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Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations, and an increased risk of developing cancer. The chromatin-binding special AT-rich sequence-binding protein-1 (SATB1) is downregulated in megakaryocyte/erythroid progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors heat shock protein 70 (HSP70) and GATA1 which accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes promotes chromatin loops that are required for the induction of HSP70, which, in turn, promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.
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Anemia de Diamond-Blackfan , Proteínas de Ligação à Região de Interação com a Matriz , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Megacariócitos/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Diferenciação Celular/genética , Fatores de Transcrição/metabolismo , Anemia de Diamond-Blackfan/metabolismo , Cromatina/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismoRESUMO
Diamond-Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because more than 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is due predominantly to a block and delay in early committed erythropoiesis with reduced megakaryocyte/erythroid progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA sequencing on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19 insufficiency. However, SATB1 expression did not affect expansion of committed erythroid progenitors, indicating ribosomal insufficiency affects multiple stages during erythroid differentiation.
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Anemia de Diamond-Blackfan , Eritropoese , Proteínas de Ligação à Região de Interação com a Matriz , MicroRNAs , Anemia de Diamond-Blackfan/patologia , Regulação para Baixo , Eritropoese/genética , Células-Tronco Hematopoéticas , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Megacariócitos/citologia , MicroRNAs/genética , Proteínas RibossômicasRESUMO
Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3-7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.
Assuntos
Distrofina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Adolescente , Adulto , Fatores Etários , Biópsia , Códon sem Sentido/genética , Distrofina/antagonistas & inibidores , Éxons/genética , Feminino , Fibroblastos/patologia , Genótipo , Humanos , Estimativa de Kaplan-Meier , Tempo de Internação , Masculino , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mioblastos/patologia , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Cultura Primária de Células , Sistema de Registros , Deleção de Sequência/genética , Caracteres Sexuais , Adulto JovemRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1000832.].
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BACKGROUND: There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres. METHODS: Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated. RESULTS: Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled 13C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS. CONCLUSIONS: These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable metabolically by analyzing expression profiles and glucose consumption. Our results also highlight important differences in nucleotide synthesis utilization and DNA repair capacity that could be exploited for therapy. Altogether, this study demonstrates that IDH1 mutant gliomas are a distinct subclass of glioma with a less malignant, but also therapy-resistant, metabolic profile that will likely require distinct modes of therapy.
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Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type-specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1, which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type-selective actions of nuclear receptors in physiology and disease.
Assuntos
Aterosclerose/genética , Colesterol/metabolismo , RNA Helicases DEAD-box/genética , Receptores X do Fígado/genética , RNA Longo não Codificante/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Células da Medula Óssea/metabolismo , Colesterol/genética , Regulação da Expressão Gênica/genética , Humanos , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-ß signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.
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Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/genética , Receptor ErbB-3/genética , Receptores de Fator de Crescimento Neural/genética , Adulto , Idoso , Sistemas CRISPR-Cas , Diferenciação Celular , Distrofina/genética , Distrofina/metabolismo , Feminino , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/citologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mioblastos/citologia , Miosinas/genética , Miosinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Receptor ErbB-3/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. RESULTS: Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. CONCLUSIONS: Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.
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Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression, conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.
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Antineoplásicos/uso terapêutico , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Glioblastoma/tratamento farmacológico , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Neoplasias do Sistema Nervoso Central/genética , DNA/genética , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Glioblastoma/genética , Humanos , Camundongos , Mutação , Transplante de Neoplasias , Quinazolinas/uso terapêutico , Análise de Célula Única , Células Tumorais Cultivadas , Suspensão de TratamentoRESUMO
PURPOSE: The growth and survival of neurofibromatosis type 2 (NF2)-deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models. EXPERIMENTAL DESIGN: We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach. RESULTS: NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2-deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple pathways necessary for tumorigenesis. CONCLUSIONS: HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies.
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Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Lactonas/farmacologia , Neurofibromatose 2/tratamento farmacológico , Oximas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neurofibromatose 2/metabolismo , Proteólise , Transcriptoma/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.
