Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. The exact mechanisms related to hypoxia‐induced invasion and metastasis remain unclear. We ...elucidated the mechanism by which the nuclear‐damage–associated molecular pattern molecule, high‐mobility group box 1 (HMGB1), released under hypoxic stress, can induce an inflammatory response to promote invasion and metastasis in hepatocellular carcinoma (HCC) cells. Caspase‐1 activation was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of HMGB1 and subsequent activation of both Toll‐like receptor 4 (TLR4)‐ and receptor for advanced glycation endproducts (RAGE)‐signaling pathways. Downstream from hypoxia‐induced caspase‐1 activation, cleavage and release of proinflammatory cytokines interleukin (IL)‐1β and ‐18 occurred. We further demonstrate that overexpression of HMGB1 or treatment with recombinant HMGB1 enhanced the invasiveness of HCC cells, whereas stable knockdown of HMGB1 remarkably reduced HCC invasion. Moreover, in a murine model of HCC pulmonary metastasis, stable knockdown of HMGB1 suppressed HCC invasion and metastasis. Conclusion: These results suggest that in hypoxic HCC cells, HMGB1 activates TLR4‐ and RAGE‐signaling pathways to induce caspase‐1 activation with the subsequent production of multiple inflammatory mediators, which, in turn, promote cancer invasion and metastasis. (HEPATOLOGY 2012;55:1866–1875)
Abstract 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?
Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, ...but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/β-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of β-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.
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.
Wnt/β‐catenin signaling is emerging as a forerunner for its critical roles in many facets of human biology. Its roles in embryogenesis, organogenesis, and maintaining tissue and organ homeostasis ...demonstrate its munificent character. Its roles in pathological conditions such as cancer and other human disorders such as inflammatory disorders and fibrosis reveal its villainous disposition. In liver, it also maintains its dual personality and is clearly of essence in several physiological events such as development, regeneration, and growth. Its aberrant activation is also evident in many different tumors of the liver, and recent studies are beginning to identify its role in additional hepatic pathological conditions. It is contributing to liver physiology and pathology by regulating various basic cellular events, including differentiation, proliferation, survival, oxidative stress, morphogenesis, and others. This review discusses the contribution of the Wnt/β‐catenin signaling pathway in these events and simultaneously provides an essential overview of the major developments in the field of Wnt/β‐catenin and liver pathobiology. In addition, areas that are currently deficient or understudied are identified and discussed along with the avenues of translational and clinical relevance. (HEPATOLOGY 2007;45:1298–1305.)
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/β-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. β-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 β-catenin for 1 to 12 hours. These data were also verified by increased expression of the β-catenin surrogate target glutamine synthetase. β-Catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3β and an increase in levels of casein kinase 2α, and led to increased cyclin-D1, another known β-catenin target. These observations were next substantiated in β-catenin conditional-null mice (β-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in β-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in β-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear β-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of β-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.
Although the role of Wnt/β-catenin signaling in liver growth and development is well established, its contribution in non-neoplastic hepatic pathologies has not been investigated. Here, we examine ...the role of β-catenin in a murine model of diet-induced liver injury. Mice with hepatocyte-specific β-catenin deletion (KO) and littermate controls were fed the steatogenic methionine and choline-deficient (MCD) diet or the corresponding control diet for 2 weeks and characterized for histological, biochemical, and molecular changes. KO mice developed significantly higher steatohepatitis and fibrosis on the MCD diet compared with wild-type mice. Both wild-type and KO livers accumulated triglyceride on the MCD diet but, unexpectedly, higher hepatic cholesterol levels were observed in KO livers on both control and MCD diets. Gene expression analysis showed that hepatic cholesterol accumulation in KO livers was not attributable to increased synthesis or uptake. KO mice had lower expression of bile acid synthetic enzymes but exhibited higher hepatic bile acid and serum bilirubin levels, suggesting defects in bile export. Therefore, loss of β-catenin in the liver leads to defective cholesterol and bile acid metabolism in the liver and increased susceptibility to developing steatohepatitis in the face of metabolic stress.
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, the central component of the canonical Wnt pathway, plays important roles in the processes of liver regeneration, growth, and cancer. Previously, we identified temporal expression of ...β‐catenin during liver development. Here, we characterize the hepatic phenotype, resulting from the successful deletion of β‐catenin in the developing hepatoblasts utilizing Foxa3‐cyclization recombination and floxed‐β‐catenin (exons 2 through 6) transgenic mice. β‐Catenin loss in developing livers resulted in significantly underdeveloped livers after embryonic day 12 (E12) with lethality occurring at around E17 stages. Histology revealed an overall deficient hepatocyte compartment due to (1) increased cell death due to oxidative stress and apoptosis, and (2) diminished expansion secondary to decreased cyclin‐D1 and impaired proliferation. Also, the remnant hepatocytes demonstrated an immature phenotype as indicated by high nuclear to cytoplasmic ratio, poor cell polarity, absent glycogen, and decreased expression of key liver‐enriched transcription factors: CCAAT‐enhancer binding protein‐α and hepatocyte nuclear factor‐4α. A paucity of primitive bile ducts was also observed. While the stem cell assays demonstrated no intrinsic defect in hematopoiesis, distorted hepatic architecture and deficient hepatocyte compartments resulted in defective endothelial cell organization leading to overall fetal pallor. Conclusion: β‐Catenin regulates multiple, critical events during the process of hepatic morphogenesis, including hepatoblast maturation, expansion, and survival, making it indispensable to survival. (HEPATOLOGY 2008.)
Abstract Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is ...particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.
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