RESUMEN
Endometriosis is a chronic disease in which lesions resembling endometrial tissue are found outside the uterus, mainly in the pelvis or abdomen. It may affect 10% of women of childbearing age. It is the cause of a significant alteration in quality of life and a major cost to the health system. Few research teams are working on this subject, and its pathophysiology is still poorly understood. This article proposes avenues of reflection for research on endometriosis in France, notably based on the mobilization of related scientific communities (involved in cancer, development, epigenetics, and neurosciences research studies).
Title: Des pistes de réflexion pour la recherche sur l'endométriose en France. Abstract: L'endométriose est une maladie chronique dans laquelle des lésions ressemblant à du tissu endométrial se retrouvent hors de l'utérus, principalement dans la cavité abdomino-pelvienne. Cette maladie pourrait toucher 10 % des femmes en âge de procréer. Elle est à l'origine d'une importante altération de la qualité de vie et d'un coût majeur pour le système de santé. Peu d'équipes de recherche sont mobilisées sur ce sujet, et la physiopathologie de la maladie reste mal comprise. Nous proposons dans cet article des pistes de réflexion pour la recherche sur l'endométriose en France, fondées notamment sur la mobilisation de communautés scientifiques connexes (notamment celles impliquées dans la recherche sur le cancer, la biologie du développement, l'épigénétique, les neurosciences).
Asunto(s)
Endometriosis , Endometriosis/genética , Endometriosis/patología , Endometrio/patología , Endometrio/fisiología , Epigénesis Genética , Femenino , Humanos , Calidad de Vida , ÚteroRESUMEN
OBJECTIVE: The AAA+ ATPase Reptin is overexpressed in hepatocellular carcinoma and preclinical studies indicate that it could be a relevant therapeutic target. However, its physiological and pathophysiological roles in vivo remain unknown. This study aimed to determine the role of Reptin in mammalian adult liver. DESIGN AND RESULTS: We generated an inducible liver-specific Reptin knockout (RepinLKO ) mouse model. Following Reptin invalidation, mice displayed decreased body and fat mass, hypoglycaemia and hypolipidaemia. This was associated with decreased hepatic mTOR protein abundance. Further experiments in primary hepatocytes demonstrated that Reptin maintains mTOR protein level through its ATPase activity. Unexpectedly, loss or inhibition of Reptin induced an opposite effect on mTORC1 and mTORC2 signalling, with: (1) strong inhibition of hepatic mTORC1 activity, likely responsible for the reduction of hepatocytes cell size, for decreased de novo lipogenesis and cholesterol transcriptional programmes and (2) enhancement of mTORC2 activity associated with inhibition of the gluconeogenesis transcriptional programme and hepatic glucose production. Consequently, the role of hepatic Reptin in the pathogenesis of insulin resistance (IR) and non-alcoholic fatty liver disease consecutive to a high-fat diet was investigated. We found that Reptin deletion completely rescued pathological phenotypes associated with IR, including glucose intolerance, hyperglycaemia, hyperlipidaemia and hepatic steatosis. CONCLUSION: We show here that the AAA +ATPase Reptin is a regulator of mTOR signalling in the liver and global glucido-lipidic homeostasis. Inhibition of hepatic Reptin expression or activity represents a new therapeutic perspective for metabolic syndrome.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/fisiología , ADN Helicasas/fisiología , Glucosa/metabolismo , Metabolismo de los Lípidos/fisiología , Adenosina Trifosfatasas/fisiología , Animales , Peso Corporal/fisiología , ADN Helicasas/deficiencia , ADN Helicasas/genética , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Metabolismo Energético/fisiología , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado Graso/prevención & control , Intolerancia a la Glucosa/fisiopatología , Intolerancia a la Glucosa/prevención & control , Hepatocitos/metabolismo , Resistencia a la Insulina/fisiología , Lipogénesis/fisiología , Hígado/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
