RESUMO
Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.
Assuntos
Carcinogênese , Carcinoma Hepatocelular , Células Estreladas do Fígado , Neoplasias Hepáticas , Animais , Carcinogênese/patologia , Carcinoma Hepatocelular/patologia , Proliferação de Células , Colágeno Tipo I/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Progressão da Doença , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos , Humanos , Cirrose Hepática/complicações , Neoplasias Hepáticas/patologia , Camundongos , Miofibroblastos/patologiaRESUMO
Cholangiocarcinoma (CCA) comprises diverse tumors of the biliary tree and is characterized by late diagnosis, short-term survival, and chemoresistance. CCAs are mainly classified according to their anatomical location and include diverse molecular subclasses harboring inter-tumoral and intratumoral heterogeneity. Besides the tumor cell component, CCA is also characterized by a complex and dynamic tumor microenvironment where tumor cells and stromal cells crosstalk in an intricate network of interactions. Cancer-associated fibroblasts, one of the most abundant cell types in the tumor stroma of CCA, are actively involved in cholangiocarcinogenesis by participating in multiple aspects of the disease including extracellular matrix remodeling, immunomodulation, neo-angiogenesis, and metastasis. Despite their overall tumor-promoting role, recent evidence indicates the presence of transcriptional and functional heterogeneous CAF subtypes with tumor-promoting and tumor-restricting properties. To elucidate the complexity and potentials of cancer-associated fibroblasts as therapeutic targets in CCA, this review will discuss the origin of cancer-associated fibroblasts, their heterogeneity, crosstalk, and role during tumorigenesis, providing an overall picture of the present and future perspectives toward cancer-associated fibroblasts targeting CCA.
Assuntos
Aracnodactilia , Neoplasias dos Ductos Biliares , Sistema Biliar , Fibroblastos Associados a Câncer , Colangiocarcinoma , Contratura , Humanos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos , Microambiente TumoralRESUMO
BACKGROUND AND AIMS: Fibrosis is the common end point for all forms of chronic liver injury, and the progression of fibrosis leads to the development of end-stage liver disease. Activation of HSCs and their transdifferentiation into myofibroblasts results in the accumulation of extracellular matrix proteins that form the fibrotic scar. Long noncoding RNAs regulate the activity of HSCs and provide targets for fibrotic therapies. APPROACH AND RESULTS: We identified long noncoding RNA TILAM located near COL1A1 , expressed in HSCs, and induced with liver fibrosis in humans and mice. Loss-of-function studies in human HSCs and human liver organoids revealed that TILAM regulates the expression of COL1A1 and other extracellular matrix genes. To determine the role of TILAM in vivo, we annotated the mouse ortholog ( Tilam ), generated Tilam- deficient green fluorescent protein-reporter mice, and challenged these mice in 2 different models of liver fibrosis. Single-cell data and analysis of single-data and analysis of Tilam-deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Tilam -deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Furthermore, loss of Tilam expression attenuated the development of fibrosis in the setting of in vivo liver injury. Finally, we found that TILAM interacts with promyelocytic leukemia nuclear body scaffold protein to regulate a feedback loop by which TGF-ß2 reinforces TILAM expression and nuclear localization of promyelocytic leukemia nuclear body scaffold protein to promote the fibrotic activity of HSCs. CONCLUSIONS: TILAM is activated in HSCs with liver injury and interacts with promyelocytic leukemia nuclear body scaffold protein to drive the development of fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end-stage liver disease.
