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Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive hepatic lipid accumulation, which can progress to nonalcoholic steatohepatitis (NASH). Histone deacetylase Sirtuin 6 (SIRT6) regulates NAFLD by regulating metabolism-related gene expression, but an extrachromosomal role for SIRT6 in NAFLD development remains elusive. We investigated whether SIRT6 functions on NAFLD in the cytoplasm. We found that SIRT6 binds saturated fatty acids, especially palmitic acid. This binding leads to its nuclear export, where it deacetylates long-chain acyl-CoA synthase 5 (ACSL5), thereby facilitating fatty acid oxidation. High-fat diet-induced NAFLD is suppressed by ACSL5 hepatic overexpression but is exacerbated by its depletion. As confirmation, overexpression of a deacetylated ACSL5 mimic attenuated NAFLD in Sirt6 liver-specific knockout mice. Moreover, NASH-hepatic tissues from both patients and diet-fed mice exhibited significantly reduced cytoplasmic SIRT6 levels and increased ACSL5 acetylation. The SIRT6/ACSL5 signaling pathway has a critical role in NAFLD progression and might constitute an avenue for therapeutic intervention.
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Enfermedad del Hígado Graso no Alcohólico , Sirtuinas , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Acilcoenzima A/metabolismo , Ratones Endogámicos C57BL , Hígado/metabolismo , Metabolismo de los Lípidos , Ratones Noqueados , Ácidos Grasos/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismo , Citoplasma/metabolismoRESUMEN
OBJECTIVE: Altered metabolites are important for the tumourigenicity of hepatocellular carcinoma (HCC). We performed integrative metabolomics analysis of the metabolites changes in portal venous blood and in comparison with the metabolites changes in liver tissues and stool samples of HCC patients and healthy liver donors. DESIGN: Serum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines. RESULTS: Detailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (p<0.001). HCC patients had significantly higher levels of portal vein serum and HCC tissue metabolites of DL-3-phenyllactic acid, L-tryptophan, glycocholic acid and 1-methylnicotinamide than healthy controls, which were associated with impaired liver function and poor survival. On the other hand, HCC patients had lower levels of linoleic acid and phenol in portal vein and stool samples than healthy controls. Linoleic acid and phenol significantly inhibited HCC proliferation, inferring their anti-HCC function as protective metabolites. CONCLUSIONS: The integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Humanos , Ácido Linoleico , Neoplasias Hepáticas/metabolismo , Metaboloma , Metabolómica/métodos , Fenoles , Vena PortaRESUMEN
OBJECTIVE: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. DESIGN: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined. RESULTS: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model. CONCLUSION: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy.
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Carcinoma Hepatocelular/terapia , Inmunoterapia/métodos , Neoplasias Hepáticas/terapia , Animales , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/inmunología , Reprogramación Celular/inmunología , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Células Estrelladas Hepáticas/inmunología , Humanos , Tolerancia Inmunológica , Cirrosis Hepática/complicaciones , Cirrosis Hepática/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas Experimentales/etiología , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/terapia , Masculino , Ratones Endogámicos C57BL , Monocitos/inmunología , Células Supresoras de Origen Mieloide/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Microambiente Tumoral , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
BACKGROUND: Tumor-associated lymphangiogenesis is considered significant in number of solid malignancies. However, its impact on prognosis of intrahepatic cholangiocarcinoma (ICC) after resection remains further confirmation. Herein, we conducted this study to evaluate prognostic impact of tumor-associated lymphangiogenesis in patients with ICC. METHODS: Extent of tumor-associated lymphangiogenesis of ICC was evaluated by quantifying microlymphatic vessel density (MLVD) from immunohistochemical staining of a lymphatic endothelial-specific antibody (podoplanin). Clinicopathological characteristics were comprehensively analyzed to identify MLVD-associated factors. The patients were stratified into high and low MLVD groups according to the distinctive correlation between the MLVD and overall survival using the Spearman's correlation test. Kaplan-Meier estimation was performed to confirm prognostic impact of MLVD in patients with ICC. Univariate and multivariate analyses were performed using the Cox proportional hazard model. RESULTS: The MLVD between 4 to 12 counts showed inverse proportion to the overall survival (Spearman's r = - 0.66; 95% confidence interval [CI], - 0.82 to - 0.39; p < 0.0001), which was set as a cut-off for the high MLVD group, whereas the MLVD between 13 to 25 showed no correlation to the overall survival (r = - 0.11; 95% CI, - 0.38 to 0.19; p = 0.4791). The high MLVD group showed more frequent lymph node metastasis (p < 0.001) and were more likely to suffer from recurrence of the tumor compared to the low MLVD group (p < 0.001). The high MLVD was found to be independently associated with reduced overall and recurrence-free survival. The 5-year overall survival of the patients with high MLVD was significantly lower compared to those with low MLVD (0% vs 48%). CONCLUSIONS: Our study reveals that tumor-associated lymphangiogenesis is significantly associated with increased lymphatic metastasis, recurrence of the tumor, and reduced overall survival in patients with ICC, thus providing guidance when estimating postresection prognosis.
