RESUMO
Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.
Assuntos
Antígeno 12E7 , Proteínas de Fusão Oncogênica , Oncogenes , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Sarcoma de Ewing , Proteína GLI1 em Dedos de Zinco , Zixina , Animais , Humanos , Camundongos , Antígeno 12E7/metabolismo , Camundongos Nus , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Transfecção , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Zixina/genéticaRESUMO
Appropriate tools for monitoring sarcoma progression are still limited. The aim of the present study was to investigate the value of miR-34a-5p (miR34a) as a circulating biomarker to follow disease progression and measure the therapeutic response. Stable forced re-expression of miR34a in Ewing sarcoma (EWS) cells significantly limited tumor growth in mice. Absolute quantification of miR34a in the plasma of mice and 31 patients showed that high levels of this miRNA inversely correlated with tumor volume. In addition, miR34a expression was higher in the blood of localized EWS patients than in the blood of metastatic EWS patients. In 12 patients, we followed miR34a expression during preoperative chemotherapy. While there was no variation in the blood miR34a levels in metastatic patients at the time of diagnosis or after the last cycle of preoperative chemotherapy, there was an increase in the circulating miR34a levels in patients with localized tumors. The three patients with the highest fold-increase in the miR levels did not show evidence of metastasis. Although this analysis should be extended to a larger cohort of patients, these findings imply that detection of the miR34a levels in the blood of EWS patients may assist with the clinical management of EWS.
RESUMO
The identification of new therapeutic strategies against osteosarcoma, the most common primary bone tumor, continues to be a primary goal to improve the outcomes of patients refractory to conventional chemotherapy. Osteosarcoma originates from the transformation of mesenchymal stem cells (MSC) and/or osteoblast progenitors, and the loss of differentiation is a common biological osteosarcoma feature, which has strong significance in predicting tumor aggressiveness. Thus, restoring differentiation through epigenetic reprogramming is potentially exploitable for therapeutic benefits. Here, we demonstrated that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 affected tumor proliferation by blocking osteosarcoma cells in G1 or G2-M phases and induced osteoblastic differentiation through the specific reexpression of genes regulating this physiologic process. Although MC3343 has a similar antiproliferative effect as 5azadC, the conventional FDA-approved nucleoside inhibitor of DNA methylation, its effects on cell differentiation are distinct. Induction of the mature osteoblast phenotype coupled with a sustained cytostatic response was also confirmed in vivo when MC3343 was used against a patient-derived xenograft (PDX). In addition, MC3343 displayed synergistic effects with doxorubicin and cisplatin (CDDP), two major chemotherapeutic agents used to treat osteosarcoma. Specifically, MC3343 increased stable doxorubicin bonds to DNA, and combined treatment resulted in sustained DNA damage and increased cell death. Overall, this nonnucleoside DNMTi is an effective novel agent and is thus a potential therapeutic option for patients with osteosarcoma who respond poorly to preadjuvant chemotherapy. Mol Cancer Ther; 17(9); 1881-92. ©2018 AACR.
Assuntos
Aminoquinolinas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Osteossarcoma/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Aminoquinolinas/administração & dosagem , Animais , Benzamidas/administração & dosagem , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Doxorrubicina/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Knockout , Osteossarcoma/genética , Osteossarcoma/metabolismo , Quinolinas/química , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.
Assuntos
Antígeno 12E7/imunologia , Anticorpos Monoclonais/farmacologia , Neoplasias Ósseas/patologia , Morte Celular/efeitos dos fármacos , Sarcoma de Ewing/patologia , Antígeno 12E7/antagonistas & inibidores , Antígeno 12E7/fisiologia , Antineoplásicos Imunológicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Células Cultivadas , Genes ras/fisiologia , Células HEK293 , Humanos , Pinocitose/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Sarcoma de Ewing/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.
Assuntos
Antígeno 12E7/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose , Sobrevivência Celular/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/genética , Sarcoma de Ewing/tratamento farmacológico , Anticorpos de Cadeia Única/genéticaRESUMO
PURPOSE: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. EXPERIMENTAL DESIGN: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. RESULTS: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS. CONCLUSIONS: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death.
