RESUMO
Activating mutations in the mitogen-activated protein kinase (MAPK) cascade, also known as the RAS-MEK-extracellular signal-related kinase (ERK1/2) pathway, are an underlying cause of >70% of human cancers. While great strides have been made toward elucidating the cytoplasmic components of MAPK signaling, the key downstream coactivators that coordinate the transcriptional response have not been fully illustrated. Here, we demonstrate that the MAPK transcriptional response in human cells is funneled through Integrator, an RNA polymerase II-associated complex. Integrator depletion diminishes ERK1/2 transcriptional responsiveness and cellular growth in human cancers harboring activating mutations in MAPK signaling. Pharmacological inhibition of the MAPK pathway abrogates the stimulus-dependent recruitment of Integrator at immediate early genes and their enhancers. Following epidermal growth factor (EGF) stimulation, activated ERK1/2 is recruited to immediate early genes and phosphorylates INTS11, the catalytic subunit of Integrator. Importantly, in contrast to the broad effects of Integrator knockdown on MAPK responsiveness, depletion of a number of critical subunits of the coactivator complex Mediator alters only a few MAPK-responsive genes. Finally, human cancers with activating mutations in the MAPK cascade, rendered resistant to targeted therapies, display diminished growth following depletion of Integrator. We propose Integrator as a crucial transcriptional coactivator in MAPK signaling, which could serve as a downstream therapeutic target for cancer treatment.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais , Células A549 , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/metabolismo , Endorribonucleases , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Indazóis/farmacologia , Sistema de Sinalização das MAP Quinases/genética , Neoplasias/enzimologia , Neoplasias/fisiopatologia , Fosforilação , Piperazinas/farmacologia , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
PURPOSE: To compare the immune response and survival after size-matched radiofrequency (RF) ablation and a proprietary form of pulsed electric field (PEF) ablation in murine tumors. MATERIAL AND METHODS: Orthotopically inoculated EMT6 or 4T1 murine tumors received sham, RF ablation, or PEF ablation. 4T1 tumor ablations included subgroups with intraperitoneal checkpoint inhibition immunotherapy (αPD-1). Blood was collected for cytokine profiling and flow cytometry. Tumor size was measured and survival was monitored. Tumor samples were processed for histology, immunohistochemistry, flow cytometry, and cytokine profiling. Lungs were collected from 4T1-bearing mice for hematoxylin and eosin histology to assess metastatic spread and abscopal effect induced by ablation. RESULTS: PEF elicited distinct immunomodulatory effects, with clear differences in serum and tumor cytokine profiles compared with RF ablation, including intratumoral downregulation of vascular endothelial growth factor, hypoxia-inducible factor 1α, c-MET, interleukin-10, Ki67, and tumor necrosis factor-α (all P < .05). PEF increased innate immune activation, with enhanced recruitment of dendritic cells, M1 macrophages, and natural killer cells coupled with a reduction in M2 macrophages and myeloid-derived suppressor cells (all P < .05). Concurrently, PEF strengthened adaptive immunity compared with RF ablation, characterized by increased antigen-specific T cells and decreased regulatory T cells (all P < .05). PEF stalled tumor growth and increased survival at the end of the study (≥4× versus RFA). Finally, PEF promoted an abscopal effect of clearing metastases in the lungs, which was stronger in combination with αPD-1 than with PEF alone. CONCLUSIONS: The proprietary form of PEF used in this study evoked a preferential immunostimulatory profile versus RF ablation thermal ablation in mice, with implications for enhancing the therapeutic effectiveness of checkpoint inhibition immunotherapy for immunotherapy-unresponsive tumors.
