RESUMO
PAX5-JAK2 has recently been identified as a novel recurrent fusion gene in B-cell precursor acute lymphoblastic leukemia, but the function of the encoded chimeric protein has not yet been characterized in detail. Herein we show that the PAX5-JAK2 chimera, which consists of the DNA-binding paired domain of PAX5 and the active kinase domain of JAK2, is a nuclear protein that has the ability to bind to wild-type PAX5 target loci. Moreover, our data provide compelling evidence that PAX5-JAK2 functions as a nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream signal transducers and activators of transcription (STATs) in an apparently noncanonical mode. The chimeric protein also enables cytokine-independent growth of Ba/F3 cells and therefore possesses transforming potential. Importantly, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. Together, our data show that PAX5-JAK2 simultaneously deregulates the PAX5 downstream transcriptional program and activates the Janus kinase-STAT signaling cascade and thus, by interfering with these two important pathways, may promote leukemogenesis.
Assuntos
Janus Quinase 2/genética , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Janus Quinase 2/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição STAT/metabolismo , Transcriptoma , Células Tumorais CultivadasRESUMO
Cell cycle progression is orchestrated by E2F factors. We previously reported that in ETS-driven cancers of the bone and prostate, activating E2F3 cooperates with ETS on target promoters. The mechanism of target co-regulation remained unknown. Using RNAi and time-resolved chromatin-immunoprecipitation in Ewing sarcoma we report replacement of E2F3/pRB by constitutively expressed repressive E2F4/p130 complexes on target genes upon EWS-FLI1 modulation. Using mathematical modeling we interrogated four alternative explanatory models for the observed EWS-FLI1/E2F3 cooperation based on longitudinal E2F target and regulating transcription factor expression analysis. Bayesian model selection revealed the formation of a synergistic complex between EWS-FLI1 and E2F3 as the by far most likely mechanism explaining the observed kinetics of E2F target induction. Consequently we propose that aberrant cell cycle activation in Ewing sarcoma is due to the de-repression of E2F targets as a consequence of transcriptional induction and physical recruitment of E2F3 by EWS-FLI1 replacing E2F4 on their target promoters.
Assuntos
Fator de Transcrição E2F3/metabolismo , Fator de Transcrição E2F4/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Algoritmos , Teorema de Bayes , Ciclo Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F4/genética , Humanos , Immunoblotting , Modelos Genéticos , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Proteína EWS de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologiaRESUMO
Deregulated E2F transcription factor activity occurs in the vast majority of human tumors and has been solidly implicated in disturbances of cell cycle control, proliferation, and apoptosis. Aberrant E2F regulatory activity is often caused by impairment of control through pRB function, but little is known about the interplay of other oncoproteins with E2F. Here we show that ETS transcription factor fusions resulting from disease driving rearrangements in Ewing sarcoma (ES) and prostate cancer (PC) are one such class of oncoproteins. We performed an integrative study of genome-wide DNA-binding and transcription data in EWSR1/FLI1 expressing ES and TMPRSS2/ERG containing PC cells. Supported by promoter activity and mutation analyses, we demonstrate that a large fraction of E2F3 target genes are synergistically coregulated by these aberrant ETS proteins. We propose that the oncogenic effect of ETS fusion oncoproteins is in part mediated by the disruptive effect of the E2F-ETS interaction on cell cycle control. Additionally, a detailed analysis of the regulatory targets of the characteristic EWSR1/FLI1 fusion in ES identifies two functionally distinct gene sets. While synergistic regulation in concert with E2F in the promoter of target genes has a generally activating effect, EWSR1/FLI1 binding independent of E2F3 is predominantly associated with repressed differentiation genes. Thus, EWSR1/FLI1 appears to promote oncogenesis by simultaneously promoting cell proliferation and perturbing differentiation.
Assuntos
Fator de Transcrição E2F3/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F3/metabolismo , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Ligação Proteica , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Regulador Transcricional ERGRESUMO
UNLABELLED: Adenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells. IMPORTANCE: Viral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.
Assuntos
Adenovírus Humanos/genética , Células Epiteliais/virologia , Regulação da Expressão Gênica , Inativação Gênica , Interações Hospedeiro-Patógeno , MicroRNAs/genética , RNA Viral/genética , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , RNA Viral/metabolismoAssuntos
Antígenos de Diferenciação/metabolismo , Biomarcadores Tumorais , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Antígenos de Diferenciação/genética , Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnósticoRESUMO
Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P=.01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n=3), glucocorticoid receptor NR3C1 (n=4), and components of the mismatch repair pathways (n=3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P=.02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.
