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Background & Aims: The homeostasis of the cellular transcriptome depends on transcription and splicing mechanisms. Moreover, the fidelity of gene expression, essential to preserve cellular identity and function is secured by different quality control mechanisms including nonsense-mediated RNA decay (NMD). In this context, alternative splicing is coupled to NMD, and several alterations in these mechanisms leading to the accumulation of aberrant gene isoforms are known to be involved in human disease including cancer. Methods: RNA sequencing, western blotting, qPCR and co-immunoprecipitation were performed in multiple silenced culture cell lines (replicates n ≥4), primary hepatocytes and samples of animal models (Jo2, APAP, Mdr2 -/- mice, n ≥3). Results: Here we show that in animal models of liver injury and in human HCC (TCGA, non-tumoral = 50 vs. HCC = 374), the process of NMD is inhibited. Moreover, we demonstrate that the splicing factor SLU7 interacts with and preserves the levels of the NMD effector UPF1, and that SLU7 is required for correct NMD. Our previous findings demonstrated that SLU7 expression is reduced in the diseased liver, contributing to hepatocellular dedifferentiation and genome instability during disease progression. Here we build on this by providing evidence that caspases activated during liver damage are responsible for the cleavage and degradation of SLU7. Conclusions: Here we identify the downregulation of UPF1 and the inhibition of NMD as a new molecular pathway contributing to the malignant reshaping of the liver transcriptome. Moreover, and importantly, we uncover caspase activation as the mechanism responsible for the downregulation of SLU7 expression during liver disease progression, which is a new link between apoptosis and hepatocarcinogenesis. Impact and implications: The mechanisms involved in reshaping the hepatocellular transcriptome and thereby driving the progressive loss of cell identity and function in liver disease are not completely understood. In this context, we provide evidence on the impairment of a key mRNA surveillance mechanism known as nonsense-mediated mRNA decay (NMD). Mechanistically, we uncover a novel role for the splicing factor SLU7 in the regulation of NMD, including its ability to interact and preserve the levels of the key NMD factor UPF1. Moreover, we demonstrate that the activation of caspases during liver damage mediates SLU7 and UPF1 protein degradation and NMD inhibition. Our findings identify potential new markers of liver disease progression, and SLU7 as a novel therapeutic target to prevent the functional decay of the chronically injured organ.
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BACKGROUND AND AIMS: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol. METHODS: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot. RESULTS: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet. CONCLUSIONS: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.
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Ácidos y Sales Biliares , Colesterol 7-alfa-Hidroxilasa , Factores de Crecimiento de Fibroblastos , Hepatectomía , Regeneración Hepática , Humanos , Animales , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/biosíntesis , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Regeneración Hepática/efectos de los fármacos , Colesterol 7-alfa-Hidroxilasa/metabolismo , Colesterol 7-alfa-Hidroxilasa/genética , Ratones , Masculino , Femenino , Adulto , Persona de Mediana Edad , Hígado/metabolismo , Ratones Endogámicos C57BL , Trasplante de Hígado , Lipopolisacáridos/farmacologíaRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition with a complex etiology. Its incidence is increasing globally in parallel with the obesity epidemic, and it is now considered the most common liver disease in Western countries. The precise mechanisms underlying the development and progression of NAFLD are complex and still poorly understood. The dysregulation of epigenetic and epitranscriptomic mechanisms is increasingly recognized to play pathogenic roles in multiple conditions, including chronic liver diseases. Here, we have performed a comprehensive analysis of the expression of epigenetic and epitranscriptomic genes in a total of 903 liver tissue samples corresponding to patients with normal liver, obese patients, and patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), advancing stages in NAFLD progression. We integrated ten transcriptomic datasets in an unbiased manner, enabling their robust analysis and comparison. We describe the complete landscape of epigenetic and epitranscriptomic genes' expression along the course of the disease. We identify signatures of genes significantly dysregulated in association with disease progression, particularly with liver fibrosis development. Most of these epigenetic and epitranscriptomic effectors have not been previously described in human NAFLD, and their altered expression may have pathogenic implications. We also performed a comprehensive analysis of the expression of enzymes involved in the metabolism of the substrates and cofactors of epigenetic and epitranscriptomic effectors. This study provides novel information on NAFLD pathogenesis and may also guide the identification of drug targets to treat this condition and its progression towards hepatocellular carcinoma.