Assuntos
Neoplasias , Ablação por Radiofrequência , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linfócitos T/metabolismo , Citocinas/metabolismoRESUMO
OBJECTIVES: Sensitivity to moral and conventional rules (SMCR) is supported by bilateral brain networks and psychosocial input both of which may be altered in temporal lobe epilepsy (TLE). This study evaluated the components of SMCR in patients with TLE, aiming to clarify their preservation and link to psychopathological and cognitive aspects. METHODS: Adult patients with unilateral TLE and healthy controls were evaluated using neuropsychological tests for SMCR, memory, language, and executive functions, the Empathy Questionnaire (EQ), and the Symptom Checklist-90-R (SCL-90-R). RESULTS: The SMCR test items showed good reliability and validity, yielding the Severity and Rules factors distinct from the Executive, Lexical and Memory factors. Patients with right TLE scored worse in moral rules recognition than controls, but this difference was nullified by a significant influence for age and sex. The Severity and Rules factors related to semantic fluency and age and, respectively, TLE side and psychoticism. However, these factors did predict TLE membership. CONCLUSIONS: In adult patients with TLE, the SMCR test reflects a distinct cognitive domain. Conventional rules are well-retained, while moral reasoning may be only affected in right TLE if unfavorable demographics coexist. Although age, TLE side, semantic abilities, and psychoticism cooperate to determine SMCR, impairment of such domain is not a distinctive feature of TLE.
RESUMO
OBJECTIVE: Encephaloceles (ENCs) may cause clinical complications, including drug-resistant epilepsy that can be cured with epilepsy surgery. METHODS: We describe clinical, diagnostic, and neuropathological findings of 12 patients with temporal ENC and epilepsy evaluated for surgery and compare them with a control group of 26 temporal lobe epilepsy (TLE) patients. RESULTS: Six patients had unilateral and 6 bilateral temporal ENCs. Compared to TLEs, ENCs showed i) later epilepsy onset, ii) higher prevalence of psychiatric comorbidities, iii) no history of febrile convulsions, and iv) ictal semiology differences. Seven patients had MRI signs of gliosis, and 9 of intracranial hypertension. Interictal EEG analysis in ENCs demonstrated significant differences with controls: prominent activity in the beta/gamma frequency bands in frontal regions, interictal short sequences of low-voltage fast activity, and less frequent and more localized interictal epileptiform discharges. Ictal EEG patterns analyzed in 9 ENCs showed delayed and slower contralateral spread compared to TLEs. All ENCs that underwent surgery (7 lobectomies and 1 lesionectomy) are in Engel class I. Neuropathological examination revealed 4 patterns: herniated brain fragments, focal layer I distortion, white matter septa extending into the cortex, and altered gyral profile. CONCLUSIONS AND SIGNIFICANCE: The described peculiarities might help clinicians to suspect the presence of largely underdiagnosed ENCs.
Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Humanos , Eletroencefalografia/métodos , Encefalocele/complicações , Encefalocele/diagnóstico por imagem , Epilepsia/diagnóstico por imagem , Epilepsia/etiologia , Epilepsia do Lobo Temporal/diagnóstico por imagem , Epilepsia do Lobo Temporal/cirurgia , Neuroimagem , Imageamento por Ressonância Magnética/métodosRESUMO
OBJECTIVE: To assess seizure and cognitive outcomes and their predictors in children (<16 years at surgery) and adults undergoing temporal lobe epilepsy (TLE) surgery in eight Italian centers. METHODS: This is a retrospective multicenter study. We performed a descriptive analysis and subsequently carried out multivariable mixed-effect models corrected for multiple comparisons. RESULTS: We analyzed data from 511 patients (114 children) and observed significant differences in several clinical features between adults and children. The possibility of achieving Engel class IA outcome and discontinuing antiepileptic drugs (AEDs) at last follow-up (FU) was significantly higher in children (P = .006 and < .0001). However, percentages of children and adults in Engel class I at last FU (mean ± SD, 45.9 ± 17 months in children; 45.9 ± 20.6 months in adults) did not differ significantly. We identified different predictors of seizure outcome in children vs adults and at short- vs long-term FU. The only variables consistently associated with class I outcome over time were postoperative electroencephalography (EEG) in adults (abnormal, improved,odds ratio [OR] = 0.414, P = .023, Q = 0.046 vs normal, at 2-year FU and abnormal, improved, OR = 0.301, P = .001, Q = 0.002 vs normal, at last FU) and the completeness of resection of temporal magnetic resonance (MR) abnormalities other than hippocampal sclerosis in children (OR = 7.93, P = .001, Q = 0.003, at 2-year FU and OR = 45.03, P < .0001, Q < 0.0001, at last FU). Cognitive outcome was best predicted by preoperative performances in either age group. SIGNIFICANCE: Clinical differences between adult and pediatric patients undergoing TLE surgery are reflected in differences in long-term outcomes and predictors of failures. Children are more likely to achieve sustained seizure freedom and withdraw AEDs after TLE surgery. Earlier referral should be encouraged as it can improve surgical outcome.
