Neuroblastoma Differentiation Research Articles

Transcriptome 3\u2032end organization by PCF11 links alternative polyadenylation to formation and neuronal differentiation of neuroblastoma

Diversification at the transcriptome 3′end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes. Here, we identify extensive transcriptome 3′end-alterations in neuroblastoma, a tumour entity with a paucity of recurrent somatic mutations and an unusually high frequency of spontaneous regression. Utilising extensive RNAi-screening we reveal the landscape and drivers of transcriptome 3′end-diversification, discovering PCF11 as critical regulator, directing alternative polyadenylation (APA) of hundreds of transcripts including a differentiation RNA-operon. PCF11 shapes inputs converging on WNT-signalling, and governs cell cycle, proliferation, apoptosis and neurodifferentiation. Postnatal PCF11 down-regulation induces a neurodifferentiation program, and low-level PCF11 in neuroblastoma associates with favourable outcome and spontaneous tumour regression. Our findings document a critical role for APA in tumorigenesis and describe a novel mechanism for cell fate reprogramming in neuroblastoma with potentially important clinical implications. We provide an interactive data repository of transcriptome-wide APA covering > 170 RNAis, and an APA-network map with regulatory hubs.

DUSP5 expression associates with poor prognosis in human neuroblastoma

Regulation of growth and differentiation of neuroblastoma (NB) cells is the rational of some maintenance therapies for high-risk NB. MAP kinase phosphatases (MKPs) are potential physiologic regulators of neuronal differentiation and survival, but their expression patterns in NB are scarcely known. Here, an expression analysis of the MKP family has been performed using human NB tumor samples and human NB cell lines (SH-SY5Y, SMS-KCNR, and IMR-32) undergoing retinoic acid (RA)-induced differentiation or subjected to stimuli that activate the MAPK ERK1/2 pathway. We have identified candidate MKPs that could modulate differentiation and growth of NB cells. pERK1/2 high expression correlated with high expression of the MKP DUSP5 in NB tumors, and was associated with poor prognosis. ERK1/2 activation on SH-SY5Y cells was accompanied by increased cell proliferation, and correlated with the expression levels of DUSP5. Accordingly, siRNA knock-down of DUSP5 augmented proliferation of SH-SY5Y cells. Our findings provide insights into the dynamic expression of MKPs in NB cells, disclose DUSP5 as a potential marker of NB poor prognosis, and suggest a role for DUSP5 in limiting ERK1/2-mediated NB proliferation.

Biological and Genetic Features of Neuroblastoma and Their Clinical Importance.

Neuroblastoma derived from primitive cells of the sympathetic nervous system typically develops in the adrenal medulla or paraspinal ganglia. Neuroblastoma usually occurs sporadically, but familial cases are also observed. ALK and PHOX2B germline mutations can cause hereditary neuroblastoma, while a common genetic variation in chromosome 6p22 is associated to sporadic neuroblastoma. However, the aetiology of sporadic neuroblastoma is not exactly known. This embryonic malignancy generally represents the second most common solid tumour after central nervous system tumours throughout the world in childhood. Neuroblastoma is a complex disease that has different clinical courses, from metastatic spread to spontaneous regression. Spontaneous regression can occur without therapy in primary or metastatic site. Potential regression mechanisms primarily involve apoptosis, hypermethylation of subtelomeric DNA, immune response and Nerve Growth Factor (NGF) deprivation. Neuroblastoma is a heterogeneous tumour that can show many different chromosomal abnormalities; e.g. MYCN amplification, 1p deletion, unbalanced translocations involving chromosome 17, aneuploidies and Loss of Heterozygosity (LOH) events. Tyrosine kinase receptors TrkA, TrkB and TrkC, their ligands NGF, Brain-derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT-3), and Aurora Kinase A (AURKA) play a regulatory role in differentiation, apoptosis, cell proliferation, tumourigenesis, angiogenesis or metastasis of neuroblastoma. TrkA expression is associated with differentiation or regression, depending on presence or absence of NGF, whereas TrkB and BDNF are mostly expressed in aggressive neuroblastomas with MYCN amplification. MYCN is amplified in 18-38% of neuroblastoma cases. MYCN amplification mechanism remains to be completely clarified. This paper reviews the biological/genetic features and their clinical importance in neuroblastoma.

