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1.
Cancers (Basel) ; 13(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918978

RESUMO

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

2.
Cancer Res ; 80(19): 4129-4144, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32816860

RESUMO

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Cobre/metabolismo , Neuroblastoma/imunologia , Evasão Tumoral/fisiologia , Animais , Antígeno B7-H1/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Quelantes/farmacologia , Transportador de Cobre 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Células Matadoras Naturais , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos BALB C , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Trietilenofosforamida/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Theranostics ; 10(14): 6411-6429, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32483461

RESUMO

Targeting glutamine metabolism has emerged as a potential therapeutic strategy for Myc overexpressing cancer cells. Myc proteins contribute to an aggressive neuroblastoma phenotype. Radiotherapy is one of the treatment modalities for high-risk neuroblastoma patients. Herein, we investigated the effect of glutamine deprivation in combination with irradiation in neuroblastoma cells representative of high-risk disease and studied the role of Myc member interplay in regulating neuroblastoma cell radioresistance. Methods: Cell proliferation and viability assays were used to establish the effect of glutamine deprivation in neuroblastoma cells expressing c-Myc or MycN. Gene silencing and overexpression were used to modulate the expression of Myc genes to determine their role in neuroblastoma radioresistance. qPCR and western blot investigated interplay between expression of Myc members. The impact of glutamine deprivation on cell response following irradiation was explored using a radiobiological 3D colony assay. DNA repair gene pathways as well as CSC-related genes were studied by qPCR array. Reactive Oxygen Species (ROS) and glutathione (GSH) levels were detected by fluorescence and luminescence probes respectively. Cancer-stem cell (CSC) properties were investigated by sphere-forming assay and flow cytometry to quantify CSC markers. Expression of DNA repair genes and CSC-related genes was analysed by mining publicly available patient datasets. Results: Our results showed that glutamine deprivation decreased neuroblastoma cell proliferation and viability and modulated Myc member expression. We then demonstrated for the first time that combined glutamine deprivation with irradiation led to a selective radioresistance of MYCN-amplified neuroblastoma cells. By exploring the underlying mechanism of neuroblastoma radioresistance properties, our results highlight interplay between c-Myc and MycN expression suggesting compensatory mechanisms in Myc proteins leading to radioresistance in MYCN-amplified cells. This result was associated with the ability of MYCN-amplified cells to dysregulate the DNA repair gene pathway, maintain GSH and ROS levels and to increase the CSC-like population and properties. Conversely, glutamine deprivation led to radiosensitization in non-MYCN amplified cell lines through a disruption of the cell redox balance and a trend to decrease in the CSC-like populations. Mining publicly available gene expression dataset obtained from pediatric neuroblastoma patients, we identified a correlation pattern between Myc members and CSC-related genes as well as a specific group of DNA repair gene pathways. Conclusions: This study demonstrated that MycN and c-Myc tightly cooperate in regulation of the neuroblastoma CSC phenotypes and radioresistance upon glutamine deprivation. Pharmacologically, strategies targeting glutamine metabolism may prove beneficial in Myc-driven tumors. Consideration of MycN/c-Myc status in selecting neuroblastoma patients for glutamine metabolism treatment will be important to avoid potential radioresistance.


Assuntos
Glutamina/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/terapia , Radioterapia/métodos
4.
Molecules ; 25(9)2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32365886

RESUMO

Carbon nanostructures (CN) are emerging valuable materials for the assembly of highly engineered multifunctional nanovehicles for cancer therapy, in particular for counteracting the insurgence of multi-drug resistance (MDR). In this regard, carbon nanotubes (CNT), graphene oxide (GO), and fullerenes (F) have been proposed as promising materials due to their superior physical, chemical, and biological features. The possibility to easily modify their surface, conferring tailored properties, allows different CN derivatives to be synthesized. Although many studies have explored this topic, a comprehensive review evaluating the beneficial use of functionalized CNT vs G or F is still missing. Within this paper, the most relevant examples of CN-based nanosystems proposed for MDR reversal are reviewed, taking into consideration the functionalization routes, as well as the biological mechanisms involved and the possible toxicity concerns. The main aim is to understand which functional CN represents the most promising strategy to be further investigated for overcoming MDR in cancer.


Assuntos
Antineoplásicos/química , Carbono/química , Resistencia a Medicamentos Antineoplásicos , Nanoestruturas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Resistência a Múltiplos Medicamentos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia
5.
ACS Pharmacol Transl Sci ; 3(1): 148-160, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32259094