Assuntos
Antígenos CD/biossíntese , Apoptose/efeitos dos fármacos , Moléculas de Adesão Celular/biossíntese , Sarcoma de Ewing/tratamento farmacológico , Anticorpos de Cadeia Única/administração & dosagem , Proteína Supressora de Tumor p53/biossíntese , Antígeno 12E7 , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Antígenos CD/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99-transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane-bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21(WAF1/CIP1) , leading to G0 /G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate.
Assuntos
Neoplasias Ósseas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/biossíntese , Osteossarcoma/metabolismo , Antígeno 12E7 , Antígenos CD , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas de Neoplasias/genética , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/terapiaRESUMO
BACKGROUND: Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones. METHODS: Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumour efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts. RESULTS: Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system. CONCLUSIONS: Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients.
Assuntos
Antineoplásicos/farmacologia , Sarcoma/genética , Sarcoma/metabolismo , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/farmacologia , Xenoenxertos , Humanos , Concentração Inibidora 50 , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Sarcoma/tratamento farmacológico , Sarcoma/patologiaRESUMO
The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14-6.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation.
Assuntos
Códon , Genótipo , Neuroblastoma/genética , Proteína Supressora de Tumor p53/genética , Adulto , Envelhecimento/genética , Apoptose/genética , Linhagem Celular Tumoral , Pré-Escolar , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Polimorfismo de Nucleotídeo Único , Prognóstico , Isoformas de Proteínas/genética , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismoRESUMO
The CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3-ITD receptor tyrosine kinase, MAP kinase-dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBPα to induce granulocytic differentiation. To assess whether this post-translational modification also modulates the activity of C/EBPα in BCR/ABL-expressing cells, we tested the biological effects of wild-type and mutant C/EBPα mimicking phosphorylated or non-phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen-regulated C/EBPα-ER chimeric proteins. We show here that S21D C/EBPα-ER induced terminal granulocytic differentiation of K562 cells almost as well as wild-type C/EBPα-ER, while S21A C/EBPα-ER was less efficient. Furthermore, wild-type C/EBPα suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBPα mutants had more modest anti-proliferative effects. Both mutants were less effective than wild-type C/EBPα in suppressing endogenous E2F-dependent transactivation and bound less E2F-2 and/or E2F-3 proteins in anti-C/EBPα immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti-proliferative effects of C/EBPα due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation-inducing effects of C/EBPα in K562 cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/química , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Benzamidas/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Primers do DNA/genética , Fatores de Transcrição E2F/metabolismo , Granulócitos/metabolismo , Granulócitos/patologia , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Fosforilação , Regiões Promotoras Genéticas , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Ensaio Tumoral de Célula-TroncoRESUMO
The transcription factor C/EBPα is more potent than C/EBPß in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a "domain swapping" approach to assess biological effects, modulation of gene expression, and binding to C/EBPα-regulated promoters by wild-type and chimeric C/EBPα/C/EBPß proteins. Wild-type and N-C/EBPα+ C/EBPß-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPß and N-C/EBPß+C/EBPα-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPα and N-C/EBPα+C/EBPß-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPα or C/EBPß inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPα. Gene expression profiles induced by C/EBPα resembled those modulated by N-C/EBPα+C/EBPß-DBD, whereas C/EBPß induced a pattern similar to that of N-C/EBPß+C/EBPα-DBD. C/EBPα activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPα-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Apoptose/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Células K562 , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
PURPOSE: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors (osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma). EXPERIMENTAL DESIGN: Antiproliferative activity as well as the effects on migration and metastasis were evaluated in a panel of osteosarcoma, Ewing's sarcoma, as well as rhabdomyosarcoma cell lines. Moreover, simultaneous and sequential treatments were done in association with two of the most important conventional drugs in the treatment of sarcoma, doxorubicin and vincristine. RESULTS: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the experimental conditions tested, the compound induced disease stasis, by arresting cells in G(1) phase of cell cycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitation of its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxic agents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phosphoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellular signal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signaling toward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235 with other small signaling inhibitors. Here, we showed synergistic effects when the compound was associated with a anti-insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 also inhibited cell migration and metastasis. Combination with vincristine further potentiated the antimetastatic effects. CONCLUSIONS: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advanced cancer patients.
