RESUMO
High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/patologia , Neoplasias Encefálicas/metabolismo , Glioma/genética , Prognóstico , DNA , Isocitrato Desidrogenase/genética , MutaçãoRESUMO
Microglia, resident myeloid cells of the central nervous system (CNS), act as immune sentinels that contribute to maintenance of physiological homeostasis and respond to any perturbation in CNS. Microglia could be polarized by various stimuli to perform dedicated functions and instigate inflammatory or pro-regenerative responses. Microglia and peripheral macrophages accumulate in glioblastomas (GBMs), malignant brain tumors, but instead of initiating antitumor responses, these cells are polarized to the pro-invasive and immunosuppressive phenotype which persists for a long time and contributes to a "cold" immune microenvironment of GBMs. Molecular mechanisms underlying this long-lasting "microglia memory" are unknown. We hypothesized that this state may rely on epigenetic silencing of inflammation-related genes. In this study, we show that cultured microglia pre-exposed to glioma-conditioned medium (GCM) acquire a "transcriptional memory" and display reduced expression of inflammatory genes after re-stimulation with lipopolysaccharide. Unstimulated microglia have unmethylated DNA and active histone marks at selected gene promoters indicating chromatin accessibility. Adding GCM increases expression and enzymatic activity of histone deacetylases (Hdac), leading to erasure of histone acetylation at tested genes. Later inflammatory genes acquire repressive histone marks (H3K27 trimethylation), which correlates with silencing of their expression. GCM induced genes acquire active histone marks. Hdac inhibitors block GCM-induced changes of histone modifications and restore microglia ability to initiate effective inflammatory responses. Altogether, we show a scenario of distinct histone modifications underlying polarization of microglia by glioma. We demonstrate contribution of epigenetic mechanisms to glioma-induced "transcriptional memory" in microglia resulting in the tumor-supportive phenotype.
Assuntos
Glioma , Microglia , Meios de Cultivo Condicionados/farmacologia , Epigênese Genética , Glioma/genética , Código das Histonas , Humanos , Lipopolissacarídeos/farmacologia , Microambiente TumoralRESUMO
Signal transduction pathways directly communicate and transform chromatin to change the epigenetic landscape and regulate gene expression. Chromatin acts as a dynamic platform of signal integration and storage. Histone modifications and alteration of chromatin structure play the main role in chromatin-based gene expression regulation. Alterations in genes coding for histone modifying enzymes and chromatin modifiers result in malfunction of proteins that regulate chromatin modification and remodeling. Such dysregulations culminate in profound changes in chromatin structure and distorted patterns of gene expression. Gliomagenesis is a multistep process, involving both genetic and epigenetic alterations. Recent applications of next generation sequencing have revealed that many chromatin regulation-related genes, including ATRX, ARID1A, SMARCA4, SMARCA2, SMARCC2, BAF155 and hSNF5 are mutated in gliomas. In this review we summarize newly identified mechanisms affecting expression or functions of selected histone modifying enzymes and chromatin modifiers in gliomas. We focus on selected examples of pathogenic mechanisms involving ATRX, histone methyltransferase G9a, histone acetylases/deacetylases and chromatin remodeling complexes SMARCA2/4. We discuss the impact of selected epigenetics alterations on glioma pathobiology, signaling and therapeutic responses. We assess the attempts of targeting defective pathways with new inhibitors.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Cromatina/metabolismo , Glioma/tratamento farmacológico , Glioma/patologia , Histonas/metabolismo , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Glioma/enzimologia , Glioma/genética , Código das Histonas/efeitos dos fármacos , Histonas/química , HumanosRESUMO
Transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers and promotes uncontrolled tumor growth and progression through multiple mechanisms. Compelling evidence shows tissue and cell-specific sets of STAT3 targets. Transcriptional targets of STAT3 in melanoma cells are largely unknown. Malignant melanoma is a deadly disease with highly aggressive and drug-resistant behavior. Less than 10% of patients with advanced melanomas reach the 5-year survival, partly due to the aggressive character of the tumor and ineffectiveness of current therapeutics for treating metastatic melanoma. STAT3 is constitutively activated in melanoma cells and plays important roles in its growth and angiogenesis in tumor xenograft studies. Moreover, highly metastatic melanoma cells have higher levels of active STAT3 than poorly metastatic ones. To identify genes that are driven by STAT3 in human melanoma cells, we performed JAK/STAT signaling specific and global gene expression profiling of human melanoma cells with silenced STAT3 expression. For selected genes, we performed computational identification of putative STAT3-binding sites and validated direct interactions STAT3 with defined promoters by using chromatin immunoprecipitation followed by qPCR. We found that STAT3 knockdown does not affect human melanoma cell viability, proliferation, or response to chemotherapeutics. We show that STAT3 regulates a discrete set of genes in melanoma cells, including SERPINA3, a novel STAT3 target gene, which is functionally involved in regulation of melanoma migration and invasion. Knockdown of STAT3 impaired cell migration and invasion, in part via regulation of its transcriptional target SERPINA3. Our results present novel targets and functions of STAT3 in melanoma cells.