RESUMO
OBJECTIVE: The diversity of the tumour microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA) has not been comprehensively assessed. We aimed to generate a novel molecular iCCA classifier that incorporates elements of the stroma, tumour and immune microenvironment ('STIM' classification). DESIGN: We applied virtual deconvolution to transcriptomic data from ~900 iCCAs, enabling us to devise a novel classification by selecting for the most relevant TME components. Murine models were generated through hydrodynamic tail vein injection and compared with the human disease. RESULTS: iCCA is composed of five robust STIM classes encompassing both inflamed (35%) and non-inflamed profiles (65%). The inflamed classes, named immune classical (~10%) and inflammatory stroma (~25%), differ in oncogenic pathways and extent of desmoplasia, with the inflammatory stroma showing T cell exhaustion, abundant stroma and KRAS mutations (p<0.001). Analysis of cell-cell interactions highlights cancer-associated fibroblast subtypes as potential mediators of immune evasion. Among the non-inflamed classes, the desert-like class (~20%) harbours the lowest immune infiltration with abundant regulatory T cells (p<0.001), whereas the hepatic stem-like class (~35%) is enriched in 'M2-like' macrophages, mutations in IDH1/2 and BAP1, and FGFR2 fusions. The remaining class (tumour classical: ~10%) is defined by cell cycle pathways and poor prognosis. Comparative analysis unveils high similarity between a KRAS/p19 murine model and the inflammatory stroma class (p=0.02). The KRAS-SOS inhibitor, BI3406, sensitises a KRAS-mutant iCCA murine model to anti-PD1 therapy. CONCLUSIONS: We describe a comprehensive TME-based stratification of iCCA. Cross-species analysis establishes murine models that align closely to human iCCA for the preclinical testing of combination strategies.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Animais , Camundongos , Modelos Animais de Doenças , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Microambiente TumoralRESUMO
BACKGROUND & AIMS: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. METHODS: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. RESULTS: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. CONCLUSIONS: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. IMPACT AND IMPLICATIONS: Our results indicate that neutrophils are highly plastic and can have an extended lifespan. Moreover, we identify a new role of neutrophils as triggers of expansion of the biliary epithelium. Overall, the results of this study indicate that ductular reaction-associated neutrophils (or DRANs) are new players in the maladaptive tissue-healing response in chronic liver injury and may be a potential target for therapeutic interventions to reduce ductular reaction expansion and promote tissue repair in advanced liver disease.
Assuntos
Hepatopatias , Neutrófilos , Animais , Camundongos , Fígado , Proliferação de Células , EpitélioRESUMO
BACKGROUND & AIMS: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood. METHODS: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity. RESULTS: Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression. CONCLUSIONS: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH. IMPACT AND IMPLICATIONS: Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair.
Assuntos
Reprogramação Celular , Hepatite Alcoólica , Animais , Camundongos , Hepatite Alcoólica/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Fígado/patologiaRESUMO
BACKGROUND & AIMS: YES-associated protein (YAP) aberrant activation is implicated in intrahepatic cholangiocarcinoma (iCCA). Transcriptional enhanced associate domain (TEAD)-mediated transcriptional regulation is the primary signaling event downstream of YAP. The role of Wnt/ß-Catenin signaling in cholangiocarcinogenesis remains undetermined. Here, we investigated the possible molecular interplay between YAP and ß-Catenin cascades in iCCA. METHODS: Activated AKT (Myr-Akt) was coexpressed with YAP (YapS127A) or Tead2VP16 via hydrodynamic tail vein injection into mouse livers. Tumor growth was monitored, and liver tissues were collected and analyzed using histopathologic and molecular analysis. YAP, ß-Catenin, and TEAD interaction in iCCAs was investigated through coimmunoprecipitation. Conditional Ctnnb1 knockout mice were used to determine ß-Catenin function in murine iCCA models. RNA sequencing was performed to analyze the genes regulated by YAP and/or ß-Catenin. Immunostaining of total and nonphosphorylated/activated ß-Catenin staining was performed in mouse and human iCCAs. RESULTS: We discovered that TEAD factors are required for YAP-dependent iCCA development. However, transcriptional activation of TEADs did not fully recapitulate YAP's activities in promoting