RESUMEN
Hepatocellular carcinoma is a frequent and fatal disease. Recent researches on rodent models and human hepatocarcinogenesis contributed to unravel the molecular mechanisms of hepatocellular carcinoma dedifferentiation and progression, and allowed the discovery of several alterations underlying the deregulation of cell cycle and signalling pathways. This review provides an interpretive analysis of the results of these studies. Mounting evidence emphasises the role of up-regulation of RAS/ERK, P13K/AKT, IKK/NF-kB, WNT, TGF-ß, NOTCH, Hedgehog, and Hippo signalling pathways as well as of aberrant proteasomal activity in hepatocarcinogenesis. Signalling deregulation often occurs in preneoplastic stages of rodent and human hepatocarcinogenesis and progressively increases in carcinomas, being most pronounced in more aggressive tumours. Numerous changes in signalling cascades are involved in the deregulation of carbohydrate, lipid, and methionine metabolism, which play a role in the maintenance of the transformed phenotype. Recent studies on the role of microRNAs in signalling deregulation, and on the interplay between signalling pathways led to crucial achievements in the knowledge of the network of signalling cascades, essential for the development of adjuvant therapies of liver cancer. Furthermore, the analysis of the mechanisms involved in signalling deregulation allowed the identification of numerous putative prognostic markers and novel therapeutic targets of specific hepatocellular carcinoma subtypes associated with different biologic and clinical features. This is of prime importance for the selection of patient subgroups that are most likely to obtain clinical benefit and, hence, for successful development of targeted therapies for liver cancer.
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Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Transducción de SeñalRESUMEN
UNLABELLED: Down-regulation of the liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and up-regulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Here we found Mat1A:Mat2A switch and low SAM levels, associated with CpG hypermethylation and histone H4 deacetylation of Mat1A promoter, and prevalent CpG hypomethylation and histone H4 acetylation in Mat2A promoter of fast-growing HCC of F344 rats, genetically susceptible to hepatocarcinogenesis. In HCC of genetically resistant BN rats, very low changes in the Mat1A:Mat2A ratio, CpG methylation, and histone H4 acetylation occurred. The highest MAT1A promoter hypermethylation and MAT2A promoter hypomethylation occurred in human HCC with poorer prognosis. Furthermore, levels of AUF1 protein, which destabilizes MAT1A messenger RNA (mRNA), Mat1A-AUF1 ribonucleoprotein, HuR protein, which stabilizes MAT2A mRNA, and Mat2A-HuR ribonucleoprotein sharply increased in F344 and human HCC, and underwent low/no increase in BN HCC. In human HCC, Mat1A:MAT2A expression and MATI/III:MATII activity ratios correlated negatively with cell proliferation and genomic instability, and positively with apoptosis and DNA methylation. Noticeably, the MATI/III:MATII ratio strongly predicted patient survival length. Forced MAT1A overexpression in HepG2 and HuH7 cells led to a rise in the SAM level, decreased cell proliferation, increased apoptosis, down-regulation of Cyclin D1, E2F1, IKK, NF-κB, and antiapoptotic BCL2 and XIAP genes, and up-regulation of BAX and BAK proapoptotic genes. In conclusion, we found for the first time a post-transcriptional regulation of MAT1A and MAT2A by AUF1 and HuR in HCC. Low MATI/III:MATII ratio is a prognostic marker that contributes to determine a phenotype susceptible to HCC and patients' survival. CONCLUSION: Interference with cell cycle progression and I-kappa B kinase (IKK)/nuclear factor kappa B (NF-κB) signaling contributes to the antiproliferative and proapoptotic effect of high SAM levels in HCC.