Assuntos
Imidazóis/uso terapêutico , Neoplasias Musculares/tratamento farmacológico , Quinolinas/uso terapêutico , Sarcoma/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Neoplasias Musculares/patologia , Metástase Neoplásica , Quinolinas/farmacologia , Sarcoma/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. RESULTS: We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. CONCLUSION: We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.
Assuntos
Neoplasias da Mama/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoprecipitação , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Fator 1 de Elongação de Peptídeos/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNARESUMO
PURPOSE: MDM2 is a key negative regulator of p53 activity, and a single nucleotide polymorphism (SNP309, T>G change; rs 2279744) in its promoter increases the affinity for the transcription factor SP1, enhancing MDM2 expression. We carried out a pilot study to investigate the effect of this polymorphism on development and behavior of neuroblastoma, an extracranial pediatric tumor with unfrequent genetic inactivation of p53. EXPERIMENTAL DESIGN: We genotyped the MDM2-SNP309 alleles of tumor DNA from 239 neuroblastoma patients and peripheral blood DNA from 237 controls. In 40 of 239 neuroblastomas, the MDM2-SNP309 alleles were also genotyped in peripheral blood DNA. Data were analyzed by two-sided Fisher's exact test, log-rank test, and Kaplan-Meier statistics. Where appropriate, data are reported with 95% confidence intervals (CI). RESULTS: The frequency of both the T/G and G/G genotypes or the G/G or T/G genotype only was higher in neuroblastoma DNA samples than in controls: 60.3% (95% CI, 54.1-66.5) versus 47.3% (95% CI, 40.9-53.6), 30.4% (95% CI, 22.4-37.8) versus 15.0% (95% CI, 9.2-20.7), and 52.0% (95% CI, 45.0-59.9) versus 41.9% (95% CI, 35.3-48.5), respectively; Two-Sided Fisher's Exact Test P values were 0.006, 0.003, and 0.048, respectively; Odds ratios were 1.69 (95% CI, 1.18-2.43), 2.45 (95% CI, 1.37-4.39) and 1.51 (95% CI, 1.02-2.22), respectively. A significant association (P = 0.016) between heterozygous (T/G)/homozygous (G/G) genotypes at SNP309 and advanced clinical stages was also shown. Homozygous/heterozygous SNP309 variant carriers had a shorter 5-year overall survival than patients with the wild-type allele (P = 0.046; log-rank test). A shorter overall survival in patients with heterozygous/homozygous SNP309 was also observed in the subgroups with age at diagnosis >1 year and adrenal primary tumor (P = 0.024 and P = 0.014, respectively). CONCLUSIONS: Data from this pilot study suggest that the MDM2 G/G and T/G-SNP309 alleles are markers of increased predisposition to tumor development and disease aggressiveness in neuroblastoma. However, additional studies with larger patient cohorts are required for a definitive assessment of the clinical relevance of these data.
Assuntos
Predisposição Genética para Doença , Neuroblastoma/genética , Neuroblastoma/patologia , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/genética , Criança , Pré-Escolar , Humanos , Lactente , Estimativa de Kaplan-Meier , Neuroblastoma/mortalidade , Projetos Piloto , Reação em Cadeia da PolimeraseRESUMO
C/EBPalpha and C/EBPbeta, two transcription factors of the C/EBP family play important roles in the proliferation and differentiation of various cell types including myeloid progenitors. Expression of C/EBPalpha and C/EBPbeta is repressed in myeloid blast crisis of Chronic Myelogenous Leukemia by mechanisms that involve translation repression which depends on the interaction of RNA-binding proteins with conserved binding sites in the 5'UTR of c/ebpalpha and c/ebpbeta mRNA. Ectopic expression of C/EBPalpha and C/EBPbeta in myeloid progenitors expressing the BCR/ABL oncogene inhibits proliferation, induces differentiation and suppresses leukemogenesis in mice, but C/EBPalpha is markedly more effective than C/EBPbeta. The more potent effects of C/EBPalpha probably depends on protein-protein interaction with cell-cycle regulatory proteins, but the pattern of genes modulated by C/EBPalpha and C/EBPbeta is not completely overlapping. This suggests that transcription-dependent and -independent effects are both involved and support the therapeutic potential of reactivating C/EBPalpha and C/EBPbeta expression in leukemic cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana/metabolismoRESUMO
Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPalpha, which were more potent than those of K298E C/EBPalpha, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Diferenciação Celular/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Crise Blástica/tratamento farmacológico , Crise Blástica/genética , Crise Blástica/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Granulócitos/metabolismo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Mutação , Piperazinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/metabolismo , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebpbeta mRNA. Constitutive expression or conditional activation of wild-type CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because expression of CEBPB (and CEBPA) is low in the blast crisis (BC) stage of chronic myelogenous leukemia (CML) and is inversely correlated with BCR/ABL tyrosine kinase levels, these findings point to the therapeutic potential of restoring C/EBP activity in CML-BC and, perhaps, other types of acute leukemia.