Assuntos
Cognição , Epilepsia do Lobo Temporal/cirurgia , Procedimentos Neurocirúrgicos , Adolescente , Adulto , Fatores Etários , Anticonvulsivantes/uso terapêutico , Criança , Pré-Escolar , Intervenção Médica Precoce , Eletroencefalografia , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/psicologia , Feminino , Hipocampo/patologia , Humanos , Masculino , Malformações do Desenvolvimento Cortical/patologia , Testes Neuropsicológicos , Complicações Pós-Operatórias/epidemiologia , Prognóstico , Estudos Retrospectivos , Esclerose , Adulto JovemRESUMO
Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.-Zub, E., Canet, G., Garbelli, R., Blaquiere, M., Rossini, L., Pastori, C., Sheikh, M., Reutelingsperger, C., Klement, W., de Bock, F., Audinat, E., Givalois, L., Solito, E., Marchi, N. The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.
Assuntos
Anexina A1/metabolismo , Epilepsia , Receptores de Glucocorticoides/metabolismo , Animais , Anexina A1/genética , Contagem de Células Sanguíneas , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Corticosterona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Ácido Caínico/administração & dosagem , Ácido Caínico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glucocorticoides/genéticaRESUMO
OBJECTIVE: Previous studies of frontal lobe epilepsy (FLE) have documented different impairments of theory of mind (ToM), while the study of frontal lobe (FL) lesion without seizures has produced inconsistent results. Given the role played by the FLs in ToM, we evaluated this and other functions in patients with FLE with and without FL lesions. The main objective was to clarify the salience of ToM impairment in the cognitive pattern of FLE and its capacity to discriminate these patients from healthy subjects. The effects of FL lesions on ToM were also explored. METHODS: Seventy-five adult patients with FLE (40 cases with FL lesions) were compared with 42 healthy controls. The Faux Pas Task (FPT) and other neuropsychological tests were utilized to assess ToM, reasoning, language, memory, praxis, attention, and executive abilities. RESULTS: The patients obtained lower z scores for the FPT than for other tests. The ToM, Executive, and Verbal factors discriminated patients from healthy subjects. The patients with or without FL lesion showed significant impairments in recognizing and understanding others' epistemic and affective mental states, but adequate capacity to exclude inexistent mental states was retained. In comparison with controls, the patients with FL lesions obtained lower scores for lexical, memory, praxis, attention, and executive functions, whereas those without lesion only showed attention and initiative deficits. Schooling was the major predictor of ToM, whereas the capacity to exclude inexistent mental states was related to seizure onset age and epilepsy duration. Other cognitive functions were related to schooling, age, or FLE laterality. SIGNIFICANCE: Impaired understanding of real mental states is a specific, salient, and discriminating cognitive aspect of FLE. Poor education is a risk factor for ToM deficit, whereas the clinical variables and FL lesions have no impact. These results suggest that impaired ToM may be a marker of FLE neurobehavioral phenotype.