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Colangiocarcinoma/mortalidad , Colangiocarcinoma/patología , Vasos Linfáticos/patología , Neovascularización Patológica , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Biomarcadores de Tumor , Colangiocarcinoma/metabolismo , Colangiocarcinoma/terapia , Comorbilidad , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Pronóstico , Carga TumoralRESUMEN
OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) contribute to tumour immunosuppressive microenvironment and immune-checkpoint blockade resistance. Emerging evidence highlights the pivotal functions of cyclin-dependent kinases (CDKs) in tumour immunity. Here we elucidated the role of tumour-intrinsic CDK20, or cell cycle-related kinase (CCRK) on immunosuppression in hepatocellular carcinoma (HCC). DESIGN: Immunosuppression of MDSCs derived from patients with HCC and relationship with CCRK were determined by flow cytometry, expression analyses and co-culture systems. Mechanistic studies were also conducted in liver-specific CCRK-inducible transgenic (TG) mice and Hepa1-6 orthotopic HCC models using CRISPR/Cas9-mediated Ccrk depletion and liver-targeted nanoparticles for interleukin (IL) 6 trapping. Tumorigenicity and immunophenotype were assessed on single or combined antiprogrammed death-1-ligand 1 (PD-L1) therapy. RESULTS: Tumour-infiltrating CD11b+CD33+HLA-DR- MDSCs from patients with HCC potently inhibited autologous CD8+T cell proliferation. Concordant overexpression of CCRK and MDSC markers (CD11b/CD33) positively correlated with poorer survival rates. Hepatocellular CCRK stimulated immunosuppressive CD11b+CD33+HLA-DR- MDSC expansion from human peripheral blood mononuclear cells through upregulating IL-6. Mechanistically, CCRK activated nuclear factor-κB (NF-κB) via enhancer of zeste homolog 2 (EZH2) and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. Hepatic CCRK induction in TG mice activated the EZH2/NF-κB/IL-6 cascade, leading to accumulation of polymorphonuclear (PMN) MDSCs with potent T cell suppressive activity. In contrast, inhibiting tumorous Ccrk or hepatic IL-6 increased interferon γ+tumour necrosis factor-α+CD8+ T cell infiltration and impaired tumorigenicity, which was rescued by restoring PMN-MDSCs. Notably, tumorous Ccrk depletion upregulated PD-L1 expression and increased intratumorous CD8+ T cells, thus enhancing PD-L1 blockade efficacy to eradicate HCC. CONCLUSION: Our results delineate an immunosuppressive mechanism of the hepatoma-intrinsic CCRK signalling and highlight an overexpressed kinase target whose inhibition might empower HCC immunotherapy.
