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BACKGROUND AND AIMS: Antimicrobial proteins of the regenerating family member 3 alpha (REG3A) family provide a first line of protection against infections and transformed cells. Their expression is inducible by inflammation, which makes their role in cancer biology less clear since an immune-inflammatory context may preexist or coexist with cancer, as occurs in HCC. The aim of this study is to clarify the role of REG3A in liver carcinogenesis and to determine whether its carbohydrate-binding functions are involved. APPROACH AND RESULTS: This study provides evidence for a suppressive role of REG3A in HCC by reducing O -GlcNAcylation in 2 mouse models of HCC, in vitro cell studies, and clinical samples. REG3A expression in hepatocytes significantly reduced global O -GlcNAcylation and O -GlcNAcylation of c-MYC in preneoplastic and tumor livers and markedly inhibited HCC development in REG3A-c-MYC double transgenic mice and mice exposed to diethylnitrosamine. REG3A modified O -GlcNAcylation without altering the expression or activity of O-linked N-acetylglucosaminyltransferase, O-linked N-acetylglucosaminyl hydrolase, or glutamine fructose-6-phosphate amidotransferase. Reduced O -GlcNAcylation was consistent with decreased levels of UDP-GlcNAc in precancerous and cancerous livers. This effect was linked to the ability of REG3A to bind glucose and glucose-6 phosphate, suggested by a REG3A mutant unable to bind glucose and glucose-6 phosphate and alter O -GlcNAcylation. Importantly, patients with cirrhosis with high hepatic REG3A expression had lower levels of O -GlcNAcylation and longer cancer-free survival than REG3A-negative cirrhotic livers. CONCLUSIONS: REG3A helps fight liver cancer by reducing O -GlcNAcylation. This study suggests a new paradigm for the regulation of O -GlcNAc signaling in cancer-related pathways through interactions with the carbohydrate-binding function of REG3A.
RESUMO
BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. RESULTS: Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. CONCLUSIONS: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).
Assuntos
Apoptose , Carcinogênese/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Hepáticas/prevenção & controle , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Animais , Apoptose/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). However, very little is known about the replication of HBV in HCC tissues. We analyzed viral and cellular parameters in HCC (T) and nontumor liver (NT) samples from 99 hepatitis B surface antigen (HBsAg)-positive, virologically suppressed patients treated by tumor resection or liver transplantation. We examined total HBV DNA and RNA as well as covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA), which are considered as markers of active HBV replication. Total HBV DNA and RNA were detected in both T and NT samples in a majority of cases, but only a subset of tumors harbored detectable levels of HBV cccDNA and pgRNA (39% and 67%) compared to NT livers (66% and 90%; P < 0.01). Further evidence for HBV replication in tumor tissues was provided by sequencing of the X gene derived from episomal forms, showing that HBV genotypes differed between T and matched NT samples in 11 cases. The detection of pgRNA and cccDNA in tumors was correlated to the absence of tumorous microvascular invasion and to better patient survival. Analysis of gene expression profiles by Agilent microarrays revealed that pgRNA-positive HCCs were characterized by low levels of cell cycle and DNA repair markers and expression of the HBV receptor, sodium taurocholate cotransporting polypeptide, indicating well-differentiated tumors. CONCLUSION: HCC replicating HBV represents a subtype of weakly invasive HCC with a transcriptomic signature. pgRNA originating from nonintegrated, complete HBV genomes is a sensitive marker for viral replication and prognosis. (Hepatology 2018;67:86-96).