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Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , Metionina Adenosiltransferasa/genética , Activación Transcripcional , Animales , Sitios de Unión , Carcinoma Hepatocelular/patología , Metilación de ADN , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/patología , Metionina Adenosiltransferasa/metabolismo , Análisis Multivariante , Pronóstico , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , S-Adenosilmetionina/metabolismo , Estadísticas no Paramétricas , Células Tumorales CultivadasRESUMEN
UNLABELLED: Up-regulation of the v-Myb avian myeloblastosis viral oncogene homolog-like2 B-Myb (MYBL2) gene occurs in human hepatocellular carcinoma (HCC) and is associated with faster progression of rodent hepatocarcinogenesis. We evaluated, in distinct human HCC prognostic subtypes (as defined by patient survival length), activation of MYBL2 and MYBL2-related genes, and relationships of p53 status with MYBL2 activity. Highest total and phosphorylated protein levels of MYBL2, E2F1-DP1, inactivated retinoblastoma protein (pRB), and cyclin B1 occurred in HCC with poorer outcome (HCCP), compared to HCC with better outcome (HCCB). In HCCP, highest LIN9-MYBL2 complex (LINC) and lowest inactive LIN9-p130 complex levels occurred. MYBL2 positively correlated with HCC genomic instability, proliferation, and microvessel density, and negatively with apoptosis. Higher MYBL2/LINC activation in HCC with mutated p53 was in contrast with LINC inactivation in HCC harboring wildtype p53. Small interfering RNA (siRNA)-mediated MYBL2/LINC silencing reduced proliferation, induced apoptosis, and DNA damage at similar levels in HCC cell lines, irrespective of p53 status. However, association of MYBL2/LINC silencing with doxorubicin-induced DNA damage caused stronger growth restraint in p53(-/-) Huh7 and Hep3B cells than in p53(+/+) Huh6 and HepG2 cells. Doxorubicin triggered LIN9 dissociation from MYBL2 in p53(+/+) cell lines and increased MYBL2-LIN9 complexes in p53(-/-) cells. Doxorubicin-induced MYBL2 dissociation from LIN9 led to p21(WAF1) up-regulation in p53(+/+) but not in p53(-/-) cell lines. Suppression of p53 or p21(WAF1) genes abolished DNA damage response, enhanced apoptosis, and inhibited growth in doxorubicin-treated cells harboring p53(+/+) . CONCLUSION: We show that MYBL2 activation is crucial for human HCC progression. In particular, our data indicate that MYBL2-LIN9 complex integrity contributes to survival of DNA damaged p53(-/-) cells. Thus, MYBL2 inhibition could represent a valuable adjuvant for treatments against human HCC with mutated p53.
Asunto(s)
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/fisiología , Neoplasias Hepáticas/genética , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/fisiología , Línea Celular Tumoral , Daño del ADN , Progresión de la Enfermedad , Doxorrubicina/farmacología , Inestabilidad Genómica , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: MYBL2 is implicated in human malignancies and over expressed in hepatocellular carcinoma (HCC). We investigated Mybl2 role in the acquisition of susceptibility to HCC and tumor progression. METHODS: MYBL2 mRNA and protein levels were evaluated by quantitative RT-PCR and immunoblotting, respectively. MYBL2 expression in HCC cell lines was controlled through MYBL2 cDNA or anti-MYBL2 siRNA transfection. Gene expression profile of cells transfected with MYBL2 was analyzed by microarray. RESULTS: Low induction of Mybl2 and its target Clusterin mRNAs, in low-grade dysplastic nodules (DN), progressively increased in fast growing high-grade DN and HCC of F344 rats, susceptible to hepatocarcinogenesis, whereas no/lower increases occurred in slow growing lesions of resistant BN rats. Highest Mybl2 protein activation, prevalently nuclear, occurred in F344 than BN lesions. Highest Mybl2, Clusterin, Cdc2, and Cyclin B1 expression occurred in fast progressing DN and HCC of E2f1 transgenics, compared to c-Myc transgenics, and anti-Mybl2 siRNA had highest anti-proliferative and apoptogenic effects in cell lines from HCC of E2f1 transgenics. MYBL2 transfected HepG2 and Huh7 cells exhibited increased cell proliferation and G1-S and G2-M cell cycle phases. The opposite occurred when MYBL2 was silenced by specific siRNA. MYBL2 transfection in Huh7 cells led to upregulation of genes involved in signal transduction, cell proliferation, cell motility, and downregulation of oncosuppressor and apoptogenic genes. CONCLUSIONS: mybl2 expression and activation are under genetic control. Mybl2 upregulation induces fast growth and progression of premalignant and malignant liver, through cell cycle deregulation and activation of genes and pathways related to tumor progression.
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Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , Transactivadores/genética , Factores de Transcripción/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes myb , Predisposición Genética a la Enfermedad , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21(WAF1), p27(KIP1), p57(KIP2) and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21(WAF1), p27(KIP1), p57(KIP), p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21(WAF1), P27(KIP1), P57(KIP2) and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas/patología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Animales , Humanos , Inmunohistoquímica , Inmunoprecipitación , Neoplasias Hepáticas/metabolismo , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Procesamiento Proteico-Postraduccional , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , UbiquitinaciónRESUMEN
BACKGROUND & AIMS: The cell cycle regulators P21(WAF1), P27(KIP1), P57(KIP2), P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. METHODS: Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, <3 years survival) and HCC cell lines were analyzed. RESULTS: The promoters of P21(WAF1), P27(KIP1), and P57(KIP2) were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. CONCLUSIONS: Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.