cholangiocarcinogenesis. Notably, ß-Catenin physically interacted with YAP in human and mouse iCCA. Ctnnb1 ablation strongly suppressed human iCCA cell growth and Yap-dependent cholangiocarcinogenesis. Furthermore, RNA-sequencing analysis revealed that YAP/ transcriptional coactivator with PDZ-binding motif (TAZ) regulate a set of genes significantly overlapping with those controlled by ß-Catenin. Importantly, activated/nonphosphorylated ß-Catenin was detected in more than 80% of human iCCAs. CONCLUSION: YAP induces cholangiocarcinogenesis via TEAD-dependent transcriptional activation and interaction with ß-Catenin. ß-Catenin binds to YAP in iCCA and is required for YAP full transcriptional activity, revealing the functional crosstalk between YAP and ß-Catenin pathways in cholangiocarcinogenesis.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Proteínas de Sinalização YAP , beta Catenina , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Carcinogênese , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: We recently showed that alcoholic hepatitis (AH) is characterized by dedifferentiation of hepatocytes and loss of mature functions. Glucose metabolism is tightly regulated in healthy hepatocytes. We hypothesize that AH may lead to metabolic reprogramming of the liver, including dysregulation of glucose metabolism. METHODS: We performed integrated metabolomic and transcriptomic analyses of liver tissue from patients with AH or alcoholic cirrhosis or normal liver tissue from hepatic resection. Focused analyses of chromatin immunoprecipitation coupled to DNA sequencing was performed. Functional in vitro studies were performed in primary rat and human hepatocytes and HepG2 cells. RESULTS: Patients with AH exhibited specific changes in the levels of intermediates of glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and monosaccharide and disaccharide metabolism. Integrated analysis of the transcriptome and metabolome showed the used of alternate energetic pathways, metabolite sinks and bottlenecks, and dysregulated glucose storage in patients with AH. Among genes involved in glucose metabolism, hexokinase domain containing 1 (HKDC1) was identified as the most up-regulated kinase in patients with AH. Histone active promoter and enhancer markers were increased in the HKDC1 genomic region. High HKDC1 levels were associated with the development of acute kidney injury and decreased survival. Increased HKDC1 activity contributed to the accumulation of glucose-6-P and glycogen in primary rat hepatocytes. CONCLUSIONS: Altered metabolite levels and messenger RNA expression of metabolic enzymes suggest the existence of extensive reprogramming of glucose metabolism in AH. Increased HKDC1 expression may contribute to dysregulated glucose metabolism and represents a novel biomarker and therapeutic target for AH.
Assuntos
Desdiferenciação Celular , Metabolismo Energético , Perfilação da Expressão Gênica , Glucose/metabolismo , Hepatite Alcoólica/enzimologia , Hepatócitos/enzimologia , Hexoquinase/metabolismo , Fígado/enzimologia , Metabolômica , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/genética , Adaptação Fisiológica , Animais , Europa (Continente) , Feminino , Regulação Enzimológica da Expressão Gênica , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Células Hep G2 , Hepatite Alcoólica/genética , Hepatite Alcoólica/patologia , Hepatócitos/patologia , Hexoquinase/genética , Humanos , Fígado/patologia , Masculino , Metaboloma , Pessoa de Meia-Idade , Ratos Wistar , Transcriptoma , Estados UnidosRESUMO
BACKGROUND & AIMS: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. METHODS: Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. In vitro cell line studies and in vivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. RESULTS: FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. CONCLUSIONS: FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. LAY SUMMARY: We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in in vitro and in vivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/efeitos adversos , Proteínas de Sinalização YAP/efeitos dos fármacos , Animais , California , Colangiocarcinoma/etiologia , Estudos de Coortes , Modelos Animais de Doenças , Proteína-Tirosina Quinases de Adesão Focal/administração & dosagem , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAP/administração & dosagemRESUMO