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Carcinoma Hepatocelular/patología , Cirrosis Hepática/genética , Obesidad/genética , Obesidad/metabolismo , Neoplasias Hepáticas/patología , Epigénesis GenéticaRESUMEN
BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Animales , Aracnodactilia , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Carcinogénesis/genética , Colangiocarcinoma/patología , Contractura , Epigénesis Genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glucosa , Glicina/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Fosfoglicerato-Deshidrogenasa/genética , Proteómica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Serina/metabolismoRESUMEN
OBJECTIVE: Despite significant progresses in imaging and pathological evaluation, early differentiation between benign and malignant biliary strictures remains challenging. Endoscopic retrograde cholangiopancreatography (ERCP) is used to investigate biliary strictures, enabling the collection of bile. We tested the diagnostic potential of next-generation sequencing (NGS) mutational analysis of bile cell-free DNA (cfDNA). DESIGN: A prospective cohort of patients with suspicious biliary strictures (n=68) was studied. The performance of initial pathological diagnosis was compared with that of the mutational analysis of bile cfDNA collected at the time of first ERCP using an NGS panel open to clinical laboratory implementation, the Oncomine Pan-Cancer Cell-Free assay. RESULTS: An initial pathological diagnosis classified these strictures as of benign (n=26), indeterminate (n=9) or malignant (n=33) origin. Sensitivity and specificity of this diagnosis were 60% and 100%, respectively, as on follow-up 14 of the 26 and eight of the nine initially benign or indeterminate strictures resulted malignant. Sensitivity and specificity for malignancy of our NGS assay, herein named Bilemut, were 96.4% and 69.2%, respectively. Importantly, one of the four Bilemut false positives developed pancreatic cancer after extended follow-up. Remarkably, the sensitivity for malignancy of Bilemut was 100% in patients with an initial diagnosis of benign or indeterminate strictures. Analysis of 30 paired bile and tissue samples also demonstrated the superior performance of Bilemut. CONCLUSION: Implementation of Bilemut at the initial diagnostic stage for biliary strictures can significantly improve detection of malignancy, reduce delays in the clinical management of patients and assist in selecting patients for targeted therapies.
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Neoplasias de los Conductos Biliares , Ácidos Nucleicos Libres de Células , Colestasis , Bilis , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Colangiopancreatografia Retrógrada Endoscópica , Colestasis/etiología , Colestasis/genética , Constricción Patológica/diagnóstico , Detección Precoz del Cáncer , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Gene expression is finely and dynamically controlled through the tightly coordinated and interconnected activity of epigenetic modulators, transcription and splicing factors and post-translational modifiers. We have recently identified the splicing factor SLU7 as essential for maintaining liver cell identity and genome integrity and for securing cell division both trough transcriptional and splicing mechanisms. Now we uncover a new function of SLU7 controlling gene expression at the epigenetic level. We show that SLU7 is required to secure DNMT1 protein stability and a correct DNA methylation. We demonstrate that SLU7 is part in the chromatome of the protein complex implicated in DNA methylation maintenance interacting with and controlling the integrity of DNMT1, its adaptor protein UHRF1 and the histone methyl-transferase G9a at the chromatin level. Mechanistically, we found that SLU7 assures DNMT1 stability preventing its acetylation and degradation by facilitating its interaction with HDAC1 and the desubiquitinase USP7. Importantly, we demonstrate that this DNMT1 dependency on SLU7 occurs in a large panel of proliferating cell lines of different origins and in in vivo models of liver proliferation. Overall, our results uncover a novel and non-redundant role of SLU7 in DNA methylation and present SLU7 as a holistic regulator of gene expression.