Abstract 3467: Growth factor receptor trafficking in neuroblastoma differentiation

Abstract Background/Objectives: UBE4B is an E3/E4 ubiquitin ligase involved in growth factor receptor (GFR) trafficking. The UBE4B gene is located in the chromosome 1p36 region commonly deleted in high-risk neuroblastoma tumors. We have previously identified associations of UBE4B expression with neuroblastoma patient outcomes. However, the functional roles of UBE4B in neuroblastoma tumor differentiation are not known. Design/Methods: We analyzed the association of UBE4B gene expression with expression of known markers of neuroblastoma tumor differentiation using publicly available databases and screened cell lines and neuroblastoma tumor samples for UBE4B protein expression using Western blot and quantitative immunohistochemistry. We measured UBE4B expression by Western blot in cell lines before and after induction of differentiation with 13-cis-retinoic acid treatment and determined the effects of UBE4B overexpression and depletion on retinoic acid-induced differentiation using continuous live-cell imaging of neurite morphology, immunohistochemistry, and Western blot for markers of differentiation. Effects on signaling through the Ras/MAPK pathway were measured using Western blots. Results: UBE4B expression was associated with neuroblastoma differentiation in patient tumors, and UBE4B gene and protein expression were associated with expression levels of known markers of neuroblastoma differentiation. Retinoic acid treatment resulted in increased UBE4B expression in retinoic acid-sensitive, but not -resistant, neuroblastoma cells, and UBE4B depletion was associated with increased ERK phosphorylation, increased proliferation, and inhibition of retinoic acid-induced neuroblastoma differentiation. Conclusion: We have demonstrated associations between UBE4B expression and tumor differentiation in gene expression databases and in neuroblastoma tumor samples, and our data suggests that UBE4B expression is required for retinoic acid-induced differentiation, potentially through effects on activation of the Ras/MAPK pathway. These associations between UBE4B and neuroblastoma differentiation combined with the established role of UBE4B in GFR trafficking suggest that UBE4B-mediated receptor trafficking may contribute to the responses of neuroblastoma tumors to therapy and to the outcomes of patients with neuroblastoma. Citation Format: Jacqueline Lesperance, Divya Subramonian, Sarah Woodfield, Rongjun Guo, Andrew Bean, Dolores Lopez-Terrada, Michael Ittmann, Sara Hakim, Peter E. Zage. Growth factor receptor trafficking in neuroblastoma differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3467.

Abstract SY28-02: A phosphorylation switch regulating proliferation and differentiation in Xenopus development and neuroblastoma

