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BACKGROUND: Certain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches. METHODS: We performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing. RESULTS: Group 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo. CONCLUSIONS: RA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.
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In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.
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Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.
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Genoma Humano/genética , Genômica , Mutação/genética , Neoplasias/classificação , Neoplasias/genética , Adolescente , Adulto , Criança , Cromotripsia , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Diploide , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa/genética , Humanos , Terapia de Alvo Molecular , Taxa de Mutação , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Adulto JovemRESUMO
Alternative lengthening of telomeres (ALT) occurs in â¼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.
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Telômero/metabolismo , Linhagem Celular , DNA de Cadeia Simples/genética , Humanos , Hibridização in Situ Fluorescente , Neoplasias/genética , Telomerase/genética , Telômero/genética , Homeostase do TelômeroRESUMO
BACKGROUND: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. METHODS: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. RESULTS: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. CONCLUSIONS: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.
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Neuroblastoma , Receptores Proteína Tirosina Quinases , Criança , Humanos , Quinase do Linfoma Anaplásico/genética , Receptores Proteína Tirosina Quinases/genética , Recidiva Local de Neoplasia/genética , Neuroblastoma/genética , Neuroblastoma/patologia , GenômicaRESUMO
The childhood malignancy neuroblastoma belongs to the group of embryonal tumors and originates from progenitor cells of the sympathoadrenal lineage. Treatment options for children with high-risk and relapsed disease are still very limited. In recent years, an ever-growing molecular diversity was identified using (epi)-genetic profiling of neuroblastoma tumors, indicating that molecularly targeted therapies could be a promising therapeutic option. In this review article, we summarize the various molecular subtypes and genetic events associated with neuroblastoma and describe recent advances in targeted therapies. We lay a strong emphasis on the importance of telomere maintenance mechanisms for understanding tumor progression and risk classification of neuroblastoma.
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Neuroblastoma/genética , Homeostase do Telômero/fisiologia , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Animais , Aurora Quinase A/antagonistas & inibidores , Aberrações Cromossômicas , Genes p53 , Genes ras , Humanos , Mutação , Neuroblastoma/tratamento farmacológico , Transdução de Sinais , Homeostase do Telômero/efeitos dos fármacosRESUMO
Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.
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Neoplasias Ósseas , Neoplasias Cerebelares , Cromotripsia , Osteossarcoma , Animais , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , DNA , Reparo do DNA , Proteínas Hedgehog/genética , Humanos , Camundongos , Osteossarcoma/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
BACKGROUND: Neuroblastoma is the most common malignancy in infancy, accounting for 15% of childhood cancer deaths. Outcome for the high-risk disease remains poor. DNA-methylation patterns are significantly altered in all cancer types and can be utilised for disease stratification. METHODS: Genome-wide DNA methylation (n = 223), gene expression (n = 130), genetic/clinical data (n = 213), whole-exome sequencing (n = 130) was derived from the TARGET study. Methylation data were derived from HumanMethylation450 BeadChip arrays. t-SNE was used for the segregation of molecular subgroups. A separate validation cohort of 105 cases was studied. RESULTS: Five distinct neuroblastoma molecular subgroups were identified, based on genome-wide DNA-methylation patterns, with unique features in each, including three subgroups associated with known prognostic features and two novel subgroups. As expected, Cluster-4 (infant diagnosis) had significantly better 5-year progression-free survival (PFS) than the four other clusters. However, in addition, the molecular subgrouping identified multiple patient subsets with highly increased risk, most notably infant patients that do not map to Cluster-4 (PFS 50% vs 80% for Cluster-4 infants, P = 0.005), and allowed identification of subgroup-specific methylation differences that may reflect important biological differences within neuroblastoma. CONCLUSIONS: Methylation-based clustering of neuroblastoma reveals novel molecular subgroups, with distinct molecular/clinical characteristics and identifies a subgroup of higher-risk infant patients.
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Metilação de DNA , Neuroblastoma , Lactente , Humanos , Neuroblastoma/genética , Prognóstico , Sequenciamento do Exoma , Análise por ConglomeradosRESUMO
Current therapies for most non-infectious diseases are directed at or affect functionality of the human translated genome, barely 2% of all genetic information. By contrast, the therapeutic potential of targeting the transcriptome, ~ 70% of the genome, remains largely unexplored. RNA therapeutics is an emerging field that widens the range of druggable targets and includes elements such as microRNA. Here, we sought to screen for microRNA with tumor-suppressive functions in neuroblastoma, an aggressive pediatric tumor of the sympathetic nervous system that requires the development of new therapies. We found miR-323a-5p and miR-342-5p to be capable of reducing cell proliferation in multiple neuroblastoma cell lines in vitro and in vivo, thereby providing a proof of concept for miRNA-based therapies for neuroblastoma. Furthermore, the combined inhibition of the direct identified targets such as CCND1, CHAF1A, INCENP and BCL-XL could reveal new vulnerabilities of high-risk neuroblastoma.
