ABSTRACT
Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer1,2. The accumulation of metabolites leads to cell stress and inflammation in the liver3, but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6+ CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6+ CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6+ CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Liver/immunology , Liver/pathology , Non-alcoholic Fatty Liver Disease/immunology , Non-alcoholic Fatty Liver Disease/pathology , Receptors, CXCR6/immunology , Acetates/pharmacology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/pathology , Cell Death/drug effects , Cell Death/immunology , Diet, High-Fat/adverse effects , Disease Models, Animal , Humans , Interleukin-15/immunology , Interleukin-15/pharmacology , Liver/drug effects , Male , Mice , Mice, Inbred C57BLABSTRACT
BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the intrahepatic (iCCA) or extrahepatic (eCCA) bile ducts with poor prognosis and limited treatment options. Prior evidence highlighted a significant contribution of the non-canonical NF-κB signalling pathway in initiation and aggressiveness of different tumour types. Lymphotoxin-ß (LTß) stimulates the NF-κB-inducing kinase (NIK), resulting in the activation of the transcription factor RelB. However, the functional contribution of the non-canonical NF-κB signalling pathway via the LTß/NIK/RelB axis in CCA carcinogenesis and progression has not been established. METHODS: Human CCA-derived cell lines and organoids were examined to determine the expression of NF-κB pathway components upon activation or inhibition. Proliferation and cell death were analysed using real-time impedance measurement and flow cytometry. Immunoblot, qRT-PCR, RNA sequencing and in situ hybridization were employed to analyse gene and protein expression. Four in vivo models of iCCA were used to probe the activation and regulation of the non-canonical NF-κB pathway. RESULTS: Exposure to LTα1/ß2 activates the LTß/NIK/RelB axis and promotes proliferation in CCA. Inhibition of NIK with the small molecule inhibitor B022 efficiently suppresses RelB expression in patient-derived CCA organoids and nuclear co-translocation of RelB and p52 stimulated by LTα1/ß2 in CCA cell lines. In murine CCA, RelB expression is significantly increased and LTß is the predominant ligand of the non-canonical NF-κB signalling pathway. CONCLUSIONS: Our study confirms that the non-canonical NF-κB axis LTß/NIK/RelB drives cholangiocarcinogenesis and represents a candidate therapeutic target.
ABSTRACT
BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Microbiota , Non-alcoholic Fatty Liver Disease , Humans , Mice , Animals , Non-alcoholic Fatty Liver Disease/complications , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Mice, Inbred C57BL , Liver/pathology , Fibrosis , Liver Cirrhosis/complications , Mice, Transgenic , Immunoglobulin A/metabolism , Immunoglobulin A/pharmacology , Disease Models, Animal , Diet, High-Fat/adverse effectsABSTRACT
In association with the pandemic spreading of obesity and metabolic syndrome, the prevalence of NAFLD-related HCC is increasing almost exponentially. In recent years, many of the underlining multifactorial causes of NAFLD have been identified, and the cellular mechanisms sustaining disease development have been dissected up to the single-cell level. However, there is still an urgent need to provide clinicians with more therapeutic targets, with particular attention on NAFLD-induced HCC, where immune checkpoint inhibitors do not work as efficiently. Whereas much effort has been invested in elucidating the role of innate immune response in the hepatic NAFLD microenvironment, only in the past decade have novel critical roles been unraveled for T cells in driving chronic inflammation toward HCC. The metabolic and immune microenvironment interact to recreate a tumor-promoting and immune-suppressive terrain, responsible for resistance to anticancer therapy. In this article, we will review the specific functions of several T-cell populations involved in NAFLD and NAFLD-driven HCC. We will illustrate the cellular crosstalk with other immune cells, regulatory networks or stimulatory effects of these interactions, and role of the metabolic microenvironment in influencing immune cell functionality. Finally, we will present the pros and cons of the current therapeutic strategies against NAFLD-related HCC and delineate possible novel approaches for the future.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Carcinogenesis , Carcinoma, Hepatocellular/metabolism , Humans , Liver Neoplasms/pathology , Non-alcoholic Fatty Liver Disease/metabolism , T-Lymphocytes/metabolism , Tumor MicroenvironmentABSTRACT