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Carcinoma Hepatocelular/inmunología , Quinasas Ciclina-Dependientes/metabolismo , Neoplasias Hepáticas/inmunología , Células Supresoras de Origen Mieloide/inmunología , Animales , Western Blotting , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultivo de Célula , Citocinas/metabolismo , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunoprecipitación , Terapia de Inmunosupresión , Hígado/patología , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Pioglitazone (PGZ), a synthetic PPARγ ligand, is known to have anti-tumor activity. However, it is unclear how it acts against hepatocellular carcinoma (HCC). We hypothesized that the pathological receptor for advanced glycation end products (RAGE) is involved in the PGZ anti-tumor process. To test this notion, human primary HCC tissues and corresponding adjacent non-cancerous tissues (ANCT) from 75 consecutive cases were analyzed. The expression and clinical significance of RAGE was assessed by immunohistochemical assay through tissue microarray. After HCC cells were pretreated with different concentrations of PGZ, cell proliferation, apoptosis, cell invasion, and cell cycle distribution were evaluated by multiple assays. The results showed that, the positive expression of RAGE was significantly higher in HCC tissues than in ANCT (66.7% vs. 36.0%, P < 0.001), and was closely associated with pathological staging (P = 0.014) and lymph-vascular space invasion (P = 0.003). Moreover, PGZ inhibited proliferative activity and invasive potential, and induced apoptosis and cell cycle arrest in HCC cells resulting in increased expression of PPARγ and decreased expression of RAGE, NF-κB, HMGB1, p38MAPK, Ki-67, MMP-2, and CyclinD1. Furthermore, knockdown of RAGE or NF-κB by siRNA effectively suppressed cell proliferation and invasion, and mediated the inhibitory effects of PGZ in HCC cells. Taken together, our findings suggest that, RAGE is overexpressed in human HCC tissues, and is closely associated with the pathological staging and tumor invasion of HCC. In addition, PGZ as a PPARγ agonist may inhibit growth and invasion of HCC cells via blockade of the RAGE signaling.
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Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Invasividad Neoplásica/prevención & control , PPAR gamma/agonistas , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Tiazolidinedionas/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Pioglitazona , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Aberrant expression of MUC15 correlates with development of colorectal adenocarcinoma, and MUC15 has been reported to prevent trophoblast invasion of human placenta. However, little is known about the role of MUC15 in pathogenesis of hepatocellular carcinoma (HCC). METHODS: We analyzed HCC samples and matched nontumor liver tissues (controls) collected from 313 patients who underwent hepatectomy in Shanghai, China, from January 2006 through September 2009. Levels of messenger RNAs and proteins were determined by immunohistochemical, quantitative reverse transcription polymerase chain reaction, and immunoblot analyses. Statistical analyses were used to associate levels of MUC15 with tumor features and patient outcomes. RESULTS: Levels of MUC15 messenger RNA and protein were reduced in a greater percentage of HCC samples than control tissues. Tumors with reduced levels of MUC15 were more likely to have aggressive characteristics (eg, high levels of α-fetoprotein, vascular invasion, lack of encapsulation, and poor differentiation) than those with low levels. Patients whose tumors had reduced levels of MUC15 had shorter overall survival times (24 months vs 46 months for patients with tumors with high levels of MUC15) and time to disease recurrence. Stable expression of MUC15 in HCC cell lines (SMMC-7721 and HCC-LM3) reduced their proliferation and invasive features in vitro, and ability to form metastatic tumors in mice. MUC15 reduced transcription of the matrix metalloproteinases 2 and 7 increased expression of tissue inhibitor of metalloproteinase-2, which required phosphoinositide 3-kinase-v-akt murine thymoma viral oncogene homolog signaling. Physical interaction between MUC15 and epidermal growth factor receptor led to its relocation and degradation within early endosomes and was required for inactivation of phosphoinositide 3-kinase-v-akt murine thymoma viral oncogene homolog signaling. CONCLUSIONS: Reduced levels of MUC15 in HCCs are associated with shorter survival times of patients and reduced time to disease recurrence. Expression of MUC15 in HCC cells reduces their aggressive behavior in vitro and in mice by inducing dimerization of epidermal growth factor receptor and decreasing phosphoinositide 3-kinase signaling via v-akt murine thymoma viral oncogene homolog.
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Carcinoma Hepatocelular/fisiopatología , Dimerización , Receptores ErbB/fisiología , Neoplasias Hepáticas/fisiopatología , Mucinas/fisiología , Proteína Oncogénica v-akt/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Técnicas In Vitro , Hígado/patología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Ratones , Invasividad Neoplásica/fisiopatología , Valor Predictivo de las Pruebas , Pronóstico , Tasa de SupervivenciaRESUMEN
Early allograft dysfunction (EAD) is a frequent phenomenon, leading to increased graft loss and higher mortality after liver transplantation (LT). Despite significant efforts for early diagnosis of EAD, there is no existing approach that can predict EAD on the first post-operative day. The aim is to define a metabolite-based biomarker on the first day after LT complicated with EAD. Ten patients diagnosed with EAD and 26 non-EAD are recruited for the study. A HPLC-MS/MS is used to determine 14 amino acids and 15 bile acids serum concentration. Comparative analyses are conducted between EAD and non-EAD groups. Arginine is identified as the most significant metabolite distinguishing the EAD and non-EAD groups, and therefore, is identified as a potential biomarker of EAD. The optimal cut-off value for arginine is 52.09 µmol L-1, with an AUROC of 0.804 (95% confidence interval: 0.638-0.917, p < 0.001), yielding a sensitivity of 100%, specificity of 53.8%, and Youden index of 0.54, NPVof 100%, and PPV of 45.45%. In summary, the study indicated that targeted metabolomics analysis would be a promising strategy for discovering novel biomarkers to predict EAD. The identified arginine may be helpful in developing an objective diagnostic method for EAD.
