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1.
Genomics ; 113(2): 515-529, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33418078

RESUMEN

Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify many HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We correlate the discovered hypoxia-driven events with available sequencing data from pan-cancer TCGA patient cohorts to select a narrow set of putative biologically relevant splice events for experimental validation. We validate a small set of candidate HIF-dependent alternative splicing events in multiple human gastrointestinal cancer cell lines as well as patient-derived human pancreatic cancer organoids. Lastly, we report the discovery of a HIF-dependent mechanism to produce a hypoxia-dependent, long and coding isoform of the UDP-N-acetylglucosamine transporter SLC35A3.


Asunto(s)
Empalme Alternativo , Neoplasias Gastrointestinales , Humanos , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Línea Celular Tumoral , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Transcriptoma , Hipoxia Tumoral
2.
Nature ; 522(7557): 444-449, 2015 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-26083752

RESUMEN

Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth.


Asunto(s)
Cardiomiopatía Hipertrófica/metabolismo , Fructoquinasas/metabolismo , Fructosa/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Fosfoproteínas/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalme Alternativo , Animales , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Cardiomiopatía Hipertrófica/fisiopatología , Modelos Animales de Enfermedad , Fructoquinasas/deficiencia , Fructoquinasas/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Síndrome Metabólico/metabolismo , Ratones , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/deficiencia , Ribonucleoproteína Nuclear Pequeña U2/genética
3.
Circulation ; 139(24): 2778-2792, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-30922078

RESUMEN

BACKGROUND: Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. METHODS: We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. RESULTS: HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. CONCLUSIONS: HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.


Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/prevención & control , Cardiomiopatía Hipertrófica/prevención & control , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , ARN no Traducido/metabolismo , Animales , Sitios de Unión , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/patología , Regiones Promotoras Genéticas , ARN no Traducido/genética , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
4.
Genes Dev ; 26(3): 259-70, 2012 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-22302938

RESUMEN

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress ß-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD(+)-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of ß-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2-Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.


Asunto(s)
Adipocitos/metabolismo , Metabolismo Energético , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , NAD/metabolismo , Obesidad/patología , Sirtuina 2/metabolismo , Acetilación , Adipocitos/citología , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Dieta , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Alineación de Secuencia , Sirtuina 2/genética , Transactivadores/metabolismo , Factores de Transcripción
5.
Bioinformatics ; 34(1): 107-108, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-28968639

RESUMEN

Motivation: Next-generation sequencing is now an established method in genomics, and massive amounts of sequencing data are being generated on a regular basis. Analysis of the sequencing data is typically performed by lab-specific in-house solutions, but the agreement of results from different facilities is often small. General standards for quality control, reproducibility and documentation are missing. Results: We developed NGS-pipe, a flexible, transparent and easy-to-use framework for the design of pipelines to analyze whole-exome, whole-genome and transcriptome sequencing data. NGS-pipe facilitates the harmonization of genomic data analysis by supporting quality control, documentation, reproducibility, parallelization and easy adaptation to other NGS experiments. Availability and implementation: https://github.com/cbg-ethz/NGS-pipe. Contact: niko.beerenwinkel@bsse.ethz.ch.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Neoplasias/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/normas , Análisis de Secuencia de ARN/normas
6.
Eur Heart J ; 39(26): 2497-2505, 2018 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-29020416

RESUMEN

Despite strong indications that increased consumption of added sugars correlates with greater risks of developing cardiometabolic syndrome (CMS) and cardiovascular disease (CVD), independent of the caloric intake, the worldwide sugar consumption remains high. In considering the negative health impact of overconsumption of dietary sugars, increased attention is recently being given to the role of the fructose component of high-sugar foods in driving CMS. The primary organs capable of metabolizing fructose include liver, small intestine, and kidneys. In these organs, fructose metabolism is initiated by ketohexokinase (KHK) isoform C of the central fructose-metabolizing enzyme KHK. Emerging data suggest that this tissue restriction of fructose metabolism can be rescinded in oxygen-deprived environments. In this review, we highlight recent progress in understanding how fructose metabolism contributes to the development of major systemic pathologies that cooperatively promote CMS and CVD, reference recent insights into microenvironmental control of fructose metabolism under stress conditions and discuss how this understanding is shaping preventive actions and therapeutic approaches.


