RESUMO
Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. SIGNIFICANCE: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.
Assuntos
Redes Reguladoras de Genes , Neoplasias Pulmonares , Humanos , Elementos Facilitadores Genéticos/genética , Neoplasias Pulmonares/genética , Mutação , Carcinogênese/genéticaRESUMO
OBJECTIVE: 20% of sporadic MTC has no RET/RAS somatic alterations or other known gene alterations. Aim of this study was to investigate RET/RAS negative MTC for the presence of NF1 alterations. METHODS: we studied 18 sporadic RET/RAS negative MTC cases: Next generation sequencing of tumoral and blood DNA was performed using a custom panel including the entire coding region of the NF1 gene. The effect of NF1 alterations on the transcripts were characterized by RT-PCR and the loss of heterozygosity of the other NF1 allele was investigated with Multiplex Ligation-dependent Probe Amplification. RESULTS: Two cases showed bi-allelic inactivation of NF1 with a prevalence of about 11% of RET/RAS negative cases. In a patient affected by neurofibromatosis there was a somatic intronic point mutation determining the transcript alteration in one allele and a germline loss of heterozygosity (LOH) in the other. In the other case described both the point mutation and the LOH were somatic events; this latter finding shows, for the first time, a driver role of NF1 inactivation in MTC independent of RET/RAS alterations and the presence of neurofibromatosis. CONCLUSIONS: About 11% of our series of sporadic RET/RAS negative MTC harbor biallelic inactivation of NF1 suppressor gene also regardless neurofibromatosis status. According to our results, NF1 alterations should be searched in all RET/RAS negative MTC as possible driver. Moreover, this finding reduces the number of negative sporadic MTCs and may have important clinical implications in the management of these tumors.
RESUMO
Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.
Assuntos
Neoplasias Colorretais , Neoplasias Peritoneais , Humanos , Matriz Extracelular Descelularizada , Peritônio , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/terapia , Organoides , Neoplasias Colorretais/metabolismoRESUMO
The anthocyanin class of flavonoids, including cyanidin-3-glucoside (C3G) present in berries, blood oranges and pigmented cereal crops, are food bioactives with antioxidant and anti-inflammatory action, capable to reduce myocardial ischemia/reperfusion (I/R) injury by unclear mechanism. Assessing the value of sporadic beneficial diet is critical for practical application. We aimed to determine whether and how the cardioptotective effect of dietary intake of anthocyanins persists. Gene expression, histology and resistance to I/R were investigated ex vivo in hearts from mice after a month beyond the cease of the C3G-enriched diet. Cardiac injury, oxidative stress and mitochondrial damage following I/R was effectively reduced in mice fed C3G-enriched diet, even after a month of wash out with standard diet. Cardioprotection was observed also in immune-deficient mice lacking mature B and T cells indicating the anti-inflammatory activity of C3G was not involved. Moreover, the transcription reprogramming induced by the C3G-enriched diets was rescued by the wash out treatment. Instead, we found C3G-enriched diet changed the microbiome and the transplantation of the fecal microbiota transferred the cardioprotection from mice fed C3G-enriched diet to mice fed standard diet. These findings established the effect of C3G dietary intake on gut microbiota determines long lasting cardioprotection.
Assuntos
Antocianinas/administração & dosagem , Cardiotônicos , Dieta , Microbioma Gastrointestinal , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Ingestão de Alimentos , Transplante de Microbiota Fecal , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Mitocôndrias Cardíacas/metabolismoRESUMO
Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Diferenciação Celular , Proliferação de Células/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Quimera , Cromatografia Líquida , Clonagem Molecular , Dano ao DNA , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Espectrometria de Massas em TandemRESUMO
PurposeMonoallelic germ-line mutations in the BRCA1/FANCS, BRCA2/FANCD1 and PALB2/FANCN genes confer high risk of breast cancer. Biallelic mutations in these genes cause Fanconi anemia (FA), characterized by malformations, bone marrow failure, chromosome fragility, and cancer predisposition (BRCA2/FANCD1 and PALB2/FANCN), or an FA-like disease presenting a phenotype similar to FA but without bone marrow failure (BRCA1/FANCS). FANCM monoallelic mutations have been reported as moderate risk factors for breast cancer, but there are no reports of any clinical phenotype observed in carriers of biallelic mutations.MethodsBreast cancer probands were subjected to mutation analysis by sequencing gene panels or testing DNA damage response genes.ResultsFive cases homozygous for FANCM loss-of-function mutations were identified. They show a heterogeneous phenotype including cancer predisposition, toxicity to chemotherapy, early menopause, and possibly chromosome fragility. Phenotype severity might correlate with mutation position in the gene.ConclusionOur data indicate that biallelic FANCM mutations do not cause classical FA, providing proof that FANCM is not a canonical FA gene. Moreover, our observations support previous findings suggesting that FANCM is a breast cancer-predisposing gene. Mutation testing of FANCM might be considered for individuals with the above-described clinical features.