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/complicações , Hepatite B Crônica/patologia , Neoplasias Hepáticas/virologia , Carga Viral/genética , Adulto , Idoso , Biópsia por Agulha , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatite B Crônica/tratamento farmacológico , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Viral/análise , Sistema de Registros , Medição de Risco , Replicação Viral/genéticaRESUMO
Surgery and cisplatin-based treatment of hepatoblastoma (HB) currently guarantee the survival of 70%-80% of patients. However, some important challenges remain in diagnosing high-risk tumors and identifying relevant targetable pathways offering new therapeutic avenues. Previously, two molecular subclasses of HB tumors have been described, C1 and C2, with C2 being the subgroup with the poorest prognosis, a more advanced tumor stage, and the worst overall survival rate. An associated 16-gene signature to discriminate the two tumoral subgroups was proposed, but it has not been transferred into clinical routine. To address these issues, we performed RNA sequencing of 25 tumors and matched normal liver samples from patients. The transcript profiling separated HB into three distinct subgroups named C1, C2A, and C2B, identifiable by a concise four-gene signature: hydroxysteroid 17-beta dehydrogenase 6, integrin alpha 6, topoisomerase 2-alpha, and vimentin, with topoisomerase 2-alpha being characteristic for the proliferative C2A tumors. Differential expression of these genes was confirmed by quantitative RT-PCR on an expanded cohort and by immunohistochemistry. We also revealed significant overexpression of genes involved in the Fanconi anemia (FA) pathway in the C2A subgroup. We then investigated the ability of several described FA inhibitors to block growth of HB cells in vitro and in vivo. We demonstrated that bortezomib, a Food and Drug Administration-approved proteasome inhibitor, strongly impairs the proliferation and survival of HB cell lines in vitro, blocks FA pathway-associated double-strand DNA repair, and significantly impedes HB growth in vivo. CONCLUSION: The highly proliferating C2A subtype is characterized by topoisomerase 2-alpha gene up-regulation and FA pathway activation, and the HB therapeutic arsenal could include bortezomib for the treatment of patients with the most aggressive tumors. (Hepatology 2018;68:89-102).
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Hepatoblastoma/classificação , Hepatoblastoma/genética , Neoplasias Hepáticas/classificação , Neoplasias Hepáticas/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores/metabolismo , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Reparo do DNA/efeitos dos fármacos , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Perfilação da Expressão Gênica , Células Hep G2 , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/enzimologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Análise de Sequência de RNARESUMO
An increasing number of noncoding RNAs (ncRNAs) have been implicated in various human diseases including cancer; however, the ncRNA transcriptome of hepatocellular carcinoma (HCC) is largely unexplored. We used CAGE to map transcription start sites across various types of human and mouse HCCs with emphasis on ncRNAs distant from protein-coding genes. Here, we report that retroviral LTR promoters, expressed in healthy tissues such as testis and placenta but not liver, are widely activated in liver tumors. Despite HCC heterogeneity, a subset of LTR-derived ncRNAs were more than 10-fold up-regulated in the vast majority of samples. HCCs with a high LTR activity mostly had a viral etiology, were less differentiated, and showed higher risk of recurrence. ChIP-seq data show that MYC and MAX are associated with ncRNA deregulation. Globally, CAGE enabled us to build a mammalian promoter map for HCC, which uncovers a new layer of complexity in HCC genomics.
Assuntos
Carcinoma Hepatocelular/etiologia , Perfilação da Expressão Gênica , Neoplasias Hepáticas/etiologia , Regiões Promotoras Genéticas , RNA não Traduzido/genética , Sequências Repetidas Terminais , Sítio de Iniciação de Transcrição , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Viral , Biologia Computacional/métodos , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcriptoma , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5' ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers. IMPORTANCE: Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5' ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Neoplasias Hepáticas/virologia , Regiões Promotoras Genéticas , Adulto , Idoso , Animais , Mapeamento Cromossômico , Feminino , Genoma Viral , Células Hep G2 , Vírus da Hepatite B/patogenicidade , Humanos , Fígado/virologia , Masculino , Camundongos , Pessoa de Meia-Idade , Capuzes de RNA/genética , RNA Viral/genética , Sítio de Iniciação de Transcrição , TranscriptomaRESUMO