Fibrosis and cancer represent two major complications of chronic liver disease. MicroRNAs have been implicated in the development of fibrosis and cancer, thus constituting potential therapeutic targets. Here, we investigated the role of microRNA-21 (miR-21), a microRNA that has been implicated in the development of fibrosis in multiple organs and has also been suggested to act as an "oncomir." Accordingly, miR-21 was the microRNA that showed the strongest up-regulation in activated hepatic stellate cells (HSCs) in multiple models of fibrogenesis, with an 8-fold to 24-fold induction compared to quiescent HSCs. However, miR-21 antisense inhibition did not suppress the activation of murine or human HSCs in culture or in liver slices. Moreover, genetic deletion of miR-21 in two independently generated knockout mice or miR-21 antisense inhibition did not alter HSC activation or liver fibrosis in models of toxic and biliary liver injury. Despite a strong up-regulation of miR-21 in injury-associated hepatocellular carcinoma and in cholangiocarcinoma, miR-21 deletion or antisense inhibition did not reduce the development of liver tumors. As inhibition of the most up-regulated microRNA did not affect HSC activation, liver fibrosis, or fibrosis-associated liver cancer, we additionally tested the role of microRNAs in HSCs by HSC-specific Dicer deletion. Although Dicer deletion decreased microRNA expression in HSCs and altered the expression of select genes, it only exerted negligible effects on HSC activation and liver fibrosis. CONCLUSION: Genetic and pharmacologic manipulation of miR-21 does not inhibit the development of liver fibrosis and liver cancer. Moreover, suppression of microRNA synthesis does not significantly affect HSC phenotype and activation. (Hepatology 2018;67:2414-2429).
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RNA Helicases DEAD-box/fisiologia , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/etiologia , MicroRNAs/fisiologia , Ribonuclease III/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos KnockoutRESUMO
BACKGROUND & AIMS: Transforming growth factor-ß (TGFß) exerts key functions in fibrogenic cells, promoting fibrosis development in the liver and other organs. In contrast, the functions of TGFß in liver epithelial cells are not well understood, despite their high level of responsiveness to TGFß. We sought to determine the contribution of epithelial TGFß signaling to hepatic fibrogenesis and carcinogenesis. METHODS: TGFß signaling in liver epithelial cells was inhibited by albumin-Cre-, K19-CreERT-, Prom1-CreERT2-, or AAV8-TBG-Cre-mediated deletion of the floxed TGFß receptor II gene (Tgfbr2). Liver fibrosis was induced by carbon tetrachloride, bile duct ligation, or disruption of the multidrug-resistance transporter 2 gene (Mdr2). Hepatocarcinogenesis was induced by diethylnitrosamine or hepatic deletion of PTEN. RESULTS: Deletion of Tgfbr2 from liver epithelial cells did not alter liver injury, toxin-induced or biliary fibrosis, or diethylnitrosamine-induced hepatocarcinogenesis. In contrast, epithelial deletion of Tgfbr2 promoted tumorigenesis and reduced survival of mice with concomitant hepatic deletion of Pten, accompanied by an increase in tumor number and a shift from hepatocellular carcinoma to cholangiocarcinoma. Surprisingly, both hepatocyte- and cholangiocyte-specific deletion of Pten and Tgfbr2 promoted the development of cholangiocarcinoma, but with different latencies. The prolonged latency and the presence of hepatocyte-derived cholangiocytes after AAV8-TBG-Cre-mediated deletion of Tgfbr2 and Pten indicated that cholangiocarcinoma might arise from hepatocyte-derived cholangiocytes in this model. Pten deletion resulted in up-regulation of Tgfbr2, and deletion of Tgfbr2 increased cholangiocyte but not hepatocyte proliferation, indicating that the main function of epithelial TGFBR2 is to restrict cholangiocyte proliferation. CONCLUSIONS: Epithelial TGFß signaling does not contribute to the development of liver fibrosis or formation of hepatocellular carcinomas in mice, but restricts cholangiocyte proliferation to prevent cholangiocarcinoma development, regardless of its cellular origin.
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Neoplasias dos Ductos Biliares/prevenção & controle , Ductos Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colangiocarcinoma/prevenção & controle , Células Epiteliais/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Neoplasias dos Ductos Biliares/induzido quimicamente , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares/patologia , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colangiocarcinoma/induzido quimicamente , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Dietilnitrosamina , Células Epiteliais/patologia , Predisposição Genética para Doença , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Fatores de Tempo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
OBJECTIVE: Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. DESIGN: Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. RESULTS: Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. CONCLUSIONS: p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis.