Assuntos
Epilepsia do Lobo Frontal/patologia , Epilepsia do Lobo Frontal/fisiopatologia , Lobo Frontal/patologia , Teoria da Mente , Adulto , Estudos Transversais , Feminino , Humanos , Masculino , Processos Mentais/fisiologia , Pessoa de Meia-Idade , FenótipoRESUMO
OBJECTIVE: The current study aimed to describe quality of life (QoL) levels, psychiatric symptoms prevalence, and perceived stigma levels in persons with either drug-resistant epilepsy (DRE) or drug-sensitive epilepsy (DSE) and in persons with epilepsy (PwE) with DRE that underwent epilepsy surgery (DREES). METHODS: Persons with epilepsy diagnosed as having DRE according to International League Against Epilepsy (ILAE) criteria, DSE, and DREES were enrolled at the Epilepsy Unit of the Neurological Institute Carlo Besta of Milan. Sociodemographic and clinical data, Quality of Life in Epilepsy Inventory (QOLIE-31), Symptom Checklist-90 (SCL-90), and the Epilepsy Stigma Scale (ESS) were collected based on self-reported information and on medical records. RESULTS: Sociodemographic, medical, and psychological data were obtained from 181 PwE: 80 with DRE, 31 with DSE, and 70 with DREES. We found that QoL is higher and psychiatric symptoms are lower in persons with DSE compared with DRE and that patients with DREES, who were drug-resistant before surgery, are in between DSE and DRE for both measures. Perceived stigma level is different in DSE and in DRE, that report the highest levels of stigma, and is between the other two groups in DREES. SIGNIFICANCE: This study suggests that low QoL levels and high psychiatric symptoms prevalence in drug-resistant PwE may be significantly improved after epilepsy surgery and suggests the importance of a biopsychosocial approach when planning therapeutic intervention.
Assuntos
Epilepsia/psicologia , Transtornos Mentais/psicologia , Percepção , Qualidade de Vida/psicologia , Estigma Social , Adulto , Estudos Transversais , Epilepsia Resistente a Medicamentos/diagnóstico , Epilepsia Resistente a Medicamentos/epidemiologia , Epilepsia Resistente a Medicamentos/psicologia , Epilepsia/diagnóstico , Epilepsia/epidemiologia , Feminino , Humanos , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , Percepção/fisiologia , PrevalênciaRESUMO
An observational, prospective study has been conducted to evaluate the effects of adjunctive treatment with perampanel (PER) on psychological functioning and quality of life (QoL) in patients with drug-resistant focal epilepsy. Fifty-six adult patients treated with PER in addition to antiepileptic drugs (AEDs) were recruited in 2 Italian Epilepsy Centers. Irritability in Adult Patients with Epilepsy (I-EPI), Quality of Life in Epilepsy (QOLIE-31), Beck Depression Inventory II (BDI-II), and State-Trait Anxiety Inventory Y-1 and Y-2 (STAI) questionnaires were administered at baseline and 3 and 6â¯months after the treatment onset. Adverse events (AEs) were collected during the observational 6â¯months period. Retention rate of treatment with PER was 82.1% at 3â¯months and 64.3% at 6â¯months. Thirteen patients reported a significant seizure frequency reduction, and one seizure freedom case was observed after 4â¯months of PER treatment. Perampanel was stopped because of inefficacy or paradoxical effects in 28.6% of cases and because of AEs in 7.1%. The peak dose was not associated with discontinuation probability. Irritability, QoL, depression, trait, and state anxiety did not change significantly during the PER therapy. A tendency of association between higher level of irritability at baseline and PER discontinuation was found. The results of this observational study have shown that the addition of PER to AEDs may improve seizure control, does not increase levels of irritability, depression, and anxiety, and does not reduce patients' QoL. This study also confirms the importance of a comprehensive clinical assessment, including psychiatric symptoms evaluation before offering a new treatment, to improve therapy compliance.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Transtornos Mentais/tratamento farmacológico , Piridonas/uso terapêutico , Qualidade de Vida , Adulto , Epilepsia Resistente a Medicamentos/epidemiologia , Epilepsia Resistente a Medicamentos/psicologia , Feminino , Humanos , Itália/epidemiologia , Masculino , Transtornos Mentais/epidemiologia , Transtornos Mentais/psicologia , Pessoa de Meia-Idade , Nitrilas , Estudos Prospectivos , Qualidade de Vida/psicologia , Inquéritos e Questionários , Resultado do TratamentoRESUMO