The 2nd International DKFZ Conference on Cancer Prevention (CCP2020) organized by the German Cancer Research Center (DKFZ) was held as a virtual event on 17-18 September 2020. The event gathered experts on cancer prevention from around the world with the aim of generating a stimulating interchange of opinions between clinicians and basic researchers working in the field. The talks and posters of the conference fueled exciting discussions and debates about the state of the art of cancer prevention and provided a comprehensive outlook on the many aspects of the field. The program was divided into three main sessions, illustrating the most recent methodological approaches and interventions in primary, secondary and tertiary prevention, enriched by introductory lectures depicting the most relevant aspects of each session. The key concepts covered in this meeting were risk factors, early detection, improving life after cancer, cancer prevention in Europe and personalized prevention. The importance of the latter was expressly highlighted, many presentations emphasizing that in the era of personalized medicine, prevention also needs to be based on the unique genetic, epigenetic, social and behavioral characteristics of the individual to achieve maximal efficacy. In this article, we summarize the key messages emerging from each section, with particular attention on the most important challenges yet to be met in the field of cancer prevention.
Subject(s)
Neoplasms/diagnosis , Neoplasms/prevention & control , Cancer Vaccines/therapeutic use , Early Detection of Cancer , Europe , Humans , Life Style , Neoplasms/genetics , Precision Medicine , Risk FactorsABSTRACT
BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. METHODS: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. RESULTS: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-ß proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. CONCLUSIONS: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. LAY SUMMARY: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Molecular Biology/statistics & numerical data , Non-alcoholic Fatty Liver Disease/genetics , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/etiology , Female , Humans , Liver Neoplasms/etiology , Liver Neoplasms/genetics , Male , Middle Aged , Molecular Biology/methods , Non-alcoholic Fatty Liver Disease/complications , Risk FactorsABSTRACT
Preclinical models of cancer based on the use of human cancer cell lines and mouse models have enabled discoveries that have been successfully translated into patients. And yet the majority of clinical trials fail, emphasising the urgent need to improve preclinical research to better interrogate the potential efficacy of each therapy and the patient population most likely to benefit. This is particularly important for liver malignancies, which lack highly efficient treatments and account for hundreds of thousands of deaths around the globe. Given the intricate network of genetic and environmental factors that contribute to liver cancer development and progression, the identification of new druggable targets will mainly depend on establishing preclinical models that mirror the complexity of features observed in patients. The development of new 3D cell culture systems, originating from cells/tissues isolated from patients, might create new opportunities for the generation of more specific and personalised therapies. However, these systems are unable to recapitulate the tumour microenvironment and interactions with the immune system, both proven to be critical influences on therapeutic outcomes. Patient-derived xenografts, in particular with humanised mouse models, more faithfully mimic the physiology of human liver cancer but are costly and time-consuming, which can be prohibitive for personalising therapies in the setting of an aggressive malignancy. In this review, we discuss the latest advances in the development of more accurate preclinical models to better understand liver cancer biology and identify paradigm-changing therapies, stressing the importance of a bi-directional communicative flow between clinicians and researchers to establish reliable model systems and determine how best to apply them to expanding our current knowledge.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/immunology , Disease Models, Animal , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Xenograft Model Antitumor Assays/methods , Adult , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Heterografts , Humans , Liver/pathology , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Mice , Organoids , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
BACKGROUND & AIMS: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. METHODS: We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. RESULTS: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor ß signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21-4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. CONCLUSIONS: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.
Subject(s)
Anticarcinogenic Agents/pharmacology , Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/genetics , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/prevention & control , Liver Neoplasms/genetics , Transcriptome , Animals , Aspirin/pharmacology , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Case-Control Studies , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/pathology , Clopidogrel/pharmacology , Diethylnitrosamine , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Indoles/pharmacology , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Male , Mice, Inbred C57BL , Tumor Escape/genetics , Tumor MicroenvironmentABSTRACT
BACKGROUND: Drug-induced immunosuppression is necessary to prevent rejection of the foreign organ in transplanted patients, but neoplastic and virus-associated skin diseases are frequent complications. Reflectance confocal microscopy (RCM) recently emerged as a promising tool for the early diagnosis of skin lesions. MATERIALS AND METHODS: A total of 61 skin lesions, among them 20 basal cell carcinomas, six Bowen's diseases, 23 actinic keratoses, and 12 verrucae, were analyzed. All lesions were clinically evaluated followed by RCM evaluation by two independent dermatologists and histological examination. RESULTS: For the diagnosis of basal cell carcinoma, a sensitivity of 100% by both investigators (INV I + II) and a specificity of 100% by INV I and 80% by INV II were achieved. The sensitivity average rate for RCM features reached by both investigators ranged between 60% and 100%, and the specificity between 55% and 90%. For the diagnosis of actinic keratosis, a concordant sensitivity of 94.4% and a specificity of 80% (INV I) and 60% (INV II) were detected. The sensitivity average rate of specific RCM criteria ranged between 72.3% and 97.2%, whereas specificity ranged between 20% and 90%. Regarding verrucae, RCM confirmed the histological diagnosis with a sensitivity of 85.7% (INV I) and 100% (INV II), while specificity was 100% and 80%, respectively. CONCLUSION: Reflectance confocal microscopy resulted to be a reliable tool for the noninvasive diagnosis of neoplastic and virus-associated skin changes in organ transplant recipients. Nevertheless, given the frequency and diagnostic complexity of the hyperkeratotic lesions occurring post-transplantation, larger cohorts of patients are required to confirm and consolidate these findings.
Subject(s)
Immunosuppression Therapy/adverse effects , Microscopy, Confocal/methods , Skin Diseases/pathology , Skin Diseases/virology , Transplant Recipients/statistics & numerical data , Aged , Aged, 80 and over , Bowen's Disease/diagnosis , Bowen's Disease/pathology , Bowen's Disease/ultrastructure , Carcinoma, Basal Cell/diagnosis , Carcinoma, Basal Cell/pathology , Carcinoma, Basal Cell/ultrastructure , Dermatologists/statistics & numerical data , Early Diagnosis , Female , Germany/epidemiology , Humans , Keratosis, Actinic/diagnosis , Keratosis, Actinic/pathology , Male , Middle Aged , Reproducibility of Results , Sensitivity and Specificity , Skin Diseases/epidemiology , Warts/diagnosis , Warts/pathologySubject(s)
Ferroptosis , Liver Neoplasms , Humans , Dietary Supplements , Tumor Microenvironment , ImmunotherapyABSTRACT
OBJECTIVE: miR-21 is an oncomir highly upregulated in hepatocellular carcinoma and in early stages of liver diseases characterised by the presence of steatosis. Whether upregulation of miR-21 contributes to hepatic metabolic disorders and their progression towards cancer is unknown. This study aims at investigating the role of miR-21/miR-21* in early stages of metabolic liver disorders associated with diet-induced obesity (DIO). DESIGN: Constitutive miR-21/miR-21* knockout (miR21KO) and liver-specific miR-21/miR-21* knockout (LImiR21KO) mice were generated. Mice were then fed with high-fat diet (HFD) and alterations of the lipid and glucose metabolism were investigated. Serum and ex vivo explanted liver tissue were analysed. RESULTS: Under normal breeding conditions and standard diet, miR-21/miR-21* deletion in mice was not associated with any detectable phenotypic alterations. However, when mice were challenged with an obesogenic diet, glucose intolerance, steatosis and adiposity were improved in mice lacking miR-21/miR-21*. Deletion of miR-21/miR-21* specifically in hepatocytes led to similar improvements in mice fed an HFD, indicating a crucial role for hepatic miR-21/miR-21* in metabolic disorders associated with DIO. Further molecular analyses demonstrated that miR-21/miR-21* deletion in hepatocytes increases insulin sensitivity and modulates the expression of multiple key metabolic transcription factors involved in fatty acid uptake, de novo lipogenesis, gluconeogenesis and glucose output. CONCLUSIONS: Hepatic miR-21/miR-21* deficiency prevents glucose intolerance and steatosis in mice fed an obesogenic diet by altering the expression of several master metabolic regulators. This study points out miR-21/miR-21* as a potential therapeutic target for non-alcoholic fatty liver disease and the metabolic syndrome.