Abstract Neuroblastoma is associated with stalling of normal differentiation, resulting in excessive proliferation of neuroblastic precursors. Here we show that Xenopus frog embryos can be used as a rapid and versatile model to investigate the molecular regulation of proliferation versus differentiation of neuroblasts and what may go wrong with this in neuroblastoma. We find that CDK-dependent phosphorylation of the transcriptional master regulator ASCL1 plays a critical role in controlling the balance between cell proliferation and differentiation in embryonic neuroblasts and in neuroblastoma, and can be modulated to reactivate differentiation of neuroblastoma cells. ASCL1 is a master regulator proneural transcription factor that controls the balance between proliferation and differentiation of neuroblasts and is expressed transiently during formation of noradrenergic neurons arising from the neural crest. Embryos of the frog Xenopus laevis provide a versatile, rapid, and inexpensive system to study the role of ASCL1 phosphorylation by cyclin-dependent kinases in restraining neuronal differentiation. We found that enhanced cyclin-dependent kinase activity or elevated MYCN levels are sufficient to lock neuroblasts into a noradrenergic precursor-like state by multisite phosphorylation of ASCL1 protein. Conversely, preventing phosphorylation of ASCL1 can potentiate neuronal differentiation even in the face of proproliferative cues. Thus, the in vivo use of Xenopus as a model organism led us to the hypothesis that neuroblastoma arises from neuroblasts that fail to undergo the normal developmental program of differentiation due to precocious phosphorylation of ASCL1. We have gone on to test this hypothesis directly in neuroblastoma cells. We show that ASCL1 is phosphorylated by cyclin-dependent kinases and that phospho-ASCL1 supports the proproliferative program in neuroblastoma cells. However, preventing CDK-dependent phosphorylation of ASCL1 results in changes in the genome-wide transcriptional program of neuroblastoma cells, leading to suppression of proproliferative targets and simultaneous activation of genes that drive cell cycle exit and differentiation. Mechanistically, ASCL1 ChIPSeq reveals enhanced binding of un(der)phosphorylated ASCL1 at sites associated with prodifferentiation targets. Moreover, PHOX protein binding at key downstream regulatory elements is also modulated by ASCL1 phosphorylation. Finally, we also show that chemical CDK inhibition is sufficient to drive differentiation of neuroblastoma cells in a manner dependent on endogenous ASCL1. Therefore, we conclude that CDK-dependent phosphorylation of ASCL1 acts as a critical fulcrum controlling the balance between proliferation and differentiation in Xenopus and in human cells, and thus offers a novel therapeutic opportunity for neuroblastoma. Citation Format: Anna Philpott, Fahad Ali, Luke Wylie, Daniel Marcos Corchado, Igor Chernukhin, Evangelia Papachristou, Tatiana Papkovskaia, Liam Lee, Christopher Weekes, Tom Hiscock, Clive D'Santos, Suzanne Turner, Jason Carroll. A phosphorylation switch regulating proliferation and differentiation in Xenopus development and neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY28-02.

ZNF281 inhibits neuronal differentiation and is a prognostic marker for neuroblastoma

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

MicroRNA-494 Favors HO-1 Expression in Neuroblastoma Cells Exposed to Oxidative Stress in a Bach1-Independent Way.

Heme oxygenase 1 (HO-1) is crucially involved in cell adaptation to oxidative stress and has been demonstrated to play an important role in cancer progression and resistance to therapies. We recently highlighted that undifferentiated neuroblastoma (NB) cells are prone to counteract oxidative stress through the induction of HO-1. Conversely, differentiated NB cells were more sensitive to oxidative stress since HO-1 was scarcely upregulated. In this work, we investigated the role played by miR-494, which has been proved to be involved in cancer biology and in the modulation of oxidative stress, in the upregulation of HO-1. We showed that NB differentiation downregulates miR-494 level. In addition, endogenous miR-494 inhibition in undifferentiated cells impairs HO-1 induction in response to exposure to 500 µM H2O2, reducing the number of viable cells. The analysis of Bach1 expression did not reveal any significant modifications in any experimental conditions tested, proving that the impairment of HO-1 induction observed in cells treated with miR-494 inhibitor and exposed to H2O2 is independent from Bach1. Our results underline the role played by miR-494 in favoring HO-1 induction and cell adaptation to oxidative stress and contribute to the discovery of new potential pharmacological targets to improve anticancer therapies.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN-amplified neuroblastoma

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Silver Nanoparticles: Two-Faced Neuronal Differentiation-Inducing Material in Neuroblastoma (SH-SY5Y) Cells.

We have previously demonstrated the potential of biologically synthesized silver nanoparticles (AgNP) in the induction of neuronal differentiation of human neuroblastoma, SH-SY5Y cells; we aimed herein to unveil its molecular mechanism in comparison to the well-known neuronal differentiation-inducing agent, all-trans-retinoic acid (RA). AgNP-treated SH-SY5Y cells showed significantly higher reactive oxygen species (ROS) generation, stronger mitochondrial membrane depolarization, lower dual-specificity phosphatase expression, higher extracellular-signal-regulated kinase (ERK) phosphorylation, lower AKT phosphorylation, and lower expression of the genes encoding the antioxidant enzymes than RA-treated cells. Notably, pretreatment with N-acetyl-l-cysteine significantly abolished AgNP-induced neuronal differentiation, but not in that induced by RA. ERK inhibition, but not AKT inhibition, suppresses neurite growth that is induced by AgNP. Taken together, our results uncover the pivotal contribution of ROS in the AgNP-induced neuronal differentiation mechanism, which is different from that of RA. However, the negative consequence of AgNP-induced neurite growth may be high ROS generation and the downregulation of the expression of the genes encoding the antioxidant enzymes, which prompts the future consideration and an in-depth study of the application of AgNP-differentiated cells in neurodegenerative disease therapy.