BACKGROUND & AIMS: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription. METHODS: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression. RESULTS: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA. CONCLUSION: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Metiltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Northern Blotting , Southern Blotting , Células Cultivadas , DNA Circular/metabolismo , Ensaio de Imunoadsorção Enzimática , Hepatite B/metabolismo , Hepatite B/patologia , Vírus da Hepatite B/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Metiltransferases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição GênicaRESUMO
Arkadia is a RING-based ubiquitin ligase that positively regulates TGF-ß signaling by targeting several pathway components for ubiquitination and degradation. However, little is known about the mechanisms controlling Arkadia activity. Here we show that the LIM-only protein FHL2 binds and synergistically cooperates with Arkadia to activate Smad3/Smad4-dependent transcription. Knockdown of FHL2 by RNA interference decreases Arkadia level and restricts the amplitude of Arkadia-induced TGF-ß target gene responses. We found that Arkadia is ubiquitinated via K63- and K27-linked polyubiquitination. A single mutation at the RING domain that abolishes the E3 activity diminishes Arkadia ubiquitination, indicating that this modification partly involves autocatalytic process. Mutation of seven lysines at the C-terminal region of Arkadia severely impairs ubiquitination through the K27 but not the K63 linkage and slows down the turnover of Arkadia, suggesting that K27-linked polyubiquitination might promote proteolysis-dependent regulation of Arkadia. We show that FHL2 increases the half-life of Arkadia through inhibition of ubiquitin chain assembly on the protein, which provides a molecular basis for functional cooperation between Arkadia and FHL2 in enhancing TGF-ß signaling. Our study uncovers a novel regulatory mechanism of Arkadia by ubiquitination and identifies FHL2 as important regulator of Arkadia ubiquitination and TGF-ß signal transduction.
Assuntos
Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Meia-Vida , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Luciferases , Camundongos , Proteínas Musculares/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.
Assuntos
Vírus da Hepatite B/patogenicidade , Interações Hospedeiro-Patógeno , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Replicação Viral , Linhagem Celular , Imunoprecipitação da Cromatina , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Humanos , Evasão da Resposta Imune , Ligação Proteica , Proteínas Virais Reguladoras e AcessóriasRESUMO
The hepatitis B virus (HBV) is a small enveloped DNA virus that causes acute and chronic hepatitis. HBV infection is a world health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). HBV has been classified among human tumor viruses by virtue of a robust epidemiologic association between chronic HBV carriage and HCC occurrence. In the absence of cytopathic effect in infected hepatocytes, the oncogenic role of HBV might involve a combination of direct and indirect effects of the virus during the multistep process of liver carcinogenesis. Liver inflammation and hepatocyte proliferation driven by host immune responses are recognized driving forces of liver cell transformation. Genetic and epigenetic alterations can also result from viral DNA integration into host chromosomes and from prolonged expression of viral gene products. Notably, the transcriptional regulatory protein HBx encoded by the X gene is endowed with tumor promoter activity. HBx has pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis. Because hepatic tumors carry a dismal prognosis, there is urgent need to develop early diagnostic markers of HCC and effective therapies against chronic hepatitis B. Deciphering the oncogenic mechanisms that underlie HBV-related tumorigenesis might help developing adapted therapeutic strategies.
Assuntos
Carcinoma Hepatocelular/etiologia , Transformação Celular Neoplásica/patologia , Vírus da Hepatite B/patogenicidade , Hepatite B/complicações , Neoplasias Hepáticas/etiologia , Animais , HumanosRESUMO
We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.