Assuntos
Melanoma/genética , Melanoma/patologia , Fator de Transcrição STAT3/genética , Serpinas/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Melanoma/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fosforilação , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Gene expression of both normal and cancer cell is tightly regulated by specific transcription regulators and epigenetic mechanisms such as DNA methylation, histone modifications (acetylation, methylation, phosphorylation), nucleosome remodeling and non-coding RNAs. Deregulation of epigenetic mechanisms plays a pivotal role in cancer, although researchers debate if it is a cause or a consequence of oncogenic transformation. Independently from the way in which epigenetic alterations arise in cancer, downstream effects will result in profound changes in transcriptomic and subsequently proteomic profiles. In most cases, changes in expression of epigenetic genes produce functional advantages in cell proliferation, tumor growth and/or migration capacity. Most of epigenetic changes in cancer are triggered by genomic alterations in specific genes that are involved in controlling one of the epigenetic mechanisms. However, there are also mutations in cell metabolism-related genes that affect activities of DNA demethylating enzymes and histone modifiers. Histone modifications are deregulated in cancer mostly due to alterations in genes coding for enzymes that attach or remove histone modifications. Mutations in genes coding for nucleosome remodelers result in aberrant global chromatin organization and facilitate subsequent global alterations of gene copy number or translocations. Recent advancements in next generation sequencing allowed for more precise mapping of global changes in the epigenetic landscape in cancer.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Metilação de DNA , Histonas/química , Histonas/metabolismo , Humanos , ProteômicaRESUMO
The epigenetic landscape is deregulated in cancer due to aberrant activation or inactivation of enzymes maintaining and modifying the epigenome. Histone modifications and global aberrations at the histone level may result in distorted patterns of gene expression, and malfunction of proteins that regulate chromatin modification and remodeling. Recent whole genome studies demonstrated that histones and chaperone proteins harbor mutations that may result in gross alterations of the epigenome leading to genome instability. Glioma development is a multistep process, involving genetic and epigenetic alterations. This review summarizes newly identified mechanisms affecting expression/functions of histone-modifying enzymes and chromatin modifiers in gliomas. We discuss recent approaches to overcome epigenetic alterations with histone-modifying enzyme inhibitors and their prospects for glioma therapy.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Glioma/etiologia , Glioma/metabolismo , Histonas/metabolismo , Fatores Etários , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/mortalidade , Glioma/patologia , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/genética , Humanos , Mutação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Nucleossomos/metabolismoRESUMO
Gliomas attract brain-resident (microglia) and peripheral macrophages and reprogram these cells into immunosuppressive, pro-invasive cells. M-CSF (macrophage colony-stimulating factor, encoded by the CSF1 gene) has been implicated in the control of recruitment and polarization of macrophages in several cancers. We found that murine GL261 glioma cells overexpress GM-CSF (granulocyte-macrophage colony-stimulating factor encoded by the CSF2 gene) but not M-CSF when compared to normal astrocytes. Knockdown of GM-CSF in GL261 glioma cells strongly reduced microglia-dependent invasion in organotypical brain slices and growth of intracranial gliomas and extended animal survival. The number of infiltrating microglia/macrophages (Iba1(+) cells) and intratumoural angiogenesis were reduced in murine gliomas depleted of GM-CSF. M1/M2 gene profiling in sorted microglia/macrophages suggests impairment of their pro-invasive activation in GM-CSF-depleted gliomas. Deficiency of M-CSF (op/op mice) did not affect glioma growth in vivo and the accumulation of Iba1(+) cells, but impaired accumulation of Iba1(+) cells in response to demyelination. These results suggest that distinct cytokines of the CSF family contribute to macrophage infiltration of tumours and in response to injury. The expression of CSF2 (but not CSF1) was highly up-regulated in glioblastoma patients and we found an inverse correlation between CSF2 expression and patient survival. Therefore we propose that GM-CSF triggers and drives the alternative activation of tumour-infiltrating microglia/macrophages in which these cells support tumour growth and angiogenesis and shape the immune microenvironment of gliomas.