Glioblastoma multiforme (GBM) is the most common and aggressive malignant adult primary brain tumor. Despite surgical resection followed by radiotherapy and chemotherapy, the median survival rate is approximately 14 months. Although experimental therapies are in clinical trials for GBM, there is an urgent need for a peripheral GBM biomarker for measuring treatment response. As we have previously demonstrated that the long noncoding RNA HOX Transcript Antisense Intergenic RNA, or HOTAIR, is dysregulated in GBM and required for GBM cell proliferation, we hypothesized that HOTAIR expression may be utilized as a peripheral biomarker for GBM. HOTAIR expression was measured in serum from 43 GBM and 40 controls using quantitative real-time PCR (qRT-PCR). The PCR products were subsequently subcloned into pCR™4-TOPO®TA vectors for DNA sequencing. A ROC curve was also generated to examine HOTAIR's prognostic value. The amount of HOTAIR in serum exosomes and exosome-depleted supernatant was calculated by qRT-PCR. The relative HOTAIR expression was also investigated in 15 pairs of GBM serum and tumors. We detected HOTAIR in serum from GBM patients. HOTAIR levels in serum samples from GBM patients was significantly higher than in the corresponding controls (P < 0.0001). The area under the ROC curve distinguishing GBM patients from controls was 0.913 (95% CI: 0.845-0.982, P < 0.0001), with 86.1% sensitivity and 87.5% specificity at the cut-off value of 10.8. HOTAIR expression was significantly correlated with high grade brain tumors. In addition, Pearson correlation analysis indicated a medium correlation of serum HOTAIR levels and the corresponding tumor HOTAIR levels (r = 0.734, P < 0.01). We confirmed via sequencing that the amplified HOTAIR from serum contained the HOTAIR sequence and maps to the known HOTAIR locus at 12q13. The serum-derived exosomes contain HOTAIR and the purified exosomes were validated by western blot and nanoparticle tracking analysis. Importantly, our results demonstrate that serum HOTAIR can be used as a novel prognostic and diagnostic biomarker for GBM.
Assuntos
Biomarcadores Tumorais , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Ácidos Nucleicos Livres , Glioblastoma/diagnóstico , Glioblastoma/genética , RNA Longo não Codificante/genética , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/mortalidade , Exossomos , Glioblastoma/sangue , Glioblastoma/mortalidade , Humanos , Prognóstico , RNA Longo não Codificante/sangue , Curva ROCRESUMO
Bromodomain and extraterminal (BET) domain proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule inhibitors of BET bromodomain proteins reduce expression of several oncogenes required for Glioblastoma Multiforme (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression, but mechanistic insight into their expression and regulation by BET bromodomain inhibitors remains elusive. In this study, we used Helicos single molecule sequencing to comprehensively profile lncRNAs differentially expressed in GBM, and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET bromdomain inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOX transcript antisense RNA (HOTAIR) and restored the expression of several other GBM down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET151 treatment abrogates the antiproliferative activity of the BET bromodomain inhibitor. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Bromodomain Containing 4 (BRD4) to the HOTAIR promoter, suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that modulation of lncRNA networks may, in part, mediate the antiproliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.
Assuntos
Neoplasias Encefálicas/genética , Proliferação de Células/genética , Glioblastoma/genética , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Camundongos Nus , Microscopia de Fluorescência , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Triplet repeat expansions in the Fragile X mental retardation 1 (FMR1) gene cause either intellectual disability and autism, or adult-onset neurodegeneration, with poorly understood variability in presentation. Previous studies have identified several long noncoding RNAs (lncRNAs) at the FMR1 locus, including FMR4. Similarly to FMR1, FMR4 is silenced by large-repeat expansions that result in enrichment of DNA and histone methylation within the shared promoter and repeat sequence, suggesting a possible role for this noncoding RNA in the pathophysiology of Fragile X. We therefore assessed the functional role of FMR4 to gain further insight into the molecular processes in Fragile X-associated disorders. Previous work showed that FMR4 does not exhibit cis-regulation of FMR1. Here, we found that FMR4 is a chromatin-associated transcript and, using genome-wide chromatin immunoprecipitation experiments, showed that FMR4 alters the chromatin state and the expression of several hundred genes in trans. Among the genes regulated by FMR4, we found enrichment for those involved in neural development and cellular proliferation. S-phase marker assays further demonstrated that FMR4 may promote cellular proliferation, rather than differentiation, of human neural precursor cells (hNPCs). By establishing this novel function for FMR4 in hNPCs, we lend support to existing evidence of the epigenetic involvement of lncRNA in nervous system development, and increase our understanding of the complex pathogenesis underlying neurological disorders associated with FMR1 repeat expansions.