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Processamento Alternativo/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Receptores ErbB/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Interferência de RNA , RNA Interferente PequenoRESUMO
BACKGROUND: Subtypes of glioblastoma multiforme (GBM) based on genetic and molecular alterations are thought to cause alterations in anatomic MRI owing to downstream biological changes, such as edema production, blood-brain barrier breakdown, and necrosis. The purpose of the current study was to identify a potential relationship between imaging features and the mesenchymal (MES) GBM subtype, which has the worst patient prognosis. METHODS: MRIs from 46 patients with histologically confirmed GBM were retrospectively analyzed. The volume of contrast enhancement, regions of central necrosis, and hyperintensity of T2/fluid attenuated inversion recovery (FLAIR) were measured. Additionally, the ratio of T2/FLAIR hyperintense volume to the volume of contrast enhancement and necrosis was calculated. RESULTS: The volume of contrast enhancement, volume of central necrosis, combined volume of contrast enhancement and central necrosis, and the ratio of T2/FLAIR to contrast enhancement and necrosis were significantly different in MES compared with non-MES GBM (Mann-Whitney, P < .05). Receiver-operator characteristics indicated that these 4 metrics were all significant predictors of the MES phenotype. The volume ratio of T2 hyperintensity to contrast enhancement and central necrosis was significantly lower in MES vs non-MES GBM (P < .0001), was a significant predictor of the MES phenotype (area under the curve = 0.93, P < .001), and could be used to stratify short- and long-term overall survival (log-rank, P = .0064 using cutoff of 3.0). These trends were also present when excluding isocitrate dehydrogenase 1 mutant tumors and incorporating covariates such as age and KPS score. CONCLUSIONS: Results suggest that volume ratio may be a simple, cost-effective, and noninvasive biomarker for quickly identifying MES GBM.
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Biomarcadores Tumorais/análise , Neoplasias Encefálicas/patologia , Meios de Contraste , Glioblastoma/patologia , Imageamento por Ressonância Magnética , Mesoderma/patologia , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/tratamento farmacológico , Ensaios Clínicos Fase II como Assunto , Seguimentos , Glioblastoma/classificação , Glioblastoma/tratamento farmacológico , Humanos , Necrose , Prognóstico , Estudos Retrospectivos , Carga TumoralRESUMO
BACKGROUND: The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA) in gliomas has limited the implementation of targeted immune-based therapies. METHODS: We hypothesized that treatment with the demethylating agent, decitabine, would upregulate the expression of TRA on tumor cells, thereby facilitating enhanced surveillance by TRA-specific T cells. RESULTS AND DISCUSSION: Treatment of human glioma cells with decitabine increased the expression of NY-ESO-1 and other well characterized cancer testes antigens. The upregulation of NY-ESO-1 made these tumors susceptible to NY-ESO-1-specific T-cell recognition and lysis. Interestingly, decitabine treatment of T98 glioma cells also sensitized them to Fas-dependent apoptosis with an agonistic antibody, while a Fas blocking antibody could largely prevent the enhanced functional recognition by NY-ESO-1 specific T cells. Thus, decitabine treatment transformed a non-immunogenic glioma cell into an immunogenic target that was efficiently recognized by NY-ESO-1--specific T cells. CONCLUSIONS: Such data supports the hypothesis that agents which alter epigenetic cellular processes may "immunosensitize" tumor cells to tumor-specific T cell-mediated lysis.