Assuntos
Deleção de Genes , Glucocorticoides/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Transdução de Sinais/genética , Pareamento Incorreto de Bases/genética , Criança , Pré-Escolar , Células Clonais , Subunidade alfa 2 de Fator de Ligação ao Core , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Rearranjo Gênico do Linfócito T/genética , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Glucocorticoides/metabolismo , RecidivaRESUMO
Notch can act as an oncogene or as a tumour suppressor and thus can either promote or inhibit tumour cell growth. To establish Notch status in Ewing's sarcoma family of tumours (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We found that although Notch receptors were highly expressed, Notch did not appear to be active, as evidenced by the absence of Notch receptors in cell nuclei. In contrast, we show that Notch receptors known to be active in colon adenocarcinoma, hepatocarcinoma, and pancreatic carcinoma stain cell nuclei in these tumours. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of ESFT, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation and reduced their clonogenic potential in soft agar. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and support a role for Notch in ESFT tumour suppression, at least partly through the Notch effector HEY1.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Ósseas/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores Notch/metabolismo , Sarcoma de Ewing/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Núcleo Celular/metabolismo , Proliferação de Células , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Notch/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição HES-1 , Células Tumorais CultivadasRESUMO
Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/fisiologia , Família de Proteínas EGF/genética , Família de Proteínas EGF/metabolismo , Neurogênese/fisiologia , Células de Schwann/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Plasticidade Celular/fisiologia , Proliferação de Células , Técnicas de Cocultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regeneração Nervosa , Neuroblastoma/patologia , Neurogênese/genética , Traumatismos dos Nervos Periféricos , Transcriptoma , Adulto JovemRESUMO
Ewing sarcoma (EwS) is a highly metastatic bone cancer characterized by the ETS fusion oncoprotein EWS-FLI1. EwS cells are phenotypically highly plastic and switch between functionally distinct cell states dependent on EWS-FLI1 fluctuations. Whereas EWS-FLI1high cells proliferate, EWS-FLI1low cells are migratory and invasive. Recently, we reported activation of MRTFB and TEAD, effectors of RhoA and Hippo signalling, upon low EWS-FLI1, orchestrating key steps of the EwS migratory gene expression program. TEAD and its co-activators YAP and TAZ are commonly overexpressed in cancer, providing attractive therapeutic targets. We find TAZ levels to increase in the migratory EWS-FLI1low state and to associate with adverse prognosis in EwS patients. We tested the effects of the potent YAP/TAZ/TEAD complex inhibitor verteporfin on EwS cell migration in vitro and on metastasis in vivo. Verteporfin suppressed expression of EWS-FLI1 regulated cytoskeletal genes involved in actin signalling to the extracellular matrix, effectively blocked F-actin and focal-adhesion assembly and inhibited EwS cell migration at submicromolar concentrations. In a mouse EwS xenograft model, verteporfin treatment reduced relapses at the surgical site and delayed lung metastasis. These data suggest that YAP/TAZ pathway inhibition may prevent EwS cell dissemination and metastasis, justifying further preclinical development of YAP/TAZ inhibitors for EwS treatment.
RESUMO
Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a+ langerin+ cells of unknown origin. We have previously reported that the Notch signaling pathway is active in LCH lesions and that the Notch ligand Jagged2 (JAG2) induces CD1a and langerin expression in monocytes in vitro. Here we show that Notch signaling induces monocytes to acquire an LCH gene signature and that Notch inhibition suppresses the LCH phenotype. In contrast, while also CD1c+ dendritic cells or IL-4-stimulated CD14+ monocytes acquire CD1a and langerin positivity in culture, their gene expression profiles and surface phenotypes are more different from primary LCH cells. We propose a model where CD14+ monocytes serve as LCH cell precursor and JAG2-mediated activation of the Notch signaling pathway initiates a differentiation of monocytes toward LCH cells in selected niches and thereby contributes to LCH pathogenesis.
Assuntos
Diferenciação Celular , Histiocitose de Células de Langerhans/metabolismo , Proteína Jagged-2/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Antígenos CD/metabolismo , Proliferação de Células , Histiocitose de Células de Langerhans/patologia , Humanos , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Fenótipo , Receptores Notch/metabolismo , Transcrição GênicaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.
RESUMO
Since publication of the article, the authors became aware that Figure 6(A,D) contained errors in the bands and loading controls. The newly compiled Figure 6A and 6D is given below.
RESUMO
A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.