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ADN (Citosina-5-)-Metiltransferasa 1/genética , Histona Desacetilasa 1/genética , Factores de Empalme de ARN/genética , Empalme del ARN/genética , Peptidasa Específica de Ubiquitina 7/genética , Proliferación Celular/genética , Cromatina/genética , Metilación de ADN/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Histonas/genética , Humanos , Hígado/metabolismo , Hígado/patología , Procesamiento Proteico-Postraduccional/genética , Estabilidad ProteicaRESUMEN
Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy of the intrahepatic biliary tract with a very poor prognosis. Although some clinicopathological parameters can be prognostic factors for iCCA, the molecular prognostic markers and potential mechanisms of iCCA have not been well investigated. Here, we report that the Fragile X mental retardation protein (FMRP), a RNA binding protein functionally absent in patients with the Fragile X syndrome (FXS) and also involved in several types of cancers, is overexpressed in human iCCA and its expression is significantly increased in iCCA metastatic tissues. The silencing of FMRP in metastatic iCCA cell lines affects cell migration and invasion, suggesting a role of FMRP in iCCA progression. Moreover, we show evidence that FMRP is localized at the invasive front of human iCCA neoplastic nests and in pseudopodia and invadopodia protrusions of migrating and invading iCCA cancer cells. Here FMRP binds several mRNAs encoding key proteins involved in the formation and/or function of these protrusions. In particular, we find that FMRP binds to and regulates the expression of Cortactin, a critical regulator of invadopodia formation. Altogether, our findings suggest that FMRP could promote cell invasiveness modulating membrane plasticity and invadopodia formation at the leading edges of invading iCCA cells.
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Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Animales , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Línea Celular Tumoral , Plasticidad de la Célula/fisiología , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Cortactina/metabolismo , Humanos , Masculino , Ratones Desnudos , Metástasis de la Neoplasia , Podosomas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.
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Neoplasias de los Conductos Biliares , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma , ADN (Citosina-5-)-Metiltransferasa 1 , Inhibidores Enzimáticos/farmacología , Antígenos de Histocompatibilidad , N-Metiltransferasa de Histona-Lisina , Animales , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/efectos de los fármacos , Metilación de ADN/fisiología , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad/metabolismo , Código de Histonas/efectos de los fármacos , Código de Histonas/fisiología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Resultado del Tratamiento , Ubiquitina-Proteína Ligasas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.
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Proteínas Adaptadoras Transductoras de Señales/genética , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , Factores de Empalme de ARN/genética , Factores de Empalme Serina-Arginina/genética , Empalme Alternativo/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Genoma Humano/genética , Inestabilidad Genómica/genética , Células Hep G2 , Humanos , Empalme del ARN/genética , Intercambio de Cromátides Hermanas/genéticaRESUMEN
Epigenetic modifications such as DNA and histone methylation functionally cooperate in fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n = 268), peritumoral tissues (n = 154), and HCC cell lines (n = 32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cell growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1, and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. Conclusion: Combined targeting of G9a/DNMT1 with compounds such as CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC.
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Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Carcinoma Hepatocelular/enzimología , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Perros , Células Hep G2 , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Neoplasias Hepáticas Experimentales/enzimología , Células de Riñón Canino Madin Darby , Masculino , Ratones Desnudos , Ubiquitina-Proteína Ligasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Advanced hepatocellular carcinoma (HCC) is a neoplastic disease with a very bad prognosis and increasing worldwide incidence. HCCs are resistant to conventional chemotherapy and the multikinase inhibitor sorafenib is the only agent that has shown some clinical efficacy. It is therefore important to identify key molecular mechanisms driving hepatocarcinogenesis for the development of more efficacious therapies. However, HCCs are heterogeneous tumors and different molecular subclasses have been characterized. This heterogeneity may underlie the poor performance of most of the targeted therapies so far tested in HCC patients. The fibroblast growth factor 15/19 (FGF15/19), FGF receptor 4 (FGFR4) and beta-Klotho (KLB) correceptor signaling system, a key regulator of bile acids (BA) synthesis and intermediary metabolism, is emerging as an important player in hepatocarcinogenesis. Key Messages: Aberrant signaling through the FGF15/19-FGFR4 pathway participates in the neoplastic behavior of HCC cells, promotes HCC development in mice and its overexpression has been characterized in a subset of HCC tumors from patients with poorer prognosis. Pharmacological interference with FGF15/19-FGFR4 signaling inhibits experimental hepatocarcinogenesis, and specific FGFR4 inhibitors are currently being tested in selected HCC patients with tumoral FGF19-FGFR4/KLB expression. CONCLUSIONS: Interference with FGF19-FGFR4 signaling represents a novel strategy in HCC therapy. Selection of candidate patients based on tumoral FGF19-FGFR4/KLB levels as biomarkers may result in increased efficacy of FGFR4-targeted drugs. Nevertheless, attention should be paid to the potential on target toxic effects of FGFR4 inhibitors due to the key role of this signaling system in BA metabolism.