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Ativação de Macrófagos , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Mutação , Invasividade Neoplásica , Fenótipo , RNA Neoplásico/genéticaRESUMO
Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Due to its fast proliferation, diffusive growth and therapy resistance survival times are less than two years for patients with IDH-wildtype GBM. GBM is noted for the considerable cellular heterogeneity, high stemness indices and abundance of the glioma stem-like cells known to support tumor progression, therapeutic resistance and recurrence. Doublesex- and mab-3-related transcription factor a2 (DMRTA2) is involved in maintaining neural progenitor cells (NPC) in the cell cycle and its overexpression suppresses NPC differentiation. Despite the reports showing that primary GBM originates from transformed neural stem/progenitors cells, the role of DMRTA2 in gliomagenesis has not been elucidated so far. Here we show the upregulation of DMRTA2 expression in malignant gliomas. Immunohistochemical staining showed the protein concentrated in small cells with high proliferative potential and cells localized around blood vessels, where it colocalizes with pericyte-specific markers. Knock-down of DMRTA2 in human glioma cells impairs proliferation but not viability of the cells, and affects the formation of the tumor spheres, as evidenced by strong decrease in the number and size of spheres in in vitro cultures. Moreover, the knockdown of DMRTA2 in glioma spheres affects the stabilization of the glioma stem-like cell-dependent tube formation in an in vitro angiogenesis assay. We conclude that DMRTA2 is a new player in gliomagenesis and tumor neovascularization and due to its high expression in malignant gliomas could be a biomarker and potential target for new therapeutic strategies in glioblastoma.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Células-Tronco Neurais , Adulto , Humanos , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Glioblastoma/metabolismo , Glioma/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Microglia are myeloid cells residing in the central nervous system that participate in inflammatory responses and could promote injury and repair. Gliomas attract microglia and polarize them into tumor-supporting cells that participate in matrix remodeling, invasion, angiogenesis, and suppression of adaptive immunity. Although signaling pathways and critical regulators underlying classical inflammation are well established, signal transduction and transcriptional circuits underlying the alternative activation of microglia are poorly known. Using primary rat microglial cultures exposed to glioma conditioned medium or lipopolysaccharide (LPS), we demonstrate that microglia adapt different fates and polarize into pro-inflammatory or alternatively activated cells. Glioma-derived factors increased cell motility, phagocytosis, and sustained proliferation of microglial cells that was mediated by enhanced focal adhesion kinase and PI-3K/Akt signaling. The signals from glioma cells induced ERK and p38 MAPK but not JNK signaling and failed to activate pro-inflammatory Stat1 and NFκB signaling in microglial cells. Transcriptome analysis of microglial cultures at 6 h after exposure to glioma-conditioned medium or LPS revealed different patterns of gene expression. Glioma-induced activation was associated with induction of genes coding for ID (inhibitor of DNA binding) 1/3 and c-Myc, markers of the alternative phenotype Arg1, MT1-MMP, CXCL14, and numerous cytokines/chemokines implicated in immune cell trafficking. Many classical inflammation-related genes and signaling pathways failed to be induced. Our study indicates for the first time molecular pathways that direct microglia toward the pro-invasive, immunosuppressive phenotype.
