Exploitation of chick embryo environments to reprogram MYCN-amplified neuroblastoma cells to a benign phenotype, lacking detectable MYCN expression.
ABSTRACT: Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gland. Tumours that occur in patients under 18 months of age have a particularly good prognosis and frequently undergo spontaneous regression. This led to the hypothesis that developmental cues in the youngest patients may prompt belated differentiation and/or apoptosis of the tumour cells. To test our hypothesis, we have injected MYCN-amplified neuroblastoma cells into the extra embryonic veins of chick embryos at embryonic day 3 (E3) and E6 and analysed the response of these Kelly cells at E10 and E14. Amplification of the MYCN gene occurs in up to 30% of tumours and is normally associated with a very poor prognosis. Kelly cells injected at E3 follow neural crest pathways and integrate into neural locations such as SG and the enteric nervous system although never into the adrenal gland. Additionally they migrate to non-neural locations such as the heart, meninges, jaw regions and tail. The cells respond to their respective microenvironments and in SG, some cells differentiate, they show reduced cell division and crucially all cells have undetectable MYCN expression by E10. In non-neural locations, cells form more rapidly dividing clumps and continue to express MYCN. The downregulation of MYCN is dependent on continuous and direct interaction with the sympathetic ganglion environment. We propose that the MYCN-amplicon in the Kelly cells retains the ability to correctly interpret the environmental cues leading to downregulation of MYCN.
Project description:Neuroblastoma is a pediatric malignancy of the sympathetic ganglia and adrenal glands, hypothesized to originate from progenitors of the developing sympathetic nervous system. Amplification of the MYCN oncogene is a genetic marker of risk in this disease. Understanding the impact of oncogene expression on sympathoadrenal progenitor development may improve our knowledge of neuroblastoma initiation and progression. We isolated sympathoadrenal progenitor cells from the postnatal murine adrenal gland by sphere culture and found them to be multipotent, generating differentiated colonies of neurons, Schwann cells, and myofibroblasts. MYCN overexpression in spheres promoted commitment to the neural lineage, evidenced by an increased frequency of neuron-containing colonies. MYCN promoted proliferation of both sympathoadrenal progenitor spheres and differentiated neurons derived from these spheres, but there was also an increase in apoptosis. The proliferation, apoptosis, and neural lineage commitment induced by MYCN are tumor-like characteristics and thereby support the hypothesis that multipotent adrenal medullary progenitor cells are cells of origin for neuroblastoma. We find, however, that MYCN overexpression is not sufficient for these cells to form tumors in nude mice, suggesting that additional transforming mutations are necessary for tumorigenesis.
Project description:Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analog following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma.
Project description:Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto?oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c?MYB proto?oncogene transcription factor (c?myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c?Myb target genes, and the effect of c?myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN?amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre? and post?c?myb RNAi treatment groups in MYCN?amplified Kelly, IMR32, SIMA and MHH?NB?11 cell lines, normalized to those of non?MYCN?amplified SH?SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c?myb, MYB proto?oncogene like 2 (B?myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c?myb may be associated with E2F1 and L3MBTL1 histone methyl?lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B?myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c?myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c?myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally?regulated genes may be evolutionarily conserved. In conclusion, c?myb may be associated with regulating MYCN expression and amplification. c?myb, B?myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication?based mechanism.
Project description:Neuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers.In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti-MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced.We have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.
Project description:PURPOSE:MYCN oncogene amplification is an independent predictor of poor prognosis in neuroblastoma. CX-5461 is a small molecular inhibitor that prevents initiation of ribosomal RNA (rRNA) synthesis by RNA Pol I, down-regulating MYCN/MYC proteins. We hypothesize that neuroblastoma tumor growth can be suppressed by CX-5461. METHODS:MYCN-amplified (KELLY, IMR5) and nonamplified (SY5Y, SKNAS) neuroblastoma cells were treated with CX-5461. MYCN/MYC expression after 24-48 h was determined by Western blot. Orthotopic neuroblastoma tumors created in mice using KELLY cells were treated with CX-5461-loaded silk films implanted locally. Tumor growth was monitored using ultrasound. Histologic evaluation of tumors was performed. RESULTS:IC50 for KELLY, IMR5, SY5Y, and SKNAS cells to CX-5461 was 0.75 ?M, 0.02 ?M, 0.8 ?M, and 1.7 ?M, respectively. CX-5461 down-regulated MYCN and MYC proteins at 0.25-1.0 ?M on Western blot analysis. CX-5461-loaded silk film released 23.7±3 ?g of the drug in 24 h and 48.2±3.9 ?g at 120 h. KELLY tumors treated with CX-5461-loaded film reached 800 mm3 after 7.8±1.4 days, while those treated with control film reached the same size on 5.1±0.6 days (p=0.03). CX-5461-treated tumors showed collapse of nucleolar hypertrophy and MYCN protein downregulation. CONCLUSION:We demonstrated that local delivery of CX-5461 via sustained release platform can suppress orthotopic neuroblastoma tumor growth, especially those with MYCN/MYC overexpression.