Subject(s)
Diet, High-Fat , Fatty Liver , Glucose Metabolism Disorders/metabolism , Glucose/metabolism , Hepatocytes , MicroRNAs/metabolism , Obesity/metabolism , Animals , Dietary Fats/adverse effects , Disease Models, Animal , Fatty Liver/metabolism , Fatty Liver/pathology , Glucose Intolerance/metabolism , Hepatocytes/metabolism , Hepatocytes/pathology , Mice , Mice, KnockoutABSTRACT
BACKGROUND & AIMS: Progression of alcoholic liver disease (ALD) can be influenced by genetic factors, which potentially include specific oncogenes and tumor suppressors. In the present study, we tested the hypothesis that aberrant expression of the proto-oncogene c-myc might exert a crucial role in the development of ALD. METHODS: Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR and immunohistochemistry. Mice with transgenic expression of c-myc in hepatocytes (alb-myc(tg)) and wild-type (WT) controls were fed either control or ethanol (EtOH) containing Lieber-DeCarli diet for 4weeks to induce ALD. RESULTS: Hepatic c-myc was strongly upregulated in human patients with advanced ALD and in EtOH-fed WT mice. Transcriptome analysis indicated deregulation of pathways involved in ER-stress, p53 signaling, hepatic fibrosis, cell cycle regulation, ribosomal synthesis and glucose homeostasis in EtOH-fed alb-myc(tg) mice. Transgenic expression of c-myc in hepatocytes with simultaneous EtOH-uptake led to early ballooning degeneration, increased liver collagen deposition and hepatic lipotoxicity, together with excessive CYP2E1-derived reactive oxygen species (ROS) production. Moreover, EtOH-fed alb-myc(tg) mice exhibited substantial changes in mitochondrial morphology associated with energy dysfunction. Pathway analysis revealed that elevated c-myc expression and ethanol uptake synergistically lead to strong AKT activation, Mdm2 phosphorylation and as a consequence to inhibition of p53. CONCLUSIONS: Expression of c-myc and EtOH-uptake synergistically accelerate the progression of ALD most likely due to loss of p53-dependent protection. Thus, c-myc is a new potential marker for the early detection of ALD and identification of risk patients.