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Carcinogénesis/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Neoplasias Hepáticas/metabolismo , Animales , Humanos , Neoplasias Hepáticas/patología , Modelos Biológicos , Terapia Molecular Dirigida , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. DESIGN: Fgf15-/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. RESULTS: Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15-/- mice. Hepatic expression of Pparγ2 was elevated in Fgf15-/- mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. CONCLUSIONS: FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.
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Estrés del Retículo Endoplásmico/efectos de los fármacos , Hígado Graso/genética , Hígado Graso/prevención & control , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Regeneración Hepática/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/metabolismo , Línea Celular , Dieta Alta en Grasa , Estrés del Retículo Endoplásmico/genética , Hígado Graso/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Semivida , Hepatectomía , Humanos , Íleon/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Regeneración Hepática/genética , Masculino , Ratones , Ratones Obesos , PPAR gamma/genética , PPAR gamma/metabolismo , Ácido Palmítico/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Regulación hacia ArribaRESUMEN
Fibroblast growth factor 15 (FGF15), FGF19 in humans, is a gut-derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF15 also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell growth. Here we addressed for the first time the role of endogenous Fgf15 in hepatocarcinogenesis. Fgf15(+/+) and Fgf15(-/-) mice were subjected to a clinically relevant model of liver inflammation and fibrosis-associated carcinogenesis. Fgf15(-/-) mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf15(+/+) animals. Importantly, ileal Fgf15 mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF15 protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf15(-/-) mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP). Interestingly, lack of FGF15 resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF15/FGF19. Conversely we demonstrate that FGF15/FGF19 induces the expression of the pro-fibrogenic and pro-tumorigenic connective tissue growth factor (CTGF) in hepatocytes. These findings suggest the existence of an FGF15-triggered CTGF-mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF15 via CTGF production. In summary, our observations indicate that ileal FGF15 may contribute to HCC development in a context of chronic liver injury and fibrosis.
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Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Íleon/metabolismo , Cirrosis Hepática Experimental/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Factores de Crecimiento de Fibroblastos/sangre , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/sangre , Cirrosis Hepática Experimental/patología , Neoplasias Hepáticas Experimentales/patología , RatonesRESUMEN
BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a chemoresistant tumor strongly associated with chronic hepatitis. Identification of molecular links connecting inflammation with cell growth/survival, and characterization of pro-tumorigenic intracellular pathways is therefore of therapeutic interest. The epidermal growth factor receptor (EGFR) signaling system stands at a crossroad between inflammatory signals and intracellular pathways associated with hepatocarcinogenesis. We investigated the regulation and activity of different components of the EGFR system, including the EGFR ligand amphiregulin (AR) and its sheddase ADAM17, and the modulation of intracellular EGFR signaling by a novel mechanism involving protein methylation. METHODS: ADAM17 protein expression was examined in models of liver injury and carcinogenesis. Crosstalk between tumor necrosis factor (TNF)-α, AR and EGFR signaling was evaluated in human HCC cells and mouse hepatocytes. Modulation of EGFR signaling and biological responses by methylation reactions was evaluated in AML12 mouse hepatocytes. RESULTS: ADAM17 was upregulated in liver injury and hepatocarcinogenesis. TNF-α triggered AR shedding and EGFR transactivation in HCC cells. AR was necessary for TNF-α activation of ERK1/2 and Akt signaling in hepatocytes. Inhibition of methylation reactions increased the ERK1/2 signal amplitude triggered by AR/EGFR and reduced DNA synthesis in AML12 cells. CONCLUSIONS: Increased ADAM17 in pre-neoplastic liver injury further supports its implication in hepatocarcinogenesis. AR release and EGFR transactivation by TNF-α constitutes a novel link between inflammatory signals and pro-tumorigenic mechanisms in liver cells. Finally, the identification of a new mechanism controlling growth factor signaling, and biological responses, involving methylation reactions within the RAS/RAF/MEK/ERK pathway, exposes a new target for antineoplastic intervention.
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Carcinoma Hepatocelular/metabolismo , Receptores ErbB/metabolismo , Inflamación/metabolismo , Neoplasias Hepáticas/metabolismo , Transducción de Señal/fisiología , Proteínas ADAM/metabolismo , Proteína ADAM17 , Anfirregulina , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Familia de Proteínas EGF , Regulación de la Expresión Génica/fisiología , Glicoproteínas/metabolismo , Hepatocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/lesiones , Hígado/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Metilación , Ratones , Proteínas Proto-Oncogénicas c-akt , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFß2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis.