Assuntos
Carcinogênese/genética , Movimento Celular/fisiologia , Glioma/patologia , Microglia/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Carcinogênese/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Lipopolissacarídeos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Quinase Induzida por NF-kappaBRESUMO
High-grade gliomas (HGGs), the most common and aggressive primary brain tumors in adults, inevitably recur due to incomplete surgery or resistance to therapy. Intratumoral genomic and cellular heterogeneity of HGGs contributes to therapeutic resistance, recurrence, and poor clinical outcomes. Transcriptomic profiles of HGGs at recurrence have not been investigated in detail. Using targeted sequencing of cancer-related genes and transcriptomics, we identified single nucleotide variations, small insertions and deletions, copy number aberrations (CNAs), as well as gene expression changes and pathway deregulation in 16 pairs of primary and recurrent HGGs. Most of the somatic mutations identified in primary HGGs were not detected after relapse, suggesting a subclone substitution during the tumor progression. We found a novel frameshift insertion in the ZNF384 gene which may contribute to extracellular matrix remodeling. An inverse correlation of focal CNAs in EGFR and PTEN genes was detected. Transcriptomic analysis revealed downregulation of genes involved in messenger RNA splicing, cell cycle, and DNA repair, while genes related to interferon signaling and phosphatidylinositol (PI) metabolism are upregulated in secondary HGGs when compared to primary HGGs. In silico analysis of the tumor microenvironment identified M2 macrophages and immature dendritic cells as enriched in recurrent HGGs, suggesting a prominent immunosuppressive signature. Accumulation of those cells in recurrent HGGs was validated by immunostaining. Our findings point to a substantial transcriptomic deregulation and a pronounced infiltration of immature dendritic cells in recurrent HGG, which may impact the effectiveness of frontline immunotherapies in the GBM management. KEY MESSAGES: Most of the somatic mutations identified in primary HGGs were not detected after relapse. Focal CNAs in EGFR and PTEN genes are inversely correlated in primary and recurrent HGGs. Transcriptomic changes and distinct immune-related signatures characterize HGG recurrence. Recurrent HGGs are characterized by a prominent infiltration of immature dendritic and M2 macrophages.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Glioma/genética , Glioma/imunologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Adulto , Idoso , Variações do Número de Cópias de DNA , Células Dendríticas/imunologia , Receptores ErbB/genética , Feminino , Humanos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , PTEN Fosfo-Hidrolase/genética , Transativadores/genética , Transcriptoma , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
To address the role of chromatin structure in the establishment of hematopoietic stem cell (HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed histone modifications at a panel of lymphoid- and myeloid-affiliated genes in multipotent and lineage-committed hematopoietic cells isolated from human cord blood. Our results show that many B- and T-lymphoid genes, although silent in HSCs, are associated with acetylated histones H3 and H4. We also detected histone H3 lysine 4 methylation but not repressive lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure. Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific loci was high in multipotent CD34(+)CD38(lo) progenitors and decreased as they become actively transcribed in B-lineage cells. In vitro differentiation of CD34(+) cells toward the erythroid, granulocyte, and T-cell lineages resulted in a loss of histone acetylation at nonlineage-associated genes. This study provides evidence that histone modifications involved in chromatin decondensation are already in place at lymphoid-specific genes in primary human HSCs, supporting the idea that these genes are "primed" for expression before lineage commitment. This permissive chromatin structure is progressively lost as the stem cell differentiates.
Assuntos
Genes , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Linfócitos/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Acetilação , Antígenos CD19/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem da Célula , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Linfócitos/citologia , Células Mieloides/citologia , Células Mieloides/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismoRESUMO
To further clarify the contribution of nuclear architecture in the regulation of gene expression patterns during differentiation of human multipotent cells, we analyzed expression status, histone modifications, and subnuclear positioning relative to repressive compartments, of hematopoietic loci in multipotent and lineage-committed primary human hematopoietic progenitors. We report here that positioning of lineage-affiliated loci relative to pericentromeric heterochromatin compartments (PCH) is identical in multipotent cells from various origins and is unchanged between multipotent and lineage-committed hematopoietic progenitors. However, during differentiation of multipotent hematopoietic progenitors, changes in gene expression and histone modifications at these loci occur in committed progenitors, prior to changes in gene positioning relative to pericentromeric heterochromatin compartments, detected at later stages in precursor and mature cells. Therefore, during normal human hematopoietic differentiation, changes in gene subnuclear location relative to pericentromeric heterochromatin appear to be dictated by whether the gene will be permanently silenced or activated, rather than being predictive of commitment toward a given lineage.
