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1.
J Neurosci ; 35(50): 16531-44, 2015 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-26674877

RESUMO

The RNA binding protein Lin28B is expressed in developing tissues and sustains stem and progenitor cell identity as a negative regulator of the Let-7 family of microRNAs, which induces differentiation. Lin28B is activated in neuroblastoma (NB), a childhood tumor in sympathetic ganglia and adrenal medulla. Forced expression of Lin28B in embryonic mouse sympathoadrenal neuroblasts elicits postnatal NB formation. However, the normal function of Lin28B in the development of sympathetic neurons and chromaffin cells and the mechanisms involved in Lin28B-induced tumor formation are unclear. Here, we demonstrate a mirror-image expression of Lin28B and Let-7a in developing chick sympathetic ganglia. Lin28B expression is not restricted to undifferentiated progenitor cells but, is observed in proliferating noradrenergic neuroblasts. Lin28 knockdown in cultured sympathetic neuroblasts decreases proliferation, whereas Let-7 inhibition increases the proportion of neuroblasts in the cell cycle. Lin28B overexpression enhances proliferation, but only during a short developmental period, and it does not reduce Let-7a. Effects of in vivo Lin28B overexpression were analyzed in the LSL-Lin28B(DBHiCre) mouse line. Sympathetic ganglion and adrenal medulla volume and the expression level of Let-7a were not altered, although Lin28B expression increased by 12- to 17-fold. In contrast, Let-7a expression was strongly reduced in LSL-Lin28B(DbhiCre) NB tumor tissue. These data demonstrate essential functions for endogenous Lin28 and Let-7 in neuroblast proliferation. However, Lin28B overexpression neither sustains neuroblast proliferation nor affects let-7 expression. Thus, in contrast to other pediatric tumors, Lin28B-induced NB is not due to expansion of proliferating embryonic neuroblasts, and Let-7-independent functions are implicated during initial NB development. SIGNIFICANCE STATEMENT: Lin28A/B proteins are highly expressed in early development and maintain progenitor cells by blocking the biogenesis and differentiation function of Let-7 microRNAs. Lin28B is aberrantly upregulated in the childhood tumor neuroblastoma (NB). NB develops in sympathetic ganglia and adrenal medulla and is elicited by forced Lin28B expression. We demonstrate that Lin28A/B and Let-7 are essential for sympathetic neuroblast proliferation during normal development. Unexpectedly, Lin28B upregulation in a mouse model does not affect neuroblast proliferation, ganglion size, and Let-7 expression during early postnatal development. Lin28B-induced NB, in contrast to other pediatric cancers, does not evolve from neuroblasts that continue to divide and involves Let-7-independent functions during initial development.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Sistema Nervoso Simpático/crescimento & desenvolvimento , Glândulas Suprarrenais/metabolismo , Animais , Proliferação de Células , Embrião de Galinha , Proteínas de Ligação a DNA/fisiologia , Gânglios Simpáticos/patologia , Camundongos , Camundongos da Linhagem 129 , MicroRNAs/fisiologia , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Proteínas de Ligação a RNA , Células-Tronco/metabolismo , Sistema Nervoso Simpático/fisiologia
2.
FASEB J ; 28(2): 1010-21, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24174424

RESUMO

O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic ß-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 ß cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Glucose/farmacologia , Imunoprecipitação , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Ratos
3.
Blood ; 119(6): 1532-42, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22160620

