RESUMO
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.
RESUMO
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.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Carcinoma Hepatocelular/patologia , Cirrose Hepática/genética , Obesidade/genética , Obesidade/metabolismo , Neoplasias Hepáticas/patologia , Epigênese GenéticaRESUMO
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.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Animais , Aracnodactilia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Carcinogênese/genética , Colangiocarcinoma/patologia , Contratura , Epigênese Genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glucose , Glicina/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Fosfoglicerato Desidrogenase/genética , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Serina/metabolismoRESUMO
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.
Assuntos
Neoplasias dos Ductos Biliares , Ácidos Nucleicos Livres , Colestase , Bile , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Colangiopancreatografia Retrógrada Endoscópica , Colestase/etiologia , Colestase/genética , Constrição Patológica/diagnóstico , Detecção Precoce de Câncer , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Plasticidade Celular/fisiologia , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Cortactina/metabolismo , Humanos , Masculino , Camundongos Nus , Metástase Neoplásica , Podossomos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Neoplasias dos Ductos Biliares , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma , DNA (Citosina-5-)-Metiltransferase 1 , Inibidores Enzimáticos/farmacologia , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/fisiologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade/metabolismo , Código das Histonas/efeitos dos fármacos , Código das Histonas/fisiologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Resultado do Tratamento , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Genoma Humano/genética , Instabilidade Genômica/genética , Células Hep G2 , Humanos , Splicing de RNA/genética , Troca de Cromátide Irmã/genéticaRESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/enzimologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Cães , Células Hep G2 , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Neoplasias Hepáticas Experimentais/enzimologia , Células Madin Darby de Rim Canino , Masculino , Camundongos Nus , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Carcinogênese/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Humanos , Neoplasias Hepáticas/patologia , Modelos Biológicos , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Íleo/metabolismo , Cirrose Hepática Experimental/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Fatores de Crescimento de Fibroblastos/sangue , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/sangue , Cirrose Hepática Experimental/patologia , Neoplasias Hepáticas Experimentais/patologia , CamundongosRESUMO
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.
Assuntos
Carcinoma Hepatocelular/metabolismo , Receptores ErbB/metabolismo , Inflamação/metabolismo , Neoplasias Hepáticas/metabolismo , Transdução de Sinais/fisiologia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Anfirregulina , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Família de Proteínas EGF , Regulação da Expressão Gênica/fisiologia , Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fígado/lesões , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Metilação , Camundongos , Proteínas Proto-Oncogênicas c-akt , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adenosina/análogos & derivados , Fibrose/tratamento farmacológico , Hepatopatias/genética , Hepatopatias/patologia , Tionucleosídeos/administração & dosagem , Adenosina/administração & dosagem , Animais , Ciclina D1/metabolismo , Citocinas/metabolismo , Progressão da Doença , Fibroblastos/metabolismo , Inflamação , Fígado/patologia , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
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.
Assuntos
Carcinoma Hepatocelular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Genes do Tumor de Wilms , Neoplasias Hepáticas/genética , Idoso , Apoptose/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/cirurgia , Diferenciação Celular , Divisão Celular/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição GênicaRESUMO
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.
Assuntos
Receptores ErbB/metabolismo , Glicoproteínas/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Cirrose Hepática/metabolismo , Fígado/metabolismo , Anfirregulina , Animais , Apoptose/fisiologia , Tetracloreto de Carbono , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Família de Proteínas EGF , Matriz Extracelular/metabolismo , Glicoproteínas/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligantes , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Proliferação de Células , Cromatina/metabolismo , Fatores de Transcrição E2F/metabolismo , Fígado/metabolismo , Metionina Adenosiltransferase/genética , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Cromatina/genética , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Fase G1/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/fisiologia , Regeneração Hepática/genética , Masculino , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Wistar , Fase S/genética , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Regulação para CimaRESUMO
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.
Assuntos
Antígenos de Superfície/metabolismo , Citoplasma/metabolismo , Hepatócitos/metabolismo , Complexos Multienzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas de Ligação a RNA/metabolismo , S-Adenosilmetionina/farmacologia , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Antígenos de Superfície/efeitos dos fármacos , Antígenos de Superfície/genética , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Immunoblotting , Imunoprecipitação , Técnicas In Vitro , Masculino , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleotídeos/farmacologiaRESUMO
5'-methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti-inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro-inflammatory cytokines. MTA administration completely prevented LPS-induced lethality. The life-sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor-alpha (TNF-alpha), inducible NO synthase (iNOS) expression, and by the stimulation of IL-10 synthesis. These responses to MTA were also observed in LPS-treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro-inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen-activated protein kinase (MAPK), c-jun phosphorylation, inhibitor kappa B alpha (IkappaBalpha) degradation, and nuclear factor kappaB (NFkappaB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti-inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Desoxiadenosinas/farmacocinética , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Lipopolissacarídeos/farmacologia , Choque Séptico/tratamento farmacológico , Tionucleosídeos/farmacocinética , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Hepatócitos/citologia , Humanos , Células de Kupffer/citologia , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , S-Adenosilmetionina/metabolismo , Choque Séptico/imunologiaRESUMO
BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Metionina Adenosiltransferase/genética , Divisão Celular/genética , DNA/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Fígado/citologia , Fígado/enzimologia , Cirrose Hepática/enzimologia , Cirrose Hepática/fisiopatologia , Fenótipo , Transfecção , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/enzimologiaRESUMO
In mammals, methionine adenosyltransferase (MAT), the enzyme responsible for S-adenosylmethionine (AdoMet) synthesis, is encoded by two genes, MAT1A and MAT2A. In liver, MAT1A expression is associated with high AdoMet levels and a differentiated phenotype, whereas MAT2A expression is associated with lower AdoMet levels and a dedifferentiated phenotype. In the current study, we examined regulation of MAT2A gene expression by l-methionine availability using HepG2 cells. In l-methionine-deficient cells, MAT2A gene expression is rapidly induced, and methionine adenosyltransferase activity is increased. Restoration of l-methionine rapidly down-regulates MAT2A mRNA levels; for this effect, l-methionine needs to be converted into AdoMet. This novel action of AdoMet is not mediated through a methyl transfer reaction. MAT2A gene expression was also regulated by 5'-methylthioadenosine, but this was dependent on 5'-methylthioadenosine conversion to methionine through the salvage pathway. The transcription rate of the MAT2A gene remained unchanged during l-methionine starvation; however, its mRNA half-life was significantly increased (from 100 min to more than 3 h). The effect of l-methionine withdrawal on MAT2A mRNA stabilization requires both gene transcription and protein synthesis. We conclude that MAT2A gene expression is modulated as an adaptive response of the cell to l-methionine availability through its conversion to AdoMet.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina Adenosiltransferase/genética , Metionina/metabolismo , S-Adenosilmetionina/fisiologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis (MAT1A(-/-)) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when MAT1A(-/-) expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at approximately 8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase beta-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in obob mice and obese patients who are at risk for nonalcoholic steatohepatitis.