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Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Adenosina/análogos & derivados , Fibrosis/tratamiento farmacológico , Hepatopatías/genética , Hepatopatías/patología , Tionucleósidos/administración & dosificación , Adenosina/administración & dosificación , Animales , Ciclina D1/metabolismo , Citocinas/metabolismo , Progresión de la Enfermedad , Fibroblastos/metabolismo , Inflamación , Hígado/patología , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
The Wilms' tumor 1 gene (WT1) encodes a transcription factor involved in cell growth and development. As we previously reported, WT1 expression is hardly detectable in normal hepatic tissue but is induced in liver cirrhosis. Although WT1 has been found to be overexpressed in a number of malignancies, the role of WT1 in hepatocarcinogenesis has not been clarified. We found that WT1 is expressed in several human hepatocellular carcinoma (HCC) cell lines, including PLC/PRF/5 and HepG2, and in HCC tumor tissue in 42% of patients. WT1 small interfering RNAs did not affect proliferation rate of HCC cells but abrogated their resistance to anoikis. Transcriptome analysis of PLC/PRF/5 cells after WT1 knockdown showed up-regulation of 251 genes and down-regulation of 321. Ninety percent of the former corresponded to metabolic genes, mostly those characterizing the mature hepatocyte phenotype. On the contrary, genes that decreased upon WT1 inhibition were mainly related to defense against apoptosis, cell cycle, and tumor progression. In agreement with these findings, WT1 expression increased the resistance of liver tumor cells to doxorubicin, a compound used to treat HCC. Interestingly, doxorubicin strongly enhanced WT1 expression in both HCC cells and normal human hepatocytes. Among different chemotherapeutics, induction of WT1 transcription was restricted to topoisomerase 2 inhibitors. When WT1 expression was prohibited, doxorubicin caused a marked increase in caspase-3 activation. In conclusion, WT1 is expressed in a substantial proportion of HCC contributing to tumor progression and resistance to chemotherapy, suggesting that WT1 may be an important target for HCC treatment.
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Carcinoma Hepatocelular/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Genes del Tumor de Wilms , Neoplasias Hepáticas/genética , Anciano , Apoptosis/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/cirugía , Diferenciación Celular , División Celular/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Interferente Pequeño/genética , Transcripción GenéticaRESUMEN
UNLABELLED: The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a condition characterized by excessive deposition of extracellular matrix. Fibrogenic cells, including hepatic stellate cells and myofibroblasts, are activated in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin (AR) is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. Here, we have addressed the in vivo role of AR in experimental liver fibrosis. To this end, liver fibrosis was induced in AR+/+ and AR-/- mice by chronic CCl(4) administration. Histological and molecular markers of hepatic fibrogenesis were measured. Additionally, the response of cultured human and mouse liver fibrogenic cells to AR was evaluated. We observed that AR was expressed in isolated Kupffer cells and liver fibrogenic cells in response to inflamatory stimuli and platelet-derived growth factor, respectively. We demonstrate that the expression of alpha-smooth muscle actin and collagen deposition were markedly reduced in AR-/- mice compared to AR+/+ animals. AR-/- mice also showed reduced expression of tissue inhibitor of metalloproteinases-1 and connective tissue growth factor, two genes that responded to AR treatment in cultured fibrogenic cells. AR also stimulated cell proliferation and exerted a potent antiapoptotic effect on isolated fibrogenic cells. CONCLUSION: These results indicate that among the different EGFR ligands, AR plays a specific role in liver fibrosis. AR may contribute to the expression of fibrogenic mediators, as well as to the growth and survival of fibrogenic cells. Additionally, our data lend further support to the role of the EGFR system in hepatic fibrogenesis.