Assuntos
Células da Medula Óssea/citologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas de Fusão bcr-abl/genética , Células da Medula Óssea/fisiologia , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Luciferases/genética , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Repetições de TrinucleotídeosRESUMO
Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.
Assuntos
Células Mieloides/citologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/fisiologia , Proteínas/genética , Proteínas/fisiologia , Animais , Sequência de Bases , Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , DNA Complementar/genética , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/farmacologia , Leucemia Mieloide/etiologia , Camundongos , Modelos Biológicos , Células Mieloides/efeitos dos fármacos , Células Mieloides/fisiologia , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/fisiologia , Transdução GenéticaRESUMO
In a BCR/ABL-expressing myeloid precursor cell line, p53 levels were markedly downmodulated. Expression of MDM2, the negative regulator of p53, was upregulated in a tyrosine kinase-dependent manner in growth factor-independent BCR/ABL-expressing cells, and in accelerated phase and blast crisis CML samples. Increased MDM2 expression was associated with enhanced mdm2 mRNA translation, which required the interaction of the La antigen with mdm2 5' UTR. Expression of MDM2 correlated with that of La and was suppressed by La siRNAs and by a dominant negative La mutant, which also enhanced the susceptibility to drug-induced apoptosis of BCR/ABL-transformed cells. By contrast, La overexpression led to increased MDM2 levels and enhanced resistance to apoptosis. Thus, La-dependent activation of mdm2 translation might represent an important molecular mechanism involved in BCR/ABL leukemogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Fusão bcr-abl/fisiologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autoantígenos , Northern Blotting , Western Blotting , Resistencia a Medicamentos Antineoplásicos , Proteína Adaptadora GRB2 , Substâncias de Crescimento/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Biossíntese de Proteínas , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Antígeno SS-BRESUMO
hnRNP A1 is a nucleocytoplasmic shuttling heterogeneous nuclear ribonucleoprotein that accompanies eukaryotic mRNAs from the active site of transcription to that of translation. Although the importance of hnRNP A1 as a regulator of nuclear pre-mRNA and mRNA processing and export is well established, it is unknown whether this is relevant for the control of proliferation, survival, and differentiation of normal and transformed cells. We show here that hnRNP A1 levels are increased in myeloid progenitor cells expressing the p210(BCR/ABL) oncoprotein, in mononuclear cells from chronic myelogenous leukemia (CML) blast crisis patients, and during disease progression. In addition, in myeloid progenitor 32Dcl3 cells, BCR/ABL stabilizes hnRNP A1 by preventing its ubiquitin/proteasome-dependent degradation. To assess the potential role of hnRNP A1 nucleocytoplasmic shuttling activity in normal and leukemic myelopoiesis, a mutant defective in nuclear export was ectopically expressed in parental and BCR/ABL-transformed myeloid precursor 32Dcl3 cells, in normal murine marrow cells, and in mononuclear cells from a CML patient in accelerated phase. In normal cells, expression of this mutant enhanced the susceptibility to apoptosis induced by interleukin-3 deprivation, suppressed granulocytic differentiation, and induced massive cell death of granulocyte colony-stimulating factor-treated cultures. In BCR/ABL-transformed cells, its expression was associated with suppression of colony formation and reduced tumorigenic potential in vivo. Moreover, interference with hnRNP A1 shuttling activity resulted in downmodulation of C/EBPalpha, the major regulator of granulocytic differentiation, and Bcl-X(L), an important survival factor for hematopoietic cells. Together, these results suggest that the shuttling activity of hnRNP A1 is important for the nucleocytoplasmic trafficking of mRNAs that encode proteins influencing the phenotype of normal and BCR/ABL-transformed myeloid progenitors.