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Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Portadoras/fisiología , Quinasas Ciclina-Dependientes/fisiología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Quinasas Asociadas a Fase-S/fisiología , Quinasas CDC2-CDC28 , Técnicas de Cultivo de Célula , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , UbiquitinaciónRESUMEN
Comparative analysis of hepatocellular carcinoma (HCC) in rat strains that are either susceptible or resistant to the induction of HCC has allowed the mapping of genes responsible for inherited predisposition to HCC. These studies show that the activity of several low penetrance genes and a predominant susceptibility gene regulate the development of hepatocarcinogenesis in rodents. These studies shed light on the epidemiology of human HCC. The identified genes regulate resistance to hepatocarcinogenesis by affecting the capacity of the initiated cells to grow autonomously and to progress to HCC. Analysis of the molecular alterations showed highest iNos cross-talk with IKK/NF-kB and RAS/ERK pathways in most aggressive liver lesions represented by HCC in the susceptible F344 rats. Unrestrained extracellular signal-regulated kinase (Erk) activity linked to proteasomal degradation of dual-specificity phosphatase 1 (Dusp1), a specific ERK inhibitor, by the CKS1-SKP2 ubiquitin ligase complex was highest in more aggressive HCC of genetically susceptible rats. Furthermore, deregulation of G1 and S phases of the cell cycle occurs in HCC of susceptible F344 rats, leading to pRb hyperphosphorylation and elevated DNA synthesis, whereas a block to G1-S transition is present in the HCC of resistant BN rats. Importantly, similar alterations in the signaling pathways that regulate cell cycle progression were found in human HCC with poorer prognosis (as defend by patients' survival length), whereas human HCC with better prognosis had molecular characteristics similar to the lesions in the HCC of resistant rat strains. This review discusses the role of molecular alterations involved in the acquisition of resistance or susceptibility to HCC and the importance of genetically susceptible and resistant rat models for the identification of prognostic markers, and chemopreventive or therapeutic targets for the biological network therapy of human disease.
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Carcinoma Hepatocelular/genética , Epigénesis Genética/genética , Neoplasias Hepáticas/genética , Animales , Ciclo Celular/fisiología , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad/genética , Humanos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Transducción de Señal/fisiologíaRESUMEN
Hepatocellular carcinoma (HCC) is characterized by the down-regulation of the liver-specific methyladenosyltransferase 1A (MAT1A) gene, encoding the S-adenosylmethionine synthesizing isozymes MATI/III, and the up-regulation of the widely expressed methyladenosyltransferase 2A (MAT2A), encoding MATII isozyme, and methyladenosyltransferase 2B (MAT2B), encoding a ß-subunit without catalytic action that regulates MATII enzymatic activity. Different observations showed hepatocarcinogenesis inhibition by miR-203. We found that miR-203 expression in HCCs is inversely correlated with HCC proliferation and aggressiveness markers, and with MAT2A and MAT2B levels. MiR-203 transfection in HepG2 and Huh7 liver cancer cells targeted the 3'-UTR of MAT2A and MAT2B, inhibiting MAT2A and MAT2B mRNA levels and MATα2 and MATß2 protein expression. These molecular events were paralleled by an increase in SAM content and were associated with growth restraint and apoptosis, inhibition of cell migration and invasiveness, and suppression of the expression of CD133 and LIN28B stemness markers. In contrast, MAT2B transfection in the same cell lines led to a rise of both MATß2 and MATα2 expression, associated with increases in cell growth, migration, invasion and overexpression of stemness markers and p-AKT. Altogether, our results indicate that the miR-203 oncosuppressor activity may at least partially depend on its inhibition of MAT2A and MAT2B and show, for the first time, an oncogenic activity of MAT2B linked to AKT activation.