Assuntos
Compartimento Celular , Cromatina/metabolismo , Ordem dos Genes , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Multipotentes/metabolismo , Acetilação , Animais , Diferenciação Celular/genética , Linhagem da Célula , Células Eritroides/metabolismo , Globinas/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Histonas/metabolismo , Humanos , Cadeias kappa de Imunoglobulina/genética , Lactente , Camundongos , Células-Tronco Multipotentes/citologiaRESUMO
BACKGROUND: The diagnosis of glioblastoma (GBM), a most aggressive primary brain tumor with a median survival of 14.6 months, carries a dismal prognosis. GBMs are characterized by numerous genetic and epigenetic alterations, affecting patient survival and treatment response. Epigenetic mechanisms are deregulated in GBM as a result of aberrant expression/activity of epigenetic enzymes, including histone deacetylases (HDAC) which remove acetyl groups from histones regulating chromatin accessibility. Nevertheless, the impact of class/isoform-selective HDAC inhibitors (HDACi) on glioma cells, including glioma stem cells, had not been systematically determined. RESULTS: Comprehensive analysis of the public TCGA dataset revealed the increased expression of HDAC 1, 2, 3, and 7 in malignant gliomas. Knockdown of HDAC 1 and 2 in human GBM cells significantly decreased cell proliferation. We tested the activity of 2 new and 3 previously described HDACi with different class/isoform selectivity on human GBM cells. All tested compounds exerted antiproliferative properties on glioma cells. However, the HDACi 1 and 4 blocked proliferation of glioblastoma cells leading to G2/M growth arrest without affecting astrocyte survival. Moreover, 1 and 4 at low micromolar concentrations displayed cytotoxic and antiproliferative effects on sphere cultures enriched in glioma stem cells. CONCLUSIONS: We identified two selective HDAC inhibitors that blocked proliferation of glioblastoma cells, but did not affect astrocyte survival. These new and highly effective inhibitors should be considered as promising candidates for further investigation in preclinical GBM models.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA/efeitos dos fármacos , Glioma/genética , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Neoplásicas/citologia , Esferoides Celulares/citologia , Benzamidas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Epigênese Genética/efeitos dos fármacos , Glioma/tratamento farmacológico , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Histona Desacetilases/genética , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Pirimidinas/farmacologia , Esferoides Celulares/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Aim: Alterations of microglia, the brain-resident macrophages, are associated with numerous brain pathologies. Genetic manipulation of microglia in diseases using small interfering RNA (siRNA) is hampered by the lack of safe and efficient siRNA delivery methods. We assessed the amphiphilic dendrimer (AD) for functional siRNA delivery and gene knockdown in primary microglia. Materials & methods: We characterized the ability of AD to form nanoparticles with siRNA, and studied their size, surface potential, cell uptake and gene silencing in rodent microglia. Results: AD effectively delivered siRNA to primary microglia and decreased target gene and protein expression, leading to transcriptomic changes without affecting basal microglial functions. Conclusion: The dendrimer AD promises to be an innocuous carrier for siRNA delivery into microglia.
Assuntos
Dendrímeros/química , Portadores de Fármacos/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Animais , Células Cultivadas , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , RNA Interferente Pequeno/genética , Ratos WistarRESUMO
Gliosarcoma is a very rare brain tumor reported to be a variant of glioblastoma (GBM), IDH-wildtype. While differences in molecular and histological features between gliosarcoma and GBM were reported, detailed information on the genetic background of this tumor is lacking. We intend to fill in this knowledge gap by the complex analysis of somatic mutations, indels, copy number variations, translocations and gene expression patterns in gliosarcomas. Using next generation sequencing, we determined somatic mutations, copy number variations (CNVs) and translocations in 10 gliosarcomas. Six tumors have been further subjected to RNA sequencing analysis and gene expression patterns have been compared to those of GBMs. We demonstrate that gliosarcoma bears somatic alterations in gene coding for PI3K/Akt (PTEN, PI3K) and RAS/MAPK (NF1, BRAF) signaling pathways that are crucial for tumor growth. Interestingly, the frequency of PTEN alterations in gliosarcomas was much higher than in GBMs. Aberrations of PTEN were the most frequent and occurred in 70% of samples. We identified genes differentially expressed in gliosarcoma compared to GBM (including collagen signature) and confirmed a difference in the protein level by immunohistochemistry. We found several novel translocations (including translocations in the RABGEF1 gene) creating potentially unfavorable combinations. Collected results on genetic alterations and transcriptomic profiles offer new insights into gliosarcoma pathobiology, highlight differences in gliosarcoma and GBM genetic backgrounds and point out to distinct molecular cues for targeted treatment.