RESUMO

Normal human erythroid cell maturation requests the transcription factor GATA-1 and a transient activation of caspase-3, with GATA-1 being protected from caspase-3-mediated cleavage by interaction with the chaperone heat shock protein 70 (Hsp70) in the nucleus. Erythroid cell dysplasia observed in early myelodysplastic syndromes (MDS) involves impairment of differentiation and excess of apoptosis with a burst of caspase activation. Analysis of gene expression in MDS erythroblasts obtained by ex vivo cultures demonstrates the down-regulation of a set of GATA-1 transcriptional target genes, including GYPA that encodes glycophorin A (GPA), and the up-regulation of members of the HSP70 family. GATA-1 protein expression is decreased in MDS erythroblasts, but restores in the presence of a pan-caspase inhibitor. Expression of a mutated GATA-1 that cannot be cleaved by caspase-3 rescues the transcription of GATA-1 targets, and the erythroid differentiation, but does not improve survival. Hsp70 fails to protect GATA-1 from caspases because the protein does not accumulate in the nucleus with active caspase-3. Expression of a nucleus-targeted mutant of Hsp70 protects GATA-1 and rescues MDS erythroid cell differentiation. Alteration of Hsp70 cytosolic-nuclear shuttling is a major feature of MDS that favors GATA-1 cleavage and differentiation impairment, but not apoptosis, in dysplastic erythroblasts.


Assuntos
Núcleo Celular/metabolismo , Eritropoese/genética , Fator de Transcrição GATA1/genética , Proteínas de Choque Térmico HSP70/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Eritroblastos/metabolismo , Células Eritroides/metabolismo , Feminino , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Immunoblotting , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937
4.
Clin Cancer Res ; 29(7): 1317-1331, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36602782

RESUMO

PURPOSE: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.


Assuntos
Neoplasias Pulmonares , Neuroblastoma , Camundongos , Animais , Humanos , Quinase do Linfoma Anaplásico/genética , Aminopiridinas/uso terapêutico , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico
5.
Nat Commun ; 14(1): 2575, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-37142597

RESUMO

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.


Assuntos
Plasticidade Celular , Neuroblastoma , Humanos , Neuroblastoma/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral/genética
6.
Cancers (Basel) ; 14(11)2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35681734

RESUMO

Neuroblastoma arising from the adrenal differ from ganglionic neuroblastoma both genetically and clinically, with adrenal tumors being associated with a more severe prognosis. The different tumor properties may be linked to specific tumor founder cells in adrenal and sympathetic ganglia. To address this question, we first set up cultures of mouse sympathetic neuroblasts and adrenal chromaffin cells. These cultures were then treated with various proliferation inhibitors to identify lineage-specific responses. We show that neuroblast and chromaffin cell proliferation was affected by WNT, ALK, IGF1, and PRC2/EZH2 signaling inhibitors to a similar extent. However, differential effects were observed in response to bromodomain and extraterminal (BET) protein inhibitors (JQ1, GSK1324726A) and to the CDK-7 inhibitor THZ1, with BET inhibitors preferentially affecting chromaffin cells, and THZ1 preferentially affecting neuroblasts. The differential dependence of chromaffin cells and neuroblasts on BET and CDK signaling may indicate different mechanisms during tumor initiation in sympathetic ganglia and adrenal.

7.
J Immunother Cancer ; 10(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-36054452

RESUMO

BACKGROUND: High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS: We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS: We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS: Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment.


Assuntos
Neuroblastoma , Transcriptoma , Animais , Criança , Ecossistema , Humanos , Camundongos , Recidiva Local de Neoplasia , Neuroblastoma/patologia , Microambiente Tumoral/genética
8.
Cancer Res ; 81(19): 4994-5006, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34341072

RESUMO

Ewing sarcoma is characterized by pathognomonic translocations, most frequently fusing EWSR1 with FLI1. An estimated 30% of Ewing sarcoma tumors also display genetic alterations in STAG2, TP53, or CDKN2A (SPC). Numerous attempts to develop relevant Ewing sarcoma models from primary human cells have been unsuccessful in faithfully recapitulating the phenotypic, transcriptomic, and epigenetic features of Ewing sarcoma. In this study, by engineering the t(11;22)(q24;q12) translocation together with a combination of SPC mutations, we generated a wide collection of immortalized cells (EWIma cells) tolerating EWSR1-FLI1 expression from primary mesenchymal stem cells (MSC) derived from a patient with Ewing sarcoma. Within this model, SPC alterations strongly favored Ewing sarcoma oncogenicity. Xenograft experiments with independent EWIma cells induced tumors and metastases in mice, which displayed bona fide features of Ewing sarcoma. EWIma cells presented balanced but also more complex translocation profiles mimicking chromoplexy, which is frequently observed in Ewing sarcoma and other cancers. Collectively, these results demonstrate that bone marrow-derived MSCs are a source of origin for Ewing sarcoma and also provide original experimental models to investigate Ewing sarcomagenesis. SIGNIFICANCE: These findings demonstrate that Ewing sarcoma can originate from human bone-marrow-derived mesenchymal stem cells and that recurrent mutations support EWSR1-FLI1 translocation-mediated transformation.