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Background & Aims: Recent studies demonstrated the importance of fibrosis in promoting an immunosuppressive liver microenvironment and thereby aggressive hepatocellular carcinoma (HCC) growth and resistance to immune checkpoint blockade (ICB), particularly via monocyte-to-monocytic myeloid-derived suppressor cell (M-MDSC) differentiation triggered by hepatic stellate cells (HSCs). We thus aimed to identify druggable targets in these immunosuppressive myeloid cells for HCC therapy. Methods: M-MDSC signature genes were identified by integrated transcriptomic analysis of a human HSC-monocyte culture system and tumor-surrounding fibrotic livers of patients with HCC. Mechanistic and functional studies were conducted using in vitro-generated and patient-derived M-MDSCs. The therapeutic efficacy of a M-MDSC targeting approach was determined in fibrosis-associated HCC mouse models. Results: We uncovered over-expression of protein phosphatase 1 regulatory subunit 15A (PPP1R15A), a myeloid cell-enriched endoplasmic reticulum stress modulator, in human M-MDSCs that correlated with poor prognosis and ICB non-responsiveness in patients with HCC. Blocking TGF-ß signaling reduced PPP1R15A expression in HSC-induced M-MDSCs, whereas treatment of monocytes by TGF-ß upregulated PPP1R15A, which in turn promoted ARG1 and S100A8/9 expression in M-MDSCs and reduced T-cell proliferation. Consistently, lentiviral-mediated knockdown of Ppp1r15a in vivo significantly reduced ARG1+S100A8/9+ M-MDSCs in fibrotic liver, leading to elevated intratumoral IFN-γ+GZMB+CD8+ T cells and enhanced anti-tumor efficacy of ICB. Notably, pharmacological inhibition of PPP1R15A by Sephin1 reduced the immunosuppressive potential but increased the maturation status of fibrotic HCC patient-derived M-MDSCs. Conclusions: PPP1R15A+ M-MDSC cells are involved in immunosuppression in HCC development and represent a novel potential target for therapies. Impact and implications: Our cross-species analysis has identified PPP1R15A as a therapeutic target governing the anti-T-cell activities of fibrosis-associated M-MDSCs (monocytic myeloid-derived suppressor cells). The results from the preclinical models show that specific inhibition of PPP1R15A can break the immunosuppressive barrier to restrict hepatocellular carcinoma growth and enhance the efficacy of immune checkpoint blockade. PPP1R15A may also function as a prognostic and/or predictive biomarker in patients with hepatocellular carcinoma.
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Massive expansion of immature and suppressive myeloid cells is a common feature of malignant solid tumors. Over-expression of cyclin-dependent kinase 20, also known as cell cycle-related kinase (CCRK), in hepatocellular carcinoma (HCC) correlates with reduced patient survival and low immunotherapy responsiveness. Beyond tumor-intrinsic oncogenicity, here we demonstrated that CCRK is upregulated in myeloid cells in tumor-bearing mice and in patients with HCC. Intratumoral injection of Ccrk-knockdown myeloid-derived suppressor cells (MDSCs) increased tumor-infiltrating CD8+T cells and suppressed HCC tumorigenicity. Using an indel mutant transgenic model, we showed that Ccrk inactivation in myeloid cells conferred a mature phenotype with elevated IL-12 production, driving Th1 responses and CD8+T cell cytotoxicity to reduce orthotopic tumor growth and prolong survival. Mechanistically, CCRK activates STAT3/E4BP4 signaling in MDSCs to acquire immunosuppressive activity through transcriptional IL-10 induction and IL-12 suppression. Taken together, our findings unravel mechanistic insights into MDSC-mediated immunosuppression and offer a therapeutic kinase-target for cancer immunotherapy.