Assuntos
Alelos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Fragilidade Cromossômica , DNA Helicases/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Predisposição Genética para Doença , Mutação , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Consanguinidade , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Estudos de Associação Genética , Genótipo , Mutação em Linhagem Germinativa , Humanos , Masculino , Linhagem , Fenótipo , Medição de Risco , Fatores de RiscoRESUMO
UNLABELLED: The canonical Wnt/ß-catenin signaling pathway is crucial for blood-brain barrier (BBB) formation in brain endothelial cells. Although glucose transporter 1, claudin-3, and plasmalemma vesicular-associated protein have been identified as Wnt/ß-catenin targets in brain endothelial cells, further downstream targets relevant to BBB formation and function are incompletely explored. By Affymetrix expression analysis, we show that the cytochrome P450 enzyme Cyp1b1 was significantly decreased in ß-catenin-deficient mouse endothelial cells, whereas its close homolog Cyp1a1 was upregulated in an aryl hydrocarbon receptor-dependent manner, hence indicating that ß-catenin is indispensable for Cyp1b1 but not for Cyp1a1 expression. Functionally, Cyp1b1 could generate retinoic acid from retinol leading to cell-autonomous induction of the barrier-related ATP-binding cassette transporter P-glycoprotein. Cyp1b1 could also generate 20-hydroxyeicosatetraenoic acid from arachidonic acid, decreasing endothelial barrier function in vitro In mice in vivo pharmacological inhibition of Cyp1b1 increased BBB permeability for small molecular tracers, and Cyp1b1 was downregulated in glioma vessels in which BBB function is lost. Hence, we propose Cyp1b1 as a target of ß-catenin indirectly influencing BBB properties via its metabolic activity, and as a potential target for modulating barrier function in endothelial cells. SIGNIFICANCE STATEMENT: Wnt/ß-catenin signaling is crucial for blood-brain barrier (BBB) development and maintenance; however, its role in regulating metabolic characteristics of endothelial cells is unclear. We provide evidence that ß-catenin influences endothelial metabolism by transcriptionally regulating the cytochrome P450 enzyme Cyp1b1 Furthermore, expression of its close homolog Cyp1a1 was inhibited by ß-catenin. Functionally, Cyp1b1 generated retinoic acid as well as 20-hydroxyeicosatetraenoic acid that regulated P-glycoprotein and junction proteins, respectively, thereby modulating BBB properties. Inhibition of Cyp1b1 in vivo increased BBB permeability being in line with its downregulation in glioma endothelia, potentially implicating Cyp1b1 in other brain pathologies. In conclusion, Wnt/ß-catenin signaling regulates endothelial metabolic barrier function through Cyp1b1 transcription.
Assuntos
Barreira Hematoencefálica/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Caderinas/genética , Caderinas/metabolismo , Permeabilidade Capilar/genética , Imunoprecipitação da Cromatina , Citocromo P-450 CYP1B1/genética , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/genética , Glioma/metabolismo , Glioma/patologia , Histonas/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Masculino , Camundongos , Camundongos Nus , Modelos Biológicos , Transplante de Neoplasias , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
Oxidative stress is a determining factor of cellular senescence and aging and a potent inducer of the tumour-suppressor p53. Resistance to oxidative stress correlates with delayed aging in mammals, in the absence of accelerated tumorigenesis, suggesting inactivation of selected p53-downstream pathways. We investigated p53 regulation in mice carrying deletion of p66, a mutation that retards aging and confers cellular resistance and systemic resistance to oxidative stress. We identified a transcriptional network of ~200 genes that are repressed by p53 and encode for determinants of progression through mitosis or suppression of senescence. They are selectively down-regulated in cultured fibroblasts after oxidative stress, and, in vivo, in proliferating tissues and during physiological aging. Selectivity is imposed by p66 expression and activation of p44/p53 (also named Delta40p53), a p53 isoform that accelerates aging and prevents mitosis after protein damage. p66 deletion retards aging and increases longevity of p44/p53 transgenic mice. Thus, oxidative stress activates a specific p53 transcriptional response, mediated by p44/p53 and p66, which regulates cellular senescence and aging.