PACAP and VIP regulate hypoxia-inducible factors in neuroblastoma cells exposed to hypoxia

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two related peptides acting as neurotransmitters/neuromodulators in central and peripheral nervous system. They are also involved in cancer showing a controversial role. Particulary, they are implicated in neuroblastoma differentiation (NB). This pediatric tumor can evolve to a malignant metastatic disease or spontaneously regress towards a benign form, known as ganglioneuroblastoma/ganglioneuroma. A negative hallmark of neoplasia progression is represented by hypoxic microenvironment. Low oxygen tension induces activation of hypoxia-inducible factors (HIFs) promoting cells proliferation and metastasis formation. Moreover, HIFs trigger vascular endothelial growth factor (VEGF) release favouring high-risk NB phenotype development.In the present work, we have investigated for the first time, if PACAP and VIP interfere with NB differentiation through modulation of hypoxic/angiogenic process. To this end, we analyzed their effect in malignant undifferentiated and all-trans retinoic acid (RA) differentiated SH-SY5Y cells, representing the benign form of this tumor.Our results have suggested tha both peptides, but predominantly VIP, induce NB differentiation into benign form by regulating HIFs, VEGF and VEGFRs expression and distribution.All these data give new insight regarding PACAP/VIP regulatory role in NB progression.

Investigation of the Roles of Novel Endogenous Ligand of Aryl Hydrocarbon Receptor in Neural Development

Activation of aryl hydrocarbon receptor (AHR) by xenobiotic toxic chemicals such as 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin regulates a variety of cellular processes including embryogenesis, tumorigenesis and inflammation. However, the identity of endogenous AHR ligands remains elusive. A potential AHR ligand, compound X, was identified from zebrafish embryos using previous developed cell free bioassay for dioxin‐like compounds. In this study, we aim to investigate the effect of compound X in AHR activation and further explore its physiological effects. Compound X induces AHR translocation into nucleus and upregulates downstream cytochrome P450 1A1 (CYP1A1) in vitro. It also promotes neuronal differentiation in neuroblastoma (NB) cells, which is consistent with our previous studies that overexpression of AHR leads to NB cells differentiation. Furthermore, compound X enhances zCyp1a, myelin‐associated myelin basic protein (zMbp) and SRY (sex determining region Y)‐box 10 (zSox10) expression and improves the mobility of zebrafish larvae via Ahr2 pathway. Therefore, our results suggest that compound X is an endogenous AHR ligand, and plays a critical role during neural differentiation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wnt Signalling Drives Context-Dependent Differentiation or Proliferation in Neuroblastoma

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 β-catenin immunohistochemistry shows high levels of β-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.

Inhibition of Sirtuin 6 Induces Neuroblastoma Differentiation.

Sirtuins (SIRTs) play crucial roles in various signaling pathways that modulate differentiation and proliferation. We sought to elucidate the role of SIRTs in differentiation and proliferation of human neuroblastoma (NB). NB cells were treated with nicotinamide (NAM), a non-specific SIRT inhibitor, SIRT-targeted short hairpin RNAs, and retinoic acid to assess cell growth and differentiation. SIRTs are involved in proliferation and differentiation using NAM in BE(2)-C cells. Specifically, SIRT6 knockdown in BE(2)-C cells reduced cell proliferation, induced neurite extension, corresponding with induction of p21CIP1 expression and G1 cell-cycle arrest. These effects were rescued by forced re-overexpression of SIRT6. SIRT6 expression was reduced in differentiated human NB sections, and RA-induced differentiation in BE(2)-C cells. SIRTs have important oncogenic properties in NB beyond its established functions in aging and genome stability. SIRT6 may represent a novel target for developing future therapeutics for the treatment of aggressive NBs.

Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery

BackgroundNeuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. Retinoic acid (RA) promotes differentiation of some neuroblastoma tumours and cell lines, and is successfully used clinically, supporting the view that differentiation therapy is a promising strategy for treatment of neuroblastoma. To improve treatment of a wider range of tumour types, development and testing of novel differentiation agents is essential. New pre-clinical models are therefore required to test therapies in a rapid cost effective way in order to identify the most useful agents.MethodsAs a proof of principle, differentiation upon ATRA treatment of two MYCN-amplified neuroblastoma cell lines, IMR32 and BE2C, was measured both in cell cultures and in tumours formed on the chick chorioallantoic membrane (CAM). Differentiation was assessed by 1) change in cell morphology, 2) reduction in cell proliferation using Ki67 staining and 3) changes in differentiation markers (STMN4 and ROBO2) and stem cell marker (KLF4). Results were compared to MLN8237, a classical Aurora Kinase A inhibitor. For the in vivo experiments, cells were implanted on the CAM at embryonic day 7 (E7), ATRA treatment was between E11 and E13 and tumours were analysed at E14.ResultsTreatment of IMR32 and BE2C cells in vitro with 10 μM ATRA resulted in a change in cell morphology, a 65% decrease in cell proliferation, upregulation of STMN4 and ROBO2 and downregulation of KLF4. ATRA proved more effective than MLN8237 in these assays. In vivo, 100 μM ATRA repetitive treatment at E11, E12 and E13 promoted a change in expression of differentiation markers and reduced proliferation by 43% (p < 0.05). 40 μM ATRA treatment at E11 and E13 reduced proliferation by 37% (p < 0.05) and also changed cell morphology within the tumour.ConclusionDifferentiation of neuroblastoma tumours formed on the chick CAM can be analysed by changes in cell morphology, proliferation and gene expression. The well-described effects of ATRA on neuroblastoma differentiation were recapitulated within 3 days in the chick embryo model, which therefore offers a rapid, cost effective model compliant with the 3Rs to select promising drugs for further preclinical analysis.

Long Non-coding RNAs, Novel Culprits, or Bodyguards in Neurodegenerative Diseases.

Long non-coding RNA (lncRNA) is a kind of non-coding RNA (ncRNA), with a length of 200 nt to 100 kb, that lacks a significant open reading frame (ORF) encoding a protein. lncRNAs are widely implicated in various physiological and pathological processes, such as epigenetic regulation, cell cycle regulation, cell differentiation regulation, cancer, and neurodegenerative diseases, through their interactions with chromatin, protein, and other RNAs. Numerous studies have suggested that lncRNAs are closely linked with the occurrence and development of a variety of diseases, especially neurodegenerative diseases, of which the etiologies are complicated and the underlying mechanisms remain elusive. Determining the roles of lncRNA in the pathogenesis of neurodegenerative diseases will not only deepen understanding of the physiological and pathological processes that occur in those diseases but also provide new ideas and solutions for their diagnosis and prevention. This review aims to highlight the progress of lncRNA research in the pathological and behavioral changes of neurodegenerative diseases. Specifically, we focus on how lncRNA dysfunctions are involved in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis.

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Legumain correlates with neuroblastoma differentiation and can be used in prodrug design.

Neuroblastoma (NB) is a highly malignant solid tumor in children. The cysteine endopeptidase legumain is expressed in adult solid tumors, but its expression in NB has not been examined. In this study, we assayed legumain expression in two NB cell lines and in microarrays of tumor tissues collected from 46 children with undifferentiated NB, differentiated NB, and ganglioneuroblastoma. Correlation analyses showed that legumain was expressed in all NB cell lines tested and that expression correlated with the degree of tumor differentiation. The efficacy, specificity, and toxicity of EMC-AANL-DOX, a novel doxorubicin-based legumain-activated prodrug, were then evaluated in mouse model of NB. Compared with DOX, EMC-AANL-DOX showed greater inhibition of tumor growth and a lower toxicity at high doses. Neither leukocyte or platelet counts nor renal function or cardiac anatomy differed significantly between the EMC-AANL-DOX and control groups (p>.05), suggesting that the prodrug caused minimal bone marrow depression and did not induce renal or cardiac damage. The good specificity and efficacy of EMC-AANL-DOX and low toxicity recommend its use in the treatment of NB. Correlation analyses of NB differentiation and legumain expression may reveal a novel anti-tumor-related functions of the drug and a new strategy for the treatment of pediatric solid tumors.

CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

Autophagy is a degradative pathway that delivers cellular components to the lysosome for degradation. The role of autophagy in cell differentiation is poorly understood. Here we show that CaMKII can directly phosphorylate Beclin 1 at Ser90 to promote K63-linked ubiquitination of Beclin 1 and activation of autophagy. Meanwhile, CaMKII can also promote K63-linked ubiquitination of inhibitor of differentiation 1/2 (Id-1/2) by catalyzing phosphorylation of Id proteins and recruiting TRAF-6. Ubiquitinated Id-1/Id-2 can then bind to p62 and be transported to autolysosomes for degradation. Id degradation promotes the differentiation of neuroblastoma cells and reduces the proportion of stem-like cells. Our study proposes a mechanism by which autophagic degradation of Id proteins can regulate cell differentiation. This suggests that targeting of CaMKII and the regulation of autophagic degradation of Id may be an effective therapeutic strategy to induce cell differentiation in neuroblastoma.

Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation

Neuroblastoma is the most common extra-cranial solid tumor in children. Its broad spectrum of clinical outcomes reflects the underlying inherent cellular heterogeneity. As current treatments often do not lead to tumor eradication, there is a need to better define therapy-resistant neuroblastoma and to identify new modulatory molecules. To this end, we performed the first comprehensive flow cytometric characterization of surface molecule expression in neuroblastoma cell lines. Exploiting an established clustering algorithm (SPADE) for unbiased visualization of cellular subsets, we conducted a multiwell screen for small molecule modulators of neuroblastoma phenotype. In addition to SH-SY5Y cells, the SH-EP, BE(2)-M17 and Kelly lines were included in follow-up analysis as in vitro models of neuroblastoma. A combinatorial detection of glycoprotein epitopes (CD15, CD24, CD44, CD57, TrkA) and the chemokine receptor CXCR4 (CD184) enabled the quantitative identification of SPADE-defined clusters differentially responding to small molecules. Exposure to bone morphogenetic protein (BMP)-4 was found to enhance a TrkAhigh/CD15−/CD184− neuroblastoma cellular subset, accompanied by a reduction in doublecortin-positive neuroblasts and of NMYC protein expression in SH-SY5Y cells. Beyond yielding novel marker candidates for studying neuroblastoma pathology, our approach may provide tools for improved pharmacological screens towards developing novel avenues of neuroblastoma diagnosis and treatment.

MicroRNAs in neuroblastoma differentiation and differentiation therapy

&lt;p class="ArticleTitle"&gt;&lt;span lang="EN-US"&gt;Neuroblastoma is one of the most common and aggressive types of pediatric cancers, making up about 7% of all childhood cancers. Neuroblastoma arises from the failure of neural crest cell precursors to differentiate, and inducing cell differentiation is one of the most important treatment approaches for neuroblastoma. MicroRNAs regulate gene expression by performing post-transcriptional gene modification by mainly translational suppression and mRNA degradation. The dysregulation of these molecules has been shown to be related to tumor development, tumor metastasis and drug resistance, and the promise of developing microRNA-based therapeutics for cancers has been demonstrated. Many recent studies have also provided evidence for the involvement of microRNAs in differentiation of neuroblastoma cells, suggesting the potential of developing microRNA-based differentiation therapies for neuroblastoma. Here we review the recent findings on the role of microRNAs in regulating cell differentiation, with a main focus on neuroblastoma cells. The investigations on the therapeutic potential of microRNAs in neuroblastoma therapy and differentiation therapy are also reviewed.&lt;/span&gt;&lt;/p&gt;