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Hepatitis B virus X protein (HBx) is implicated in the pathogenesis of hepatitis B virus (HBV)-associated liver diseases. However, whether HBx has the ability to disturb the susceptibility of hepatocytes to common chemotherapeutic agents remains incompletely understood. Here we demonstrate that HBx enhances cisplatin-induced hepatotoxicity by a mechanism involving degradation of Mcl-1, an antiapoptotic member of the Bcl-2 family. Ectopic expression of HBx sensitized hepatocytes to cisplatin-induced apoptosis, which was accompanied by a marked downregulation of Mcl-1 but not of Bcl-2 or Bcl-xL. Overexpression of Mcl-1 prevented HBx-induced proapoptotic and proinflammatory effects during cisplatin treatment both in vitro and in vivo. HBx-induced dysregulation of Mcl-1 resulted mainly from posttranslational degradation rather than transcription repression. Moreover, a caspase-3 inhibitor effectively abrogated HBx-enhanced Mcl-1 degradation and cell death. Importantly, antioxidants blocked activation of caspase-3 and acceleration of Mcl-1 loss, as well as cell death, in HBx-expressing hepatocytes upon cisplatin exposure in vitro and in vivo. Collectively, these data implicate oxidative stress-dependent caspase-3-mediated degradation of Mcl-1 as a mechanism contributing to HBx-mediated sensitization of cisplatin-induced hepatotoxicity. A combination of cisplatin and antioxidants might provide more advantage than cisplatin alone in the treatment of cancer patients with chronic HBV infection.
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Cisplatino/toxicidad , Virus de la Hepatitis B/patogenicidad , Hepatocitos/patología , Hepatocitos/virología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/metabolismo , Factores de Virulencia/metabolismo , Animales , Apoptosis , Caspasa 3/metabolismo , Células Cultivadas , Humanos , Hígado/patología , Hígado/virología , Ratones , Ratones Desnudos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Estrés Oxidativo , Ratas , Proteínas Reguladoras y Accesorias ViralesRESUMEN
UNLABELLED: One of the challenges surrounding nonalcoholic fatty liver disease (NAFLD) is to discover the mechanisms that underlie the initiation of it. The aim of the present study was to elucidate the effects of Toll-like receptor 4 (TLR4) signaling in liver parenchymal cells during the early stage of NAFLD. Male TLR4-wildtype, TLR4-knockout, TLR2-knockout, MyD88-knockout, and TRIF-knockout mice were fed a normal diet or high-fat diet (HFD). Liver steatosis, alanine aminotransferase levels, nuclear translocation of nuclear factor kappa B (NF-κB) (p65), macrophage accumulation, and neutrophil infiltration were assessed. Using Kupffer cell depletion or bone marrow transplantation, we examined the potential role of Kupffer cells and myeloid infiltrating cells during the initiation of NAFLD. Immunohistochemistry and western blotting were implemented to determine the release of high-mobility group box1 (HMGB1). The neutral-antibody against HMGB1 was used to block the activity of free HMGB1. Here we report that the activation of TLR4 signaling in hepatocytes, accompanied with the relocation of P65 in nucleus, was proven to play an important role during the initiation of NAFLD. Importantly, HMGB1 releasing from hepatocytes in response to free fatty acid (FFA) infusion was first reported as the key molecule for the TLR4/MyD88 activation and cytokines expression in vitro and in vivo. Treatment with neutralizing antibody to HMGB1 protects against FFA-induced tumor necrosis factor alpha and interleukin-6 production. CONCLUSION: Our study supports the notion that TLR4/MyD88 signaling in liver parenchymal cells plays a pivotal role during the early progression of HFD-induced NAFLD, in which free HMGB1 served as a positive component mediating TLR4 activation.
