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Wilms tumour (WT), an embryonal kidney cancer, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of foetal kidney with two WT cell lines, filtering our results to remove common cancer-associated epigenetic changes and to enrich for genes involved in early kidney development. This identified four hypermethylated genes, of which ESRP2 (epithelial splicing regulatory protein 2) was the most promising for further study. ESRP2 was commonly repressed by DNA methylation in WT, and this occurred early in WT development (in nephrogenic rests). ESRP2 expression was reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was overexpressed in WT cell lines, it inhibited cellular proliferation in vitro, and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets. We propose a model in which epigenetic inactivation of ESRP2 disrupts the mesenchymal to epithelial transition in early kidney development to generate WT.
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Neoplasias Renales , Tumor de Wilms , Animales , Línea Celular Tumoral , ADN/metabolismo , Metilación de ADN/genética , Genes Supresores de Tumor , Humanos , Neoplasias Renales/genética , Ratones , Ratones Desnudos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Tumor de Wilms/genéticaRESUMEN
The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma, where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated overexpression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms' tumours with poorer outcomes, including tumours with diffuse anaplasia. Surprisingly, however, the expression and functions of the MYCN protein in Wilms' tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms' tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms' tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms' tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes including the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms' tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of RE1-Silencing Transcription factor (REST) target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor-prognosis Wilms' tumour.
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Three strains of novel bacteria were isolated from oil-contaminated sediment from the Arabian Gulf (Brevibacillus brevis T2C2008, Proteus mirabilis T2A12001, and Rhodococcus quinshengi TA13008). The isolated strains were tested for their degrading efficacy of low and high molecular hydrocarbon (naphthalene and pyrene). The efficacy of the two-hydrocarbon degradation by the isolates bacterial was determined at a temperature of 25 °C and 37 °C and pH of 5.0 and 9.0. In inoculated media at 37 °C, Rhodococcus qinshengi fully metabolized naphthalene and degrade 56% of pyrene. Brevibacillus brevis break down over 80% of naphthalene at room temperatures (25 °C). However, it was found that P. mirabilis and R. qinshengi biodegraded nearly 94% of naphthalene in the incubated media. The capacity for pyrene and naphthalene degradation in varying pH and temperature conditions was shown to be significant in Rhodococcus qinshengi because of its mineralization exceeding 50% across the tested pH and temperature. This implies that the isolated strains are ideal for biodegradation of contaminated sediment with naphthalene and pyrene.
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Organismos Acuáticos , Bacterias , Sedimentos Geológicos/microbiología , Naftalenos/metabolismo , Contaminación por Petróleo , Organismos Acuáticos/clasificación , Organismos Acuáticos/aislamiento & purificación , Organismos Acuáticos/metabolismo , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Biodegradación Ambiental , Océanos y MaresRESUMEN
Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a pediatric solid tumor with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumors is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638, and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumor suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2, and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumors.
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Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.
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The Wnt and bone morphogenetic protein (BMP) signaling pathways are known to be crucial in the development of neural crest lineages, including the sympathetic nervous system. Surprisingly, their role in paediatric neuroblastoma, the prototypic tumor arising from this lineage, remains relatively uncharacterised. We previously demonstrated that Wnt/b-catenin signaling can have cell-type-specific effects on neuroblastoma phenotypes, including growth inhibition and differentiation, and that BMP4 mRNA and protein were induced by Wnt3a/Rspo2. In this study, we characterised the phenotypic effects of BMP4 on neuroblastoma cells, demonstrating convergent induction of MSX homeobox transcription factors by Wnt and BMP4 signaling and BMP4-induced growth suppression and differentiation. An immunohistochemical analysis of BMP4 expression in primary neuroblastomas confirms a striking absence of BMP4 in poorly differentiated tumors, in contrast to a high expression in ganglion cells. These results are consistent with a tumor suppressive role for BMP4 in neuroblastoma. RNA sequencing following BMP4 treatment revealed induction of Notch signaling, verified by increases of Notch3 and Hes1 proteins. Together, our data demonstrate, for the first time, Wnt-BMP-Notch signaling crosstalk associated with growth suppression of neuroblastoma.