Assuntos
Transformação Celular Neoplásica , Suscetibilidade a Doenças , Células-Tronco Mesenquimais/metabolismo , Sarcoma de Ewing/etiologia , Sarcoma de Ewing/metabolismo , Animais , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Biologia Computacional/métodos , Modelos Animais de Doenças , Edição de Genes , Perfilação da Expressão Gênica , Rearranjo Gênico , Marcação de Genes , Xenoenxertos , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Células-Tronco Mesenquimais/patologia , Camundongos , Mutação , Sarcoma de Ewing/patologia , Translocação Genética
9.
Haematologica ; 95(11): 1964-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20823131

RESUMO

Serum erythropoietin level less than 100U/L and a transfusion requirement of less than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes. To investigate the factors influencing the response to erythropoiesis-stimulating agents, we enrolled 127 low/int-1 myelodysplastic syndrome patients at diagnosis in a biological study of erythropoiesis. The 54 non-responders had a significantly lower number of burst-forming unit-erythroid and colony-forming unit-erythroid than responders. Erythropoietin-dependent proliferation and survival, and phospho (p)-ERK1/2 expression in steady state and after erythropoietin stimulation were defective in cultured erythroblasts. By flow cytometry, p-ERK1/2 was significantly lower in bone marrow CD45(-)/CD71(+)/GPA(-)cells from non-responders compared to responders or controls. Receiver Operator Characteristic curve analysis showed that this flow cytometry test was a sensitive biomarker for predicting the response to erythropoiesis-stimulating agents.


Assuntos
Eritroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Hematínicos/uso terapêutico , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Síndromes Mielodisplásicas/enzimologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Eritroblastos/patologia , Eritropoese/efeitos dos fármacos , Eritropoetina/sangue , Eritropoetina/farmacologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia
10.
Oncotarget ; 10(48): 4937-4950, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31452835

RESUMO

The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

11.
Oncogene ; 37(11): 1417-1429, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29321660

RESUMO

Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinogênese/genética , Mutação com Ganho de Função , Neuroblastoma/genética , Quinase do Linfoma Anaplásico/metabolismo , Animais , Carcinogênese/patologia , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Feminino , Mutação com Ganho de Função/fisiologia , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-ret/fisiologia , Transdução de Sinais/genética , Fatores de Transcrição/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Sci Rep ; 7(1): 16901, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29203791

RESUMO

Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptor de Insulina/metabolismo , Sulfanilamidas/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Insulina/metabolismo , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Receptor de Insulina/química , Transdução de Sinais/efeitos dos fármacos , Sulfanilamidas/metabolismo , Sulfanilamidas/farmacologia
14.
Nat Genet ; 49(9): 1408-1413, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28740262

RESUMO

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.


Assuntos
Linhagem da Célula/genética , Regulação Neoplásica da Expressão Gênica/genética , Neuroblastoma/genética , Fatores de Transcrição/genética , Animais , Western Blotting , Linhagem Celular Tumoral/classificação , Linhagem da Célula/efeitos dos fármacos , Doxiciclina/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Interferência de RNA , Terapêutica com RNAi , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Célula Única , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
15.
Arch Physiol Biochem ; 122(2): 54-60, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26707268