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Hígado Graso/metabolismo , Proteína HMGB1/metabolismo , Hepatocitos/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Citocinas/metabolismo , Hígado Graso/patología , Hígado Graso/fisiopatología , Proteína HMGB1/genética , Hepatocitos/patología , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/metabolismo , Células Mieloides/patología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Receptor Toll-Like 4/genéticaRESUMEN
UNLABELLED: Gankyrin is a critical oncoprotein overexpressed in human hepatocellular carcinoma (HCC). However, the mechanism underlying gankyrin-mediated hepatocarcinogenesis remains elusive. Herein, we provide evidence that gankyrin expression was progressively elevated in liver fibrosis, cirrhosis, and HCC. Levels of gankyrin expression were closely associated with the dedifferentiation status of hepatoma in patients. Decrease of hepatocyte characteristic markers and increase of cholangiocyte-specific markers were observed in rat primary hepatocytes with enforced gankyrin expression and diethylnitrosamine (DEN)-triggered rat hepatocarcinogenesis. Overexpression of gankyrin also attenuated the hepatic function of primary hepatocytes, which further suggests that gankyrin promotes the dedifferentiation of hepatocytes. Moreover, elevated expression of gankyrin closely correlated with the expression of HCC stem/progenitor cell markers in DEN-triggered hepatocarcinogenesis and human HCCs. Hepatoma cells derived from suspension-cultured spheroids exhibited a higher gankyrin level, and enforced gankyrin expression in hepatoma cells remarkably enhanced cluster of differentiation (CD)133, CD90, and epithelial cellular adhesion molecule expression, indicating a role of gankyrin in hepatoma cell dedifferentiation and the generation of hepatoma stem/progenitor cells. In contrast, down-regulation of gankyrin in hepatoma cells by lentivirus-mediated microRNA delivery significantly improved their differentiation status and attenuated malignancy. Interference of gankyrin expression in hepatoma cells also diminished the proportion of cancer stem/progenitor cells and their self-renewal capacity. Furthermore, gankyrin was found to bind hepatocyte nuclear factor 4α (HNF4α), which determines hepatocyte differentiation status and enhances proteasome-dependent HNF4α degradation in hepatoma cells. The inverse correlation of gankyrin and HNF4α was further confirmed in primary hepatocytes, DEN-induced hepatocarcinogenesis, and human HCCs. CONCLUSION: Gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells via, at least partially, down-regulation of HNF4α facilitates HCC development, and interference of gankyrin expression could be a novel strategy for HCC prevention and differentiation therapy.
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Diferenciación Celular/fisiología , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/citología , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas/genética , Distribución Aleatoria , Ratas , Ratas Wistar , Células Madre/metabolismoRESUMEN
Background: Hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) have conventionally been regarded as a contraindication for liver transplantation (LT). However, the outcomes of deceased donor liver transplantation (DDLT) in patients with segmental PVTT remain unknown. The aim of this study is to evaluate the feasibility and effectiveness of DDLT in the treatment of HCC with segmental PVTT. Methods: We retrospectively analyzed 254 patients who underwent DDLT for HCC in our institution from January 2015 to November 2019. To assess the risks of PVTT, various clinicopathological variables were evaluated. Overall (OS) and recurrence-free survival (RFS) analyses based on different PVTT types were performed in HCC patients. Results: Of the 254 patients, a total of 46 patients had PVTT, of whom 35 had lobar PVTT and 11 had segmental PVTT in second-order branches or below. Alpha-fetoprotein (AFP) level, tumor maximal diameter, histological grade, micro-vascular invasion (MVI), RFS, and OS were significantly different between the control and PVTT groups. Lobar PVTT was associated with unfavorable 5-year RFS and OS compared with MVI group (28.6% and 17.1%, respectively). Instead, no significant difference was observed between the segmental PVTT and MVI group in terms of 5-year RFS and OS (RFS: 36.4% vs. 40.4%, p=0.667; OS: 54.5% vs. 45.1%, p=0.395). Further subgroup analysis showed segmental PVTT with AFP levels ≤100 ng/ml presented significantly favorable RFS and OS rates than those with AFP level >100 ng/ml (p=0.050 and 0.035, respectively). Conclusions: In summary, lobar PVTT remains a contraindication to DDLT. HCC patients with segmental PVTT and AFP level ≤100 ng/ml may be acceptable candidates for DDLT.