Assuntos
Senescência Celular/fisiologia , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Células Cultivadas , Senescência Celular/genética , Hepatócitos/metabolismo , Longevidade , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Timo/metabolismo , Fatores de Transcrição , Transcrição Gênica , Proteína Supressora de Tumor p53/genéticaRESUMO
Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions.
Assuntos
Antígenos de Neoplasias/metabolismo , Cromossomos Fúngicos , Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Loci Gênicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Regiões Terminadoras Genéticas , Antígenos de Neoplasias/genética , Divisão Celular , Fragilidade Cromossômica , Quebras de DNA , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Fúngico/química , Proteínas de Ligação a DNA/genética , Fase G2 , Rearranjo Gênico , Mutação , Conformação de Ácido Nucleico , Fase S , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Previous studies showed that p66(Shc-/-) mice on a very-high-fat diet (HFD) had reduced oxidative stress, foam cell, and early atherosclerotic lesion formation. Here, the authors have used hypercholesterolemic apolipoprotein E (ApoE(-/-)) mice to investigate the role of p66Shc deletion in advanced atheroma. The authors generated mice deficient of both ApoE and p66Shc genes (ApoE(-/-) /p66(Shc-/-)). They used microsatellite polymerase chain reaction (PCR) analysis to analyze the genetic background and considered only animals with a constant percentages of C57B6L and 129SV background strands (it was obtained the 50.3% +/- 6.4% of C57B6L background). Computer-assisted analysis revealed that advanced atherosclerotic lesions in ApoE(-/-)/p66(Shc+/+) were significantly larger than those observed in ApoE(-/-)/p66(Shc-/-). Accordingly, the lipid-laden macrophage foam cells and oxidation-specific epitopes in ApoE(-/-)/p66(shc+/+) HFD-treated groups were higher than those observed in normal diet (ND)-treated groups. Thus, p66(Shc-/-) plays an important protective role also against advanced atherosclerotic lesion formation. Finally, the authors have used microarray to investigate major changes in gene expression in aortas of mice with ApoE(-/-)/p66(Shc-/-) background treated with a very HFD in comparison to ApoE(-/-)/p66(Shc+/+) (these data have been confirmed by by real-time PCR and immunohistochemistry). DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis revealed that CD36 antigen (CD36), tissue inhibitor of metalloproteinase 2 (TIMP2), apolipoprotein E (ApoE), acetyl-coenzyme A acetyltransferase 1 (ACAT1), and thrombospondin 1 (THBS1) can be involved in p66 deletion-dependent vascular protection through the adipocytokine/lipid signaling pathway.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Adipocinas/genética , Adipocinas/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Apolipoproteínas E/metabolismo , Aterosclerose/fisiopatologia , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Regulação para Baixo/genética , Células Espumosas/metabolismo , Células Espumosas/patologia , Alimentos Formulados/efeitos adversos , Deleção de Genes , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Genótipo , Hipercolesterolemia/fisiopatologia , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Fusão Oncogênica/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromossomos Humanos Par 19/genética , Células HeLa , Humanos , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Transcrição Gênica , Células U937RESUMO
Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G(1) phase of the cell cycle or at the G(2)/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.
Assuntos
Histona Desacetilases/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Acetilação , Morte Celular , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Análise por Conglomerados , Inibidor de Quinase Dependente de Ciclina p21 , Fase G1 , Fase G2 , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilase 2 , Histona Desacetilases/deficiência , Histonas/metabolismo , Humanos , Neoplasias/genética , Fosforilação , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismoRESUMO
PURPOSE: To delineate clinically relevant molecular signatures of intracranial ependymoma. MATERIALS AND METHODS: We analyzed 24 primary intracranial ependymomas. For genomic profiling, microarray-based comparative genomic hybridization (CGH) was used and results were validated by fluorescent in situ hybridization and loss of heterozygosity mapping. We performed gene expression profiling using microarrays, real-time quantitative reverse transcriptase polymerase chain reaction, and methylation analysis of selected genes. We applied class comparison analyses to compare both genomic and expression profiling data with clinical characteristics. RESULTS: A variable number of genomic imbalances were detected by array CGH, revealing multiple regions of recurrent gain (including 2q23, 7p21, 12p, 13q21.1, and 20p12) and loss (including 5q31, 6q26, 7q36, 15q21.1, 16q24, 17p13.3, 19p13.2, and 22q13.3). An ependymoma-specific gene expression signature was characterized by the concurrent abnormal expression of developmental and differentiation pathways, including NOTCH and sonic hedgehog signaling. We identified specific differentially imbalanced genomic clones and gene expression signatures significantly associated with tumor location, patient age at disease onset, and retrospective risk for relapse. Integrated genomic and expression profiling allowed us to identify genes of which the expression is deregulated in intracranial ependymoma, such as overexpression of the putative proto-oncogene YAP1 (located at 11q22) and downregulation of the SULT4A1 gene (at 22q13.3). CONCLUSION: The present exploratory molecular profiling study allowed us to refine previously reported intervals of genomic imbalance, to identify novel restricted regions of gain and loss, and to identify molecular signatures correlating with various clinical variables. Validation of these results on independent data sets represents the next step before translation into the clinical setting.