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Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias do Sistema Nervoso/genética , Neuroblastoma/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Criança , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Neoplasias do Sistema Nervoso/mortalidade , Neoplasias do Sistema Nervoso/patologia , Neoplasias do Sistema Nervoso/terapia , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neuroblastoma/terapia , Neurônios/metabolismo , Neurônios/patologia , Ligação Proteica , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Supplementation of micronutrients like folate is a double-edged sword in terms of their ambivalent role in cell metabolism. Although several epidemiological studies support a protective role of folate in carcinogenesis, there are also data arguing for an opposite effect. To address this issue in the context of human papillomavirus (HPV)-induced transformation, the molecular events of different folate availability on human keratinocytes immortalized by HPV16 E6 and E7 oncoproteins were examined. Several sublines were established: Control (4.5 µM folate), folate deficient (0.002 µM folate), and repleted cells (4.5 µM folate). Cells were analyzed in terms of oncogene expression, DNA damage and repair, karyotype changes, whole-genome sequencing, and transcriptomics. Here we show that folate depletion irreversibly induces DNA damage, impairment of DNA repair fidelity, and unique chromosomal alterations. Repleted cells additionally underwent growth advantage and enhanced clonogenicity, while the above mentioned impaired molecular properties became even more pronounced. Overall, it appears that a period of folate deficiency followed by repletion can shape immortalized cells toward an anomalous phenotype, thereby potentially contributing to carcinogenesis. These observations should elicit questions and inquiries for broader additional studies regarding folate fortification programs, especially in developing countries with micronutrient deficiencies and high HPV prevalence.
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Deficiência de Ácido Fólico/genética , Papillomavirus Humano 16/genética , Infecções por Papillomavirus/genética , Transcrição Gênica , Carcinogênese/genética , Dano ao DNA/ética , Reparo do DNA/genética , Ácido Fólico/genética , Deficiência de Ácido Fólico/patologia , Deficiência de Ácido Fólico/virologia , Genômica , Papillomavirus Humano 16/patogenicidade , Humanos , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genéticaRESUMO
High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Neuroblastoma/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Histona Desacetilases/metabolismo , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Tretinoína/administração & dosagem , Células Tumorais CultivadasRESUMO
BACKGROUND: Biological systems and processes are highly dynamic. To gain insights into their functioning time-resolved measurements are necessary. Time-resolved gene expression data captures temporal behaviour of the genes genome-wide under various biological conditions: in response to stimuli, during cell cycle, differentiation or developmental programs. Dissecting dynamic gene expression patterns from this data may shed light on the functioning of the gene regulatory system. The present approach facilitates this discovery. The fundamental idea behind it is the following: there are change-points (switches) in the gene behaviour separating intervals of increasing and decreasing activity, whereas the intervals may have different durations. Elucidating the switch-points is important for the identification of biologically meanigfull features and patterns of the gene dynamics. RESULTS: We developed a statistical method, called SwitchFinder, for the analysis of time-series data, in particular gene expression data, based on a change-point model. Fitting the model to the gene expression time-courses indicates switch-points between increasing and decreasing activities of each gene. Two types of the model - based on linear and on generalized logistic function - were used to capture the data between the switch-points. Model inference was facilitated with the Bayesian methodology using Markov chain Monte Carlo (MCMC) technique Gibbs sampling. Further on, we introduced features of the switch-points: growth, decay, spike and cleft, which reflect important dynamic aspects. With this, the gene expression profiles are represented in a qualitative manner - as sets of the dynamic features at their onset-times. We developed a Web application of the approach, enabling to put queries to the gene expression time-courses and to deduce groups of genes with common dynamic patterns. SwitchFinder was applied to our original data - the gene expression time-series measured in neuroblastoma cell line upon treatment with all-trans retinoic acid (ATRA). The analysis revealed eight patterns of the gene expression responses to ATRA, indicating the induction of the BMP, WNT, Notch, FGF and NTRK-receptor signaling pathways involved in cell differentiation, as well as the repression of the cell-cycle related genes. CONCLUSIONS: SwitchFinder is a novel approach to the analysis of biological time-series data, supporting inference and interactive exploration of its inherent dynamic patterns, hence facilitating biological discovery process. SwitchFinder is freely available at https://newbioinformatics.eu/switchfinder.
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Perfilação da Expressão Gênica/métodos , Animais , Ciclo Celular , Humanos , Internet , Modelos Teóricos , Método de Monte Carlo , Proteínas/genética , SoftwareRESUMO
Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.