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Hepatite Alcoólica/complicações , Hepatite Alcoólica/enzimologia , Cirrose Hepática/etiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-IdadeRESUMO
UNLABELLED: Alcoholic hepatitis (AH) frequently progresses to multiple organ failure (MOF) and death. However, the driving factors are largely unknown. At admission, patients with AH often show criteria of systemic inflammatory response syndrome (SIRS) even in the absence of an infection. We hypothesize that the presence of SIRS may predispose to MOF and death. To test this hypothesis, we studied a cohort including 162 patients with biopsy-proven AH. The presence of SIRS and infections was assessed in all patients, and multivariate analyses identified variables independently associated with MOF and 90-day mortality. At admission, 32 (19.8%) patients were diagnosed with a bacterial infection, while 75 (46.3%) fulfilled SIRS criteria; 58 patients (35.8%) developed MOF during hospitalization. Short-term mortality was significantly higher among patients who developed MOF (62.1% versus 3.8%, P < 0.001). The presence of SIRS was a major predictor of MOF (odds ratio = 2.69, P = 0.025) and strongly correlated with mortality. Importantly, the course of patients with SIRS with and without infection was similar in terms of MOF development and short-term mortality. Finally, we sought to identify serum markers that differentiate SIRS with and without infection. We studied serum levels of high-sensitivity C-reactive protein, procalcitonin, and lipopolysaccharide at admission. All of them predicted mortality. Procalcitonin, but not high-sensitivity C-reactive protein, serum levels identified those patients with SIRS and infection. Lipopolysaccharide serum levels predicted MOF and the response to prednisolone. CONCLUSION: In the presence or absence of infections, SIRS is a major determinant of MOF and mortality in AH, and the mechanisms involved in the development of SIRS should be investigated; procalcitonin serum levels can help to identify patients with infection, and lipopolysaccharide levels may help to predict mortality and the response to steroids.
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Hepatite Alcoólica/sangue , Hepatite Alcoólica/mortalidade , Lipopolissacarídeos/sangue , Insuficiência de Múltiplos Órgãos/mortalidade , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/mortalidade , Biomarcadores/sangue , Proteína C-Reativa/análise , Calcitonina/sangue , Peptídeo Relacionado com Gene de Calcitonina , Estudos de Coortes , Progressão da Doença , Feminino , Hepatite Alcoólica/complicações , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/fisiopatologia , Análise Multivariada , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Precursores de Proteínas/sangue , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Espanha , Análise de Sobrevida , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologiaRESUMO
UNLABELLED: Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1ß). However, HNF1ß expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1ß-expressing cells we used a tamoxifen-inducible Hnf1ßCreER/R26R(Yfp/LacZ) mouse to lineage-trace Hnf1ß(+) biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1ß(+) cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1ß(+) cells to hepatocyte was detected. We next assessed the contribution of Hnf1ß(+) -derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1ß(+) cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1ß(+) cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1ß(+) -derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. CONCLUSION: HNF1ß(+) biliary duct cells are the origin of LPC. HNF1ß(+) cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration.
Assuntos
Ductos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Células Epiteliais/patologia , Hepatócitos/patologia , Regeneração Hepática/fisiologia , Fígado/patologia , Células-Tronco/patologia , Acetaminofen/efeitos adversos , Animais , Ductos Biliares/metabolismo , Tetracloreto de Carbono/efeitos adversos , Diferenciação Celular/fisiologia , Linhagem da Célula , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Dieta/efeitos adversos , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Fator 1-beta Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Homeostase/fisiologia , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco/metabolismoRESUMO
OBJECTIVE: Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. DESIGN: CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. RESULTS: CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. CONCLUSIONS: Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.