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Proteína Morfogenética Ósea 4/metabolismo , Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Factor de Transcripción MSX1/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptor Notch3/metabolismo , Proteínas Wnt/metabolismo , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Neuroblastoma/genética , Pronóstico , Transducción de Señal , Transcriptoma/genéticaRESUMEN
To discover epigenetic changes that may underly neuroblastoma pathogenesis, we identified differentially methylated genes in neuroblastoma cells compared to neural crest cells, the presumptive precursors cells for neuroblastoma, by using genome-wide DNA methylation analysis. We previously described genes that were hypermethylated in neuroblastoma; in this paper we report on 67 hypomethylated genes, which were filtered to select genes that showed transcriptional over-expression and an association with poor prognosis in neuroblastoma, highlighting GATA3 for detailed studies. Specific methylation assays confirmed the hypomethylation of GATA3 in neuroblastoma, which correlated with high expression at both the RNA and protein level. Demethylation with azacytidine in cultured sympathetic ganglia cells led to increased GATA3 expression, suggesting a mechanistic link between GATA3 expression and DNA methylation. Neuroblastomas that had completely absent GATA3 methylation and/or very high levels of protein expression, were associated with poor prognosis. Knock-down of GATA3 in neuroblastoma cells lines inhibited cell proliferation and increased apoptosis but had no effect on cellular differentiation. These results identify GATA3 as an epigenetically regulated component of the neuroblastoma transcriptional control network, that is essential for neuroblastoma proliferation. This suggests that the GATA3 transcriptional network is a promising target for novel neuroblastoma therapies.
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Metilación de ADN , ADN de Neoplasias/metabolismo , Epigénesis Genética , Factor de Transcripción GATA3/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neuroblastoma/metabolismo , Línea Celular Tumoral , ADN de Neoplasias/genética , Factor de Transcripción GATA3/genética , Humanos , Proteínas de Neoplasias/genética , Neuroblastoma/diagnóstico , Neuroblastoma/genética , Neuroblastoma/patología , PronósticoRESUMEN
In the abstract, the statement "The GHG emissions avoidance expected to be achieved by the GCC countries will vary between 5 and 247 million tons of CO equivalent by 2030."
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There is a growing focus on the role of renewable energy (RE) policies such as feed-in tariffs (FITs), renewable portfolio standards (RPSs), subsidies, incentives, and research and development in the global energy policy mix and in promoting environmental sustainability. Although most developed countries have well-formulated RE policies, in developing countries, such policies face many barriers. This study analyzes the policies, drivers, and barriers to RE deployment for fostering environmental sustainability in the Gulf Cooperation Council (GCC) countries. In the GCC region, the need for economic diversification to reduce dependency on single resource, diminishing hydrocarbon reserve, loss of oil export revenue, climate change mitigation pledges, and abundant solar energy resource are the key drivers for diversifying energy sources to include RE. However, the apparent lack of consolidated policy framework for wide-scale RE utilization calls for a well-articulated policy to advance RE development in each member state. Although FIT and RPS approaches could be effective for initial deployment of small-scale RE projects, a competitive tendering and auctioning mechanisms are more suitable for large-scale projects. Whereas, developing effective energy codes could successfully promote RE deployment, the increased share of RE in energy supply would have synergistic impacts on the region. The GHG emissions avoidance expected to be achieved by the GCC countries will vary between 5 and 247 million tons of CO2 equivalent by 2030. The fulfillment of inspirational RE targets for 2030 would contribute in fulfilling climate change mitigation pledges, environmental sustainability, economic growth, and generating new jobs.