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Receptores ErbB/metabolismo , Glicoproteínas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Anfirregulina , Animales , Apoptosis/fisiología , Tetracloruro de Carbono , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Familia de Proteínas EGF , Matriz Extracelular/metabolismo , Glicoproteínas/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ligandos , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine, the main methyl donor. Two MAT-encoding genes (MAT1A, MAT2A) are found in mammals. The latter is expressed in proliferating liver, dedifferentiation and cancer, whereas MAT1A is expressed in adult quiescent hepatocytes. Here, we report studies on the molecular mechanisms controlling the induction of MAT2A in regenerating rat liver and in proliferating hepatocytes. The MAT2A is up-regulated at two discrete moments during liver regeneration, as confirmed by RNApol-ChIP analysis. The first one coincides with hepatocyte priming (i.e. G0-G1 transition), while the second one takes place at the G1-S interface. Electrophoretic mobility shift assays showed that a putative E2F sequence present in MAT2A promoter binds this factor and ChIP assays confirmed that E2F1, E2F3 and E2F4, as well as the pocket protein p130, are bound to the promoter in quiescent liver. MAT2A activation is accompanied by changes in the binding of histone-modifying enzymes to the promoter. Interestingly, p130 is not displaced from MAT2A promoter during hepatocyte priming, but it is in the late expression of the gene at the G1-S transition. Finally, the transcription factor Sp1 seems to play a decisive role in MAT2A induction, as it binds the promoter when the gene is being actively transcribed. In summary, the present work shows that the molecular mechanism of MAT2A expression is different during G0-G1 or G1-S transition and this may be related to the distinct requirements of S-adenosylmethionine during liver regeneration.
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Proliferación Celular , Cromatina/metabolismo , Factores de Transcripción E2F/metabolismo , Hígado/metabolismo , Metionina Adenosiltransferasa/genética , Factor de Transcripción Sp1/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Cromatina/genética , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Fase G1/genética , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/citología , Hígado/fisiología , Regeneración Hepática/genética , Masculino , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Ratas Wistar , Fase S/genética , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Regulación hacia ArribaRESUMEN
Various immediate early genes (IEGs) upregulated during the early process of liver regeneration are transcriptional targets of the serum response factor (SRF). We show here that the expression of SRF is rapidly induced in rodent liver after partial hepatectomy. Because the inactivation of the SRF gene in mice is embryonic lethal, the in vivo role of SRF in liver regeneration after partial hepatectomy was analyzed in mutant mice conditionally deleted for SRF in the liver. We demonstrate that SRF is not an essential factor for liver ontogenesis. However, adult mutant mice show impaired liver regeneration after partial hepatectomy, associated with a blunted upregulation of various SRF target IEGs. In conclusion, our work suggests that SRF is an early response transcription factor that may contribute to the initial phases of liver regeneration through its activation of IEGs.
Asunto(s)
Regeneración Hepática , Hígado/metabolismo , Factor de Respuesta Sérica/metabolismo , Animales , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , ADN/biosíntesis , Hepatectomía , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Hígado/citología , Hígado/fisiología , Hígado/cirugía , Regeneración Hepática/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Factor de Respuesta Sérica/deficiencia , Factor de Respuesta Sérica/genética , Factores de Tiempo , Activación TranscripcionalRESUMEN
BACKGROUND & AIMS: After liver injury, hepatic S-adenosylmethionine (SAM) content decreases, and the blockage this molecule imposes on hepatocyte proliferation is released, facilitating liver regeneration. This activity of SAM is important for normal liver function because mice deficient in hepatic SAM display abnormal liver regeneration and develop hepatocellular carcinoma. How SAM regulates hepatocyte growth is unclear, but because SAM blocks hepatocyte growth factor (HGF)-induced cyclin D1 expression and DNA synthesis without affecting HGF-induced extracellular signal-regulated kinase phosphorylation, the mitogen-activated protein kinase (MAPK) pathway is probably not the target. METHODS: The effects of SAM on AMPK, HuR localization were assessed in rat hepatocytes after HGF, AICAR, and SAM treatment. RESULTS: We show here that HGF and 5-aminoimidazole-4-carboxamide-riboside (AICAR), an activator of AMP-activated protein kinase (AMPK), induce the phosphorylation of AMPK in hepatocytes and that SAM blocks this process. We also show that HGF- and AICAR-induced AMPK activation stimulate the transport from nucleus to cytoplasm of HuR, an RNA-binding protein that increases the half-life of target mRNA such as cyclin A2, and that SAM blocks this process. We found that, in hepatocytes, AICAR increases HuR binding to cyclin A2 messenger RNA (mRNA) as well as the expression and stability of this mRNA and that SAM blocks these events. Consistently, we found that AICAR induces hepatocyte proliferation and that SAM blocks this effect. Finally, we found that liver AMPK phosphorylation, cytoplasmic HuR, and binding of HuR to HuR-target mRNA and the steady-state levels of these mRNA are increased in knockout mice deficient in hepatic SAM. CONCLUSIONS: Our results yield novel insights about the mechanism by which SAM inhibits cell-cycle progression in the liver.