RESUMO
Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Furanos/farmacologia , Meduloblastoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Furanos/uso terapêutico , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Meduloblastoma/genética , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Mutação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polo-like kinase 1 (PLK1) is a serine/threonine kinase that promotes G2/M-phase transition, is expressed in elevated levels in high-risk neuroblastomas and correlates with unfavorable patient outcome. Recently, we and others have presented PLK1 as a potential drug target for neuroblastoma, and reported that the BI2536 PLK1 inhibitor showed antitumoral actvity in preclinical neuroblastoma models. Here we analyzed the effects of GSK461364, a competitive inhibitor for ATP binding to PLK1, on typical tumorigenic properties of preclinical in vitro and in vivo neuroblastoma models. GSK461364 treatment of neuroblastoma cell lines reduced cell viability and proliferative capacity, caused cell cycle arrest and massively induced apoptosis. These phenotypic consequences were induced by treatment in the low-dose nanomolar range, and were independent of MYCN copy number status. GSK461364 treatment strongly delayed established xenograft tumor growth in nude mice, and significantly increased survival time in the treatment group. These preclinical findings indicate PLK1 inhibitors may be effective for patients with high-risk or relapsed neuroblastomas with upregulated PLK1 and might be considered for entry into early phase clinical trials in pediatric patients.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Dosagem de Genes , Humanos , Concentração Inibidora 50 , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR-34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR-34a in vivo. We generated mice with a constitutive deletion of the miR-34a gene. These mice were devoid of mir-34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re-expression of miR-34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR-34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR-34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR-34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR-34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re-express miR-34a in tumors could, therefore, represent an efficient therapeutic option.
Assuntos
Neoplasias Cerebelares/patologia , Cerebelo/metabolismo , Meduloblastoma/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Transdução de SinaisRESUMO
Medulloblastoma is the most common malignant brain tumor of childhood, and represents a significant clinical challenge in pediatric oncology, since overall survival currently remains under 70%. Patients with tumors overexpressing MYC or harboring a MYC oncogene amplification have an extremely poor prognosis. Pharmacologically inhibiting MYC expression may, thus, have clinical utility given its pathogenetic role in medulloblastoma. Recent studies using the selective small molecule BET inhibitor, JQ1, have identified BET bromodomain proteins, especially BRD4, as epigenetic regulatory factors for MYC and its targets. Targeting MYC expression by BET inhibition resulted in antitumoral effects in various cancers. Our aim here was to evaluate the efficacy of JQ1 against preclinical models for high-risk MYC-driven medulloblastoma. Treatment of medulloblastoma cell lines with JQ1 significantly reduced cell proliferation and preferentially induced apoptosis in cells expressing high levels of MYC. JQ1 treatment of medulloblastoma cell lines downregulated MYC expression and resulted in a transcriptional deregulation of MYC targets, and also significantly altered expression of genes involved in cell cycle progression and p53 signalling. JQ1 treatment prolonged the survival of mice harboring medulloblastoma xenografts and reduced the tumor burden in these mice. Our preclinical data provide evidence to pursue testing BET inhibitors, such as JQ1, as molecular targeted therapeutic options for patients with high-risk medulloblastomas overexpressing MYC or harboring MYC amplifications.
Assuntos
Azepinas/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Genes myc , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Distribuição Aleatória , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Medulloblastoma is a leading cause of childhood cancer-related deaths. Current aggressive treatments frequently lead to cognitive and neurological disabilities in survivors. Novel targeted therapies are required to improve outcome in high-risk medulloblastoma patients and quality of life of survivors. Targeting enzymes controlling epigenetic alterations is a promising approach recently bolstered by the identification of mutations in histone demethylating enzymes in medulloblastoma sequencing efforts. Hypomethylation of lysine 4 in histone 3 (H3K4) is also associated with a dismal prognosis for medulloblastoma patients. Functional characterization of important epigenetic key regulators is urgently needed. RESULTS: We examined the role of the H3K4 modifying enzyme, KDM1A, in medulloblastoma, an enzyme also associated with malignant progression in the closely related tumor, neuroblastoma. Re-analysis of gene expression data and immunohistochemistry of tissue microarrays of human medulloblastomas showed strong KDM1A overexpression in the majority of tumors throughout all molecular subgroups. Interestingly, KDM1A knockdown in medulloblastoma cell lines not only induced apoptosis and suppressed proliferation, but also impaired migratory capacity. Further analyses revealed bone morphogenetic protein 2 (BMP2) as a major KDM1A target gene. BMP2 is known to be involved in development and differentiation of granule neuron precursor cells (GNCPs), one potential cell of origin for medulloblastoma. Treating medulloblastoma cells with the specific KDM1A inhibitor, NCL-1, significantly inhibited growth in vitro. CONCLUSION: We provide the first evidence that a histone demethylase is functionally involved in the regulation of the malignant phenotype of medulloblastoma cells, and lay a foundation for future evaluation of KDM1A-inihibiting therapies in combating medulloblastoma.
Assuntos
Histona Desmetilases/metabolismo , Meduloblastoma/enzimologia , Oxirredutases N-Desmetilantes/metabolismo , Animais , Apoptose/fisiologia , Benzamidas/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclopropanos/farmacologia , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Camundongos TransgênicosRESUMO
LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.
Assuntos
Proteínas de Ligação a DNA/genética , MicroRNAs , Neuroblastoma , Proteínas Nucleares , Proteínas Oncogênicas , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a RNA , Transdução de SinaisRESUMO
Activating anaplastic lymphoma kinase (ALK) mutations were recently detected in most familial and 10% of sporadic neuroblastomas. However, the role of mutated ALK in tumorigenesis remains elusive. We demonstrate that targeted expression of the most frequent and aggressive variant, ALK(F1174L), is tumorigenic in mice. Tumors resembled human neuroblastomas in morphology, metastasis pattern, gene expression, and the presence of neurosecretory vesicles as well as synaptic structures. This ALK-driven neuroblastoma mouse model precisely recapitulated the genetic spectrum of the disease. Chromosomal aberrations were syntenic to those in human neuroblastoma, including 17q gain and MYCN oncogene amplification. Targeted ALK(F1174L) and MYCN coexpression revealed a strong synergism in inducing neuroblastoma with minimal chromosomal aberrations, suggesting that fewer secondary hits are required for tumor induction if both oncoproteins are targeted. Treatment of ALK(F1174L) transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALK(F1174L) activity. We conclude that an activating mutation within the ALK kinase domain is sufficient to induce neuroblastoma development, and ALK inhibitors show promise for treating human neuroblastomas harboring ALK mutations.
Assuntos
Neuroblastoma/etiologia , Neuroblastoma/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Humanos , Camundongos , Camundongos Transgênicos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%-70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.
Assuntos
Neoplasias Cerebelares/prevenção & controle , Imidazóis/farmacologia , Meduloblastoma/prevenção & controle , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Estudos de Casos e Controles , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Cerebelo/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Mutação/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Análise Serial de Tecidos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
For neuroblastoma, the most common extracranial tumour of childhood, identification of new biomarkers and potential therapeutic targets is mandatory to improve risk stratification and survival rates. MicroRNAs are deregulated in most cancers, including neuroblastoma. In this study, we analysed 430 miRNAs in 69 neuroblastomas by stem-loop RT-qPCR. Prediction of event-free survival (EFS) with support vector machines (SVM) and actual survival times with Cox regression-based models (CASPAR) were highly accurate and were independently validated. SVM-accuracy for prediction of EFS was 88.7% (95% CI: 88.5-88.8%). For CASPAR-based predictions, 5y-EFS probability was 0.19% (95% CI: 0-38%) in the CASPAR-predicted short survival group compared with 0.78% (95%CI: 64-93%) in the CASPAR-predicted long survival group. Both classifiers were validated on an independent test set yielding accuracies of 94.74% (SVM) and 5y-EFS probabilities as 0.25 (95% CI: 0.0-0.55) for short versus 1 ± 0.0 for long survival (CASPAR), respectively. Amplification of the MYCN oncogene was highly correlated with deregulation of miRNA expression. In addition, 37 miRNAs correlated with TrkA expression, a marker of excellent outcome, and 6 miRNAs further analysed in vitro were regulated upon TrkA transfection, suggesting a functional relationship. Expression of the most significant TrkA-correlated miRNA, miR-542-5p, also discriminated between local and metastatic disease and was inversely correlated with MYCN amplification and event-free survival. We conclude that neuroblastoma patient outcome prediction using miRNA expression is feasible and effective. Studies testing miRNA-based predictors in comparison to and in combination with mRNA and aCGH information should be initiated. Specific miRNAs (e.g., miR-542-5p) might be important in neuroblastoma tumour biology, and qualify as potential therapeutic targets.
Assuntos
MicroRNAs/biossíntese , Neuroblastoma/genética , Algoritmos , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , MicroRNAs/genética , Neuroblastoma/metabolismo , Receptor trkA/biossíntese , Receptor trkA/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Small non-coding RNAs, in particular microRNAs(miRNAs), regulate fine-tuning of gene expression and can act as oncogenes or tumor suppressor genes. Differential miRNA expression has been reported to be of functional relevance for tumor biology. Using next-generation sequencing, the unbiased and absolute quantification of the small RNA transcriptome is now feasible. Neuroblastoma(NB) is an embryonal tumor with highly variable clinical course. We analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads. MiRNA expression profiles obtained by deep sequencing correlated well with real-time PCR data. Cluster analysis differentiated between favorable and unfavorable NBs, and the miRNA transcriptomes of these two groups were significantly different. Oncogenic miRNAs of the miR17-92 cluster and the miR-181 family were overexpressed in unfavorable NBs. In contrast, the putative tumor suppressive microRNAs, miR-542-5p and miR-628, were expressed in favorable NBs and virtually absent in unfavorable NBs. In-depth sequence analysis revealed extensive post-transcriptional miRNA editing. Of 13 identified novel miRNAs, three were further analyzed, and expression could be confirmed in a cohort of 70 NBs.