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Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Leucemia , MicroRNAs/genética , Neoplasias , Adenocarcinoma/metabolismo , Animais , Linhagem Celular Tumoral , Dosagem de Genes , Humanos , Leucemia/genética , Leucemia/metabolismo , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
UNLABELLED: Chronic hepatitis B virus (HBV) infection is a major risk factor for developing liver cancer, and the HBV X protein (pX) has been implicated as a cofactor in hepatocyte transformation. We have shown that HBV replication as well as in vitro transformation by pX are associated with induction of the mitotic polo-like kinase 1 (Plk1) and down-regulation of the chromatin remodeling components Suz12 and Znf198. Herein, we demonstrate the same inverse relationship between Plk1 and Suz12/Znf198 in liver tumors from X/c-myc bitransgenic mice and woodchuck hepatitis virus (WHV)-infected woodchucks. Employing these animal models and the HBV replicating HepAD38 cells we examined the effect of Suz12/Znf198 down-regulation on gene expression. Genes analyzed include hepatic cancer stem cell markers BAMBI, DKK1,2, DLK1, EpCAM, MYC, and proliferation genes CCNA1, CCND2, IGFII, MCM4-6, PLK1, RPA2, and TYMS. Suz12 occupancy at the promoters of BAMBI, CCND2, DKK2, DLK1, EpCAM, and IGFII was demonstrated by chromatin immunoprecipitation in untransformed hepatocytes, but was markedly reduced in pX-transformed and Suz12 knockdown cells. Accordingly, we refer to these genes as "Suz12 repressed" genes in untransformed hepatocytes. The Suz12 repressed genes and proliferation genes were induced in HBV-replicating HepAD38 cells and, interestingly, they exhibited distinct expression profiles during hepatocellular carcinoma (HCC) progression in X/c-myc bitransgenics. Specifically, CCND2, EpCAM, and IGFII expression was elevated at the proliferative and preneoplastic stages in X/c-myc bitransgenic livers, whereas BAMBI and PLK1 were overexpressed in hepatic tumors from X/c-myc bitransgenics and WHV-infected woodchucks. Importantly, most of these genes were selectively up-regulated in HBV-induced HCCs. CONCLUSION: The distinct expression profile of the identified Suz12 repressed genes in combination with the proliferation genes hold promise as biomarkers for progression of chronic HBV infection to HCC.
Assuntos
Carcinoma Hepatocelular/virologia , Proteínas de Ciclo Celular/genética , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , Complexo Repressor Polycomb 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Animais , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Regulação Viral da Expressão Gênica , Hepatite B Crônica/genética , Hepatite B Crônica/fisiopatologia , Hepatócitos/patologia , Neoplasias Hepáticas/genética , Marmota , Camundongos , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Distribuição Aleatória , Sensibilidade e Especificidade , Transativadores/metabolismo , Ativação Transcricional , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genética , Quinase 1 Polo-LikeRESUMO
Myc activation has been implicated in the pathogenesis of hepatoblastoma (HB), a rare embryonal neoplasm derived from liver progenitor cells. Here, microRNA (miR) expression profiling of 65 HBs evidenced differential patterns related to developmental stage and Myc activity. Undifferentiated aggressive HBs overexpressed the miR-371-3 cluster with concomitant down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster, evoking an ES cell expression profile. ChIP and Myc inhibition assays in hepatoma cells demonstrated that both miR clusters are regulated by Myc in an opposite manner. We show that the two miR clusters exert antagonistic effects on cell proliferation and tumorigenicity. Moreover, their combined deregulation cooperated in modulating the hepatic tumor phenotype, implicating stem cell-like regulation of Myc-dependent miRs in poorly differentiated HBs. Importantly, a four-miR signature representative of these clusters efficiently stratified HB patients, and when applied to 241 hepatocellular carcinomas (HCCs), it identified invasive tumors with a poor prognosis. Our data argue that Myc-driven reprogramming of miR expression patterns contributes to the aggressive phenotype of liver tumors originating from hepatic progenitor cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , França , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND & AIMS: The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2). METHODS: We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice. RESULTS: Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/ß-catenin signaling in Apc(lox/lox) animals. CONCLUSIONS: Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Homeostase/fisiologia , Proteínas com Homeodomínio LIM/metabolismo , Fígado/metabolismo , Fígado/patologia , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/fisiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Proliferação de Células , Ciclina D1/metabolismo , Modelos Animais de Doenças , Feminino , Hepatectomia , Humanos , Proteínas com Homeodomínio LIM/genética , Fígado/cirurgia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Regeneração Hepática/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The hepatitis B virus (HBV) is a small enveloped DNA virus, which primarily infects hepatocytes and causes acute and persistent liver disease. Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma, but the molecular mechanisms underlying virally-induced tumourigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular cancer-related genes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long-term effects of viral proteins in enhancing immune-mediated liver disease. Recent genetic studies indicate that HBV-related tumours display a distinctive profile with a high rate of chromosomal alterations and low frequency of beta-catenin mutations. This review will discuss the evidence implicating chronic HBV infection as a causal risk factor of primary liver cancer. It will also discuss the molecular mechanisms that are critical for the tumourigenic process due to long lasting infection with HBV.