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Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , RNA Longo não Codificante/genética , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Genes Controladores do Desenvolvimento , Células HEK293 , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese , RNA Longo não Codificante/metabolismoRESUMO
Epigenetic enzymes modulate signal transduction pathways in different biological contexts. We reasoned that epigenetic regulators might modulate the Hedgehog (HH) signaling pathway, a main driver of cell proliferation in various cancers including medulloblastoma. To test this hypothesis, we performed an unbiased small-molecule screen utilizing an HH-dependent reporter cell line (Light2 cells). We incubated Light2 cells with small molecules targeting different epigenetic modulators and identified four histone deacetylase inhibitors and a bromodomain and extra terminal domain (BET) protein inhibitor (I-BET151) that attenuate HH activity. I-BET151 was also able to inhibit the expression of HH target genes in Sufu(-/-) mouse embryonic fibroblasts, in which constitutive Gli activity is activated in a Smoothened (Smo)-independent fashion, consistent with it acting downstream of Smo. Knockdown of Brd4 (which encodes one of the BET proteins) phenocopies I-BET151 treatment, suggesting that Brd4 is a regulator of the HH signaling pathway. Consistent with this suggestion, Brd4 associates with the proximal promoter region of the Gli1 locus, and does so in a manner that can be reversed by I-BET151. Importantly, I-BET151 also suppressed the HH activity-dependent growth of medulloblastoma cells, in vitro and in vivo. These studies suggest that BET protein modulation may be an attractive therapeutic strategy for attenuating the growth of HH-dependent cancers, such as medulloblastoma.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/genética , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Meduloblastoma/prevenção & controle , Receptores Acoplados a Proteínas G/genética , Animais , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos Knockout , Camundongos Nus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor Smoothened , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5'-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called "Deep-RACE", which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3' region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes.
Assuntos
Ataxia/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , RNA Longo não Codificante/isolamento & purificação , Tremor/genética , Regiões 5' não Traduzidas , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Regulação da Expressão Gênica , Loci Gênicos , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Expansão das Repetições de TrinucleotídeosRESUMO
Presurgical monitoring with intracerebral electrodes in patients with drug-resistant focal epilepsy represents a standard invasive procedure to localize the sites of seizures origin, defined as the epileptogenic zone (EZ). During presurgical evaluation, intracerebral single-pulse electrical stimulation (SPES) is performed to define the boundaries of eloquent areas and to evoke seizure-associated symptoms. Extensive intracranial exploration and stimulation generate a large dataset on brain connectivity that can be used to improve EZ detection and to understand the organization of the human epileptic brain. We developed a protocol to analyse field responses evoked by intracranial stimulation. Intracerebral recordings were performed with 105-162 recording sites positioned in fronto-temporal regions in 12 patients with pharmacoresistant focal epilepsy. Recording sites were used for bipolar SPES at 1 Hz. Reproducible early and late phases (<60 ms and 60-500 ms from stimulus artefact, respectively) were identified on averaged evoked responses. Phase 1 and 2 responses recorded at all and each recording sites were plotted on a 3D brain reconstructions. Based on connectivity properties, electrode contacts were primarily identified as receivers, mainly activators or bidirectional. We used connectivity patterns to construct networks and applied cluster partitioning to study the proprieties between potentials evoked/stimulated in different regions. We demonstrate that bidirectional connectivity during phase 1 is a prevalent feature that characterize contacts included in the EZ. This study shows that the application of an analytical protocol on intracerebral stimulus-evoked recordings provides useful information that may contribute to EZ detection and to the management of surgical-remediable epilepsies.