RESUMEN
Mounting evidence underlines the role of inducible nitric oxide synthase (iNOS) in hepatocellular carcinoma (HCC) development, but its functional interactions with pathways involved in HCC progression remain uninvestigated. Here, we analyzed in preneoplastic and neoplastic livers from Fisher 344 and Brown Norway rats, possessing different genetic predisposition to HCC, in transforming growth factor-alpha (TGF-alpha) and c-Myc-TGF-alpha transgenic mice, characterized by different susceptibility to HCC, and in human HCC: (i) iNOS function and interactions with nuclear factor-kB (NF-kB) and Ha-RAS/extracellular signal-regulated kinase (ERK) during hepatocarcinogenesis; (ii) influence of genetic predisposition to liver cancer on these pathways and role of these cascades in determining a susceptible or resistant phenotype and (iii) iNOS prognostic value in human HCC. We found progressive iNos induction in rat and mouse liver lesions, always at higher levels in the most aggressive models represented by HCC of rats genetically susceptible to hepatocarcinogenesis and c-Myc-TGF-alpha transgenic mice. iNOS, inhibitor of kB kinase/NF-kB and RAS/ERK upregulation was significantly higher in HCC with poorer prognosis (as defined by patients' survival length) and positively correlated with tumor proliferation, genomic instability and microvascularization and negatively with apoptosis. Suppression of iNOS signaling by aminoguanidine led to decreased HCC growth and NF-kB and RAS/ERK expression and increased apoptosis both in vivo and in vitro. Conversely, block of NF-kB signaling by sulfasalazine or short interfering RNA (siRNA) or ERK signaling by UO126 caused iNOS downregulation in HCC cell lines. These findings indicate that iNOS cross talk with NF-kB and Ha-RAS/ERK cascades influences HCC growth and prognosis, suggesting that key component of iNOS signaling could represent important therapeutic targets for human HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Óxido Nítrico Sintasa de Tipo II/genética , Transducción de Señal/fisiología , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/epidemiología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Predisposición Genética a la Enfermedad , Humanos , Incidencia , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Transgénicos , Pronóstico , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344RESUMEN
Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.
Asunto(s)
Apoptosis , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Transducción de Señal/fisiología , Proteínas ras/fisiología , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Animales , Proliferación Celular , Fosfatasa 1 de Especificidad Dual/análisis , MAP Quinasa Quinasa Quinasa 5/fisiología , Lesiones Precancerosas/patología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344RESUMEN
Sporadic colorectal cancer (CRC) is a major health concern worldwide. Epidemiologic evidence suggests a polygenic predisposition to CRC, but the genes responsible remain unknown. Here, we performed genome-wide scanning of male (ACI/SegHsd x Wistar-Furth)F2 (AWF2) rats to map susceptibility genes influencing the evolution of early colorectal lesions to adenocarcinoma following 1,2-dimethylhydrazine administration. Phenotypic analysis revealed higher incidence/multiplicity and lower size of adenomas in ACI/SegHsd (ACI) and (ACI/SegHsd x Wistar-Furth)F1 (AWF1) than Wistar-Furth (WF) rats and higher incidence/multiplicity of poorly differentiated adenocarcinomas in WF than ACI rats, with intermediate values in AWF1 rats. Linkage analysis of 138 AWF2 rats identified three loci on chromosomes 4, 15 and 18 in significant linkage with lesion multiplicity that were identified as rat Colon cancer resistance (rCcr) 1, rCcr2 and rCcr3, respectively. Seven other loci on chromosomes 5, 6, 15, 17, 18 and 20 were in suggestive linkage with adenoma/adenocarcinoma multiplicity/surface area. Six of them were identified as rCcr4-9 and a locus on chromosome 5 was identified as a susceptibility locus, rCcs1. Significant interactions between rCcr3 and rCcr6, rCcr6 and rCcr8 and rCcr5 and rCcr9, and four novel epistatic loci controlling multiplicity/size of colorectal lesions were discovered. Apc, located at rCcr3, did not show functional promoter polymorphisms. However, influence of susceptibility/resistance genes on Wnt/beta-catenin pathway was shown by defective beta-catenin inactivation in WF but not in ACI and AWF1 rat adenocarcinomas. These data indicate that inheritance of predisposition to CRC depends on interplays of several genetic factors, and suggest a possible mechanism of polygenic control of CRC progression.
