliferation. b-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 upregulation of b-catenin, cyclin-D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. Conclusion: b-Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, b-catenin overexpression makes TG mice susceptible to DEN-induced HCC. (HEPATOLOGY 2010;51:1603-1613 W nt/b-catenin signaling is an evolutionarily well-conserved pathway and important in liver health and repair.1 In adult liver, bcatenin signaling is essentially quiescent, with active bcatenin restricted to hepatocytes in the centrizonal area where it regulates expression of genes such as glutamine synthetase (GS) and others involved in xenobiotic metabolism.2 In other hepatocytes, b-catenin steady state is achieved by phosphorylation at key serine/threonine residues and subsequent degradation, and is predominantly localized to membrane to mediate cell-cell adhesion by forming a bridge between E-cadherin and actin cytoskeleton. Activation of b-catenin signaling during liver regeneration has been reported in rats and mice. [4][5][6][7] Although a positive regulator in the activity of normal liver growth, aberrant activation of the Wnt/bcatenin pathway is implicated in hepatocarcinogenesis,

β‐Catenin in hepatocytes, under the control of Wnts, regulates pericentral gene expression. It also contributes to liver regeneration (LR) after partial hepatectomy (PH) by regulating cyclin‐D1 gene expression as shown in the β‐catenin and Wnt coreceptors low‐density lipoprotein receptor‐related protein 5/6 conditional knockouts (KO). However, conditional deletion of Wntless (Wls), required for Wnt secretion, in hepatocytes, cholangiocytes, or macrophages lacked any impact on zonation, while Wls deletion in macrophages only marginally affected LR. Here, we address the contribution of hepatic endothelial cells (ECs) in zonation and LR by characterizing EC‐Wls‐KO generated by interbreeding Wls‐floxed and lymphatic vessel endothelial hyaluronan receptor (Lyve1)‐cre mice. These mice were also used to study LR after PH. While Lyve1 expression in adult liver is limited to sinusoidal ECs only, Lyve1‐cre mice bred to ROSA26‐Stopflox/flox‐enhanced yellow fluorescent protein (EYFP) mice showed EYFP labeling in sinusoidal and central vein ECs. EC‐Wls‐KO mice showed decreased liver weights; lacked glutamine synthetase, cytochrome P450 2e1, and cytochrome P450 1a2; and were resistant to acetaminophen‐induced liver injury. After PH, EC‐Wls‐KO showed quantitative and qualitative differences in cyclin‐D1 expression at 24‐72 hours, which led to a lower hepatocyte proliferation at 40 hours but a rebound increase by 72 hours. ECs and macrophages isolated from regenerating livers at 12 hours showed significant up‐regulation of Wnt2 and Wnt9b messenger RNA; these are the same two Wnts involved in baseline β‐catenin activity in pericentral hepatocytes. Conclusion: At baseline, ECs secrete Wnt proteins essential for β‐catenin activation in pericentral hepatocytes. During LR, sinusoidal and central vein ECs and secondarily macrophages secrete Wnt2, while predominantly central vein ECs and secondarily macrophages are the likely source of Wnt9b. This process spatiotemporally regulates β‐catenin activation in hepatocytes to induce cell proliferation. (Hepatology Communications 2018;2:845‐860)

Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to poorly understood molecular basis. In the current study, we investigate 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 CTNNB1 mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we co-expressed hMet and β-catenin point-mutants (S33Y or S45Y) in hepatocytes using sleeping beauty (SB) 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. Co-expression 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 MAPK/ERK, AKT/Ras/mTOR activation. Introduction of dominant-negative TCF4 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. In conclusion, we have identified co-operation 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 towards precision medicine.

Acute liver failure (ALF) remains a disease with poor patient outcome. Improved prognosis is associated with spontaneous liver regeneration, which supports the relevance of exploring 'regenerative' therapies. Therefore, the role of the Wnt/␤-catenin pathway in liver regeneration following ALF was investigated. ALF was induced in mice by acetaminophen overdose, which is also a leading cause of liver failure in patients. ␤-catenin distribution was also studied in liver sections from acetaminophen-induced ALF patients. A nonlethal dose of acetaminophen, which induces liver regeneration, led to stabilization and activation of ␤-catenin for 1 to 12 hours. These data were also verified by increased expression of the ␤-catenin surrogate target glutamine synthetase. ␤-Catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3␤ and an increase in levels of casein kinase 2␣, and led to increased cyclin-D1, another known ␤-catenin target. These observations were next substantiated in ␤-catenin conditional-null mice (␤-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in ␤-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in ␤-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear ␤-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of ␤-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.