Project description:The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on MycN have been conducted in human tumor cells, thus, the role of MycN in normal human neural crest development is completely unknown. In the present study, we determined the role of MycN in hNCSCs isolated from in vitro-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of MycN in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of MycN in hNCSCs increases the expression of Cdkn1a, Cdkn2a and Cdkn2b, which encodes the cyclin-dependent kinases p21CIP1, p16 INK4a and p15INK4b. In addition, MycN is involved in the regulation of human sympathetic neurogenesis, as knockdown of MycN enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including Phox2a, Phox2b, Mash1, Hand2 and Gata3. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases.
Project description:MYCN, a proto-oncogene normally expressed in the migrating neural crest, is in its amplified state a key factor in the genesis of human neuroblastoma (NB). However, the mechanisms underlying MYCN-mediated NB progression are poorly understood. Here, we present a MYCN-induced miRNA signature in human NB involving the activation and transrepression of several miRNA genes from paralogous clusters. Several family members derived from the miR-17 approximately 92 cluster, including miR-18a and miR-19a, were among the up-regulated miRNAs. Expression analysis of these miRNAs in NB tumors confirmed increased levels in MYCN-amplified samples. Specifically, we show that miR-18a and miR-19a target and repress the expression of estrogen receptor-alpha (ESR1), a ligand-inducible transcription factor implicated in neuronal differentiation. Immunohistochemical staining demonstrated ESR1 expression in human fetal sympathetic ganglia, suggesting a role for ESR1 during sympathetic nervous system development. Concordantly, lentiviral restoration of ESR1 in NB cells resulted in growth arrest and neuronal differentiation. Moreover, lentiviral-mediated inhibition of miR-18a in NB cells led to severe growth retardation, outgrowth of varicosity-containing neurites, and induction of neuronal sympathetic differentiation markers. Bioinformatic analyses of microarray data from NB tumors revealed that high ESR1 expression correlates with increased event-free survival in NB patients and favorable disease outcome. Thus, MYCN amplification may disrupt estrogen signaling sensitivity in primitive sympathetic cells through deregulation of ESR1, thereby preventing the normal induction of neuroblast differentiation. Collectively, our findings demonstrate the molecular consequences of abnormal miRNA transcription in a MYCN-driven tumor and offer unique insights into the pathology underlying MYCN-amplified NB.
Project description:Introduction:Neuroblastoma is an embryonal tumor of the sympathetic nervous system. The MYCN oncogene is amplified in some neuroblastoma patients and correlated with poor prognosis. However, less is known regarding the relationship between MYCN gene single-nucleotide polymorphisms (SNPs) and neuroblastoma risk. Patients and methods:To investigate the contribution of MYCN gene polymorphisms to neuroblastoma risk, we performed a 3-center case-control study by genotyping 4 SNPs in the MYCN gene from 429 cases and 884 controls. Results:The results showed that only rs57961569 G>A was associated with neuroblastoma risk (GA vs GG: adjusted odds ratio =0.76, 95% confidence interval =0.60-0.98, P=0.033), while the other 3 SNPs were not (rs9653226 T>C, rs13034994 A>G, and rs60226897 G>A). Stratified analysis revealed that rs57961569 GG carriers were more likely to develop neuroblastoma in the following subgroups: children older than 18 months, tumor derived from the adrenal gland, and clinical stages III + IV. The increased neuroblastoma risk associated with the rs9653226 variant CC genotypes was more evident in the following subgroups: females, tumor derived from the adrenal gland, and clinical stages III + IV. The presence of 2-3 risk genotypes had a significant relationship with the following subgroups: tumor derived from the adrenal gland and clinical stages III + IV. Conclusion:This study demonstrates a weak impact of MYCN gene polymorphisms on neuroblastoma risk, which should be further validated.
Project description:Neuroblastoma is the most common pediatric cancer, arising from the neural crest cells of the sympathetic nervous system. Its most aggressive subtype, characterized by the amplification of the MYCN oncogene, has a dismal prognosis and no effective treatment is available. Understanding the alterations induced by the tumor on the various layers of gene expression is therefore important for a complete characterization of this neuroblastoma subtype and for the discovery of new therapeutic opportunities. Here we describe the profiling of 13 MYCN-amplified neuroblastoma cell lines at the genome (copy number), transcriptome, translatome and miRome levels (GEO series GSE56654, GSE56552 and GSE56655). We provide detailed experimental and data analysis procedures by means of which we derived the results described in .
Project description:MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation.A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3'-untranslated region (3'UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3'UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line.The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells.The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.