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Antígenos de Neoplasias/imunologia , Azacitidina/análogos & derivados , Proteína Ligante Fas/metabolismo , Glioma/tratamento farmacológico , Glioma/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/efeitos dos fármacos , Receptor fas/metabolismo , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Decitabina , Glioma/patologia , Glioma/cirurgia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Terapia de Alvo Molecular , Retroviridae/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética , Regulação para Cima/efeitos dos fármacosRESUMO
Glioblastoma stem cells (GSC) express both radial glial cell and neural crest cell (NCC)-associated genes. We report that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSCs. Serum-induced proliferative differentiation rapidly decreased EDN3 production and downregulated the expression of stemness-associated genes, and reciprocally, two glioblastoma markers, EDN1 and YKL-40 transcripts, were induced. Correspondingly, patient glioblastoma tissues express low levels of EDN3 mRNA and high levels of EDN1 and YKL-40 mRNA. Blocking EDN3/EDN receptor B (EDNRB) signaling by an EDNRB antagonist (BQ788), or EDN3 RNA interference (siRNA), leads to cell apoptosis and functional impairment of tumor sphere formation and cell spreading/migration in culture and loss of tumorigenic capacity in animals. Using exogenous EDN3 as the sole mitogen in culture does not support GSC propagation, but it can rescue GSCs from undergoing cell apoptosis. Molecular analysis by gene expression profiling revealed that most genes downregulated by EDN3/EDNRB blockade were those involved in cytoskeleton organization, pause of growth and differentiation, and DNA damage response, implicating the involvement of EDN3/EDNRB signaling in maintaining GSC migration, undifferentiation, and survival. These data suggest that autocrine EDN3/EDNRB signaling is essential for maintaining GSCs. Incorporating END3/EDNRB-targeted therapies into conventional cancer treatments may have clinical implication for the prevention of tumor recurrence.
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Comunicação Autócrina/genética , Neoplasias Encefálicas/genética , Endotelina-3/metabolismo , Glioblastoma/metabolismo , Crista Neural/metabolismo , Receptor de Endotelina B/metabolismo , Animais , Apoptose , Neoplasias Encefálicas/metabolismo , Diferenciação Celular , Movimento Celular , Transformação Celular Neoplásica , Endotelina-3/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Genoma Humano , Glioblastoma/genética , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas , Crista Neural/citologia , Crista Neural/crescimento & desenvolvimento , RNA Interferente Pequeno , Receptor de Endotelina B/genéticaRESUMO
PURPOSE: To report three low-passage cell lines from primary choroidal melanoma with metastatic outcome, which were stable for cytogenetic patterns and expression profiles of the primary melanoma. METHODS: In patients with choroidal melanoma, transscleral fine needle aspiration biopsy (FNAB) was performed immediately before plaque placement for (125)iodine brachytherapy or immediately after enucleation. Cells were examined for cytopathology, evaluated by fluorescence in-situ hybridization (FISH) for the centromere of chromosome 3, analyzed by 250K whole genome Mapping Array and U133 plus 2.0 Expression Array, and placed in cell culture. At passage 3, the cell lines were analyzed by Mapping Array and Expression Array. RESULTS: Three cell lines were propagated from primary choroidal melanomas in three patients who subsequently developed metastasis. Two cell lines were stable for the entire chromosomal aberration pattern of the respective primary tumor. In the third, necrotic material from the biopsy prevented further analysis, yet resulted in a stable cell line. Each cell line had chromosome 3 loss, 6q loss, 8p loss, multiple 8q gain, and 16q loss. Additionally, two cell lines had chromosome 6p gain. Two cell lines had RNA expression profiles similar to the respective primary tumors; the third cell line had a similar RNA expression profile relative to the other two cell lines. CONCLUSIONS: FNAB of primary choroidal melanomas resulted in highly characterized, low-passage cell lines, which were stable for the cytogenetic patterns and expression profiles found in the primary tumor. These cell lines represent novel tools for the study of metastatic choroidal melanoma biology.