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/fisiopatologia , Carcinoma Hepatocelular/virologia , Genes Supressores de Tumor/fisiologia , Hepatite B Crônica/complicações , Hepatite B Crônica/genética , Hepatite B Crônica/fisiopatologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia , Neoplasias Hepáticas/virologia , Oncogenes/fisiologiaRESUMO
UNLABELLED: Chronic hepatitis B virus (HBV) infection is linked to development of hepatocellular carcinoma (HCC). The HBV X protein (pX) is implicated in HCC pathogenesis acting as a weak oncogene or a cofactor in hepatocarcinogenesis. pX induces DNA re-replication, DNA damage, and partial polyploidy in a poorly differentiated, immortalized hepatocyte cell line. In this study we employed sorted, pX-induced polyploid cells to investigate their growth and oncogenic transformation potential over the course of 70 cell doublings. Immediately after live cell-sorting, nearly 40% of pX-induced polyploid cells undergo apoptosis, whereas the surviving cells exhibit proliferation sensitive to p53. After 40 cell generations the pX-expressing polyploid cultures exhibit loss of p53 function and become growth factor- and anchorage-independent, indicative of oncogenic transformation. The pX-induced polyploid cultures in the course of 70 cell generations undergo progressively increasing DNA damage, propagate damaged DNA to daughter cells, and display increased expression of a cluster of proliferation genes shown to be elevated in human HCC, including HBV-HCC. One of these genes is the mitotic kinase Polo-like kinase 1 (Plk1). Oncogenic transformation is suppressed in the absence of pX expression, and significantly, by inhibition of Plk1. These results identify Plk1 as crucial in pX-mediated oncogenic transformation. CONCLUSION: Partial polyploidy induced by pX is not immediately associated with oncogenic transformation. Continued DNA damage for 40 cell generations is reproducibly associated with loss of p53 function, enhanced expression of Plk1, and oncogenic transformation. Because Plk1 expression is also elevated in HBV-HCC tumors, this in vitro cellular model simulates liver cancer progression and pathogenesis in chronic HBV patients. Inhibition of Plk1 activity suppresses pX-mediated oncogenic transformation, identifying Plk1 as a promising therapeutic target for HBV-mediated HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica , Neoplasias Hepáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Apoptose , Carcinoma Hepatocelular/virologia , Linhagem Celular , Proliferação de Células , Dano ao DNA , Replicação do DNA , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Hepatite B Crônica/complicações , Neoplasias Hepáticas/virologia , Camundongos , Poliploidia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais Reguladoras e Acessórias , Quinase 1 Polo-LikeRESUMO
AIM: Stratification of hepatoblastoma (HB) patients is based on clinical and imaging characteristics obtained at the time of diagnosis. We aim to integrate biomarkers into a tool that accurately predicts survival of HB patients. METHODS: We retrospectively analysed 174 HB patients for the presence of four biomarkers and explored their prognostic potential by correlating with overall survival (OS) and event-free survival (EFS). RESULTS: Mutations of CTNNB1, NFE2L2 and TERT were found in 135 (78%), 10 (6%) and 10 (6%) patients, respectively, and the adverse C2 subtype of the 16-gene signature in 63 (36%) patients. C2-patients had more frequent metastatic disease, higher alpha-fetoprotein levels, non-fetal histology and significantly worse 3-year OS (68% versus 95%) and EFS (63% versus 87%) than C1-patients. Patients carrying a NFE2L2 mutation had a significantly worse 3-year OS (57% versus 88%) than NFE2L2 wild-type patients and were more likely to have vessel invasive growth and non-fetal histology. TERT mutations were almost exclusively found in older patients, whereas CTNNB1 mutations showed no association with any clinical feature or outcome. In a multivariable analysis, the C2 subtype remained a significant predictor of poor outcome with hazard ratios of 6.202 and 3.611 for OS and EFS, respectively. When added to the Children's Hepatic tumors International Collaboration risk stratification, the presence of the C2 subtype identified a group of high-risk patients with a very poor outcome. CONCLUSION: We propose a new stratification system based on the combination of clinical factors and the 16-gene signature, which may facilitate a risk-adapted management of HB patients.