RESUMO

MYCN-amplified neuroblastoma is one of the deadliest forms of childhood cancer and remains a significant clinical challenge. Direct pharmacological inhibition of MYCN is not currently achievable. One strategy could be to target the AKT/GSK3ß pathway, which directly regulates the stability of the MYCN protein. Numerous potent and isoform-specific small-molecule AKT inhibitors have been developed. However, the selection of the right drug combinations in the relevant indication will have a significant impact on AKT inhibitor clinical success. To maximally exploit the potential of AKT inhibitors, a better understanding of AKT isoform functions in cancer is crucial. Here using RNAi to downregulate specific AKT isoforms, we demonstrated that loss of total AKT activity rather than isoform-specific expression was necessary to decrease MYCN expression and cause a significant decrease in neuroblastoma cell proliferation. Consistent with these observations, isoform-specific pharmacological inhibition of AKT was substantially less effective than pan-AKT inhibition in combination with cytotoxic drugs in MYCN-amplified neuroblastoma. The allosteric pan-AKT inhibitor perifosine had promising in vitro and in vivo activity in combination with conventional cytotoxic drugs in MYCN-amplified neuroblastoma cells. Our results demonstrated that perifosine drug combination was able to induce apoptosis and downregulate ABC transporter expression. Collectively, this study shows that selecting pan-AKT inhibitors rather than isoform-specific drugs to synergize with first-line chemotherapy treatment should be considered for clinical trials for aggressive neuroblastoma and, potentially, other MYCN -driven cancers.

6.
Int J Cancer ; 147(8): 2225-2238, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32277480

RESUMO

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.


Assuntos
Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Genes myc/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Prognóstico , RNA Interferente Pequeno/genética , Taxa de Sobrevida
7.
Oncogene ; 39(17): 3555-3570, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32123312

RESUMO

Amplification of the MYCN oncogene occurs in ~25% of primary neuroblastomas and is the single most powerful biological marker of poor prognosis in this disease. MYCN transcriptionally regulates a range of biological processes important for cancer, including cell metabolism. The MYCN-regulated metabolic gene SLC16A1, encoding the lactate transporter monocarboxylate transporter 1 (MCT1), is a potential therapeutic target. Treatment of neuroblastoma cells with the MCT1 inhibitor SR13800 increased intracellular lactate levels, disrupted the nicotinamide adenine dinucleotide (NADH/NAD+) ratio, and decreased intracellular glutathione levels. Metabolite tracing with 13C-glucose and 13C-glutamine following MCT1 inhibitor treatment revealed increased quantities of tricarboxylic acid (TCA) cycle intermediates and increased oxygen consumption rate. MCT1 inhibition was highly synergistic with vincristine and LDHA inhibition under cell culture conditions, but this combination was ineffective against neuroblastoma xenografts. Posttreatment xenograft tumors had increased synthesis of the MCT1 homolog MCT4/SLC16A, a known resistance factor to MCT1 inhibition. We found that MCT4 was negatively regulated by MYCN in luciferase reporter assays and its synthesis in neuroblastoma cells was increased under hypoxic conditions and following hypoxia-inducible factor (HIF1) induction, suggesting that MCT4 may contribute to resistance to MCT1 inhibitor treatment in hypoxic neuroblastoma tumors. Co-treatment of neuroblastoma cells with inhibitors of MCT1 and LDHA, the enzyme responsible for lactate production, resulted in a large increase in intracellular pyruvate and was highly synergistic in decreasing neuroblastoma cell viability. These results highlight the potential of targeting MCT1 in neuroblastoma in conjunction with strategies that involve disruption of pyruvate homeostasis and indicate possible resistance mechanisms.


Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Transportadores de Ácidos Monocarboxílicos , Proteínas de Neoplasias , Neuroblastoma , Simportadores , Vincristina/farmacocinética , Animais , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Simportadores/antagonistas & inibidores , Simportadores/genética , Simportadores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Materials (Basel) ; 12(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500165

RESUMO

A hybrid system composed of multi-walled carbon nanotubes coated with chitosan was proposed as a pH-responsive carrier for the vectorization of methotrexate to lung cancer. The effective coating of the carbon nanostructure by chitosan, quantified (20% by weight) by thermogravimetric analysis, was assessed by combined scanning and transmission electron microscopy, and X-ray photoelectron spectroscopy (N1s signal), respectively. Furthermore, Raman spectroscopy was used to characterize the interaction between polysaccharide and carbon counterparts. Methotrexate was physically loaded onto the nanohybrid and the release profiles showed a pH-responsive behavior with higher and faster release in acidic (pH 5.0) vs. neutral (pH 7.4) environments. Empty nanoparticles were found to be highly biocompatible in either healthy (MRC-5) or cancerous (H1299) cells, with the nanocarrier being effective in reducing the drug toxicity on MRC-5 while enhancing the anticancer activity on H1299.

10.
Pharmaceuticals (Basel) ; 12(2)2019 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31109098

RESUMO

Selective vectorization of Cisplatin (CisPt) to Glioblastoma U87 cells was exploited by the fabrication of a hybrid nanocarrier composed of magnetic γ-Fe2O3 nanoparticles and nanographene oxide (NGO). The magnetic component, obtained by annealing magnetite Fe3O4 and characterized by XRD measurements, was combined with NGO sheets prepared via a modified Hummer's method. The morphological and thermogravimetric analysis proved the effective binding of γ-Fe2O3 nanoparticles onto NGO layers. The magnetization measured under magnetic fields up to 7 Tesla at room temperature revealed superparamagnetic-like behavior with a maximum value of MS = 15 emu/g and coercivity HC ≈ 0 Oe within experimental error. The nanohybrid was found to possess high affinity towards CisPt, and a rather slow fractional release profile of 80% after 250 h. Negligible toxicity was observed for empty nanoparticles, while the retainment of CisPt anticancer activity upon loading into the carrier was observed, together with the possibility to spatially control the drug delivery at a target site.