Assuntos
Insuficiência Hepática Crônica Agudizada/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Quimiocina CCL20/fisiologia , Hepatite Alcoólica/fisiopatologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Quimiocina CCL20/análise , Quimiocina CCL20/sangue , Feminino , Humanos , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Pessoa de Meia-Idade , RNA Interferente Pequeno , Regulação para Cima/fisiologiaRESUMO
UNLABELLED: We identified, in the transcriptome analysis of patients with alcoholic hepatitis (AH), osteopontin (OPN) as one of the most up-regulated genes. Here, we used a translational approach to investigate its pathogenic role. OPN hepatic gene expression was quantified in patients with AH and other liver diseases. OPN protein expression and processing were assessed by immmunohistochemistry, western blotting and enzyme-linked immunosorbent assay. OPN gene polymorphisms were evaluated in patients with alcoholic liver disease. The role of OPN was evaluated in OPN(-/-) mice with alcohol-induced liver injury. OPN biological actions were studied in human hepatic stellate cells (HSCs) and in precision-cut liver slices. Hepatic expression and serum levels of OPN were markedly increased in AH, compared to normal livers and other types of chronic liver diseases, and correlated with short-term survival. Serum levels of OPN also correlated with hepatic expression and disease severity. OPN was mainly expressed in areas with inflammation and fibrosis. Two proteases that process OPN (thrombin and matrix metalloproteinase 7) and cleaved OPN were increased in livers with AH. Patients with AH had a tendency of a lower frequency of the CC genotype of the +1239C single-nucleotide polymorphism of the OPN gene, compared to patients with alcohol abuse without liver disease. Importantly, OPN(-/-) mice were protected against alcohol-induced liver injury and showed decreased expression of inflammatory cytokines. Finally, OPN was induced by lipopolysaccharide and stimulated inflammatory actions in HSCs. CONCLUSION: Human and experimental data suggest a role for OPN in the pathogenesis of AH. Further studies should evaluate OPN as a potential therapeutic target.
Assuntos
Hepatite Alcoólica/etiologia , Osteopontina/fisiologia , Animais , Feminino , Humanos , Hepatopatias Alcoólicas/etiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Osteopontina/sangue , Osteopontina/genética , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: Alcoholic hepatitis (AH) is a severe clinical condition that needs novel therapies. The identification of targets for therapy is hampered by the lack of animal models of advanced AH. The authors performed a translational study through a transcriptome analysis in patients with AH to identify new molecular targets. DESIGN: Hepatic gene expression profiling was assessed by DNA microarray in patients with AH (n=15) and normal livers (n=7). Functional analysis was assessed by gene set enrichment analysis. Quantitative PCR was performed in patients with AH (n=40), hepatitis C (n=18), non-alcoholic steatohepatitis (n=20) and in mouse models of acute and chronic liver injury. Protein expression was assessed by immunohistochemistry and western blotting. RESULTS: Gene expression analysis showed 207 genes >5-fold differentially expressed in patients with AH and revealed seven pathways differentially regulated including 'cytokine-cytokine receptor interaction'. Several tumour necrosis factor (TNF) superfamily receptors, but not ligands, were overexpressed in AH. Importantly, Fn14 was the only TNF superfamily receptor exclusively upregulated in AH compared with other liver diseases and correlated with both 90-day mortality and severity of portal hypertension. Fn14 protein expression was detected in areas of fibrogenesis and in a population of hepatocytes. Fn14 expression was increased in experimental models of liver injury and was detected in progenitor cells. CONCLUSION: Translational research revealed that TNF superfamily receptors are overexpressed in AH. Fn14, the receptor for TNF-like weak inducer of apoptosis, is selectively upregulated in patients with AH. TNF superfamily receptors could represent a potential target for therapy.