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Linhagem Celular Tumoral/patologia , Neoplasias da Coroide/patologia , Corioide/patologia , Melanoma/patologia , Sequência de Bases , Biópsia por Agulha Fina , Corioide/metabolismo , Neoplasias da Coroide/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 3/genética , Códon/genética , Análise Mutacional de DNA , Regulação para Baixo/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Microscopia de Contraste de Fase , Dados de Sequência Molecular , Monossomia/genética , Metástase Neoplásica , Regulação para Cima/genéticaRESUMO
PURPOSE: To assess the feasibility, safety, and toxicity of autologous tumor lysate-pulsed dendritic cell (DC) vaccination and toll-like receptor (TLR) agonists in patients with newly diagnosed and recurrent glioblastoma. Clinical and immune responses were monitored and correlated with tumor gene expression profiles. EXPERIMENTAL DESIGN: Twenty-three patients with glioblastoma (WHO grade IV) were enrolled in this dose-escalation study and received three biweekly injections of glioma lysate-pulsed DCs followed by booster vaccinations with either imiquimod or poly-ICLC adjuvant every 3 months until tumor progression. Gene expression profiling, immunohistochemistry, FACS, and cytokine bead arrays were performed on patient tumors and peripheral blood mononuclear cells. RESULTS: DC vaccinations are safe and not associated with any dose-limiting toxicity. The median overall survival from the time of initial surgical diagnosis of glioblastoma was 31.4 months, with a 1-, 2-, and 3-year survival rate of 91%, 55%, and 47%, respectively. Patients whose tumors had mesenchymal gene expression signatures exhibited increased survival following DC vaccination compared with historic controls of the same genetic subtype. Tumor samples with a mesenchymal gene expression signature had a higher number of CD3(+) and CD8(+) tumor-infiltrating lymphocytes compared with glioblastomas of other gene expression signatures (P = 0.006). CONCLUSION: Autologous tumor lysate-pulsed DC vaccination in conjunction with TLR agonists is safe as adjuvant therapy in newly diagnosed and recurrent glioblastoma patients. Our results suggest that the mesenchymal gene expression profile may identify an immunogenic subgroup of glioblastoma that may be more responsive to immune-based therapies.
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Vacinas Anticâncer/metabolismo , Células Dendríticas/citologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/mortalidade , Glioblastoma/terapia , Imunoterapia/métodos , Adulto , Idoso , Complexo CD3/biossíntese , Antígenos CD8/biossíntese , Separação Celular , Feminino , Citometria de Fluxo , Glioma , Humanos , Sistema Imunitário , Imuno-Histoquímica , Leucócitos Mononucleares/citologia , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: To identify genomic targets for ciliochoroidal melanoma diagnosis, prognosis, and therapy. METHODS: Fifty-eight ciliochoroidal melanomas were analyzed by high-resolution, genome-wide, single nucleotide polymorphism (SNP) mapping arrays. The 58 SNP arrays were compared to 48 HapMap normals representing both sexes and assessed with a systematic statistical method, Genomic Identification of Significant Targets in Cancer (GISTIC), to identify significant ciliochoroidal chromosomal abnormalities including chromosome-arm-sized as well as focal events of amplification and deletion. The 58 SNP arrays were also analyzed to assess copy number. RESULTS: The 58 ciliochoroidal melanomas analyzed by GISTIC showed large regions of chromosome amplification on 6p and 8q in addition to focal amplification peaks on 1q31.3, 4p16.2, 9p23, and 9q33.1. The melanomas also showed large regions of deletion on 1p and all of 3, 6q, 8p, and 16q, as well as focal deletion peaks on 2p12, 2q14.3, 4q26, 5q21.1, 7q21.11, 8p21.3, 9p21.1, 13q21.31, 13q31.3, and 16q23.3. For each large region and focal peak, the statistical significance was computed, and known genes were specified. CONCLUSIONS: High-resolution analysis of ciliochoroidal melanoma cytogenetic aberration patterns supports the utility of systematic characterization of the cancer genome by corroborating known melanoma-related genomic aberrations and identifying additional melanoma-related genomic abnormalities that can be used to identify potential targets for diagnosis, prognosis and therapy.