Assuntos
Neoplasias Encefálicas/genética , Ependimoma/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Criança , Pré-Escolar , Metilação de DNA , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Lactente , Perda de Heterozigosidade , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Proto-Oncogene Mas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genética , Fatores de Transcrição , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
Approximately one third of acute myeloid leukemias (AMLs) are characterized by aberrant cytoplasmic localization of nucleophosmin (NPMc+ AML), consequent to mutations in the NPM putative nucleolar localization signal. These events are mutually exclusive with the major AML-associated chromosomal rearrangements, and are frequently associated with normal karyotype, FLT3 mutations, and multilineage involvement. We report the gene expression profiles of 78 de novo AMLs (72 with normal karyotype; 6 without major chromosomal abnormalities) that were characterized for the subcellular localization and mutation status of NPM. Unsupervised clustering clearly separated NPMc+ from NPMc- AMLs, regardless of the presence of FLT3 mutations or non-major chromosomal rearrangements, supporting the concept that NPMc+ AML represents a distinct entity. The molecular signature of NPMc+ AML includes up-regulation of several genes putatively involved in the maintenance of a stem-cell phenotype, suggesting that NPMc+ AML may derive from a multipotent hematopoietic progenitor.
Assuntos
Citoplasma/química , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Proteínas Nucleares/análise , Regulação para Cima/genética , Doença Aguda , Linhagem da Célula , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas , Leucemia Mieloide/classificação , Proteínas de Neoplasias/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Tirosina Quinase 3 Semelhante a fmsRESUMO
Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by a block of differentiation at the promyelocytic stage. APL patients respond to pharmacological concentrations of all-trans retinoic acid (RA) and disease remission correlates with terminal differentiation of leukemic blasts. The PML/RAR oncogenic transcription factor is responsible for both the pathogenesis of APL and for its sensitivity to RA. In order to identify physiological targets of RA therapy, we analysed gene expression profiles of RA-treated APL blasts and found 1056 common target genes. Comparing these results to those obtained in RA-treated U937 cell lines revealed that transcriptional response to RA is largely dependent on the expression of PML/RAR. Several genes involved in the control of differentiation and stem cell renewal are early targets of RA regulation, and may be important effectors of RA response. Modulation of chromatin modifying genes was also observed, suggesting that specific structural changes in local chromatin domains may be required to promote RA-mediated differentiation. Computational analysis of upstream genomic regions in RA target genes revealed nonrandom distribution of transcription factor binding sites, indicating that specific transcriptional regulatory complexes may be involved in determining RA response.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Tretinoína/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/metabolismo , Análise por Conglomerados , Éxons , Humanos , Leucemia Promielocítica Aguda/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Transcrição Gênica , Tretinoína/metabolismo , Células Tumorais Cultivadas , Células U937RESUMO
UNLABELLED: GenePicker allows efficient analysis of Affymetrix gene expression data performed in replicate, through definition of analysis schemes, data normalization, t-test/ANOVA, Change-Fold Change-analysis and yields lists of differentially expressed genes with high confidence. Comparison of noise and signal analysis schemes allows determining a signal-to-noise ratio in a given experiment. Change Call, Fold Change and Signal mean ratios are used in the analysis. While each parameter alone yields gene lists that contain up to 30% false positives, the combination of these parameters nearly eliminates the false positives as verified by northern blotting, quantitative PCR in numerous independent experiments as well as by the analysis of spike-in data. AVAILABILITY: http://www.ifom-firc.it/RESEARCH/Appl_Bioinfo/tools.html. SUPPLEMENTARY INFORMATION: http://www.ifom-firc.it/RESEARCH/Appl_Bioinfo/tools.html.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Software , Interface Usuário-Computador , Análise de Sequência com Séries de Oligonucleotídeos/instrumentaçãoRESUMO
Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.