RESUMO
Melatonin plays a pleiotropic role in the immune system of mammals and birds. Endogenous and exogenous melatonin modulates lymphocyte proliferation via specific MT(1), MT(2) and Mel(1c) membrane receptors, although the mechanisms behind this process are poorly understood. The diurnal changes in the expression and function of melatonin membrane receptors within the immune system have so far received little attention. We investigated the day/night differences in melatonin membrane receptor mRNA expression in chicken lymphoid organs and cultured splenocytes and examined the in vitro effect of melatonin and 2-iodomelatonin on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in chicken splenocytes. In whole organs, expression of all subtypes of Mel membrane receptors was observed, and the level did not change significantly with the time of day. Interestingly, we observed a significant increase in the expression of the transcripts of all receptor subtypes in cultured splenocytes isolated at night compared with cells obtained during the day. In chicken spleen leukocytes isolated during the day, melatonin and 2-iodomelatonin increased [Ca(2+)](i), with only 2-iodomelatonin being effective in the 'night' cells. Luzindole modulated the [Ca(2+)](i) increase caused by melatonin receptor agonists: it potentiated the stimulatory effect of melatonin during the day, but counteracted that evoked by 2-iodomelatonin at night. The results of this study demonstrate that melatonin can induce changes in [Ca(2+)](i) in chicken spleen leukocytes that should modulate proliferation. The effect of melatonin on [Ca(2+)](i) is less pronounced at night, possibly caused by receptor desensitization.
Assuntos
Cálcio/metabolismo , Galinhas/metabolismo , Ritmo Circadiano/fisiologia , Líquido Intracelular/metabolismo , Leucócitos/metabolismo , Melatonina/fisiologia , Baço/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Masculino , Receptores de Melatonina/biossíntese , Receptores de Melatonina/genética , Receptores de Melatonina/metabolismo , Baço/citologiaRESUMO
Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKß, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKß levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKß expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Quinase I-kappa B/metabolismo , Macrófagos/patologia , Microglia/patologia , Animais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Regulação para Baixo , Perfilação da Expressão Gênica , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/imunologia , Humanos , Quinase I-kappa B/genética , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Microglia/enzimologia , Microglia/imunologia , Microglia/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: Aberrant epigenetic histone modifications are implicated in cancer pathobiology, therefore histone modifying enzymes are emerging targets for anti-cancer therapy. There is a few evidence for deregulation of the histone modifying enzymes in glioblastomas. Glioma treatment is a clinical challenge due to its resistance to current therapies. METHODS: The effect of selected inhibitors on epigenetic modifications and viability of glioma C6 cells were studied using immunofluorescence and MTT metabolism test. RESULTS: We found that VPA and TSA increase histone H4 acetylation in glioma cells, while chaetocin and BIX01294 at low concentrations reduce H3K9me3, and 3DZNep decreases H3K27me3. Long-term treatment with some epigenetic inhibitors affects viability of glioma cells. CONCLUSIONS: We established the concentrations of selected inhibitors which in C6 glioma cells inhibit the enzyme activity, but do not decrease cell viability, hence allow to study the role of histone modifications in C6 glioma biology.
Assuntos
Neoplasias Encefálicas/enzimologia , Inibidores Enzimáticos/farmacologia , Glioma/enzimologia , Histonas/metabolismo , Acetilação , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Azepinas/farmacologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Epigênese Genética , Glioma/patologia , Ácidos Hidroxâmicos/farmacologia , Piperazinas/farmacologia , Quinazolinas/farmacologia , Ratos , Ácido Valproico/farmacologiaRESUMO
Epigenetic modifications control gene expression by regulating the access of nuclear proteins to their target DNA and have been implicated in both normal cell differentiation and oncogenic transformation. Epigenetic abnormalities can occur both as a cause and as a consequence of cancer. Oncogenic transformation can deeply alter the epigenetic information enclosed in the pattern of DNA methylation or histone modifications. In addition, in some cancers epigenetic dysfunctions can drive oncogenic transformation. Growing evidence emphasizes the interplay between metabolic disturbances, epigenomic changes and cancer, i.e., mutations in the metabolic enzymes SDH, FH, and IDH may contribute to cancer development. Epigenetic-based mechanisms are reversible and the possibility of "resetting" the abnormal cancer epigenome by applying pharmacological or genetic strategies is an attractive, novel approach. Gliomas are incurable with all current therapeutic approaches and new strategies are urgently needed. Increasing evidence suggests the role of epigenetic events in development and/or progression of gliomas. In this review, we summarize current data on the occurrence and significance of mutations in the epigenetic and metabolic enzymes in pathobiology of gliomas. We discuss emerging therapies targeting specific epigenetic modifications or chromatin modifying enzymes either alone or in combination with other treatment regimens.