Subject(s)
Genes, myc/physiology , Hepatocytes/metabolism , Liver Diseases, Alcoholic/etiology , Animals , Cell Cycle , Disease Progression , Endoplasmic Reticulum Stress , Fatty Acids, Nonesterified/metabolism , Humans , Liver Regeneration , Male , Mice , Proto-Oncogene Mas , Proto-Oncogene Proteins c-akt/metabolism , Tumor Suppressor Protein p53/physiologyABSTRACT
BACKGROUND & AIMS: Non-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver cirrhosis and hepatocellular carcinoma. HGF (hepatocyte growth factor)/mesenchymal-epithelial transition factor (c-Met) receptor signalling is known to activate distinct intracellular pathways mediating among others anti-apoptotic properties to hepatocytes. Therefore, the aim was to characterise the role of c-Met during NASH development. METHODS: Hepatocyte specific c-Met knockout mice (c-MetΔ(hepa)) using the cre-loxP system and wild type controls (c-Met(loxP/loxP)) were fed a methionine-choline deficient (MCD) diet. RESULTS: MCD feeding triggered massive steatosis, decreased survival and higher transaminases in c-MetΔ(hepa) livers compared to c-Met(loxP/loxP). Gene array analysis demonstrated that genes involved in fatty acid metabolism were strongly upregulated in c-MetΔ(hepa) livers correlating with higher amounts of hepatic free fatty acids. Consequently, c-MetΔ(hepa) mice showed significantly more TUNEL positive cells and more superoxide anion production than c-Met(loxPloxP) animals. Additionally, c-MetΔ(hepa) livers showed significantly larger fractions of infiltrating neutrophils, macrophages, and cytotoxic T cells. These changes correlated with an enhanced progression of liver fibrosis as evidenced by higher collagen deposition in c-MetΔ(hepa) livers. As increased apoptosis was a prominent feature in c-MetΔ(hepa) livers, we generated c-Met/Casp8Δ(hepa) double knockout mice. In these animals compared to c-MetΔ(hepa) animals the increase in apoptosis could be reverted. CONCLUSIONS: c-Met deletion in hepatocytes triggers NASH progression. A prominent mechanism is higher fatty acid accumulation and increased apoptosis, which in part can be reverted by blocking caspase 8.
Subject(s)
Apoptosis , Choline Deficiency , Diet , Hepatocyte Growth Factor/metabolism , Inflammation/metabolism , Liver Cirrhosis , Methionine , Non-alcoholic Fatty Liver Disease , Oxidative Stress , Proto-Oncogene Proteins c-met/metabolism , Animals , Caspase 8/metabolism , Choline Deficiency/metabolism , Diet/adverse effects , Diet/methods , Hepatocytes/metabolism , Lipotropic Agents/metabolism , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Liver Cirrhosis/prevention & control , Methionine/deficiency , Methionine/metabolism , Mice , Mice, Knockout , Neutrophil Infiltration , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathologyABSTRACT
The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 in vivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara in vivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer.
Subject(s)
Carcinoma, Hepatocellular , Fasting , Liver Neoplasms , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease , PPAR alpha , Phosphoenolpyruvate Carboxykinase (GTP) , PPAR alpha/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Humans , Mice , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Male , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , Liver/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Signal Transduction , Intermittent FastingABSTRACT
It has been recently shown that the biological effects of erythropoietin (EPO) are not limited to the hematopoietic compartment but, as pleiotropic glycoprotein, this hormone can exert pro-angiogenic and tissue-protective functions also in a wide range of non-hematopoietic organs. The role of EPO and the effective functionality of its receptor in solid tumors are still a controversial point of debate. In the present work we analyzed the gene expression of EPO and its cognate receptor (EpoR) in a rat model of thioacetamide-induced damage and tumor. An analysis of the EPO/EpoR axis was also performed on human cholangiocarcinoma (CC) cell lines. A progressive increase of EPO and EpoR mRNA can already be observed during the fibrotic-cirrhotic development with a peak of expression rising at tumor formation (24.7 ± 9.9-fold increase and 15.5 ± 1.1-fold increase, respectively, for the two genes). Co-localization studies by immunofluorescence revealed hepatocytes in the regenerative cirrhotic nodules (Hep Par-1(+)) and in the dysplastic bile duct cells (CK19(+)) as the major EPO producers in this specific condition. The same cell populations, together with endothelial cells, exhibited an increased expression of EpoR, although all the non-parenchymal cell populations in the liver exhibited modest basal mRNA levels. Challenging human CC cells, Mz-Cha-2, with a combination of EPO and SCF resulted in a synergistic effect on the gene expression of EPO, CyclinD1 and PCNA. This study suggests that the autocrine and paracrine release of endogenous EPO in the microenvironment may contribute to the development and maintenance of the CC possibly in cooperation with other signaling pathways.