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Fuentes Generadoras de Energía/estadística & datos numéricos , Política Pública , Energía Renovable , Cambio Climático , Desarrollo Económico , Política Ambiental , Gases de Efecto Invernadero , Medio Oriente , Energía Renovable/economía , Desarrollo SostenibleRESUMEN
The neural crest (NC), which has been referred to as the fourth germ layer, comprises a multipotent cell population which will specify diverse cells and tissues, including craniofacial cartilage and bones, melanocytes, the adrenal medulla and the peripheral nervous system. These cell fates are known to be determined by gene regulatory networks (GRNs) acting at various stages of NC development, such as induction, specification, and migration. Although transcription factor hierarchies and some of their interplay with morphogenetic signaling pathways have been characterized, the full complexity of activities required for regulated development remains uncharted. Deregulation of these pathways may contribute to tumorigenesis, as in the case of neuroblastoma, a frequently lethal embryonic cancer thought to arise from the sympathoadrenal lineage of the NC. In this "Hypothesis and Theory" article, we utilize the next generation sequencing data from neuroblastoma cells and tumors to evaluate the possible influences of Wnt signaling on NC GRNs and on neuroblastoma cell lineages. We propose that Wnt signaling is a major determinant of regulatory networks that underlie mesenchymal/neural crest cell (NCC)-like cell identities through PRRX1 and YAP/TAZ transcription factors. Furthermore, Wnt may also co-operate with Hedgehog signaling in driving proneural differentiation programmes along the adrenergic (ADRN) lineage. Elucidation of Signaling Regulatory Networks can augment and complement GRNs in characterizing cell identities, which may in turn contribute to the design of improved therapeutics tailored to primary and relapsing neuroblastoma.
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TITLE: Survey of the functional priorities in patients with disability due to neuromuscular disorders. OBJECTIVE: This study attempts to determine the functional priorities for patients with neuromuscular disorders. METHODS: A survey asking about functional priorities with respect to activities of daily living, ankle foot orthotic design, and assistive device design, was distributed to patients with neuromuscular disorders to assess the needs of patients from their perspectives. Descriptive statistics were used to analyse answers. RESULTS: A total of 171 subjects with neuromuscular disorders responded to the questionnaire. Of the respondents with weakness in both the upper and lower extremities, 45% stated that if they had to choose between correction of one or the other, they would prefer that of their lower extremities. Activities that patients most frequently wanted to gain independence with were mobility and transfers (46%), followed by toilet use and hygiene (32%). The most popular control mechanism of an assistive device was voice activation (35%). CONCLUSION: This study assessed the functional priorities of those with neuromuscular disorders. Although such individuals can experience a range of weakness in the upper and/or lower extremities, common functional priorities were reported: independence with mobility, transfers, toilet use and hygiene. Knowledge of these priorities will help guide development of assistive devices that will restore function in the future. Implications for Rehabilitation Neuromuscular Disorders ⢠Neuromuscular disorders result in disabling weakness; there are few cures and many are unable to carry out activities of daily living. ⢠Information that would be helpful in determining functional priorities is limited. ⢠In a survey of 171 patients with neuromuscular disorders, functional priorities included mobility and transfers (46%), followed by toilet use and hygiene (32%). ⢠Of the respondents with weakness in both the upper and lower extremities, 45% stated that if they had to choose between correction of one or the other, they would prefer that of their lower extremities. ⢠If an assistive device were to be created to help those with neuromuscular disorders, the most popular control mechanism would be voice activation (35%).