Assuntos
Mapeamento Encefálico/métodos , Eletroencefalografia/métodos , Epilepsias Parciais/fisiopatologia , Epilepsias Parciais/cirurgia , Potenciais Evocados , Cuidados Pré-Operatórios/métodos , Adolescente , Adulto , Estimulação Elétrica/métodos , Epilepsias Parciais/patologia , Estudos de Viabilidade , Feminino , Seguimentos , Lobo Frontal/patologia , Lobo Frontal/fisiopatologia , Humanos , Neuroestimuladores Implantáveis , Imageamento por Ressonância Magnética , Masculino , Modelos Neurológicos , Vias Neurais/fisiopatologia , Processamento de Sinais Assistido por Computador , Lobo Temporal/patologia , Lobo Temporal/fisiopatologia , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
OBJECTIVE: In one third of patients with a diagnosis of pharmacoresistant focal epilepsy who are candidates for therapeutic surgery, cerebral areas responsible for seizure generation can be defined exclusively with invasive intracranial recordings. A correct presurgical identification of the epileptogenic zone (EZ) with intracranial electrodes has a direct impact on postsurgical outcome. We aimed at identifying biomarkers of the EZ based on computer-assisted inspection of intracranial electroencephalography (EEG). METHODS: Computer-driven intracranial EEG analysis in the domains of time, frequency, and space was retrospectively applied to a population of 10 patients with focal epilepsy to detect EZ electrophysiologic markers. Next, a prospective study was performed on 14 surgery candidate patients. The stereo-EEG computer-assisted analysis of EZ boundaries performed blind from patients data was compared to that defined with the traditional visual inspection completed by neurophysiologists. RESULTS: In the retrospective study, the EZ was characterized by the combined detection of three biomarkers observed at seizure onset: (1) fast activity at 80-120 Hz associated with (2) very slow transient polarizing shift and (3) voltage depression (flattening). Correlations between these indexes were calculated for each seizure. In the prospective study, the quantified analysis based on the three biomarkers confirmed a complete overlap between leads within the EZ identified by expert clinicians. In 2 of 14 patients the proposed biomarkers partially identified the EZ. SIGNIFICANCE: Our findings demonstrate and validate with a prospective unbiased study the use of three neurophysiologic intracranial EEG parameters as excellent biomarkers of ictogenesis and as reliable indicators of EZ boundaries.
Assuntos
Eletroencefalografia/métodos , Epilepsias Parciais/metabolismo , Epilepsias Parciais/fisiopatologia , Técnicas Estereotáxicas , Biomarcadores/análise , Epilepsias Parciais/diagnóstico , Humanos , Estudos Prospectivos , Estudos RetrospectivosRESUMO
Chemo-immunotherapy uses combined systemic therapies for resectable and unresectable tumors. This approach is gaining clinical momentum, but survival increases leave considerable room for improvement. A novel form of Pulsed Electric Field (PEF) ablation combines focal tissue destruction with immune activation in preclinical settings. The PEFs induce lethal cell damage without requiring thermal processes, leaving cellular proteins intact. This affords PEF a favorable safety profile, improved antigenicity, and significant immunostimulatory damage-associated molecular pattern release compared to other focal therapies. Preclinical investigations demonstrate a combinatorial benefit of PEF with immunostimulation. This study evaluates whether this proprietary PEF therapy induces an immunostimulatory effect sufficient to augment systemic neoadjuvant chemotherapy and immunotherapy to reverse metastatic disease in an immune-cold murine tumor model. To determine whether PEF improves a neoadjuvant chemo-immunotherapy standard-of-care, partial PEF ablation was delivered to orthotopically inoculated 4T1 metastatic tumors in addition to combinations of cisplatin chemotherapy and/or αPD-1 immunotherapy, followed by resection. In addition, to determine whether PEF combined with chemo-immunotherapy improves local and metastatic response in unresectable populations, partial PEF ablation was added to chemo-immunotherapy in mice that did not receive resection. Blood cytokines and flow cytometry evaluated immune response. Partial PEF ablation generates an immunostimulatory tumor microenvironment, increases systemic immune cell populations, slows tumor growth, and prolongs survival relative to neoadjuvant systemic therapies-alone. These data suggest the addition of this proprietary PEF locoregional therapy may synergize with systemic standard-of-care paradigms to improve outcomes with potential or demonstrated metastatic disease in both resectable and unresectable patient cohorts.