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Background: The forecast accuracy of the European Association for the Study of the Liver-Chronic Liver Failure (EASL-CLIF) and Asian Pacific Association for the Study of the Liver (APASL) acute-on-chronic liver failure (ACLF) criteria in assessing long-term outcomes after liver transplantation (LT) is still unclear, especially when the staging of the two standards is inconsistent. Methods: A retrospective cohort (NCT05036031) including 565 patients from January 2015 to June 2021 was conducted. The 28 and 90 days, 1- and 3-years overall survival (OS) after LT were compared between different grades. Findings: Total of 162 (28.7%) and 230 (40.7%) patients met the ACLF standards. In the EASL-CLIF criteria, the 3-year OS rates were 83·0%, 80·3%, and 69·8% for ACLF1-3, respectively. In the APASL criteria, the 3-year OS rates were 85·7% for APASL ACLF Research Consortium (AARC)-1, similar to ACLF-1. The 3-year OS rates were 84·5% for AARC-2, which were slightly better than ACLF-2. Regarding AARC-3, the 3-year OS rate was 5·8% higher than ACLF-3. For patients who met neither set of criteria for ACLF, the 3-year OS rates were 89·8%. The multivariate analysis showed that alanine aminotransferase >100 U/L, respiration failure, and cerebral failure were independent risk factors for post-LT death. Interpretation: This study provides the first large-scale long-term follow-up data in Asia. Both criteria showed favorable distinguishing ability for post-LT survival. Patients with ACLF had a higher post-LT mortality risk, and ACLF-3 and AARC-3 correlated with significantly greater mortality. Funding: National Natural Science Foundation of China and Science and Technology Commission of Shanghai Municipality.
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Obesity is a major risk factor for cancers including hepatocellular carcinoma (HCC) that develops from a background of non-alcoholic fatty liver disease (NAFLD). Hypercholesterolemia is a common comorbidity of obesity. Although cholesterol biosynthesis mainly occurs in the liver, its role in HCC development of obese people remains obscure. Using high-fat high-carbohydrate diet-associated orthotopic and spontaneous NAFLD-HCC mouse models, we found that hepatic cholesterol accumulation in obesity selectively suppressed natural killer T (NKT) cell-mediated antitumor immunosurveillance. Transcriptome analysis of human liver revealed aberrant cholesterol metabolism and NKT cell dysfunction in NAFLD patients. Notably, cholesterol-lowering rosuvastatin restored NKT expansion and cytotoxicity to prevent obesogenic diet-promoted HCC development. Moreover, suppression of hepatic cholesterol biosynthesis by a mammalian target of rapamycin (mTOR) inhibitor vistusertib preceded tumor regression, which was abolished by NKT inactivation but not CD8+ T cell depletion. Mechanistically, sterol regulatory element-binding protein 2 (SREBP2)-driven excessive cholesterol production from hepatocytes induced lipid peroxide accumulation and deficient cytotoxicity in NKT cells, which were supported by findings in people with obesity, NAFLD and NAFLD-HCC. This study highlights mTORC1/SREBP2/cholesterol-mediated NKT dysfunction in the tumor-promoting NAFLD liver microenvironment, providing intervention strategies that invigorating NKT cells to control HCC in the obesity epidemic.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Células T Asesinas Naturales , Enfermedad del Hígado Graso no Alcohólico , Animales , Colesterol/metabolismo , Humanos , Hígado/patología , Mamíferos , Ratones , Monitorización Inmunológica/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/patología , Microambiente TumoralRESUMEN
BACKGROUND: Oxidative stress has been implicated in the pathogenesis of a wide spectrum of human diseases, including Hepatitis B virus (HBV)-related liver disease. Hepatitis B virus X protein (HBx) is a key regulator of HBV that exerts pleiotropic activity on cellular functions. Recent studies showed that HBx alters mitochondrial membrane potential, thereby sensitizing cells to pro-apoptotic signals. However, it remains largely unknown whether susceptibility of hepatocytes could be disturbed by HBx under oxidative stress conditions. The purpose of this study is to determine the apoptotic susceptibility of HBx-expressing hepatocytes upon exposure to pro-oxidant stimuli in vitro and in vivo and explore its underlying mechanism. RESULTS: Although expression of HBx itself did not activate apoptotic signaling, it significantly enhanced oxidative stress-induced cell death both in vitro and in vivo. Interestingly, this phenomenon was associated with a pronounced reduction of protein levels of Mcl-1, but not other anti-apoptotic Bcl-2 members. Importantly, enforced expression of Mcl-1 prevented HBx-triggered cell apoptosis; conversely, specific knockdown of Mcl-1 exacerbated HBx-induced apoptosis upon exposure to oxidative stress. Furthermore, inhibition of caspase-3 not only abrogated HBx-triggered apoptotic killing but also blocked HBx-induced Mcl-1 loss. Additionally, expression of HBx and Mcl-1 was found to be inversely correlated in HBV-related hepatocellular carcinogenesis (HCC) tissues. CONCLUSIONS: Our findings indicate that HBx exerts pro-apoptotic effect upon exposure to oxidative stress probably through accelerating the loss of Mcl-1 protein via caspase-3 cascade, which may shed a new light on the molecular mechanism of HBV-related hepatocarcinogenesis.