A paradox is a seemingly absurd or impossible concept, proposition, or theory that is often difficult to understand or explain, sometimes apparently self-contradictory, and yet ultimately correct or true. How is it possible, for example, that oxygen "a toxic environmental poison" could be also indispensable for life (Beckman and Ames Physiol Rev 78(2):547-81, 1998; Stadtman and Berlett Chem Res Toxicol 10(5):485-94, 1997)?: the so-called Oxygen Paradox (Davies and Ursini 1995; Davies Biochem Soc Symp 61:1-31, 1995). How can French people apparently disregard the rule that high dietary intakes of cholesterol and saturated fats (e.g., cheese and paté) will result in an early death from cardiovascular diseases (Renaud and de Lorgeril Lancet 339(8808):1523-6, 1992; Catalgol et al. Front Pharmacol 3:141, 2012; Eisenberg et al. Nat Med 22(12):1428-1438, 2016)?: the so-called, French Paradox. Doubtless, the truth is not a duality and epistemological bias probably generates apparently self-contradictory conclusions. Perhaps nowhere in biology are there so many apparently contradictory views, and even experimental results, affecting human physiology and pathology as in the fields of free radicals and oxidative stress, antioxidants, foods and drinks, and dietary recommendations; this is particularly true when issues such as disease-susceptibility or avoidance, "healthspan," "lifespan," and ageing are involved. Consider, for example, the apparently paradoxical observation that treatment with low doses of a substance that is toxic at high concentrations may actually induce transient adaptations that protect against a subsequent exposure to the same (or similar) toxin. This particular paradox is now mechanistically explained as "Adaptive Homeostasis" (Davies Mol Asp Med 49:1-7, 2016; Pomatto et al. 2017a; Lomeli et al. Clin Sci (Lond) 131(21):2573-2599, 2017; Pomatto and Davies 2017); the non-damaging process by which an apparent toxicant can activate biological signal transduction pathways to increase expression of protective genes, by mechanisms that are completely different from those by which the same agent induces toxicity at high concentrations. In this review, we explore the influences and effects of paradoxes such as the Oxygen Paradox and the French Paradox on the etiology, progression, and outcomes of many of the major human age-related diseases, as well as the basic biological phenomenon of ageing itself.
Asunto(s)
Adaptación Fisiológica , Envejecimiento/genética , Dieta Rica en Proteínas/estadística & datos numéricos , Hipercolesterolemia/epidemiología , Estrés Oxidativo/fisiología , Oxígeno/metabolismo , Anciano , Anciano de 80 o más Años , Envejecimiento/fisiología , Femenino , Francia , Radicales Libres/metabolismo , Evaluación Geriátrica , Humanos , Masculino , Persona de Mediana Edad , Medición de RiesgoRESUMEN
Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.
Asunto(s)
Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular Tumoral , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Doxiciclina/farmacología , Humanos , Insulina/farmacología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects.MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers.Silencing Reptin decreased Mep1A expression and activity, without affecting meprin ß. Mep1A, but not meprin ß, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted.Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Carcinoma Hepatocelular/enzimología , Proteínas Portadoras/metabolismo , Movimiento Celular , ADN Helicasas/metabolismo , Neoplasias Hepáticas/enzimología , Metaloendopeptidasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , ADN Helicasas/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metaloendopeptidasas/genética , Invasividad Neoplásica , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10-12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models.
RESUMEN
Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 µM, 30 min) or γ irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or γ irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.