Assuntos
Terapia Neoadjuvante , Neoplasias , Camundongos , Humanos , Animais , Modelos Animais de Doenças , Neoplasias/patologia , Imunoterapia , Cisplatino/uso terapêutico , Microambiente TumoralRESUMO
OBJECTIVE: To investigate the Italian experience on the surgical and radiosurgical treatment of drug-resistant epilepsy due to hypothalamic hamartoma (HH) in the period 2011-2021 in six Italian epilepsy surgery centers, and to compare safety and efficacy profiles of the different techniques. METHODS: We collected pseudo-anonymized patient's data with at least 12 months of follow-up. Surgical outcome was defined according to Engel classification of seizure outcome. Univariate analysis was performed to assess the risk of post-operative seizures, categorized in dichotomous variable as favorable and unfavorable; explanatory variables were considered. Mann-Whitney or Chi-squared test were used to assess the presence of an association between variables (p < 0.05). RESULTS: Full presurgical and postoperative data about 42 patients from 6 epilepsy surgery centers were gathered. Engel class I was reached in the 65.8% and 66.6% of patients with gelastic and non-gelastic seizures, respectively. Other than daily non-gelastic seizures were associated with seizure freedom (p = 0.01), and the radiological type presented a trend toward significance (p = 0.12). SIGNIFICANCE: Endoscopic disconnection and laser interstitial thermal therapy are effective in the treatment of HH-related epilepsy, with a tolerable safety profile. Both gelastic and non-gelastic seizures can be treated, also in patients with a long history of seizures. PLAIN LANGUAGE SUMMARY: This study collected data about 42 patients with HH-related epilepsies. Endoscopic disconnection and laser therapy are both effective and safe in the treatment of hypothalamic hamartoma-related epilepsies.
Assuntos
Hamartoma , Doenças Hipotalâmicas , Radiocirurgia , Humanos , Hamartoma/cirurgia , Doenças Hipotalâmicas/cirurgia , Radiocirurgia/métodos , Itália , Feminino , Masculino , Criança , Pré-Escolar , Adolescente , Epilepsia Resistente a Medicamentos/cirurgia , Resultado do Tratamento , Adulto , Lactente , Adulto Jovem , Estudos Retrospectivos , Procedimentos Neurocirúrgicos/métodosRESUMO
Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA-directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin-remodelling factors. Here, we show that a promoter-targeted siRNA inhibits transcription of the c-myc gene. Transcriptional silencing of c-myc did not involve changes of known epigenetic marks. Instead, the c-myc promoter-targeted siRNA interfered with transcription initiation blocking the assembly of the pre-initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter-associated RNA initiated upstream and overlapping the transcription start site. Silencing of c-myc led to growth arrest, reduced clonogenic potential and senescence of c-myc over-expressing prostate cancer cells with minimal effect on normal cells. RNA-directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases.
Assuntos
Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Proteínas Argonautas , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , RNA não Traduzido/genética , Ensaio Tumoral de Célula-TroncoRESUMO
DNA sequences associated with protein-coding genes have been the primary focus of most genetic analyses of complex human diseases. Although we are rapidly gaining a comprehensive view of the etiology of certain central nervous system disorders, major gaps in our understanding persist. Recent studies have uncovered that many human genomic sequences are transcribed but not translated, generating an astounding diversity of noncoding RNAs (ncRNAs). This awareness should be taken into account when studying human diseases and may have profound implications on the development of novel biomarkers as well as therapies.