Assuntos
Aberrações Cromossômicas , Perfilação da Expressão Gênica , Melanoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Neoplasias Uveais/genética , Neoplasias da Coroide/genética , Neoplasias da Coroide/patologia , Corpo Ciliar/patologia , DNA de Neoplasias/genética , Feminino , Genes Neoplásicos , Genômica , Genótipo , Humanos , Masculino , Melanoma/patologia , Neoplasias Uveais/patologiaRESUMO
The contribution of inflammation to neurodegenerative diseases is increasingly recognized, but the role of inflammation in sporadic amyotrophic lateral sclerosis (sALS) is not well understood and no animal model is available. We used enzyme-linked immunosorbent assays (ELISAs) to measure the cytokine interleukin-17A (IL-17A) in the serum of ALS patients (n = 32; 28 sporadic ALS (sALS) and 4 familial ALS (fALS)) and control subjects (n = 14; 10 healthy subjects and 4 with autoimmune disorders). IL-17A serum concentrations were 5767 ± 2700 pg/ml (mean ± SEM) in sALS patients and 937 ± 927 pg/ml in fALS patients in comparison to 7 ± 2 pg/ml in control subjects without autoimmune disorders (p = 0.008 ALS patients vs. control subjects by Mann-Whitney test). Sixty-four percent of patients and no control subjects had IL-17A serum concentrations > 50 pg/ml (p = 0.003 ALS patients vs. healthy subjects by Fisher's exact test). The spinal cords of sALS (n = 8), but not control subjects (n = 4), were infiltrated by interleukin-1ß- (IL-1ß-), and tumor necrosis factor-α-positive macrophages (co-localizing with neurons), IL-17A-positive CD8 cells, and IL-17A-positive mast cells. Mononuclear cells treated with aggregated forms of wild type superoxide dismutase-1 (SOD-1) showed induction of the cytokines IL-1ß, interleukin-6 (IL-6), and interleukin-23 (IL-23) that may be responsible for induction of IL-17A. In a microarray analysis of 28,869 genes, stimulation of peripheral blood mononuclear cells by mutant superoxide dismutase-1 induced four-fold higher transcripts of interleukin-1α (IL-1α), IL-6, CCL20, matrix metallopeptidase 1, and tissue factor pathway inhibitor 2 in mononuclear cells of patients as compared to controls, whereas the anti-inflammatory cytokine interleukin-10 (IL-10) was increased in mononuclear cells of control subjects. Aggregated wild type SOD-1 in sALS neurons could induce in mononuclear cells the cytokines inducing chronic inflammation in sALS spinal cord, in particular IL-6 and IL-17A, damaging neurons. Immune modulation of chronic inflammation may be a new approach to sALS.
Assuntos
Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-17 , Mastócitos/imunologia , Medula Espinal/citologia , Medula Espinal/imunologia , Superóxido Dismutase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/patologia , Estudos Transversais , Citocinas/sangue , Citocinas/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-17/sangue , Interleucina-17/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Mastócitos/citologia , Pessoa de Meia-Idade , Mutação , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30x genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate-pair information identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (<21 bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in PTEN was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions, and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date.
Assuntos
Linhagem Celular Tumoral/química , Genoma Humano , Glioma/genética , Linhagem Celular Tumoral/citologia , Genótipo , Humanos , Dados de Sequência Molecular , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Análise de Sequência de DNARESUMO
Current therapies for glioblastoma (GBM) target bulk tumor through measures such as resection and radiotherapy. However, recent evidence suggests that targeting a subset of tumor cells, so-called cancer stem cells, may be critical for inhibiting tumor growth and relapse. The subventricular zone (SVZ), which lines the ventricles of the brain, is thought to be the origin for the majority of neural stem cells and potentially cancer stem cells. Therefore, we assessed the relationship between tumor contact with the SVZ as determined by MRI, cancer stem cell gene expression and survival in 47 patients with GBM. Using DNA microarrays, we found that genes associated with cancer stem cells were not over-expressed in tumors contacting the SVZ. Contact with the SVZ trended with shorter survival (median 358 versus 644, P = 0.066). Over-expression of CD133 (prominin-1) and maternal embryonic leucine zipper kinase (MELK) was associated with shorter survival, whereas mitogen activated protein kinase 8 (MAPK8) was associated with longer survival (P values 0.008, 0.005 and 0.002 respectively). Thus we found no evidence of a stem-cell derived genetic signature specific for GBM in contact with the SVZ, but there was a relationship between stem cell gene expression and survival. More research is required to clarify the relationship between the SVZ, cancer stem cells and survival.