RESUMEN
Background & aim: Resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (CRC) is frequent and prognostic biomarkers are lacking. MicroRNAs (miR) are good candidates in this context. We aimed to characterize cetuximab and panitumumab exposure influence on miR expression in colorectal cancer cells to identify those regulating the EGFR pathway and implicated in resistance to treatment. Finally, we aimed to identify miR expression in serum of patients with advanced CRC treated with cetuximab or panitumumab. Results: Cetuximab and panitumumab exposure induced significant expression variations of 17 miR out of a miRnome panel of 752. Six of those miR interacted with at least one downstream element of the EGFR pathway. Conclusion: After the bioinformatics two-phase process, five miR rarely described before could be potential actors of anti-EGFR monoclonal antibody resistance: miR-95-3p, miR-139-5p, miR-145-5p, miR-429 and miR-1247-5p. In vivo, we detected the expression of miR-139-5p and miR-145-5p in serum of patients with metastatic CRC.
Asunto(s)
PanitumumabRESUMEN
BACKGROUND: Colorectal cancer (CRC) metastases are the main cause of CRC mortality. Intracellular Ca2+ regulates cell migration and invasion, key factors for metastases. Ca2+ also activates Ca2+-dependent potassium channels which in turn affect Ca2+ driving force. We have previously reported that the expression of the Ca2+ activated potassium channel KCNN4 (SK4) is higher in CRC primary tumors compared to normal tissues. Here, we aimed to investigate the role of SK4 in the physiology of CRC. RESULTS: SK4 protein expression is enhanced in CRC tissues compared to normal colon tissues, with a higher level of KCNN4 in CRC patients with KRAS mutations. At the cellular level, we found that SK4 regulates the membrane potential of HCT116 cells. We also found that its inhibition reduced store operated Ca2+ entry (SOCE) and constitutive Ca2+ entry (CCE), while reducing cell migration. We also found that the activity of SK4 is linked to resistance pathways such as KRAS mutation and the expression of NRF2 and HIF-1α. In addition, the pharmacological inhibition of SK4 reduced intracellular reactive oxygen species (ROS) production, NRF2 expression and HIF1α stabilization. CONCLUSION: Our results suggest that SK4 contributes to colorectal cancer cell migration and invasion by modulating both Ca2+ entry and ROS regulation. Therefore, SK4 could be a potential target to reduce metastasis in KRAS-mutated CRC.
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Calcio/metabolismo , Movimiento Celular/fisiología , Neoplasias Colorrectales/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Mutación/fisiología , Proteínas Proto-Oncogénicas p21(ras) , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Bases de Datos Genéticas , Células HCT116 , Células HT29 , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirazoles/farmacologíaRESUMEN
Human malignant gliomas exhibit acquisition of either one of two telomere maintenance mechanisms, resulting from either reactivation of telomerase expression or activation of an alternative lengthening of telomeres (ALT) mechanism. In the present study, we analyzed 63 human malignant gliomas for the presence of ALT-specific extrachromosomal circles of telomeric DNA (C-circles) and measured telomerase expression, telomeric DNA content (Telo/Alu method), and telomeric repeat-containing RNAs (TERRA) levels. We also assessed histomolecular markers routinely used in clinical practice. The presence of C-circles significantly correlated with IDH1/2 mutation, MGMT exon 1 methylation, low Ki-67 immunostaining, increased telomeric DNA content, absence of functional ATRX protein and level of HTERT gene expression. In multivariate analysis, we observed a trend to a correlation between elevated TERRA levels and increased survival. Interestingly, the C-circles assay allowed to detect ALT activation in glioblastomas exhibiting wild-type IDH1/2 and ATRX expression. These results suggest that, after the correlations uncovered here have been confirmed on larger numbers of tumors, telomeric markers might be useful in improving diagnosis. They also point out to the utility of using the specific, sensitive and quantitative C-circle and Telo/Alu assays that can work with as few as 30 ng of tumor DNA.
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Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Homeostasis del Telómero , Adulto , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/cirugía , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Línea Celular Tumoral , Estudios de Cohortes , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Femenino , Glioma/genética , Glioma/patología , Glioma/cirugía , Humanos , Isocitrato Deshidrogenasa/genética , Masculino , Persona de Mediana Edad , Clasificación del Tumor , ARN/metabolismo , Telomerasa/metabolismo , Homeostasis del Telómero/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
BACKGROUND: In budding yeast, the highly conserved Tel2 protein is part of several complexes and its main function is now believed to be in the biogenesis of phosphatidyl inositol 3-kinase related kinases. PRINCIPAL FINDINGS: To uncover potentially novel functions of Tel2, we set out to isolate temperature-sensitive (ts) mutant alleles of TEL2 in order to perform genetic screenings. MED15/GAL11, a subunit of Mediator, a general regulator of transcription, was isolated as a suppressor of these mutants. The isolated tel2 mutants exhibited a short telomere phenotype that was partially rescued by MED15/GAL11 overexpression. The tel2-15 mutant was markedly deficient in the transcription of EST2, coding for the catalytic subunit of telomerase, potentially explaining the short telomere phenotype of this mutant. In parallel, a two-hybrid screen identified an association between Tel2 and Rvb2, a highly conserved member of the AAA+ family of ATPases further found by in vivo co-immunoprecipitation to be tight and constitutive. Transiently overproduced Tel2 and Med15/Gal11 associated together, suggesting a potential role for Tel2 in transcription. Other Mediator subunits, as well as SUA7/TFIIB, also rescued the tel2-ts mutants. SIGNIFICANCE: Altogether, the present data suggest the existence of a novel role for Tel2, namely in transcription, possibly in cooperation with Rvb2 and involving the existence of physical interactions with the Med15/Gal11 Mediator subunit.