Asunto(s)
Proteínas Portadoras/genética , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , Reparación del ADN/genética , Interferencia de ARN , ATPasas Asociadas con Actividades Celulares Diversas , Antineoplásicos Fitogénicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Ensayo Cometa , ADN Helicasas/metabolismo , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Etopósido/farmacología , Rayos gamma , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Microscopía Confocal , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Ciclo Celular/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Transducción de Señal/genética , Transcripción Genética/fisiología , Respuesta de Proteína Desplegada/fisiología , Adenosina Trifosfatasas/genética , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Estrés del Retículo Endoplásmico/genética , Proteómica/métodos , Interferencia de ARN , Proteínas Represoras/metabolismo , Proteína que Contiene ValosinaRESUMEN
Accumulation of type I collagen fibrils in tumors is associated with an increased risk of metastasis. Invadosomes are F-actin structures able to degrade the extracellular matrix. We previously found that collagen I fibrils induced the formation of peculiar linear invadosomes in an unexpected integrin-independent manner. Here, we show that Discoidin Domain Receptor 1 (DDR1), a collagen receptor overexpressed in cancer, colocalizes with linear invadosomes in tumor cells and is required for their formation and matrix degradation ability. Unexpectedly, DDR1 kinase activity is not required for invadosome formation or activity, nor is Src tyrosine kinase. We show that the RhoGTPase Cdc42 is activated on collagen in a DDR1-dependent manner. Cdc42 and its specific guanine nucleotide-exchange factor (GEF), Tuba, localize to linear invadosomes, and both are required for linear invadosome formation. Finally, DDR1 depletion blocked cell invasion in a collagen gel. Altogether, our data uncover an important role for DDR1, acting through Tuba and Cdc42, in proteolysis-based cell invasion in a collagen-rich environment.
Asunto(s)
Colágeno Tipo I/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Línea Celular Tumoral , Colagenasas/metabolismo , Dipéptidos/farmacología , Receptor con Dominio Discoidina 1 , Matriz Extracelular/metabolismo , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Invasividad Neoplásica/genética , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Calnexina/genética , Calnexina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dipeptidasas/genética , Dipeptidasas/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Persona de Mediana Edad , Anotación de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Proteómica/métodos , Transducción de Señal , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteína que Contiene ValosinaRESUMEN
A virtual screening strategy, through molecular docking, for the elaboration of an electronic library of Pontin inhibitors has resulted in the identification of two original scaffolds. The chemical synthesis of four candidates allowed extensive biological evaluations for their anticancer activity. Two compounds displayed an effect on Pontin ATPase activity, and one of them also exhibited a noticeable effect on cell growth. Further biological studies revealed that the most active compound induced apoptotic cell death together with necrosis, this latter effect being likely related to the cellular balance of ATP regulation.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Portadoras/antagonistas & inhibidores , ADN Helicasas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , ATPasas Asociadas con Actividades Celulares Diversas , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Proliferación Celular/efectos de los fármacos , ADN Helicasas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HCT116 , Células HL-60 , Humanos , Células KB , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development.
Asunto(s)
Endorribonucleasas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/metabolismo , Proteínas Circadianas Period/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Secuencia de Bases , Endorribonucleasas/genética , Glioblastoma/genética , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Circadianas Period/genética , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pontin (also known as RUVBL1 and RVB1) and Reptin (also called RUVBL2 and RVB2) are related members of the large AAA+ (adenosine triphosphatase associated with diverse cellular activities) superfamily of conserved proteins. Various cellular functions depend on Pontin and Reptin, mostly because of their functions in the assembly of protein complexes that play a role in the regulation of cellular energetic metabolism, transcription, chromatin remodeling, and the DNA damage response. Little is known, though, about the interconnections between