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Inibidor de Quinase Dependente de Ciclina p19/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias do Sistema Nervoso/genética , Neuroblastoma/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Adolescente , Adulto , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Pré-Escolar , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Epigênese Genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Proteína Proto-Oncogênica N-Myc , Estadiamento de Neoplasias , Neoplasias do Sistema Nervoso/metabolismo , Neoplasias do Sistema Nervoso/mortalidade , Neoplasias do Sistema Nervoso/patologia , Neuroblastoma/metabolismo , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Transdução de Sinais , Análise de Sobrevida , Tretinoína/farmacologiaRESUMO
The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14(ARF), in controlling the TP53-MDM2 balance in neuroblastoma is unresolved. In the present study, we show that conditional p14(ARF) expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14(ARF)-deficient or MYCN-amplified neuroblastoma cell lines. Furthermore, ectopic 14(ARF) expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14(ARF) and p16(INK4A)) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14(ARF) and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14(ARF) expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14(ARF) in neuroblastoma cell lines that correlated with endogenous p14(ARF) expression but not with episomal p14(ARF) promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14(ARF) in neuroblastoma cells. Collectively, the data pinpoint p14(ARF) as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14(ARF) as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.
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Apoptose , Repressão Epigenética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Histonas/genética , Neuroblastoma/patologia , Proteína Supressora de Tumor p14ARF/genética , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Deleção de Genes , Expressão Gênica , Histonas/metabolismo , Humanos , Perda de Heterozigosidade , Masculino , Neuroblastoma/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Computational approaches for automatic analysis of image-based high-throughput and high-content screens are gaining increased importance to cope with the large amounts of data generated by automated microscopy systems. Typically, automatic image analysis is used to extract phenotypic information once all images of a screen have been acquired. However, also in earlier stages of large-scale experiments image analysis is important, in particular, to support and accelerate the tedious and time-consuming optimization of the experimental conditions and technical settings. We here present a novel approach for automatic, large-scale analysis and experimental optimization with application to a screen on neuroblastoma cell lines. Our approach consists of cell segmentation, tracking, feature extraction, classification, and model-based error correction. The approach can be used for experimental optimization by extracting quantitative information which allows experimentalists to optimally choose and to verify the experimental parameters. This involves systematically studying the global cell movement and proliferation behavior. Moreover, we performed a comprehensive phenotypic analysis of a large-scale neuroblastoma screen including the detection of rare division events such as multi-polar divisions. Major challenges of the analyzed high-throughput data are the relatively low spatio-temporal resolution in conjunction with densely growing cells as well as the high variability of the data. To account for the data variability we optimized feature extraction and classification, and introduced a gray value normalization technique as well as a novel approach for automatic model-based correction of classification errors. In total, we analyzed 4,400 real image sequences, covering observation periods of around 120 h each. We performed an extensive quantitative evaluation, which showed that our approach yields high accuracies of 92.2% for segmentation, 98.2% for tracking, and 86.5% for classification.
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Movimento Celular/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Neuroblastoma/fisiopatologia , Reconhecimento Automatizado de Padrão/métodos , Linhagem Celular Tumoral , Núcleo Celular/fisiologia , Proliferação de Células/fisiologia , Biologia Computacional/métodos , Humanos , Mitose/fisiologia , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno , Biologia de Sistemas/métodos , Proteína Supressora de Tumor p53/genéticaRESUMO
MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.
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Histona Desacetilase 2/metabolismo , MicroRNAs/metabolismo , Neuroblastoma/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Motivation: BigWig files serve as essential inputs in epigenomic data visualization. However, current R packages for visualizing these files are limited, slow, and burdened by numerous dependencies. Results: We introduce trackplot, a minimal R script designed for the rapid generation of integrative genomics viewer (IGV) style track plots, profile plots, and heatmaps from bigWig files. This script offers speed, owing to its reliance on bwtool, resulting in performance gains of several magnitudes compared to equivalent packages. The script is lightweight, requiring only the data.table and bwtool packages as primary dependencies. Notably, the plots are generated in base R graphics, eliminating the need for additional packages. trackplot queries the University of California Santa Cruz (UCSC) genome browser for gene models thereby enhancing the reproducibility of analyses. The script extends its support to general transfer format (GTF) further enhancing its versatility. This tool addresses the gaps in existing bigWig visualization approaches by offering speed, simplicity, and minimal dependencies, thereby presenting a valuable asset to researchers in the fields of epigenomics. Availability and implementation: trackplot is implemented in R is made available under MIT license at https://github.com/PoisonAlien/trackplot.