Asunto(s)
Enfermedad de la Arteria Coronaria/metabolismo , Fructosa/metabolismo , Hipoxia/metabolismo , Hígado/metabolismo , Síndrome Metabólico/metabolismo , Enfermedad de la Arteria Coronaria/epidemiología , Sacarosa en la Dieta , Epidemias , Fructoquinasas/metabolismo , Humanos , Intestino Delgado/metabolismo , Riñón/metabolismo , Metabolismo de los Lípidos , Síndrome Metabólico/epidemiología , Isoformas de Proteínas
7.
Biochim Biophys Acta ; 1863(7 Pt B): 1822-8, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26896647

RESUMEN

Pathologic cardiac growth is an adaptive response of the myocardium to various forms of systemic (e.g. pressure overload) or genetically-based (e. g. mutations in genes encoding sarcomeric proteins) stress. It represents a key aspect of different types of heart disease including aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM). While many of the pathophysiological and hemodynamical aspects of pathologic cardiac hypertrophy have been uncovered during the last decades, its underlying metabolic determinants are only beginning to come into focus. Here, we review the epidemiological evidence and pathological features of hypertrophic heart disease in AS and HCM and consider in this context the development of microenvironmental tissue hypoxia as a key component of the heart's growth response to pathologic stress. We particularly reflect on recent evidence illustrating how activation of hypoxia-inducible factor (HIF) drives glycolytic and fructolytic metabolic programs to maintain ATP generation and support anabolic growth of the pathologically-stressed heart. Finally we discuss how this metabolic programs, when protracted, deprive the heart of energy leading ultimately to heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.


Asunto(s)
Cardiomegalia/metabolismo , Microambiente Celular , Fructosa/metabolismo , Glucólisis , Miocardio/metabolismo , Adaptación Fisiológica , Adenosina Trifosfato/metabolismo , Animales , Cardiomegalia/epidemiología , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Hipoxia de la Célula , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocardio/patología , Factores de Riesgo , Transducción de Señal , Estrés Fisiológico
8.
Bioinformatics ; 32(14): 2111-9, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27153587

RESUMEN

MOTIVATION: Alternative splicing represents a prime mechanism of post-transcriptional gene regulation whose misregulation is associated with a broad range of human diseases. Despite the vast availability of transcriptome data from different cell types and diseases, bioinformatics-based surveys of alternative splicing patterns remain a major challenge due to limited availability of analytical tools that combine high accuracy and rapidity. RESULTS: We describe here a novel junction-centric method, jSplice, that enables de novo extraction of alternative splicing events from RNA-sequencing data with high accuracy, reliability and speed. Application to clear cell renal carcinoma (ccRCC) cell lines and 65 ccRCC patients revealed experimentally validatable alternative splicing changes and signatures able to prognosticate ccRCC outcome. In the aggregate, our results propose jSplice as a key analytic tool for the derivation of cell context-dependent alternative splicing patterns from large-scale RNA-sequencing datasets. AVAILABILITY AND IMPLEMENTATION: jSplice is a standalone Python application freely available at http://www.mhs.biol.ethz.ch/research/krek/jsplice CONTACT: wilhelm.krek@biol.ethz.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Empalme Alternativo , Neoplasias Renales/genética , Programas Informáticos , Transcriptoma , Algoritmos , Humanos , Reproducibilidad de los Resultados
9.
EMBO Rep ; 15(1): 77-85, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24378640

RESUMEN

Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHL-dependency of USP8 for ciliogenesis is directly linked to its function as a HIF1α deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1α, USP8 maintains a basal expression of HIF1α and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.


Asunto(s)
Cilios/fisiología , Endopeptidasas/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ubiquitina Tiolesterasa/fisiología , Ubiquitinación , Animales , Hipoxia de la Célula , Endocitosis , Células HEK293 , Humanos , Ratones , Oxígeno/metabolismo , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
10.
J Pathol ; 234(2): 239-52, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24930886

RESUMEN

Endometrial cancer is the most frequently occurring malignancy of the female genital tract in Western countries. Although in many cases surgically curable, about 30% of the tumours represent an aggressive and untreatable disease. In an attempt to establish a reliable prognostic marker for endometrial carcinomas disregarding their histological diversity, we investigated the expression of KPNA2, a mediator of nucleocytoplasmic transport, and other cell proliferation-associated proteins and their correlation with cancer progression. We analysed patient tissue microarrays (TMAs) assembled from 527 endometrial cancer tissue specimens and uterus samples from a Trp53 knockout mouse model of endometrial cancer. Our data show that KPNA2 expression was significantly up-regulated in human endometrial carcinomas and associated with higher tumour grade (p = 0.026), higher FIGO stage (p = 0.027), p53 overexpression (p < 0.001), activation of the PI3K/AKT pathway, and epithelial-mesenchymal transition. Increased nuclear KPNA2 immunoreactivity was identified as a novel predictor of overall survival, independent of well-established prognostic factors in Cox regression analyses (hazard ratio 1.7, 95% CI 1.13-2.56, p = 0.01). No significant association between KPNA2 expression and endometrial cancer subtype was detected. In the mouse model, KPNA2 showed increased expression levels from precancerous (EmgD, EIC) to far-advanced invasive lesions. We further investigated the cell proliferation capacity after siRNA-mediated KPNA2 knockdown in the human endometrial cancer cell line MFE-296. KPNA2 silencing led to decreased proliferation of the cancer cells, suggesting interplay of the protein with the cell cycle. Taken together, increased expression of KPNA2 is an independent prognostic marker for poor survival. The mechanism of enhanced nucleocytoplasmic transport by KPNA2 overexpression seems a common event in aggressive cancers since we have shown a significant correlation of KPNA2 expression and tumour aggressiveness in a large variety of other solid tumour entities. Introducing KPNA2 immunohistochemistry in routine diagnostics may allow for the identification of patients who need more aggressive treatment regimens.


Asunto(s)
Proliferación Celular , Neoplasias Endometriales/metabolismo , Proteínas Nucleares/metabolismo , alfa Carioferinas/metabolismo , Animales , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo
11.
EMBO J ; 29(2): 469-81, 2010 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-19942859

RESUMEN

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by beta-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKalpha1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKalpha1 by LKB1 in response to lipolytic signals. Activation of AMPKalpha1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKalpha1 in vitro. Functional analysis of an AMPKalpha1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA-activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adipocitos/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Lipólisis , Proteínas Quinasas Activadas por AMP/genética , Agonistas Adrenérgicos beta/farmacología , Animales , Células Cultivadas , Ácidos Grasos/metabolismo , Glicerol/metabolismo , Isoproterenol/farmacología , Ratones , Fosforilación , Mutación Puntual , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
12.
Curr Opin Cell Biol ; 19(6): 685-90, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18006292

RESUMEN

Functional inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product, pVHL, leads to cancer in humans. It is widely accepted that pVHL functions to destabilise hypoxia inducible factor alpha (HIFalpha) subunits, key effectors of the hypoxia signalling pathway. However, growing evidence indicates that tumour suppression by pVHL also involves the control of a wide variety of HIFalpha-independent processes including microtubule dynamics, primary cilium maintenance, cell proliferation, neuronal apoptosis, extracellular matrix deposition and responses to DNA damage. Moreover, it is becoming apparent that tumour initiation requires not only VHL mutation but also the alteration of additional cooperating cancer pathways. These studies are beginning to provide insights into the signalling networks involving pVHL that normally control diverse cellular processes and how disruption of these networks leads to tumour formation.


Asunto(s)
Genes Supresores de Tumor/fisiología , Neoplasias/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Transducción de Señal/fisiología
13.
Nat Cell Biol ; 9(5): 588-95, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17450132

RESUMEN

Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3beta are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3beta alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3beta is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3beta function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.


Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Enfermedades Renales Quísticas/metabolismo , Túbulos Renales/metabolismo , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/metabolismo , Animales , Línea Celular , Cilios/metabolismo , Cilios/patología , Embrión de Mamíferos/citología , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Enfermedades Renales Quísticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , Interferencia de ARN , Transfección , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/patología
14.
J Pathol ; 229(4): 525-34, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23007793

RESUMEN

Mass spectrometry analysis of renal cancer cell lines recently suggested that the protein-tyrosine phosphatase receptor type J (PTPRJ), an important regulator of tyrosine kinase receptors, is tightly linked to the von Hippel-Lindau protein (pVHL). Therefore, we aimed to characterize the biological relevance of PTPRJ for clear cell renal cell carcinoma (ccRCC). In pVHL-negative ccRCC cell lines, both RNA and protein expression levels of PTPRJ were lower than those in the corresponding pVHL reconstituted cells. Quantitative RT-PCR and western blot analysis of ccRCC with known VHL mutation status and normal matched tissues as well as RNA in situ hybridization on a tissue microarray (TMA) confirmed a decrease of PTPRJ expression in more than 80% of ccRCCs, but in only 12% of papillary RCCs. ccRCC patients with no or reduced PTPRJ mRNA expression had a less favourable outcome than those with a normal expression status (p = 0.05). Sequence analysis of 32 PTPRJ mRNA-negative ccRCC samples showed five known polymorphisms but no mutations, implying other mechanisms leading to PTPRJ's down-regulation. Selective silencing of HIF-α by siRNA and reporter gene assays demonstrated that pVHL inactivation reduces PTPRJ expression through a HIF-dependent mechanism, which is mainly driven by HIF-2α stabilization. Our results suggest PTPRJ as a member of a pVHL-controlled pathway whose suppression by HIF is critical for ccRCC development.


Asunto(s)
Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hibridación in Situ , Estimación de Kaplan-Meier , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Modelos Moleculares , Polimorfismo Genético , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Análisis de Secuencia de ADN , Análisis de Matrices Tisulares , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
15.
Proc Natl Acad Sci U S A ; 108(8): 3342-7, 2011 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-21300890

RESUMEN

A key barrier to the realization of personalized medicine for cancer is the identification of biomarkers. Here we describe a two-stage strategy for the discovery of serum biomarker signatures corresponding to specific cancer-causing mutations and its application to prostate cancer (PCa) in the context of the commonly occurring phosphatase and tensin homolog (PTEN) tumor-suppressor gene inactivation. In the first stage of our approach, we identified 775 N-linked glycoproteins from sera and prostate tissue of wild-type and Pten-null mice. Using label-free quantitative proteomics, we showed that Pten inactivation leads to measurable perturbations in the murine prostate and serum glycoproteome. Following bioinformatic prioritization, in a second stage we applied targeted proteomics to detect and quantify 39 human ortholog candidate biomarkers in the sera of PCa patients and control individuals. The resulting proteomic profiles were analyzed by machine learning to build predictive regression models for tissue PTEN status and diagnosis and grading of PCa. Our approach suggests a general path to rational cancer biomarker discovery and initial validation guided by cancer genetics and based on the integration of experimental mouse models, proteomics-based technologies, and computational modeling.


Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Próstata/diagnóstico , Proteómica/métodos , Animales , Biología Computacional , Silenciador del Gen , Glicoproteínas/sangre , Humanos , Masculino , Métodos , Ratones , Fosfohidrolasa PTEN/análisis , Fosfohidrolasa PTEN/genética
16.
Int J Cancer ; 132(2): E11-7, 2013 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22949125

RESUMEN

Although von Hippel-Lindau (VHL) tumor suppressor gene alterations dominate the genetic landscape of clear cell renal cell carcinoma (ccRCC), recent studies have identified new ccRCC genes, including SETD2, KDM6A, KDM5C, BAP1 and PBRM1. Strikingly, all these genes fall into a category of histone/chromatin regulators. Polybromo-1 (PBRM1) is the second most frequently mutated gene after VHL; however, the clinical relevance of its loss in ccRCC has not yet been reported. Here, we analyzed the expression of PBRM1, the product encoded by PBRM1, in ccRCC cell lines and in more than 300 RCC tumor samples. The data were correlated with clinicopathological parameters and VHL mutation status. We found that a significant number of ccRCC cancer cell lines lack detectable PBRM1 expression. Loss of PBRM1 was predominant in the clear cell subtype of RCC (~ 70%) and correlated with advanced tumor stage (p < 0.0001), low differentiation grade (p = 0.0002) and worse patient outcome (p = 0.025), but not with the VHL mutation status. Our results indicate a critical role for PBRM1 in the suppression of ccRCC progression. Moreover, the results suggest that functional inactivation of PBRM1 in the context of pVHL loss-of-function may represent a key event in facilitating the development of key aspects of an aggressive tumor behavior. Given the role of PBRM1 in chromatin modification, the gene expression pathways disrupted by the inactivation of this protein may lead to new treatment strategies for ccRCC.


Asunto(s)
Carcinoma de Células Renales/metabolismo , Expresión Génica , Neoplasias Renales/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proteínas de Unión al ADN , Progresión de la Enfermedad , Humanos , Estimación de Kaplan-Meier , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Mutación , Estadificación de Neoplasias , Proteínas Nucleares/genética , Análisis de Matrices Tisulares , Factores de Transcripción/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética
17.
Nat Methods ; 7(9): 761-8, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20729842

RESUMEN

Regulation of microtubule dynamics is essential for many cell biological processes and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracked all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We introduce spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. We validated the algorithm by comparing the results to data for manually tracked, homogeneously labeled microtubules and by analyzing the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells.


Asunto(s)
Biología Computacional , Proteínas Asociadas a Microtúbulos/análisis , Microtúbulos/química , Microtúbulos/metabolismo , Acetilación , Algoritmos , Biomarcadores/análisis , Biomarcadores/metabolismo , Línea Celular , Movimiento Celular , Simulación por Computador , Células Epiteliales/citología , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Reproducibilidad de los Resultados , Programas Informáticos , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
18.
J Proteome Res ; 11(3): 1598-608, 2012 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-22148984

RESUMEN

Cell surface N-glycoproteins provide a key interface of cells to their environment and therapeutic entry points for drug and biomarker discovery. Their comprehensive description denotes therefore a formidable challenge. The ß-cells of the pancreas play a crucial role in blood glucose homeostasis, and disruption of their function contributes to diabetes. By combining cell surface and whole cell capturing technologies with high-throughput quantitative proteomic analysis, we report on the identification of a total of 956 unique N-glycoproteins from mouse MIN6 ß-cells and human islets. Three-hundred-forty-nine of these proteins encompass potential surface N-glycoproteins and include orphan G-protein-coupled receptors, novel proteases, receptor protein kinases, and phosphatases. Interestingly, stimulation of MIN6 ß-cells with glucose and the hormone GLP1, known stimulators of insulin secretion, causes significant changes in surface N-glycoproteome expression. Taken together, this ß-cell N-glycoproteome resource provides a comprehensive view on the composition of ß-cell surface proteins and expands the scope of signaling systems potentially involved in mediating responses of ß-cells to various forms of (patho)physiologic stress and the extent of dynamic remodeling of surface N-glycoprotein expression associated with metabolic and hormonal stimulation. Moreover, it provides a foundation for the development of diabetes medicines that target or are derived from the ß-cell surface N-glycoproteome.


Asunto(s)
Péptido 1 Similar al Glucagón/fisiología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoma/metabolismo , Adulto , Anciano , Animales , Células Cultivadas , Expresión Génica , Regulación de la Expresión Génica , Glucosa/fisiología , Humanos , Células Secretoras de Insulina/enzimología , Islotes Pancreáticos/enzimología , Glicoproteínas de Membrana/genética , Ratones , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteoma/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Espectrometría de Masas en Tándem
19.
EMBO J ; 27(12): 1747-57, 2008 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-18497742

RESUMEN

In patients with von Hippel-Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.


Asunto(s)
Quistes/enzimología , Quistes/patología , Enfermedades Renales Quísticas/enzimología , Enfermedades Renales Quísticas/patología , Fosfohidrolasa PTEN/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Proliferación Celular , Cilios/enzimología , Cilios/patología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Distales/enzimología , Túbulos Renales Distales/patología , Sistema de Señalización de MAP Quinasas , Ratones , Mutación/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo
20.
NMR Biomed ; 24(10): 1295-301, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22223365

RESUMEN

Insulin resistance is a central feature of type II diabetes and is associated with alterations in skeletal muscle lipid metabolism, which manifest themselves, in part, in increased intramyocellular lipid (IMCL) accumulation. The objective of this study was to assess noninvasively the levels of IMCL longitudinally in the tibialis anterior muscle of Lep(ob) /Lep(ob) (ob/ob) mice, a genetic model of obesity and mild diabetes, and Lep(ob) /+ (ob/+) heterozygous control animals, using (1) H MRS at 9.4 T. The use of a cryogenic surface coil transceiver leads to significant increases in sensitivity. Method implementation included the assessment of the reproducibility and spatial heterogeneity of the IMCL signal and the determination of T(2) relaxation times, as IMCL levels were expressed relative to the total creatine signal, and therefore the signal ratios had to be corrected for differences in T(2) relaxation. IMCL levels were found to be significantly higher in ob/ob mice relative to ob/+ heterozygous control mice that do not develop disease. An increase in IMCL levels was observed for ob/ob mice until weeks 16/17; after this time point, IMCL levels decreased again, reaching final levels that were slightly higher than the initial values. These noninvasively detected alterations in skeletal muscle lipid metabolism in ob/ob mice were accompanied by a transient increase in plasma insulin concentrations. This study indicates that IMCL may be reliably assessed in mouse tibialis anterior muscle using a cryogenic surface coil, implying that (1) H MRS at 9.4 T represents a useful technology for the noninvasive measurement of changes in lipid metabolism in the skeletal muscle that accompany obesity.


Asunto(s)
Frío , Metabolismo de los Lípidos , Espectroscopía de Resonancia Magnética/instrumentación , Espectroscopía de Resonancia Magnética/métodos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Envejecimiento/metabolismo , Animales , Peso Corporal , Creatina/metabolismo , Estudios de Factibilidad , Insulina/metabolismo , Masculino , Ratones , Ratones Obesos , Marcadores de Spin , Propiedades de Superficie
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