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Apoptosis/fisiología , Caspasa 3/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/metabolismo , Análisis de Varianza , Animales , Células HEK293 , Células Hep G2 , Humanos , Hígado/irrigación sanguínea , Ratones , Ratones Transgénicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Estrés Oxidativo/fisiología , Transactivadores/biosíntesis , Transactivadores/genética , Transfección , Proteínas Reguladoras y Accesorias Virales , Isquemia TibiaRESUMEN
BACKGROUND & AIMS: Reactive oxygen species (ROS) are considered to play a prominent causative role in the development of various hepatic disorders. Antioxidants have been effectively demonstrated to protect against hepatic damage. Hydrogen (H(2)), a new antioxidant, was reported to selectively reduce the strongest oxidants, such as hydroxyl radicals (·OH) and peroxynitrite (ONOO(-)), without disturbing metabolic oxidation-reduction reactions or disrupting ROS involved in cell signaling. In place of H(2) gas, hydrogen-rich saline (HS) may be more suitable for clinical application. We herein aim to verify its protective effects in experimental models of liver injury. METHODS: H(2) concentration in vivo was detected by hydrogen microelectrode for the first time. Liver damage, ROS accumulation, cytokine levels, and apoptotic protein expression were, respectively, evaluated after GalN/LPS, CCl(4), and DEN challenge. Simultaneously, CCl(4)-induced hepatic cirrhosis and DEN-induced hepatocyte proliferation were measured. RESULTS: HS significantly increased hydrogen concentration in liver and kidney tissues. As a result, acute liver injury, hepatic cirrhosis, and hepatocyte proliferation were reduced through the quenching of detrimental ROS. Activity of pro-apoptotic players, such as JNK and caspase-3, were also inhibited. CONCLUSIONS: HS could protect against liver injury and also inhibit the processes leading to liver cirrhosis and hepatocyte compensatory proliferation.
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Antioxidantes/administración & dosificación , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hidrógeno/administración & dosificación , Cloruro de Sodio/administración & dosificación , Animales , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Hidrógeno/metabolismo , Inyecciones Intraperitoneales , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/prevención & control , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Insufficient T cell infiltration into noninflamed tumors, such as hepatocellular carcinoma (HCC), restricts the effectiveness of immune-checkpoint blockade (ICB) for a subset of patients. Epigenetic therapy provides further opportunities to rewire cancer-associated transcriptional programs, but whether and how selective epigenetic inhibition counteracts the immune-excluded phenotype remain incompletely defined. Here, we showed that pharmacological inhibition of histone deacetylase 8 (HDAC8), a histone H3 lysine 27 (H3K27)-specific isozyme overexpressed in a variety of human cancers, thwarts HCC tumorigenicity in a T cell-dependent manner. The tumor-suppressive effect of selective HDAC8 inhibition was abrogated by CD8+ T cell depletion or regulatory T cell adoptive transfer. Chromatin profiling of human HDAC8-expressing HCCs revealed genome-wide H3K27 deacetylation in 1251 silenced enhancer-target gene pairs that are enriched in metabolic and immune regulators. Mechanistically, down-regulation of HDAC8 increased global and enhancer acetylation of H3K27 to reactivate production of T cell-trafficking chemokines by HCC cells, thus relieving T cell exclusion in both immunodeficient and humanized mouse models. In an HCC preclinical model, selective HDAC8 inhibition increased tumor-infiltrating CD8+ T cells and potentiated eradication of established hepatomas by anti-PD-L1 therapy without evidence of toxicity. Mice treated with HDAC8 and PD-L1 coblockade were protected against subsequent tumor rechallenge as a result of the induction of memory T cells and remained tumor-free for greater than 15 months. Collectively, our study demonstrates that selective HDAC8 inhibition elicits effective and durable responses to ICB by co-opting adaptive immunity through enhancer reprogramming.
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Carcinoma Hepatocelular , Inhibidores de Histona Desacetilasas , Inhibidores de Puntos de Control Inmunológico , Neoplasias Hepáticas , Animales , Linfocitos T CD8-positivos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/inmunología , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Ratones , Proteínas RepresorasRESUMEN
The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.