these multiple functions, how the relevant signaling pathways are regulated, whether the interconnections are affected in human disease, and whether components of these pathways are suitable targets for therapeutic intervention. The First International Workshop on Pontin (RUVBL1) and Reptin (RUVBL2), held between 16 and 19 October 2012, discussed the nature of the oligomeric organization of these proteins, their structures, their roles as partners in various protein complexes, and their involvement in cellular regulation, signaling, and pathophysiology, as well as their potential for therapeutic targeting. A major outcome of the meeting was a general consensus that most functions of Pontin and Reptin are related to their roles as chaperones or adaptor proteins that are important for the assembly and function of large signaling protein complexes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Transducción de Señal , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/química , Proteínas Portadoras/química , Ensamble y Desensamble de Cromatina , ADN Helicasas/química , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: The heterogeneity of hepatitis C virus (HCV) infection cannot always be explained by HCV genotypes or host genetic factors, raising the issue of possible cofactors. A new form of hepatitis leading to liver cancer was discovered in 1992 in mice, owing to an infection by Helicobacter hepaticus. Moreover, several studies showed an association between the presence of HCV and Helicobacter in the liver of patients with severe liver diseases suggesting a possible synergism between the two pathogens. In an HCV transgenic mouse model with a B6C3F1 background, the combination of H. hepaticus infection and the HCV transgene resulted in a significantly greater incidence and multiplicity of preneoplastic and neoplastic liver foci in males. OBJECTIVES: Because the mouse genetic background is a major determinant in the development of liver disease, our aim was to test the synergism between HCV and H. hepaticus infection using transgenic mice with a more sensitive genetic background to H. hepaticus infection. METHODS: For this purpose, four groups of mice were followed up to 14 months, the presence of H. hepaticus was monitored by PCR and hepatic lesions were looked for. RESULTS: We found that H. hepaticus, but not the HCV transgene, increased the number of hepatic lesions. The presence of carcinoma was more likely to occur on a background of hepatitis, and the overall lesions were more frequent in the presence of steatosis. The effect of the mouse genetic background was greater than the effect of the HCV transgene and was sufficient to promote lesions particularly via its sensitivity to H. hepaticus infection. CONCLUSIONS: Genetic susceptibility may be a more important factor than expected. Indeed, the synergism between HCV and H. hepaticus infection involved in liver disease may be highly host dependent.
Asunto(s)
Infecciones por Helicobacter/patología , Helicobacter hepaticus/patogenicidad , Hepacivirus/patogenicidad , Hepatitis C/patología , Hígado/patología , Animales , Coinfección/microbiología , Coinfección/patología , Coinfección/virología , Modelos Animales de Enfermedad , Estudios de Seguimiento , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Hepatitis C/complicaciones , Hepatitis C/virología , Masculino , Ratones , Ratones TransgénicosRESUMEN
Reptin is overexpressed in most human hepatocellular carcinomas. Reptin is involved in chromatin remodeling, transcription regulation, or supramolecular complexes assembly. Its silencing leads to growth arrest and apoptosis in cultured hepatocellular carcinoma cells and stops hepatocellular carcinoma progression in xenografts. Reptin has an ATPase activity linked to Walker A and B domains. It is unclear whether every Reptin function depends on its ATPase activity. Here, we expressed Walker B ATPase-dead mutants (D299N or E300G) in hepatocellular carcinoma cells in the presence of endogenous Reptin. Then, we silenced endogenous Reptin and substituted it with siRNA-resistant wild-type (WT) or Flag-Reptin mutants. There was a significant decrease in cell growth when expressing either mutant in the presence of endogenous Reptin, revealing a dominant negative effect of the ATPase dead mutants on hepatocellular carcinoma cell growth. Substitution of endogenous Reptin by WT Flag-Reptin rescued cell growth of HuH7. On the other hand, substitution by Flag-Reptin D299N or E300G led to cell growth arrest. Similar results were seen with Hep3B cells. Reptin silencing in HuH7 cells led to an increased apoptotic cell death, which was prevented by WT Flag-Reptin but not by the D299N mutant. These data show that Reptin functions relevant for cancer are dependent on its ATPase activity, and suggest that antagonists of Reptin ATPase activity may be useful as anticancer agents.