Subject(s)
Bile Duct Neoplasms/metabolism , Cholangiocarcinoma/metabolism , Disease Models, Animal , Erythropoietin/metabolism , Liver/metabolism , Liver/pathology , Receptors, Erythropoietin/metabolism , Animals , Bile Duct Neoplasms/pathology , Cholangiocarcinoma/pathology , Erythropoietin/genetics , Humans , Liver/injuries , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Erythropoietin/genetics , Thioacetamide , Tumor Cells, CulturedABSTRACT
The last 10 years have revolutionized our basic understanding of nonalcoholic fatty liver disease and consequent liver cancer. It has become clear that several innate and adaptive immune cells play an important role in initiating, maintaining, or exacerbating nonalcoholic steatohepatitis (NASH)-a disease that has been recently defined as autoaggressive. Despite improved disease management aimed at reducing the progression of fibrosis, NASH is set to become a leading cause for hepatocellular carcinoma (HCC). Preliminary data from preclinical studies suggest that immunotherapy efficacy may be reduced in NASH-related HCC compared with viral HCC; however, conclusive evidence supporting clinical translation of these findings is lacking. Comprehensive clinical and immunologic phenotyping of mechanisms linking NASH progression with carcinogenesis and therapeutic resistance is key to prevent progression to cirrhosis, improve monitoring and stratification of NASH according to predicted cancer risk, and ultimately increase survival of patients with NASH-HCC. In this review, we summarize the state of the art in the field of NASH and NASH-HCC with focus on immunobiology. We discuss preclinical and clinical findings underpinning NASH as an immunologically distinct pro-tumorigenic disease entity, and explore areas of potential therapeutic vulnerabilities in NASH-associated HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Humans , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/complications , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/therapy , Liver Neoplasms/etiology , Liver Neoplasms/therapy , Liver Neoplasms/pathology , Liver Cirrhosis/pathology , Fibrosis , Immunotherapy/adverse effects , CarcinogenesisABSTRACT
BACKGROUND: Cyclin E1 is the regulatory subunit of cyclin-dependent kinase 2 (Cdk2) and one of the central players in cell cycle progression. We recently showed its crucial role for initiation of liver fibrosis and hepatocarcinogenesis. In the present study, we investigated the role of Cyclin E1 in the development of alcohol-associated liver disease (ALD). METHODS: Mice with constitutive (E1-/-), hepatocyte-specific (Cyclin E1Δhepa), or intestinal-epithelial-cell-specific (Cyclin E1ΔIEC) inactivation of Cyclin E1 and corresponding wild type littermate controls (WT) were administered either a Lieber-DeCarli ethanol diet (LDE) for 3 weeks or acute ethanol binges (6 g/kg) through oral gavage. Serum parameters of liver functionality were measured; hepatic tissues were collected for biochemical and histological analyses. RESULTS: The administration of acute EtOH binge and chronic LDE diet to E1-/- mice enhanced hepatic steatosis, worsened liver damage and triggered body weight loss. Similarly, in the acute EtOH binge model, Cyclin E1Δhepa mice revealed a significantly worsened liver phenotype. In contrast, inactivation of Cyclin E1 only in intestinal epithelial cell (IECs)did not lead to any significant changes in comparison to WT mice after acute EtOH challenge. Remarkably, both acute and chronic EtOH administration in E1-/- animals resulted in increased levels of ADH and decreased expression of ALDH1/2. The additional application of a pan-Cdk inhibitor (S-CR8) further promoted liver damage in EtOH-treated WT mice. CONCLUSION: Our data point to a novel unexpected role of Cyclin E1 in hepatocytes for alcohol metabolism, which seems to be independent of the canonical Cyclin E1/Cdk2 function as a cell cycle regulator.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Fatty Liver , Liver Diseases, Alcoholic , Animals , Mice , Chemical and Drug Induced Liver Injury, Chronic/metabolism , Disease Models, Animal , Ethanol/toxicity , Fatty Liver/pathology , Hepatocytes/metabolism , Liver Diseases, Alcoholic/metabolismABSTRACT
MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1ß and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.