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MYCN amplification occurs in approximately 20-30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.
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Antimitóticos , Neuroblastoma , Humanos , Camundongos , Animais , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Linhagem Celular Tumoral , Camundongos Transgênicos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.
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PURPOSE: Patients with oligometastatic prostate cancer (PC) may benefit from metastasis-directed therapy (MDT), delaying disease progression and the start of palliative systemic treatment. However, a significant proportion of oligometastatic PC patients progress to polymetastatic PC within a year following MDT, suggesting an underestimation of the metastatic load by current staging modalities. Molecular markers could help to identify true oligometastatic patients eligible for MDT. METHODS: Patients with asymptomatic biochemical recurrence following primary PC treatment were classified as oligo- or polymetastatic based on 18F-choline PET/CT imaging. Oligometastatic patients had up to three metastases at baseline and did not progress to more than three lesions following MDT or surveillance within 1 year of diagnosis of metastases. Polymetastatic patients had > 3 metastases at baseline or developed > 3 metastases within 1 year following imaging. A model aiming to prospectively distinguish oligo- and polymetastatic PC patients was trained using clinicopathological parameters and serum-derived microRNA expression profiles from a discovery cohort of 20 oligometastatic and 20 polymetastatic PC patients. To confirm the models predictive performance, it was applied on biomarker data obtained from an independent validation cohort of 44 patients with oligometastatic and 39 patients with polymetastatic disease. RESULTS: Oligometastatic PC patients had a more favorable prognosis compared to polymetastatic ones, as defined by a significantly longer median CRPC-free survival (not reached versus 38 months; 95% confidence interval 31-45 months with P < 0.001). Despite the good performance of a predictive model trained on the discovery cohort, with an AUC of 0.833 (0.693-0.973; 95% CI) and a sensitivity of 0.894 (0.714-1.000; 95% CI) for oligometastatic disease, none of the miRNA targets were found to be differentially expressed between oligo- and polymetastatic PC patients in the signature validation cohort. The multivariate model had an AUC of 0.393 (0.534 after cross-validation) and therefore, no predictive ability. CONCLUSIONS: Although PC patients with oligometastatic disease had a more favorable prognosis, no serum-derived biomarkers allowing for prospective discrimination of oligo- and polymetastatic prostate cancer patients could be identified.
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MicroRNAs/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Idoso , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Metástase Neoplásica/genética , Estudos Prospectivos , TranscriptomaRESUMO
Neuroblastoma is a pediatric cancer of the sympathetic nervous system where MYCN amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN+/+ mice to age-matched wild-type controls, we devised a predicted miRNA-mRNA interaction network. Among the miRNA-mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with MYCN-amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation in vitro and tumorigenesis in vivo Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis.Significance: Network modeling of miRNA-mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. Cancer Res; 78(12); 3122-34. ©2018 AACR.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neoplasias do Sistema Nervoso Periférico/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Conjuntos de Dados como Assunto , Feminino , Redes Reguladoras de Genes , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Oncogenes/genética , Neoplasias do Sistema Nervoso Periférico/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems (ALK, Th-MYCN, Dbh-MYCN and Lin28b). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.
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Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.
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Modelos Teóricos , Reação em Cadeia da Polimerase/métodos , DNA de Plantas/análise , DNA de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/normas , Controle de QualidadeRESUMO
Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.
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Antineoplásicos/uso terapêutico , Morfolinas/uso terapêutico , Neuroblastoma/tratamento farmacológico , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Pró-Fármacos/uso terapêutico , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Aprepitanto , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Fator de Transcrição E2F2/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular/métodos , Neuroblastoma/patologia , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.
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Regulação Neoplásica da Expressão Gênica/fisiologia , Histona Desacetilases/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/patologia , Tetraspanina 29/biossíntese , Animais , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc/genética , Invasividade Neoplásica/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Tetraspanina 29/genéticaRESUMO
The MYCN gene is amplified and overexpressed in a large proportion of high stage neuroblastoma patients and has been identified as a key driver of tumorigenesis. However, the mechanism by which MYCN promotes tumor initiation is poorly understood. Here we conducted metabolic profiling of pre-malignant sympathetic ganglia and tumors derived from the TH-MYCN mouse model of neuroblastoma, compared to non-malignant ganglia from wildtype littermates. We found that metabolites involved in the biosynthesis of glutathione, the most abundant cellular antioxidant, were the most significantly upregulated metabolic pathway at tumor initiation, and progressively increased to meet the demands of tumorigenesis. A corresponding increase in the expression of genes involved in ribosomal biogenesis suggested that MYCN-driven transactivation of the protein biosynthetic machinery generated the necessary substrates to drive glutathione biosynthesis. Pre-malignant sympathetic ganglia from TH-MYCN mice had higher antioxidant capacity and required glutathione upregulation for cell survival, when compared to wildtype ganglia. Moreover, in vivo administration of inhibitors of glutathione biosynthesis significantly delayed tumorigenesis when administered prophylactically and potentiated the anticancer activity of cytotoxic chemotherapy against established tumors. Together these results identify enhanced glutathione biosynthesis as a selective metabolic adaptation required for initiation of MYCN-driven neuroblastoma, and suggest that glutathione-targeted agents may be used as a potential preventative strategy, or as an adjuvant to existing chemotherapies in established disease.
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Carcinogênese/metabolismo , Gânglios Simpáticos/patologia , Glutationa/metabolismo , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neoplasias do Sistema Nervoso Periférico/metabolismo , Animais , Vias Biossintéticas , Carcinogênese/patologia , Modelos Animais de Doenças , Gânglios Simpáticos/citologia , Gânglios Simpáticos/metabolismo , Humanos , Metaboloma , Camundongos , Camundongos Transgênicos , Neuroblastoma/patologia , Neoplasias do Sistema Nervoso Periférico/patologiaRESUMO
PURPOSE: Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. EXPERIMENTAL DESIGN: The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. RESULTS: OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. CONCLUSIONS: We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR.
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Antineoplásicos/uso terapêutico , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Acetanilidas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Camundongos , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.
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Antineoplásicos/farmacologia , Carbazóis/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Neoplasias do Sistema Nervoso/tratamento farmacológico , Neoplasias do Sistema Nervoso/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Elongação da Transcrição/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Carbazóis/uso terapêutico , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismoRESUMO
In the last decade, the integration of molecular approaches including transcriptome and miRNome analyses uncovered pathological mechanisms involved in the progression of diabetic nephropathy (DN). Using these techniques, molecular marker candidates [both messenger RNA (mRNA) and miRNA] have also been identified which may enable the characterization of patients at high risk for progression to end-stage renal disease. The results of such studies are urgently needed for a molecular definition of DN and for targeted treatment to improve patient care. The heterogeneity of kidney tissue and the minute amounts of RNA isolated from renal biopsies remain a challenge for omics-studies. Nevertheless, several studies have succeeded in the identification of RNA expression signatures in patients with diabetes and kidney disease. These studies show a reduced expression of growth factors such as VEGF and EGF, and an increased expression of matrix components and matrix-modulating enzymes, an activation of specific NF-κB modules, inflammatory pathways and the complement system. microRNAs are involved in the fine-tuning of mRNA abundance by binding to the 3' untranslated region of a target mRNA, which leads in most cases to translational repression or mRNA cleavage and a decrease in protein output. Here, we review the platforms used for miRNA expression profiling and ways to predict miRNA targets and functions. Several miRNAs have been shown to be involved in the pathogenesis of DN (e.g. miR-21, miR-192, miR-215, miR-216a, miR-29, let-7, miR-25, miR-93, etc.). Functional studies provide evidence that miRNAs are not only diagnostic tools but also represent potential therapeutic targets in DN.
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Biomarcadores/metabolismo , Nefropatias Diabéticas/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Nefropatias Diabéticas/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
LIN28B has been identified as an oncogene in various tumor entities, including neuroblastoma, a childhood cancer that originates from neural crest-derived cells, and is characterized by amplification of the MYCN oncogene. Recently, elevated LIN28B expression levels were shown to contribute to neuroblastoma tumorigenesis via let-7 dependent de-repression of MYCN. However, additional insight in the regulation of LIN28B in neuroblastoma is lacking. Therefore, we have performed a comprehensive analysis of the regulation of LIN28B in neuroblastoma, with a specific focus on the contribution of miRNAs. We show that MYCN regulates LIN28B expression in neuroblastoma tumors via two distinct parallel mechanisms. First, through an unbiased LIN28B-3'UTR reporter screen, we found that miR-26a-5p and miR-26b-5p regulate LIN28B expression. Next, we demonstrated that MYCN indirectly affects the expression of miR-26a-5p, and hence regulates LIN28B, therefore establishing an MYCN-miR-26a-5p-LIN28B regulatory axis. Second, we provide evidence that MYCN regulates LIN28B expression via interaction with the LIN28B promoter, establishing a direct MYCN-LIN28B regulatory axis. We believe that these findings mark LIN28B as an important effector of the MYCN oncogenic phenotype and underline the importance of MYCN-regulated miRNAs in establishing the MYCN-driven oncogenic process.
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Neuroblastoma/patologia , Proteínas Nucleares/fisiologia , Proteínas Oncogênicas/fisiologia , Proteínas de Ligação a RNA/genética , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc , Transcrição GênicaRESUMO
PURPOSE: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. EXPERIMENTAL DESIGN: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALK(F1174L) or ALK(R1275Q) hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALK(F1174L) double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. RESULTS: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALK(F1174L) and ALK(R1275Q) regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. CONCLUSIONS: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies.
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Fosfatase Alcalina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular/métodos , Neuroblastoma/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Humanos , Camundongos , Camundongos Transgênicos , Neuroblastoma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transcriptoma , Regulação para CimaRESUMO
MYCN is a transcription factor that plays key roles in both normal development and cancer. In neuroblastoma, MYCN acts as a major oncogenic driver through pleiotropic effects regulated by multiple protein encoding genes as well as microRNAs (miRNAs). MYCN activity is tightly controlled at the level of transcription and protein stability through various mechanisms. Like most genes, MYCN is further controlled by miRNAs, but the full complement of all miRNAs implicated in this process has not been determined through an unbiased approach. To elucidate the role of miRNAs in regulation of MYCN, we thus explored the MYCN-miRNA interactome to establish miRNAs controlling MYCN expression levels. We combined results from an unbiased and genome-wide high-throughput miRNA target reporter screen with miRNA and mRNA expression data from patients and a murine neuroblastoma progression model. We identified 29 miRNAs targeting MYCN, of which 12 miRNAs are inversely correlated with MYCN expression or activity in neuroblastoma tumor tissue. The majority of MYCN-targeting miRNAs in neuroblastoma showed a decrease in expression during murine MYCN-driven neuroblastoma tumor development. Therefore, we provide evidence that MYCN-targeting miRNAs are preferentially downregulated in MYCN-driven neuroblastoma, suggesting that MYCN negatively controls the expression of these miRNAs, to safeguard its expression.
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Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Humanos , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma.
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Proteínas Inibidoras de Apoptose/fisiologia , MicroRNAs/fisiologia , Neuroblastoma/patologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Masculino , Camundongos , Proteína Proto-Oncogênica N-Myc , Nanopartículas , Neuroblastoma/prevenção & controle , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , SurvivinaRESUMO
Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annotation. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA sequences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annotation changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms. As a result, correct interpretation, comparison and integration of miRNA study results are compromised, which we demonstrate here by assessing the impact of ignoring sequence annotation changes. To address this problem, we developed miRBase Tracker (www.mirbasetracker.org), an easy-to-use online database that keeps track of all historical and current miRNA annotation present in the miRBase database. Three basic functionalities allow researchers to keep their miRNA annotation up-to-date, reannotate analytical miRNA platforms and link published results with outdated annotation to the latest miRBase release. We expect miRBase Tracker to increase the transparency and annotation accuracy in the field of miRNA research. DATABASE URL: www.mirbasetracker.org.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , MicroRNAs , Anotação de Sequência Molecular , Software , Animais , Sistemas de Gerenciamento de Base de Dados , Humanos , Internet , CamundongosRESUMO
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
Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Assuntos
Serviços de Informação , Reação em Cadeia da Polimerase/métodos , Coleta de DadosRESUMO
BACKGROUND: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. RESULTS: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5). CONCLUSIONS: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.