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Mapeo Cromosómico , Neoplasias Colorrectales/genética , Predisposición Genética a la Enfermedad , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Ligamiento Genético , Masculino , Sitios de Carácter Cuantitativo , Ratas , Ratas Endogámicas WF , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c-Myc and c-Myc/Tgf-alpha transgenic mice and human HCCs. S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and liver-specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c-Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patient's survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c-Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c-Myc/Tgf-alpha lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c-Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c-Myc/Tgf-alpha HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c-Myc/Tgf-alpha tumors, may develop in the absence of alterations in DNA methylation.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Metilación de ADN , Inestabilidad Genómica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metionina/metabolismo , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Proliferación Celular , ADN de Neoplasias , Genes myc , Humanos , Immunoblotting , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Ratones , Ratones Transgénicos , Pronóstico , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Factor de Crecimiento Transformador alfa/genéticaRESUMEN
It is likely that the majority of proteins will undergo post-translational modification, be it enzymatic or non-enzymatic. These modified protein(s) regulate activity, localization and interaction with other cellular molecules thereby maintaining cellular hemostasis. Alcohol exposure significantly alters several of these post-translational modifications leading to impairments of many essential physiological processes. Here, we present new insights into novel modifications following ethanol exposure and their role in the initiation and progression of liver injury. This critical review condenses the proceedings of a symposium at the European Society for the Biomedical Research on Alcoholism Meeting held September 12-15, 2015, in Valencia, Spain.
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Hepatopatías Alcohólicas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Ácidos Grasos/metabolismo , Glicosilación , Humanos , Metilación , Óxidos/metabolismo , Carbonilación Proteica , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , SumoilaciónRESUMEN
Previous studies showed that YAP1 is over-expressed in hepatocellular carcinoma (HCC). Here we observed higher expression of Yap1/Ctgf axis in dysplastic nodules and HCC chemically-induced in F344 rats, genetically susceptible to hepatocarcinogenesis, than in lesions induced in resistant BN rats. In BN rats, highest increase in Yap1-tyr357, p73 phosphorylation and Caspase 3 cleavage occurred. In human HCCs with poorer prognosis (< 3 years survival after partial liver resection, HCCP), levels of YAP1, CTGF, 14-3-3, and TEAD proteins, and YAP1-14-3-3 and YAP1-TEAD complexes were higher than in HCCs with better outcome (> 3 years survival; HCCB). In the latter, higher levels of phosphorylated YAP1-ser127, YAP1-tyr357 and p73, YAP1 ubiquitination, and Caspase 3 cleavage occurred. Expression of stemness markers NANOG, OCT-3/4, and CD133 were highest in HCCP and correlated with YAP1 and YAP1-TEAD levels. In HepG2, Huh7, and Hep3B cells, forced YAP1 over-expression led to stem cell markers expression and increased cell viability, whereas inhibition of YAP1 expression by specific siRNA, or transfection of mutant YAP1 which does not bind to TEAD, induced opposite alterations. These changes were associated, in Huh7 cells transfected with YAP1 or YAP1 siRNA, with stimulation or inhibition of cell migration and invasivity, respectively. Furthermore, transcriptome analysis showed that YAP1 transfection in Huh7 cells induces over-expression of genes involved in tumor stemness. In conclusion, Yap1 post-translational modifications favoring its ubiquitination and apoptosis characterize HCC with better prognosis, whereas conditions favoring the formation of YAP1-TEAD complexes are associated with aggressiveness and acquisition of stemness features by HCC cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Animales , Caspasa 3/metabolismo , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Células Madre Neoplásicas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Pronóstico , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Endogámicas F344 , Factores de Transcripción de Dominio TEA , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND AND AIMS: Hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DNs) and hepatocellular carcinomas (HCCs) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis. METHODS: Expression profiles were performed by microarray and validated by quantitative RT-PCR and Western blot. RESULTS: Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of both strains and BN nodules, and the second one F344 nodules and HCC of both strains. We identified a signature predicting DN and HCC progression, characterized by highest expression of oncosuppressors Csmd1, Dmbt1, Dusp1, and Gnmt, in DNs, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13 in HCCs of resistant rats. Integrated gene expression data revealed highest expression of proliferation-related CTGF, c-MYC, and PCNA, and lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressive rat and human HCC. BHMT, DUSP1, and GADD45g expression predicted patients' survival. CONCLUSIONS: Our results disclose, for the first time, a major role of oncosuppressor genes as effectors of genetic resistance to hepatocarcinogenesis. Comparative functional genomic analysis allowed discovering an evolutionarily conserved gene expression signature discriminating HCC with different propensity to progression in rat and human.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Resistencia a la Enfermedad/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Animales , Proliferación Celular , Supervivencia Celular/genética , Análisis por Conglomerados , Humanos , Hígado/metabolismo , Hígado/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Endogámicas BN , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription-PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC.