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1.
Hepatology ; 76(5): 1248-1258, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35445421

RESUMEN

BACKGROUND AND AIMS: Chloride (Cl- ) channels in the apical membrane of biliary epithelial cells (BECs), also known as cholangiocytes, provide the driving force for biliary secretion. Although two Cl- channels have been identified on a molecular basis, the Cystic Fibrosis Transmembrane Conductance Regulator and Transmembrane Member 16A, a third Cl- channel with unique biophysical properties has been described. Leucine-Rich Repeat-Containing Protein 8, subfamily A (LRRC8A) is a newly identified protein capable of transporting Cl- in other epithelium in response to cell swelling. The aim of the present study was to determine if LRRC8A represents the volume-regulated anion channel in mouse BECs. APPROACH AND RESULTS: Studies were performed in mouse small (MSC) and large (MLC) cholangiocytes. Membrane Cl- currents were measured by whole-cell patch-clamp techniques and cell volume measurements were performed by calcein-AM fluorescence. Exposure of either MSC or MLC to hypotonicity (190 mOsm) rapidly increased cell volume and activated Cl- currents. Currents exhibited outward rectification, time-dependent inactivation at positive membrane potentials, and reversal potential at 0 mV (ECl ). Removal of extracellular Cl- or specific pharmacological inhibition of LRRC8A abolished currents. LRRC8A was detected in both MSC and MLC by reverse transcription polymerase chain reaction and confirmed by western blot. Transfection with LRRC8A small interfering RNA decreased protein levels by >70% and abolished volume-stimulated Cl- currents. CONCLUSION: These results demonstrate that LRRC8A is functionally present in mouse BECs, contributes to volume-activated Cl- secretion, and, therefore, may be a target to modulate bile formation in the treatment of cholestatic liver disorders.


Asunto(s)
Canales de Cloruro , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Ratones , Animales , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Leucina , Proteínas Repetidas Ricas en Leucina , ARN Interferente Pequeño/metabolismo , Células Epiteliales/metabolismo , Proteínas de la Membrana/genética
2.
Genomics ; 114(1): 278-291, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34942352

RESUMEN

Mammalian nephrons originate from a population of nephron progenitor cells, and changes in these cells' transcriptomes contribute to the cessation of nephrogenesis, an important determinant of nephron number. To characterize microRNA (miRNA) expression and identify putative cis-regulatory regions, we collected nephron progenitor cells from mouse kidneys at embryonic day 14.5 and postnatal day zero and assayed small RNA expression and transposase-accessible chromatin. We detect expression of 1104 miRNA (114 with expression changes), and 46,374 chromatin accessible regions (2103 with changes in accessibility). Genome-wide, our data highlight processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways. Furthermore, they identify new candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in nephron progenitor cell differentiation. Finally, we associate expression-changing miRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements and target genes. These analyses highlight new putative cis-regulatory loci for miRNA in nephron progenitors.


Asunto(s)
Cromatina , MicroARNs , Animales , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Riñón/metabolismo , Mamíferos/genética , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Nefronas/metabolismo , Células Madre
3.
Am J Physiol Gastrointest Liver Physiol ; 318(2): G277-G287, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31760763

RESUMEN

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.


Asunto(s)
Conductos Biliares/metabolismo , Bilis/metabolismo , Células Epiteliales/metabolismo , Canales Catiónicos TRPV/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Conductos Biliares/citología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Mecanorreceptores/efectos de los fármacos , Mecanorreceptores/fisiología , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Canales Catiónicos TRPV/efectos adversos
4.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G763-G771, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-32090602

RESUMEN

TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.


Asunto(s)
Anoctamina-1/metabolismo , Regulación de la Expresión Génica/fisiología , Hígado/metabolismo , Receptores de Interleucina-13/metabolismo , Receptores de Interleucina-4/metabolismo , Adenosina Trifosfato/farmacología , Animales , Anoctamina-1/genética , Ácidos y Sales Biliares , Conductos Biliares/metabolismo , Línea Celular , Cloruros , Fenómenos Electrofisiológicos , Humanos , Janus Quinasa 3/genética , Janus Quinasa 3/metabolismo , Masculino , Ratones , Técnicas de Placa-Clamp , Ratas , Receptores de Interleucina-13/genética , Receptores de Interleucina-4/genética , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo
5.
RNA ; 24(8): 1056-1066, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29844105

RESUMEN

The recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.


Asunto(s)
Compuestos Epoxi/farmacología , Exones/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Biosíntesis de Proteínas/genética , Factores de Empalme de ARN/antagonistas & inhibidores , Factores de Empalme de ARN/genética , Empalme del ARN/genética , Compuestos de Espiro/farmacología , Empalmosomas/genética , Secuencia de Bases , Línea Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Sistemas de Lectura/genética , Análisis de Secuencia de ARN , Transcriptoma/genética
6.
Nature ; 506(7489): 451-5, 2014 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-24553141

RESUMEN

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.


Asunto(s)
Transformación Celular Neoplásica , Ependimoma/genética , Ependimoma/metabolismo , FN-kappa B/metabolismo , Proteínas/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 11/genética , Ependimoma/patología , Femenino , Humanos , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , FN-kappa B/genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas/genética , Factor de Transcripción ReIA/genética , Factores de Transcripción , Translocación Genética/genética , Proteínas Señalizadoras YAP
7.
Acta Neuropathol ; 137(4): 637-655, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30770999

RESUMEN

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.


Asunto(s)
Neoplasias del Tronco Encefálico/genética , Diferenciación Celular/genética , Glioma Pontino Intrínseco Difuso/genética , Histonas/genética , Mutación , Animales , Neoplasias del Tronco Encefálico/patología , Línea Celular Tumoral , Glioma Pontino Intrínseco Difuso/patología , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Ratones
9.
N Engl J Med ; 371(11): 1005-15, 2014 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-25207766

RESUMEN

BACKGROUND: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS: We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS: Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS: Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Adolescente , Adulto , Animales , Niño , Preescolar , ADN de Neoplasias/análisis , Femenino , Genoma Humano , Xenoinjertos , Humanos , Lactante , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Cromosoma Filadelfia , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transducción de Señal/genética , Análisis de Supervivencia , Adulto Joven
10.
Acta Neuropathol ; 131(6): 833-45, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26810070

RESUMEN

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.


Asunto(s)
Neoplasias Encefálicas/patología , Genes myb , Predisposición Genética a la Enfermedad , Glioma/genética , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Adolescente , Adulto , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Niño , Preescolar , Proteínas de Unión al ADN , Femenino , Ganglioglioma/genética , Ganglioglioma/patología , Glioma/patología , Humanos , Lactante , Masculino , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ARN , Transactivadores/genética , Factores de Transcripción , Adulto Joven
11.
Mol Cell ; 32(2): 180-9, 2008 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-18951086

RESUMEN

Mdm2 regulates the p53 tumor suppressor by promoting its proteasome-mediated degradation. Mdm2 and p53 engage in an autoregulatory feedback loop that maintains low p53 activity in nonstressed cells. We now report that Mdm2 regulates p53 levels also by targeting ribosomal protein L26. L26 binds p53 mRNA and augments its translation. Mdm2 binds L26 and drives its polyubiquitylation and proteasomal degradation. In addition, the binding of Mdm2 to L26 attenuates the association of L26 with p53 mRNA and represses L26-mediated augmentation of p53 protein synthesis. Under nonstressed conditions, both mechanisms help maintain low cellular p53 levels by constitutively tuning down p53 translation. In response to genotoxic stress, the inhibitory effect of Mdm2 on L26 is attenuated, enabling a rapid increase in p53 synthesis. The Mdm2-L26 interaction thus represents an additional important component of the autoregulatory feedback loop that dictates cellular p53 levels and activity.


Asunto(s)
Regulación de la Expresión Génica , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/fisiología , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular , Retroalimentación Fisiológica , Humanos , Ratones , Modelos Genéticos , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación
13.
Cell Biol Int ; 33(3): 318-24, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19135161

RESUMEN

The tightly regulated expression of p53 contributes to genomic stability and transcription of the p53 gene is induced prior to cells entering S-phase, possibly as a mechanism to insure a rapid p53 response in the event of DNA damage. We have previously described the cloning of an additional 1000bp of upstream p53 sequences that play a role in the regulated expression of p53, and identified that C/EBPbeta-2 participates in inducing p53 gene expression in a cell cycle regulated fashion. This report deals with the transcriptional regulator, RBP-Jkappa, an essential target of the Notch receptor signaling pathway. It binds to the p53 promoter in a cell cycle regulation fashion and also serves to repress p53 gene expression. We conclude from these findings that the coordinate expression of C/EBPbeta-2 and RBP-Jkappa may be linked to p53 transcription during G(0) and as cells move into S-phase. Because defects in the Notch signaling pathway have been implicated in carcinogenesis, aberrant RBP-Jkappa expression and deregulated regulation of the p53 tumor suppressor could be an important step in some forms of cancers.


Asunto(s)
Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células COS , Ciclo Celular , Línea Celular , Chlorocebus aethiops , Regulación hacia Abajo , Células HeLa , Humanos , Ratones , Regiones Promotoras Genéticas , Fase de Descanso del Ciclo Celular , Fase S , Células 3T3 Swiss , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/metabolismo
14.
Sci Rep ; 8(1): 1406, 2018 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-29362419

RESUMEN

It is well known that pancreatic recovery after a single episode of injury such as an isolated bout of pancreatitis occurs rapidly. It is unclear, however, what changes are inflicted in such conditions to the molecular landscape of the pancreas. In the caerulein hyperstimulation model of pancreatitis, the murine pancreas has the ability to recover within one week based on histological appearance. In this study, we sought to characterize by RNA-sequencing (RNA-seq) the transcriptional profile of the recovering pancreas up to two weeks post-injury. We found that one week after injury there were 319 differentially expressed genes (DEGs) compared with baseline and that after two weeks there were 53 DEGs. Forty (12.5%) of the DEGs persisted from week one to week two, and another 13 DEGs newly emerged in the second week. Amongst the top up-regulated DEGs were several trypsinogen genes (trypsinogen 4, 5, 12, 15, and 16). To our knowledge, this is the first characterization of the transcriptome during pancreatic recovery by deep sequencing, and it reveals on a molecular basis that there is an ongoing recovery of the pancreas even after apparent histological resolution. The findings also raise the possibility of an emerging novel transcriptome upon pancreatic recovery.


Asunto(s)
Ceruletida/efectos adversos , Perfilación de la Expresión Génica/métodos , Pancreatitis/genética , Regeneración , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Pancreatitis/inducido químicamente , Análisis de Secuencia de ARN/métodos
15.
Cancer Cell ; 34(3): 411-426.e19, 2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30146332

RESUMEN

Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.


Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias de los Músculos/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Rabdomiosarcoma/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Niño , Epigenómica , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Genómica , Humanos , Masculino , Ratones , Terapia Molecular Dirigida/métodos , Neoplasias de los Músculos/genética , Neoplasias de los Músculos/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Medicina de Precisión/métodos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteómica , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Respuesta de Proteína Desplegada/genética , Ensayos Antitumor por Modelo de Xenoinjerto
16.
Cell Mol Gastroenterol Hepatol ; 3(1): 119-128, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28090570

RESUMEN

BACKGROUND AND AIMS: There is a pressing need to develop effective preventative therapies for post-ERCP pancreatitis (PEP). We demonstrated that early PEP events are induced through the calcium-activated phosphatase calcineurin and that global calcineurin deletion abolishes PEP in mice. A crucial question is whether acinar cell calcineurin controls the initiation of PEP in vivo. METHODS: We used a mouse model of PEP and examined the effects of in vivo acinar cell-specific calcineurin deletion by either generating a conditional knockout line or infusing a novel AAV-Ela-iCre into the pancreatic duct of a calcineurin floxed line. RESULTS: We found that PEP is dependent on acinar cell calcineurin in vivo, and this led us to determine that calcineurin inhibitors, infused within the radiocontrast, can largely prevent PEP. CONCLUSIONS: These results provide impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent PEP.

17.
Nat Genet ; 48(12): 1481-1489, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27776115

RESUMEN

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.


Asunto(s)
Transformación Celular Neoplásica/genética , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Proteínas de Homeodominio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Transformación Celular Neoplásica/patología , Perfilación de la Expresión Génica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Isoformas de Proteínas , Regulador Transcripcional ERG/genética , Adulto Joven
18.
Nat Genet ; 48(12): 1551-1556, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27798625

RESUMEN

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.


Asunto(s)
Biomarcadores de Tumor/genética , Factores de Unión al Sitio Principal/genética , Genómica/métodos , Leucemia Mieloide Aguda/genética , Mutación/genética , Proteínas de Fusión Oncogénica/genética , Adulto , Niño , Humanos
19.
Nat Commun ; 6: 6302, 2015 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-25743702

RESUMEN

Paediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here we analyse 37 adrenocortical tumours (ACTs) by whole-genome, whole-exome and/or transcriptome sequencing. Most cases (91%) show loss of heterozygosity (LOH) of chromosome 11p, with uniform selection against the maternal chromosome. IGF2 on chromosome 11p is overexpressed in 100% of the tumours. TP53 mutations and chromosome 17 LOH with selection against wild-type TP53 are observed in 28 ACTs (76%). Chromosomes 11p and 17 undergo copy-neutral LOH early during tumorigenesis, suggesting tumour-driver events. Additional genetic alterations include recurrent somatic mutations in ATRX and CTNNB1 and integration of human herpesvirus-6 in chromosome 11p. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. Collectively, these findings demonstrate the nature, timing and potential prognostic significance of key genetic alterations in paediatric ACT and outline a hypothetical model of paediatric adrenocortical tumorigenesis.


Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Carcinoma Corticosuprarrenal/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 17/genética , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Secuencia de Bases , Niño , ADN Helicasas/genética , Perfilación de la Expresión Génica , Humanos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Pérdida de Heterocigocidad , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma X , beta Catenina/genética
20.
Nat Genet ; 47(4): 330-7, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25730765

RESUMEN

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.


Asunto(s)
Mutación , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Desequilibrio Alélico/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Frecuencia de los Genes , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Proteínas de Fusión Oncogénica/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/genética , Proteínas ras/genética , Proteínas ras/metabolismo
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