RESUMO

CONTEXT: Insulin analogues are largely used for the treatment of diabetic patients, but concerns have been raised about their mitogenic/anti-apoptotic potential. It is therefore important to evaluate these analogues in different cell systems. OBJECTIVE: The aim of this work was to establish the pharmacological profiles of insulin analogues towards PI-3 kinase/Akt pathway in INS-1 ß-pancreatic cells. METHODS: Bioluminescence Resonance Energy Transfer (BRET), in cell western and caspase 3/7 assays, was used to study the effects of ligands. RESULTS: Among the five analogues evaluated, only glargine stimulated PI-3 kinase/Akt pathway with higher efficiency than insulin, whereas glargine's metabolite M1 was less efficient. However, glargine did not show higher anti-apoptotic efficiency than insulin. CONCLUSION: Glargine was more efficient than insulin for the activation of PI-3 kinase/Akt pathway, but not for the inhibition of caspase 3/7 activity. Moreover, glargine's metabolite M1 displayed lower efficiency than insulin towards PI-3 kinase/Akt activation and caspase 3/7 inhibition.


Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Insulina/análogos & derivados , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Insulina Glargina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/biossíntese , Ratos
16.
Oncotarget ; 7(12): 14898-911, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26910909

RESUMO

CD95, a member of the death receptor family initiates a caspase-dependent apoptosis, when activated by its ligand CD95L, thought to negatively regulate erythrocyte production in the bone marrow. We have previously shown that CD95 is overexpressed in two thirds of patients with a lower risk myelodysplastic syndrome (MDS) and that resistance to erythropoiesis-stimulating agents (ESA) is linked to poor residual erythropoiesis. In the present study, we show that CD95 overexpression and previous transfusion are independent predictive factors of ESA resistance. To investigate an alternative therapeutic strategy of anemia in ESA-resistant patients, we have conducted a preclinical study of the effects of APG101, a fusion protein consisting of the extracellular domain of human CD95 and the Fc region of human IgG1 on MDS erythropoiesis in vitro. APG101 increases the number of burst-forming unit-erythroid (BFU-E) progenitors derived from CD34+ progenitors in liquid culture and improves overall proliferation rate of erythroid precursors by inhibiting apoptosis. APG101 rescues BFU-E growth in MDS patients presenting with attrition of erythroid progenitors at baseline, independently of CD95 or CD95L expression level. Our data show that overexpression of CD95 at diagnosis is a hallmark of ESA resistance and that severe impairment of erythropoiesis is predictive of erythroid response to APG101 in vitro. These data provide a rationale for further clinical investigation of APG101 in an attempt to treat anemia in lower risk MDS patients.


Assuntos
Eritropoese , Hematopoese , Imunoglobulina G/metabolismo , Síndromes Mielodisplásicas/prevenção & controle , Proteínas Recombinantes de Fusão/metabolismo , Receptor fas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Feminino , Seguimentos , Humanos , Imunoglobulina G/genética , Masculino , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Prognóstico , Proteínas Recombinantes de Fusão/genética , Taxa de Sobrevida , Receptor fas/genética
17.
PLoS One ; 9(3): e92737, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24647478

RESUMO

Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Fosfatos de Fosfatidilinositol/análise , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Humanos
18.
Oncotarget ; 5(9): 2703-13, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24811761

RESUMO

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.


Assuntos
Comportamento Animal/fisiologia , Ingestão de Alimentos , Mutação/genética , Neuroblastoma/genética , Receptores Proteína Tirosina Quinases/fisiologia , Respiração , Quinase do Linfoma Anaplásico , Animais , Animais Recém-Nascidos , Genes Letais , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fenótipo
19.
Oncotarget ; 5(9): 2688-702, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24811913

RESUMO

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.


Assuntos
Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Mutação/genética , Neuroblastoma/genética , Neurogênese , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Proteína Tirosina Quinases/genética , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Southern Blotting , Western Blotting , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Integrases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-ret/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Células Tumorais Cultivadas
20.
PLoS One ; 8(7): e69150, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23935944

RESUMO

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the ß-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.


Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Processamento de Proteína Pós-Traducional , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Antineoplásicos Hormonais/farmacologia , Vias Biossintéticas , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hexosaminas/biossíntese , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Células MCF-7 , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/análogos & derivados
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