11.
Sci Transl Med ; 11(477)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700572

RESUMO

Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.


Assuntos
Progressão da Doença , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Poliaminas/metabolismo , Animais , Vias Biossintéticas/genética , Linhagem Celular Tumoral , Estudos de Coortes , Modelos Animais de Doenças , Amplificação de Genes , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Análise Multivariada , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Prognóstico , Modelos de Riscos Proporcionais , Análise de Sobrevida , Resultado do Tratamento
12.
Eur J Cancer ; 83: 132-141, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28735070

RESUMO

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Neuroblastoma/tratamento farmacológico , Animais , Western Blotting , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Doxiciclina/farmacologia , Xenoenxertos/efeitos dos fármacos , Irinotecano , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Oncotarget ; 7(34): 54937-54951, 2016 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-27448979

RESUMO

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Perfilação da Expressão Gênica/métodos , Humanos , Estimativa de Kaplan-Meier , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Interferência de RNA , Fatores de Transcrição/metabolismo
14.
Hum Mol Genet ; 25(18): 3887-3907, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27466189

RESUMO

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.


Assuntos
Histona Desacetilases/biossíntese , Deficiência Intelectual/genética , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/genética , Espasmos Infantis/genética , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Síndromes Epilépticas , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/genética , Humanos , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/fisiopatologia , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/patologia , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/patologia
15.
J Biol Chem ; 288(29): 20817-20829, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23740250

RESUMO

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.


Assuntos
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Diferenciação Celular , Síndrome de Down/genética , Células-Tronco Neurais/patologia , Neuritos/metabolismo , Trissomia/genética , Animais , Astrócitos/metabolismo , Linhagem da Célula , Forma Celular , Modelos Animais de Doenças , Feminino , Inativação Gênica , Proteínas Hedgehog/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Neurais/metabolismo , Neurogênese , Receptores Patched , Receptor Patched-1 , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
16.
Front Oncol ; 3: 42, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23482921

RESUMO

Neuroblastoma is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during the infancy. MYCN amplification and overexpression occur in about 25% of total neuroblastoma cases and this percentage increases at 30% in advanced stage neuroblastoma. So far, MYCN expression profile is still one of the most robust and significant prognostic markers for neuroblastoma outcome. MYCN is a transcription factor that belongs to the family of MYC oncoproteins, comprising c-MYC and MYCL genes. Deregulation of MYC oncoprotein expression is a crucial event involved in the occurrence of different types of malignant tumors. MYCN, as well as c-MYC, can heterodimerize with its partner MAX and activate the transcription of several target genes containing E-Box sites in their promoter regions. However, recent several lines of evidence have revealed that MYCN can repress at least as many genes as it activates, thus proposing a novel function of this protein in neuroblastoma biology. Whereas the mechanism by which MYCN can act as a transcriptional activator is relatively well known, very few studies has been done in the attempt to explain how MYCN can exert its transcription repression function. Here, we will review current knowledge about the mechanism of MYCN-mediated transcriptional repression and will emphasize its role as a repressor in the recruitment of a precise set of proteins to form complexes capable of down-regulating specific subsets of genes whose function is actively involved in apoptosis, cell differentiation, chemosensitivity, and cell motility. The finding that MYCN can also act as a repressor has widen our view on its role in oncogenesis and has posed the bases to search for novel therapeutic drugs that can specifically target its transcriptional repression function.

17.
J Biol Chem ; 288(12): 8332-8341, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23362253

RESUMO

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5'-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.


Assuntos
Neuroblastoma/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Região 5'-Flanqueadora , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Clusterina/genética , Clusterina/metabolismo , Elementos E-Box , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/fisiologia , Proteínas Oncogênicas/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica
18.
PLoS One ; 7(9): e45328, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028937

RESUMO

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.


Assuntos
Distrofina/genética , Músculo Esquelético/metabolismo , Isoformas de RNA/genética , RNA Longo não Codificante/genética , Feminino , Humanos , Masculino , Transcrição Genética/genética
19.
Biochim Biophys Acta ; 1819(11-12): 1173-85, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22921766

RESUMO

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G(0)/G(1) phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5.


Assuntos
Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Encéfalo , Linhagem Celular Tumoral , Síndromes Epilépticas , Humanos , Camundongos , Mutação , Proteína Proto-Oncogênica N-Myc , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Espasmos Infantis/patologia
20.
PLoS One ; 7(7): e40934, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22848414

RESUMO

BACKGROUND: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. PRINCIPAL FINDINGS: Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. CONCLUSIONS: Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.


Assuntos
Deleção de Genes , Regulação Leucêmica da Expressão Gênica , Fator de Transcrição Ikaros , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Transdução de Sinais , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética
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