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Actividades Cotidianas , Personas con Discapacidad , Extremidad Inferior/fisiopatología , Evaluación de Necesidades , Enfermedades Neuromusculares/fisiopatología , Aparatos Ortopédicos , Dispositivos de Autoayuda , Adolescente , Adulto , Anciano , Niño , Preescolar , Diseño de Equipo , Femenino , Humanos , Iowa , Masculino , Persona de Mediana Edad , Encuestas y CuestionariosRESUMEN
Hypoxia is a hallmark of solid tumours and a key physiological feature distinguishing cancer from normal tissue. However, a major challenge remains in identifying tractable molecular targets that hypoxic cancer cells depend on for survival. Here, we used SILAC-based proteomics to identify the orphan G protein-coupled receptor GPRC5A as a novel hypoxia-induced protein that functions to protect cancer cells from apoptosis during oxygen deprivation. Using genetic approaches in vitro and in vivo, we reveal HIFs as direct activators of GPRC5A transcription. Furthermore, we find that GPRC5A is upregulated in the colonic epithelium of patients with mesenteric ischaemia, and in colorectal cancers high GPRC5A correlates with hypoxia gene signatures and poor clinical outcomes. Mechanistically, we show that GPRC5A enables hypoxic cell survival by activating the Hippo pathway effector YAP and its anti-apoptotic target gene BCL2L1 Importantly, we show that the apoptosis induced by GPRC5A depletion in hypoxia can be rescued by constitutively active YAP. Our study identifies a novel HIF-GPRC5A-YAP axis as a critical mediator of the hypoxia-induced adaptive response and a potential target for cancer therapy.
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Adaptación Fisiológica , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/patología , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Adaptación Fisiológica/efectos de los fármacos , Animales , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxiciclina/farmacología , Humanos , Ratones Endogámicos C57BL , Modelos Biológicos , Neoplasias/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Proteínas Señalizadoras YAP , Pez CebraRESUMEN
Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra- and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology. Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of ß-catenin immunohistochemistry shows high levels of ß-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.
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Diferenciación Celular/genética , Proliferación Celular/genética , Neuroblastoma/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Genes myc/genética , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genéticaRESUMEN
Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers.
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Antimitóticos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Indoles/farmacología , Mitosis/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/metabolismo , Azepinas/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Concentración 50 Inhibidora , Cinetocoros/efectos de los fármacos , Cinetocoros/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Paclitaxel/farmacología , Prometafase/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Huso Acromático/patología , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Vincristina/farmacologíaRESUMEN
Recent exciting work partly through The Cancer Genome Atlas has implicated epigenetic mechanisms including histone modifications in the development of both pediatric and adult high-grade glioma (HGG). Histone lysine methylation has emerged as an important player in regulating gene expression and chromatin function. Lysine (K) 27 (K27) is a critical residue in all seven histone 3 variants and the subject of posttranslational histone modifications, as it can be both methylated and acetylated. In pediatric HGG, two critical single-point mutations occur in the H3F3A gene encoding the regulatory histone variant H3.3. These mutations occur at lysine (K) 27 (K27M) and glycine (G) 34 (G34R/V), both of which are involved with key regulatory posttranscriptional modifications. Therefore, these mutations effect gene expression, cell differentiation, and telomere maintenance. In recent years, alterations in histone acetylation have provided novel opportunities to explore new pharmacological targeting, with histone deacetylase (HDAC) overexpression reported in high-grade, late-stage proliferative tumors. HDAC inhibitors have shown promising therapeutic potential in many malignancies. This review focuses on the epigenetic mechanisms propagating pediatric and adult HGGs, as well as summarizing the current advances in clinical trials using HDAC inhibitors.
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Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.
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Metilación de ADN , Genes Supresores de Tumor , Proteínas de la Membrana/genética , Neuroblastoma/genética , Línea Celular Tumoral , Niño , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Código de Histonas , HumanosRESUMEN
LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of ß-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuroblastoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vía de Señalización Wnt/genética , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Niño , Preescolar , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Lactante , Recién Nacido , Microscopía Confocal , Neuroblastoma/genética , Neuroblastoma/patología , Interferencia de ARN , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Interleucina-10/biosíntesis , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Acetilación , Traslado Adoptivo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Células Cultivadas , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Factor de Transcripción GATA3/biosíntesis , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Inflamación/inmunología , Interleucina-10/genética , Metilación , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-maf/biosíntesis , Células TH1/trasplanteRESUMEN
Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.