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Ferroptosis and apoptosis are key cell-death pathways implicated in several human diseases including cancer. Ferroptosis is driven by iron-dependent lipid peroxidation and currently has no characteristic biomarkers or gene signatures. Here a continuous phenotypic gradient between ferroptosis and apoptosis coupled to transcriptomic and metabolomic landscapes is established. The gradual ferroptosis-to-apoptosis transcriptomic landscape is used to generate a unique, unbiased transcriptomic predictor, the Gradient Gene Set (GGS), which classified ferroptosis and apoptosis with high accuracy. Further GGS optimization using multiple ferroptotic and apoptotic datasets revealed highly specific ferroptosis biomarkers, which are robustly validated in vitro and in vivo. A subset of the GGS is associated with poor prognosis in breast cancer patients and PDXs and contains different ferroptosis repressors. Depletion of one representative, PDGFA-assaociated protein 1(PDAP1), is found to suppress basal-like breast tumor growth in a mouse model. Omics and mechanistic studies revealed that ferroptosis is associated with enhanced lysosomal function, glutaminolysis, and the tricarboxylic acid (TCA) cycle, while its transition into apoptosis is attributed to enhanced endoplasmic reticulum(ER)-stress and phosphatidylethanolamine (PE)-to-phosphatidylcholine (PC) metabolic shift. Collectively, this study highlights molecular mechanisms underlying ferroptosis execution, identified a highly predictive ferroptosis gene signature with prognostic value, ferroptosis versus apoptosis biomarkers, and ferroptosis repressors for breast cancer therapy.
Assuntos
Apoptose , Biomarcadores Tumorais , Ferroptose , Ferroptose/genética , Humanos , Animais , Camundongos , Apoptose/genética , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Biomarcadores/metabolismoRESUMO
CONTEXT: Sexual dimorphism has direct consequences on the incidence and survival of cancer. Early and accurate diagnosis is crucial to improve prognosis. OBJECTIVE: This work aimed to characterized the influence of sex and adrenal asymmetry on the emergence of adrenal tumors. METHODS: We conducted a multicenter, observational study involving 8037 patients with adrenal tumors, including adrenocortical carcinoma (ACC), aldosterone-producing adenoma (APA), cortisol-secreting adrenocortical adenomas (CSAs), non-aldosterone-producing adrenal cortical adenoma (NAPACA), pheochromocytoma (PCC), and neuroblastoma (NB), and investigated tumor lateralization according to sex. Human adrenal tissues (n = 20) were analyzed with a multiomics approach that allows determination of gene expression, catecholamine, and steroid contents in a single sample. In addition, we performed a literature review of computed tomography and magnetic resonance imaging-based studies examining adrenal gland size. RESULTS: ACC (n = 1858); CSA (n = 68), NAPACA (n = 2174), and PCC (n = 1824) were more common in females than in males (female-to-male ratio: 1.1:1-3.8:1), whereas NBs (n = 2320) and APAs (n = 228) were less prevalent in females (0.8:1). ACC, APA, CSA, NAPACA, and NB occurred more frequently in the left than in the right adrenal (left-to-right ratio: 1.1:1-1.8:1), whereas PCC arose more often in the right than in the left adrenal (0.8:1). In both sexes, the left adrenal was larger than the right adrenal; females have smaller adrenals than males. CONCLUSION: Adrenal asymmetry in both sexes may be related to the pathogenesis of adrenal tumors and should be considered during the diagnosis of these tumors.
Assuntos
Neoplasias do Córtex Suprarrenal , Neoplasias das Glândulas Suprarrenais , Adenoma Adrenocortical , Carcinoma Adrenocortical , Feocromocitoma , Feminino , Humanos , Masculino , Corticosteroides , Neoplasias do Córtex Suprarrenal/genética , Glândulas Suprarrenais/diagnóstico por imagem , Glândulas Suprarrenais/metabolismo , Adenoma Adrenocortical/metabolismo , Aldosterona/metabolismo , Feocromocitoma/metabolismo , Caracteres SexuaisRESUMO
Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction, including autoimmune diseases, chronic infections, and cancer. Co-inhibitory "checkpoint molecules," such as programmed cell death protein-1, balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of natural killer (NK) cell recognition on T cells, we identified the immunoglobulin superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR) T cells. Unlike other checkpoint molecules, B7H6 mediated NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells were prevalent in the tissue and blood of several diseases, and their abundance in tumor tissue positively correlated with clinical response in a cohort of patients with immune checkpoint inhibitor-treated esophageal cancer. In humanized mouse models, NK cell surveillance via B7H6 limited the persistence and antitumor activity of CAR T cells, and its genetic deletion enhanced T cell proliferation and persistence. Together, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as a therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.