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Carcinoma Hepatocelular/enzimología , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Neoplasias Hepáticas/enzimología , ATPasas Asociadas con Actividades Celulares Diversas , Apoptosis/fisiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , ADN Helicasas/biosíntesis , ADN Helicasas/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Mutación , TransfecciónRESUMEN
BACKGROUND INFORMATION: Podosomes are actin-based structures involved in cell adhesion, migration, invasion and extracellular matrix degradation. They have been described in large vessel endothelial cells, but nothing is known concerning microvascular endothelial cells. Here, we focussed on liver sinusoidal endothelial cells (LSECs), fenestrated microvascular cells that play major roles in liver physiology. Liver fibrosis induces a dedifferentiation of LSECs leading notably to a loss of fenestrae. Because liver fibrosis is associated with increased matrix stiffness, and because substrate stiffness is known to regulate the actin cytoskeleton, we investigated the impact of matrix rigidity on podosome structures in LSECs. RESULTS: Using primary LSECs, we demonstrated that microvascular endothelial cells are able to form constitutive podosomes. Podosome presence in LSECs was independent of cytokines such as transforming growth factor-ß or vascular endothelial growth factor, but could be modulated by matrix stiffness. As expected, LSECs lost their differentiated phenotype during cell culture, which was paralleled by a loss of podosomes. LSECs however retained the capacity to form active podosomes following detachment/reseeding or actin-destabilising drug treatments. Finally, constitutive podosomes were also found in primary microvascular endothelial cells from other organs. CONCLUSIONS: Our results show that microvascular endothelial cells are able to form podosomes without specific stimulation. Our data suggest that the major determinant of podosome induction in these cells is substrate rigidity.
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Citoesqueleto de Actina/metabolismo , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Hígado/metabolismo , Microvasos/metabolismo , Transducción de Señal/fisiología , Adhesión Celular/fisiología , Humanos , Hígado/irrigación sanguínea , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
RhoGTPases are GDP/GTP molecular switches that control a wide variety of cellular processes, thereby contributing to many diseases, including cancer. As a consequence, there is great interest in the identification of small-molecule inhibitors of RhoGTPases. In the present paper, using the property of GTP-loaded RhoGTPases to bind to their effectors, we describe a miniaturized and robust assay to monitor Rac1 GTPase activation that is suitable for large-scale high-throughput screening. A pilot compound library screen revealed that the topoisomerase II poison MTX (mitoxantrone) is an inhibitor of Rac1, and also inhibits RhoA and Cdc42 in vitro. We show that MTX prevents GTP binding to RhoA/Rac1/Cdc42 in vitro. Furthermore, MTX strongly inhibits RhoGTPase-mediated F-actin (filamentous actin) reorganization and cell migration. Hence, we report a novel biochemical assay yielding the identification of RhoGTPase inhibitors and we present a proof-of-concept validation with the identification of MTX as a novel pan-RhoGTPase inhibitor.
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Mitoxantrona/farmacología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Animales , Movimiento Celular , Células Endoteliales/fisiología , Ensayos Analíticos de Alto Rendimiento , Humanos , Transducción de Señal , Porcinos , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA⺠ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients.
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Adenosina Trifosfatasas/antagonistas & inhibidores , Carcinoma Hepatocelular/tratamiento farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida , Niacinamida/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Vías Secretoras/efectos de los fármacos , SorafenibRESUMEN
Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe³âº and TiO2 (respectively IMAC and MOAC media), using a simple mixture of peptides from casein-albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe³âº and TiO2 were pH dependant. In the case of liver extract, selectivity increased from 12-18% to 58-60% when loading buffer contained 0.1 M acetic acid or 0.1 M trifluoroacetic acid, respectively. However, with POROS-Fe³âº column, the number of identifications decreased from 356 phosphopeptides with 0.1 M acetic acid to 119 phosphopeptides with 0.1 M TFA. This decrease of binding capacity of POROS-Fe³âº was associated with strong Fe³âº leaching. Furthermore, repetitive use of IMAC-Fe³âº with the 0.5 M NH4OH solution required for phosphopeptide elution induced Fe2O3 accumulation in the column. By comparison, MOAC columns packed with TiO2 support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.