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Epistasis Genética/fisiología , Complejo Mediador/genética , Complejo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Helicasas/fisiología , Prueba de Complementación Genética , Inmunoprecipitación , Organismos Modificados Genéticamente , Unión Proteica/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Especificidad por Sustrato/genética , Telómero/metabolismo , Temperatura , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.
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Proteínas Portadoras/metabolismo , Endocitosis/fisiología , Receptores de Leptina/metabolismo , Animales , Proteínas Portadoras/genética , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/genética , Lisosomas/metabolismo , Mutación , Transporte de Proteínas/fisiología , Ratas , Receptores de Leptina/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Menin, the protein encoded by the Multiple Endocrine Neoplasia type 1 gene, is involved in the cell cycle control through its participation in functional dynamics of chromatin and regulation of transcription. RB, the protein of the retinoblastoma gene RB1, controls the progression of the cell cycle and is regulated in its activity by means of a feedback by phosphorylation. Studies in double heterozygous knockout mice for Men1 and the Retinoblastoma gene Rb1 have recently indicated that both genes may be implicated in the same pathways. In the course of our studies on Menin, we found that after suppression or in absence of Menin, RB1 expression was strongly reduced in a posttranscriptional manner. Under conditions of growth arrest, the hyperphosphorylated form of RB was most strongly affected, whereas its hypophosphorylated form was less or not at all reduced. Our findings confirm the hypothesis that the pathways of two tumor suppressor genes are connected.
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Proteínas Proto-Oncogénicas/fisiología , Proteína de Retinoblastoma/metabolismo , Animales , Ciclo Celular/genética , Humanos , Ratones , Proteínas Proto-Oncogénicas/genética , Proteína de Retinoblastoma/genéticaRESUMEN
Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this "leptin resistance" is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.
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Núcleo Arqueado del Hipotálamo/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Leptina/metabolismo , Obesidad/prevención & control , Receptores de Leptina/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dieta , Grasas de la Dieta/administración & dosificación , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lentivirus/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Oligonucleótidos Antisentido/genética , Receptores de Leptina/antagonistas & inhibidores , Receptores de Leptina/genética , Transducción de SeñalRESUMEN
Animal studies have illustrated the importance of the expression in adipose tissue of the leptin receptor (OB-R), and of SOCS3 an inhibitor of the leptin signaling pathway, in body weight regulation. The aim of the present study was to investigate in human adipose tissues of the same patients the OB-R isoforms and SOCS3 expression. Subcutaneous and omental adipose tissues were obtained from 6 lean and 18 morbidly obese women. The long isoform OB-Rb mRNA mediating leptin signaling, and SOCS3 mRNA are abundantly present in the subcutaneous fat of lean women, but are 90% and 70% decreased (P<0.0001) in obese women. In visceral fat from lean and obese women, both OB-Rb and SOCS3 mRNA are detected at very low levels. Subcutaneous/visceral ratios for OB-Ra the short OB-R isoform, OB-Rb, and SOCS3 mRNA abundance strongly correlate with the insulin sensitivity index, HOMA-% S, (r=0.49, P<0.0001, r=0.42, P=0.0002 and r=0.38, P=0.0002, respectively) in both lean and obese patients without type 2 diabetes. The near absence of OB-Rb mRNA and the similarly decreased SOCS3 expression in obese adipose tissue may reflect a defective leptin signaling pathway that could play a role in the impairment of insulin sensitivity associated with excess adiposity.
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Grasa Intraabdominal/metabolismo , Obesidad/metabolismo , Receptores de Superficie Celular/metabolismo , Grasa Subcutánea/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Adulto , Femenino , Regulación de la Expresión Génica , Humanos , Insulina/análisis , Persona de Mediana Edad , Obesidad/diagnóstico , Obesidad/genética , Epiplón/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Leptina , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Delgadez/diagnóstico , Delgadez/genética , Delgadez/metabolismoRESUMEN
The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11-12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of gamma-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C>A and +83897 T>A (OR = 0.81, 95% CI [0.681-0.972], p = 0.0049) and an at-risk SNP (-243 A>G) for morbid obesity (OR = 1.3, 95% CI [1.053-1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C>A and +83897 T>A haplotype (chi(2) = 7.637, p = 0.02). In the murine insulinoma cell line betaTC3, the G at-risk allele of SNP -243 A>G increased six times GAD2 promoter activity (p < 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The -243 A>G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic beta cells, we analyzed GAD65 antibody level as a marker of beta-cell activity and of insulin secretion. In the control group, -243 A>G, +61450 C>A, and +83897 T>A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T>A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of beta-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.