Assuntos
Regulação da Expressão Gênica/fisiologia , Hepatite Alcoólica/genética , Receptores do Fator de Necrose Tumoral/genética , Animais , Western Blotting , Análise por Conglomerados , Citocinas/genética , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Hepatite Alcoólica/tratamento farmacológico , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Prognóstico , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Receptor de TWEAK , Regulação para CimaRESUMO
Biliary tract cancers (BTCs) are rare and aggressive malignancies with an increasing incidence and poor prognosis. The standard systemic treatment for BTCs has evolved to include immune checkpoint inhibitors associated with gemcitabine-cisplatin as first-line therapies. However, survival rates remain low, highlighting the critical need for personalized treatment strategies based on molecular profiling. Currently, significant advancements have been made in the molecular characterization of BTCs, where genetic alterations, such as IDH1 mutations and FGFR2 fusions, provide targets for therapy. Molecular profiling is crucial early in the management process to identify potential candidates for clinical trials and guide treatment strategy. The integration of these molecular insights into clinical practice has allowed for the development of targeted therapies, although many of them are still in the phase 2 trial stage without definitive survival benefits demonstrated in phase 3 trials. This integration of comprehensive molecular profile insights with traditional treatment approaches offers a new horizon in the personalized medicine landscape for BTCs, with the aim of significantly improving patient outcomes through precision oncology.
Assuntos
Neoplasias do Sistema Biliar , Medicina de Precisão , Humanos , Neoplasias do Sistema Biliar/tratamento farmacológico , Neoplasias do Sistema Biliar/genética , Neoplasias do Sistema Biliar/terapia , Medicina de Precisão/métodos , Terapia de Alvo Molecular/métodosRESUMO
Accumulation of fibroblasts in the premalignant or malignant liver is a characteristic feature of liver cancer, but has not been therapeutically leveraged despite evidence for pathophysiologically relevant roles in tumour growth. Hepatocellular carcinoma is a largely non-desmoplastic tumour, in which fibroblasts accumulate predominantly in the pre-neoplastic fibrotic liver and regulate the risk for hepatocellular carcinoma development through a balance of tumour-suppressive and tumour-promoting mediators. By contrast, cholangiocarcinoma is desmoplastic, with cancer-associated fibroblasts contributing to tumour growth. Accordingly, restoring the balance from tumour-promoting to tumour-suppressive fibroblasts and mediators might represent a strategy for hepatocellular carcinoma prevention, whereas in cholangiocarcinoma, fibroblasts and their mediators could be leveraged for tumour treatment. Importantly, fibroblast mediators regulating hepatocellular carcinoma development might exert opposite effects on cholangiocarcinoma growth. This Review translates the improved understanding of tumour-specific, location-specific, and stage-specific roles of fibroblasts and their mediators in liver cancer into novel and rational therapeutic concepts.
Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Fibroblastos/patologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Colangiocarcinoma/terapia , Colangiocarcinoma/patologia , Ductos Biliares Intra-Hepáticos/patologiaRESUMO
In the past 5 years, important advances have been made in the scientific understanding and clinical management of cholangiocarcinoma (CCA). The cellular immune landscape of CCA has been characterized and tumour subsets with distinct immune microenvironments have been defined using molecular approaches. Among these subsets, the identification of 'immune-desert' tumours that are relatively devoid of immune cells emphasizes the need to consider the tumour immune microenvironment in the development of immunotherapy approaches. Progress has also made in identifying the complex heterogeneity and diverse functions of cancer-associated fibroblasts in this desmoplastic cancer. Assays measuring circulating cell-free DNA and cell-free tumour DNA are emerging as clinical tools for detection and monitoring of the disease. Molecularly targeted therapy for CCA has now become a reality, with three drugs targeting oncogenic fibroblast growth factor receptor 2 (FGFR2) fusions and one targeting neomorphic, gain-of-function variants of isocitrate dehydrogenase 1 (IDH1) obtaining regulatory approval. By contrast, immunotherapy using immune-checkpoint inhibitors has produced disappointing results in patients with CCA, underscoring the requirement for novel immune-based treatment strategies. Finally, liver transplantation for early stage intrahepatic CCA under research protocols is emerging as a viable therapeutic option in selected patients. This Review highlights and provides in-depth information on these advances.