Assuntos
Neoplasias Ósseas/genética , Proteínas dos Microtúbulos/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Doxiciclina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Masculino , Proteínas dos Microtúbulos/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Interferência de RNA , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/mortalidade , Sarcoma de Ewing/patologia , Repetições WD40RESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disease in which multiple genetic abnormalities cooperate in the malignant transformation of T-lymphoid progenitors. Although in pediatric T-ALL, CDKN1B deletions occur in about 12% of the cases and represent one of the most frequent copy number alterations, neither their association with other genetic alterations nor the clinical characteristics of these patients have been determined yet. In this study, we show that loss of CDKN1B increased the prevalence of cell cycle regulator defects in immature T-ALL, usually only ascribed to CDKN2A/B deletions, and that CDKN1B deletions frequently coincide with expression of MEF2C, considered as one of the driving oncogenes in immature early T-cell precursor (ETP) ALL. However, MEF2C-dysregulation was only partially associated with the immunophenotypic characteristics used to define ETP-ALL. Furthermore, MEF2C expression levels were significantly associated with or may even be predictive of the response to glucocorticoid treatment.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Deleção de Genes , Regulação Leucêmica da Expressão Gênica , Variantes Farmacogenômicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Biomarcadores , Linhagem Celular , Criança , Pré-Escolar , Análise por Conglomerados , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Glucocorticoides/uso terapêutico , Humanos , Imunofenotipagem , Lactente , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Resultado do TratamentoRESUMO
Maintaining dendritic cells (DC) in a state of dysfunction represents a key mechanism by which tumour cells evade recognition and elimination by the immune system. Limited knowledge about the intracellular mediators of DC dysfunction restricts success of therapies aimed at reactivating a DC-driven anti-tumour immune response. Using a cell type-specific murine knock-out model, we have identified MAPK-activated protein kinase 2 (MK2) as a major guardian of a suppressive DC phenotype in the melanoma tumour microenvironment. MK2 deletion in CD11c+ cells led to an expansion of stimulatory CD103+ DCs, mounting a potent CD8+ T cell response that resulted in elimination of highly aggressive B16-F10 tumours upon toll-like receptor (TLR) activation in the presence of tumour antigen. Moreover, tumour infiltration by suppressive myeloid cells was strongly diminished. These insights into the regulation of DC functionality reveal MK2 as a targetable pathway for DC-centred immunomodulatory cancer therapies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Melanoma Experimental/imunologia , Proteínas Serina-Treonina Quinases/deficiência , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Células Dendríticas/enzimologia , Células Dendríticas/patologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3'UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing sarcoma. MiR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be among the top EWS-FLI1 activated miRNAs. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3'UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling. Testing for SMAD phosphorylation, we identify quiet but activatable TGFB signaling and cell autonomous activity of the BMP pathway resulting in the activation of the stemness regulatory transcriptional repressors ID1 and ID3. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway.
Assuntos
Regiões 3' não Traduzidas/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Predisposição Genética para Doença/genética , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Longo não Codificante , Proteína EWS de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Análise de Sequência de RNA , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.
Assuntos
Acrilamidas/farmacologia , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Piperidinas/farmacologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HeLa , Humanos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/enzimologia , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologiaRESUMO
The ETV6/RUNX1 gene fusion defines the largest genetic subgroup of childhood ALL with overall rapid treatment response. However, up to 15% of cases relapse. Because an impaired glucocorticoid pathway is implicated in disease recurrence we studied the impact of genetic alterations by SNP array analysis in 31 relapsed cases. In 58% of samples, we found deletions in various glucocorticoid signaling pathway-associated genes, but only NR3C1 and ETV6 deletions prevailed in minimal residual disease poor responding and subsequently relapsing cases (p<0.05). To prove the necessity of a functional glucocorticoid receptor, we reconstituted wild-type NR3C1 expression in mutant, glucocorticoid-resistant REH cells and studied the glucocorticoid response in vitro and in a xenograft mouse model. While these results prove that glucocorticoid receptor defects are crucial for glucocorticoid resistance in an experimental setting, they do not address the essential clinical situation where glucocorticoid resistance at relapse is rather part of a global drug resistance.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Glucocorticoides/metabolismo , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de Sinais , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Terapia Combinada , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Proteínas de Fusão Oncogênica/metabolismo , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Receptores de Glucocorticoides/metabolismo , Recidiva , Deleção de Sequência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth-arrest and act immune-stimulatory, while others are tumor-promoting. We evaluated whether metronomic, i.e. long-term, repetitive low-dose, drug treatment induces senescence in vitro and in vivo. And importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified.We demonstrate that metronomic application of chemotherapeutic drugs induces therapy-induced senescence, characterized by cell cycle arrest, p21(WAF/CIP1) up-regulation and DNA double-strand breaks selectively in MYCN-amplified NB. Low-dose topotecan (TPT) was identified as an inducer of a favorable SASP while lacking NFKB1/p50 activation. In contrast, Bromo-deoxy-uridine induced senescent NB-cells secret a tumor-promoting SASP in a NFKB1/p50-dependent manner. Importantly, TPT-treated senescent tumor cells act growth-inhibitory in a dose-dependent manner on non-senescent tumor cells and MYCN expression is significantly reduced in vitro and in vivo. Furthermore, in a mouse xenotransplant-model for MYCN-amplified NB metronomic TPT leads to senescence selectively in tumor cells, complete or partial remission, prolonged survival and a favorable SASP.This new mode-of-action of metronomic TPT treatment, i.e. promoting a tumor-inhibiting type of senescence in MYCN-amplified tumors, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles.