Neuroblastoma is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during the infancy. MYCN amplification and overexpression occur in about 25% of total neuroblastoma cases and this percentage increases at 30% in advanced stage neuroblastoma. So far, MYCN expression profile is still one of the most robust and significant prognostic markers for neuroblastoma outcome. MYCN is a transcription factor that belongs to the family of MYC oncoproteins, comprising c-MYC and MYCL genes. Deregulation of MYC oncoprotein expression is a crucial event involved in the occurrence of different types of malignant tumors. MYCN, as well as c-MYC, can heterodimerize with its partner MAX and activate the transcription of several target genes containing E-Box sites in their promoter regions. However, recent several lines of evidence have revealed that MYCN can repress at least as many genes as it activates, thus proposing a novel function of this protein in neuroblastoma biology. Whereas the mechanism by which MYCN can act as a transcriptional activator is relatively well known, very few studies has been done in the attempt to explain how MYCN can exert its transcription repression function. Here, we will review current knowledge about the mechanism of MYCN-mediated transcriptional repression and will emphasize its role as a repressor in the recruitment of a precise set of proteins to form complexes capable of down-regulating specific subsets of genes whose function is actively involved in apoptosis, cell differentiation, chemosensitivity, and cell motility. The finding that MYCN can also act as a repressor has widen our view on its role in oncogenesis and has posed the bases to search for novel therapeutic drugs that can specifically target its transcriptional repression function.
MYCN; neuroblastoma; transcriptional repression; cell differentiation; apoptosis; cell cycle
Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors.
We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions.
Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the activity of individual genes, and that of a mediator of global chromatin structure.
MYCN oncogene amplification is the most important prognostic factor in neuroblastoma. 25% neuroblastoma tumors have somatic amplifications at this locus but little is known about its constitutional aberrations and their potential role in carcinogenesis. Here, we have performed an array-CGH and qPCR characterization of two patients with constitutional partial 2p trisomy including MYCN genomic region.
One of the patients had congenital neuroblastoma and showed presence of minute areas of gains and losses within the common fragile site FRA2C at 2p24 encompassing MYCN. The link between 2p24 germline rearrangements and neuroblastoma development was reassessed by reviewing similar cases in the literature.
It appears that constitutional rearrangements involving chromosome 2p24 may play role in NB development.
Neuroblastoma; MYCN; FRA2C; Partial 2p trisomy
Neuroblastoma is one of the most common solid tumors of childhood, arising from immature sympathetic nervous system cells. The clinical course of patients with neuroblastoma is highly variable, ranging from spontaneous regression to widespread metastatic disease. Although the outcome for children with cancer has improved considerably during the past decades, the prognosis of children with aggressive neuroblastoma remains dismal. The clinical heterogeneity of neuroblastoma mirrors the biological and genetic heterogeneity of these tumors. Ploidy and MYCN amplification have been used as genetic markers for risk stratification and therapeutic decision making, and, more recently, gene expression profiling and genome-wide DNA copy number analysis have come into the picture as sensitive and specific tools for assessing prognosis. The applica tion of new genetic tools also led to the discovery of an important familial neuroblastoma cancer gene, ALK, which is mutated in approximately 8% of sporadic tumors, and genome-wide association studies have unveiled loci with risk alleles for neuroblastoma development. For some of the genomic regions that are deleted in some neuroblastomas, on 1p, 3p and 11q, candidate tumor suppressor genes have been identified. In addition, evidence has emerged for the contribution of epigenetic disturbances in neuroblastoma oncogenesis. As in other cancer entities, altered microRNA expression is also being recognized as an important player in neuroblastoma. The recent successes in unraveling the genetic basis of neuroblastoma are now opening opportunities for development of targeted therapies.
Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping. © 2000 Cancer Research Campaign
prognosis; fluorescence in situ hybridization (FISH); MYCN; del(1p); ploidy
Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high-risk tumors; yet, only ~25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. Since aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether it or the other family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma.
Aminoacylase expression was examined in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN amplified (SK-N-AS, SK-N-SH, SH-SY5Y, and SH-EP) neuroblastoma cell lines. Each aminoacylase exhibited distinct spatial localization (i.e., cytosolic ACY1, membrane-associated ASPA, and nuclear ACY3). When SK-N-SH cells were treated with neural differentiation agents (e.g., retinoic acid, cAMP) in media containing 10% serum ACY1 was the only aminoacylase whose expression was up-regulated. ASPA was primarily expressed in SH-EP cells of a glial sublineage. ACY3 was more highly expressed in the TrkB-positive, MYCN-amplified lines. All three aminoacylases were expressed in normal human adrenal gland, a common site of neuroblastoma origin, but only ACY1 and ACY3 displayed detectable expression in primary neuroblastoma tumor. Bioinformatics data mining of Kaplan-Meier survival revealed that high ACY3 expression is correlated with poor prognosis; while, low expression of ACY1 or ASPA is correlated with poor prognosis. These data suggest that aminoacylase expression is dysregulated in neuroblastoma.
MYCN is a well-known oncogene over-expressed in different human malignancies including neuroblastoma (NB), rhabdomyosarcoma, medulloblastoma, astrocytoma, Wilms’ tumor, and small cell lung cancer. In the case of NB, MYCN amplification is an established biomarker of poor-prognosis. MYCN belongs to a family of transcription factors (the most important of which is C-MYC) that show a high degree of homology. Down-regulation of MYC protein expression leads to tumor regression in animal models, indicating that MYC proteins represent interesting therapeutic targets. Pre-requisites for a candidate tumor-associated antigen (TAA) to be targeted by immunotherapeutic approaches are the following, (i) expression should be tumor-restricted, (ii) the putative TAA should be up-regulated in cancer cells, and (iii) protein should be processed into immunogenic peptides capable of associating to major histocompatibility complex molecules with high affinity. Indeed, the MYCN protein is not expressed in human adult tissues and up-regulated variably in NB cells, and MYCN peptides capable of associating to HLA-A1 or HLA-A2 molecules with high affinity have been identified. Thus the MYCN protein qualifies as putative TAA in NB. Additional issues that determine the feasibility of targeting a putative TAA with cytotoxic T lymphocytes (CTLs) and will be here discussed are the following, (i) the inadequacy of tumor cells per se to act as antigen-presenting cells witnessed, in the case of NB cells, by the low to absent expression of HLA class I molecules, the lack of co-stimulatory molecules and multiple defects in the HLA class I related antigen processing machinery, and (ii) the immune evasion mechanisms operated by cancer cells to fool the host immune system, such as up-regulation of soluble immunosuppressive molecules (e.g., soluble MICA and HLA-G in the case of NB) or generation of immunosuppressive cells in the tumor microenvironment. A final issue that deserves consideration is the strategy used to generate CTL.
MYCN oncoprotein; neuroblastoma; tumor antigen; cytotoxic T cells; immunotherapy
Most children with neuroblastoma presenting after infancy have metastatic, chemoresistant disease. Amplification of the MYCN protooncogene is a significant marker of these poor-prognosis neuroblastoma tumors. Recent studies suggest that MYCN may function in part by promoting angiogenesis via vascular endothelial growth factor (VEGF). VEGF blockade has been validated as a therapeutic strategy in adult cancers. In these studies, we asked whether inhibition of VEGF signaling via VEGFR2 blockade in established MYCN-amplified neuroblastoma xenografts would: 1) restrict tumor growth; 2) induce hypoxia; and 3) alter tumor vasculature. The MYCN-amplified neuroblastoma human cell line NGP was implanted intrarenally in athymic female mice. After 5 weeks, mice with established tumors were selected, a cohort euthanized to provide Day 0 controls, and the rest assigned to receive biweekly injections of DC101 (anti-murine VEGFR2 antibody) or vehicle. DC101 treatment did not inhibit progressive tumor growth in established NGP xenografts. Although tumor vasculature was not significantly disrupted, a modest increase in tumor hypoxia was demonstrated by pimonidazole staining, and expression of a previously described hypoxia metagene was increased by gene set enrichment analysis (GSEA) in DC101-treated tumors. Intriguingly, DC101 treatment elicited increased: 1) expression of VEGFR1 and its ligand placental growth factor; and 2) increased Notch activation in tumor vasculature concurrent with expression of the Notch ligand Jagged1. This result suggests that established MYCN-amplified neuroblastoma tumors are relatively VEGF-independent, and display the ability to rapidly up-regulate hypoxia-responsive alternative proangiogenic mechanisms that may stabilize vasculature when VEGF is deficient.
angiogenesis; neuroblastoma; Notch; placental growth factor; vascular endothelial growth factor
Neuroblastoma is the most common extra-cranial solid tumor of childhood. Despite significant advances, it currently still remains one of the most difficult childhood cancers to cure, with less than 40% of patients with high-risk disease being long-term survivors. MYCN is a proto-oncogene implicated to be directly involved in neuroblastoma development. Amplification of MYCN is associated with rapid tumor progression and poor prognosis. Novel therapeutic strategies which can improve the survival rates whilst reducing the toxicity in these patients are therefore required. Here we discuss genes regulated by MYCN in neuroblastoma, with particular reference to p53, SKP2, and DKK3 and strategies that may be employed to target them.
neuroblastoma; MYCN; p53; SKP2; DKK3; MDM2-p53 antagonists
Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that regulates both cellular adhesion and apoptosis. FAK is overexpressed in a number of human tumors including neuroblastoma. Previously, we have shown that the MYCN oncogene, the primary adverse prognostic indicator in neuroblastoma, regulates the expression of FAK in neuroblastoma. In this study, we have examined the effects of FAK inhibition upon neuroblastoma using a small molecule [1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)] to inhibit FAK expression and the phosphorylation of FAK at the Y397 site. Utilizing both non-isogenic and isogenic MYCN+/MYCN− neuroblastoma cell lines, we found that Y15 effectively diminished phosphorylation of the Y397 site of FAK. Treatment with Y15 resulted in increased detachment, decreased cell viability and increased apoptosis in the neuroblastoma cell lines. We also found that the cell lines with higher MYCN are more sensitive to Y15 treatment than their MYCN negative counterparts. In addition, we have shown that treatment with Y15 in vivo leads to less tumor growth in nude mouse xenograft models, again with the greatest effects seen in MYCN+ tumor xenografts. The results of the current study suggest that FAK and phosphorylation at the Y397 site plays a role in neuroblastoma cell survival, and that the FAK Y397 phosphorylation site is a potential therapeutic target for this childhood tumor.
neuroblastoma; focal adhesion kinase; FAK; MYCN; pediatric; Y397; apoptosis
Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G1 cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27Kip1 protein and causes p27Kip1/Rb-coupled G1 cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27Kip1 phosphorylation at residues Ser10 and Thr198. While p27Kip1 is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27Kip1 as a regulator of cell migration and invasion. In the present study we found that p27Kip1 regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27Kip1. DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-β (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27Kip1 mRNA correlates with clinical features and the survival probability of NB patients.
DFMO; Kaplan-Meier survival plot; metastasis; MYCN; neuroblastoma; ornithine decarboxylase; polyamines; p27Kip
Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased neoangiogenesis mark high-risk disease. Because angiogenesis is regulated by phosphatidylinositol 3-kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis and improved survival in both primary human (highly pretreated recurrent MYCN-amplified orthotopic xenograft) and transgenic mouse models for MYCN-driven neuroblastoma. Using both gain- and loss-of-function approaches, we demonstrated that the anti-angiogenic efficacy of NVP-BEZ235 depended critically on MYCN in vitro and in vivo. Thus, clinical PI3K/mammalian target of rapamycin inhibitors drove degradation of MYCN in tumor cells, with secondary paracrine blockade of angiogenesis. Our data demonstrated significantly improved survival in treated animals and suggest that NVP-BEZ235 should be tested in children with high-risk, MYCN-amplified neuroblastoma.
Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.
class I HOX cluster; Hox-C9; neuroblastoma; apoptosis; differentiation; spontaneous regression
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.
Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Comparison of mRNA from benign neuroblastic tumors and neuroblastomas revealed that expression of the type III TGF-β receptor (TGFBR3) decreases with advancing stage of neuroblastoma and this loss correlates with a poorer prognosis. Patients with MYCN oncogene amplification and low TGFBR3 expression were more likely to have an adverse outcome. In vitro, TβRIII expression was epigenetically suppressed by MYCN-mediated recruitment of histone deacetylases to regions of the TGFBR3 promoter. TβRIII bound FGF2 and exogenous FGFR1, which promoted neuronal differentiation of neuroblastoma cells. TβRIII and FGF2 cooperated to induce expression of the transcription factor inhibitor of DNA binding 1 via Erk MAPK. TβRIII-mediated neuronal differentiation suppressed cell proliferation in vitro as well as tumor growth and metastasis in vivo. These studies characterize a coreceptor function for TβRIII in FGF2-mediated neuronal differentiation, while identifying potential therapeutic targets and clinical biomarkers for neuroblastoma.
Myc proteins (c-myc, Mycn and Mycl) target proliferative and apoptotic pathways vital for progression in cancer. Amplification of the MYCN gene has emerged as one of the clearest indicators of aggressive and chemotherapy-refractory disease in children with neuroblastoma, the most common extracranial solid tumor of childhood. Phosphorylation and ubiquitin-mediated modulation of Myc protein influence stability and represent potential targets for therapeutic intervention. Phosphorylation of Myc proteins is controlled in-part by the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt/mTOR signaling, with additional contributions from Aurora A kinase. Myc proteins regulate apoptosis in part through interactions with the p53/Mdm2/Arf signaling pathway. Mutation in p53 is commonly observed in patients with relapsed neuroblastoma, contributing to both biology and therapeutic resistance. This review examines Myc function and regulation in neuroblastoma, and discusses emerging therapies that target Mycn.
myc; mycn; neuroblastoma; N-myc; mTor; PI3K
Amplification of the MYCN gene in neuroblastoma (NB) is associated with a poor prognosis. However, MYCN-amplification does not automatically result in higher expression of MYCN in children with NB. We hypothesized that the discrepancy between MYCN gene expression and prognosis in these children might be explained by the expression of either MYCN-opposite strand (MYCNOS) or the shortened MYCN-isoform (ΔMYCN) that was recently identified in fetal tissues. Both MYCNOS and ΔMYCN are potential inhibitors of MYCN either at the mRNA or at the protein level.
Expression of MYCN, MYCNOS and ΔMYCN was measured in human NB tissues of different stages. Transcript levels were quantified using a real-time reverse transcriptase polymerase chain reaction assay (QPCR). In addition, relative expression of these three transcripts was compared to the number of MYCN copies, which was determined by genomic real-time PCR (gQPCR).
Both ΔMYCN and MYCNOS are expressed in all NBs examined. In NBs with MYCN-amplification, these transcripts are significantly higher expressed. The ratio of MYCN:ΔMYCN expression was identical in all tested NBs. This indicates that ΔMYCN and MYCN are co-regulated, which suggests that ΔMYCN is not a regulator of MYCN in NB. However, the ratio of MYCNOS:MYCN expression is directly correlated with NB disease stage (p = 0.007). In the more advanced NB stages and NBs with MYCN-amplification, relatively more MYCNOS is present as compared to MYCN. Expression of the antisense gene MYCNOS might be relevant to the progression of NB, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level.
The MYCNOS:MYCN-ratio in NBs is significantly correlated with both MYCN-amplification and NB-stage. Our data indicate that in NB, MYCN expression levels might be influenced by MYCNOS but not by ΔMYCN.
Differences in MYCN/c-MYC target gene expression are associated with distinct neuroblastoma subtypes and clinical outcome.
Amplified MYCN oncogene resulting in deregulated MYCN transcriptional activity is observed in 20% of neuroblastomas and identifies a highly aggressive subtype. In MYCN single-copy neuroblastomas, elevated MYCN mRNA and protein levels are paradoxically associated with a more favorable clinical phenotype, including disseminated tumors that subsequently regress spontaneously (stage 4s-non-amplified). In this study, we asked whether distinct transcriptional MYCN or c-MYC activities are associated with specific neuroblastoma phenotypes.
We defined a core set of direct MYCN/c-MYC target genes by applying gene expression profiling and chromatin immunoprecipitation (ChIP, ChIP-chip) in neuroblastoma cells that allow conditional regulation of MYCN and c-MYC. Their transcript levels were analyzed in 251 primary neuroblastomas. Compared to localized-non-amplified neuroblastomas, MYCN/c-MYC target gene expression gradually increases from stage 4s-non-amplified through stage 4-non-amplified to MYCN amplified tumors. This was associated with MYCN activation in stage 4s-non-amplified and predominantly c-MYC activation in stage 4-non-amplified tumors. A defined set of MYCN/c-MYC target genes was induced in stage 4-non-amplified but not in stage 4s-non-amplified neuroblastomas. In line with this, high expression of a subset of MYCN/c-MYC target genes identifies a patient subtype with poor overall survival independent of the established risk markers amplified MYCN, disease stage, and age at diagnosis.
High MYCN/c-MYC target gene expression is a hallmark of malignant neuroblastoma progression, which is predominantly driven by c-MYC in stage 4-non-amplified tumors. In contrast, moderate MYCN function gain in stage 4s-non-amplified tumors induces only a restricted set of target genes that is still compatible with spontaneous regression.
Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that is overexpressed in a number of human tumors including neuroblastoma, and regulates both cellular adhesion and survival. We have studied the effects of FAK inhibition upon neuroblastoma using adenovirus-containing FAK-CD (AdFAK-CD). Utilizing an isogenic MYCN+ / MYCN− neuroblastoma cell line, we found that the MYCN+ cells are more sensitive to FAK inhibition with AdFAK-CD than their MYCN negative counterparts. In addition, we have shown that phosphorylation of Src is increased in the untreated isogenic MYCN− neuroblastoma cells, and that the decreased sensitivity of the MYCN− neuroblastoma cells to FAK inhibition with AdFAK-CD is abrogated by the addition of the Src family kinase inhibitor, PP2. The results of the current study suggest that both FAK and Src play a role in protecting neuroblastoma cells from apoptosis, and that dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor.
Pediatric; Cancer; MYCN oncogene
MYCN amplification occurs in around 25% of neuroblastomas, and is associated with rapid tumor progression and poor prognosis. MYCN plays a paradoxical role in driving cellular proliferation and inducing apoptosis. We previously observed nuclear p53 accumulation in neuroblastoma and hypothesize that MYCN regulates p53 in neuroblastoma. Immunohistochemical analysis of 82 neuroblastoma tumors demonstrated an association between high p53 expression and MYCN expression and MYCN amplification. In a panel of 5 MYCN amplified and 5 non-amplified neuroblastoma cell lines and also the Tet21N regulatable MYCN expression system there was a correlation between p53 expression and MYCN expression. Knockdown of MYCN in 2 MYCN amplified cell lines led to a decrease in p53 expression. Tet21N MYCN+ cells expressed higher p53 mRNA and protein, and had greater p53 transcriptional activity, in comparison with Tet21N MYCN− cells. Using chromatin immunoprecipitation and reporter gene assays, MYCN was found to bind directly to an E-Box motif located close to the transcriptional start site within the p53 promoter and initiate transcription. Mutation of the E-Box led to a decrease in MYCN driven transcriptional activity. Microarray analysis of Tet21N MYCN+/− cells showed that several p53 regulated genes were upregulated in the presence of MYCN, including MDM2 and PUMA. Knockdown of MYCN and p53 in a MYCN amplified cell line led to reduced PUMA levels and other markers of apoptosis. We conclude that MYCN transcriptionally upregulates p53 expression in neuroblastoma and may be an important mechanism by which MYCN induces apoptosis.
p53; MYCN; neuroblastoma; chromatin immunoprecipitation; microarray
Neuroblastoma is the most common extracranial pediatric solid cancer. Lung metastasis is rarely detected in children with newly diagnosed neuroblastoma. We aimed to describe the incidence, clinical characteristics, and outcome of patients with lung metastasis at initial diagnosis using a large international database.
The subset of patients from the International Neuroblastoma Risk Group database with INSS stage 4 neuroblastoma and known data regarding lung metastasis at diagnosis was selected for analysis. Clinical and biological characteristics were compared between patients with and without lung metastasis. Survival for patients with and without lung metastasis was estimated by Kaplan-Meier methods. Cox proportional hazards methods were used to determine the independent prognostic value of lung metastasis at diagnosis.
Of the 2,808 patients with INSS stage 4 neuroblastoma diagnosed between 1990 and 2002, 100 patients (3.6%) were reported to have lung metastasis at diagnosis. Lung metastasis was more common among patients with MYCN amplified tumors, adrenal primary tumors, or elevated lactate dehydrogenase (LDH) levels (p < 0.02 in each case). Five-year overall survival ± standard error for patients with lung metastasis was 34.5% ± 6.8% compared to 44.7% ± 1.3% for patients without lung metastasis (p=0.0002). However, in multivariable analysis, the presence of lung metastasis was not independently predictive of outcome.
Lung metastasis at initial diagnosis of neuroblastoma is associated with MYCN amplification and elevated LDH levels. Although lung metastasis at diagnosis was not independently predictive of outcome in this analysis, it remains a useful prognostic marker of unfavorable outcome.
Neuroblastoma; Lung Metastases; Pulmonary; MYCN
The transcription factor MycN is the prototypical neuroblastoma oncogene and a potential therapeutic target. However, its strong expression caused by gene amplification in about 30% of neuroblastoma patients is a considerable obstacle to the development of therapeutic approaches aiming at eliminating its tumourigenic activity. We have previously reported that B-Myb is essentially required for transcription of the MYCN amplicon and have also shown that B-MYB and MYCN are engaged in a feed forward loop promoting the survival/proliferation of neuroblastoma cells. We postulated that pharmacological strategies breaking the B-MYB/MYCN axis should result in clinically desirable effects. Thus, we implemented a high throughput chemical screen, using a curated library of ~1500 compounds from the National Cancer Institute, whose endpoint was the identification of small molecules that inhibited B-Myb. At the end of the screening, we found that the compounds pinafide, ellipticine and camptothecin inhibited B-Myb transcriptional activity in luciferase assays. One of the compounds, the topoisomerase-1 inhibitor camptothecin, is of considerable clinical interest since its derivatives topotecan and irinotecan are currently used as first and second line treatment agents for various types of cancer, including neuroblastoma. We found that neuroblastoma cells with amplification of MYCN are more sensitive than MYCN negative cells to camptothecin and topotecan killing. Campothecin and topotecan caused selective down-regulation of B-Myb and MycN expression in neuroblastoma cells. Notably, forced overexpression of B-Myb could antagonize the killing effect of topotecan and camptothecin, demonstrating that the transcription factor is a key target of the drugs. These results suggest that camptothecin and its analogues should be more effective in patients whose tumours feature amplification of MYCN and/or overexpression of B-MYB.
Neuroblastoma; chemotherapeutic drug; oncogene; transcription
Amplification of the MYCN oncogene is strongly associated with poor prognosis in neuroblastoma (NB). In addition to MYCN amplification, many studies have focused on identifying patients with a poor prognosis based on gene expression profiling. The majority of prognostic signatures today are comprised of large gene lists limiting their clinical application. In addition, although of prognostic significance, most of these signatures fail to identify cellular processes that can explain their relation to prognosis. Here, we determined prognostically predictive genes in a data set containing 251 NBs. Gene Ontology analysis was performed on significant genes with a positive hazard ratio to search for cellular processes associated with poor prognosis. An enrichment in ribonucleoproteins (RNPs) was found. Genes involved in the stabilization and formation of the central small nucleolar RNP (snoRNP) complex were scrutinized using a backward conditional Cox regression resulting in an snoRNP signature consisting of three genes: DKC1, NHP2, and GAR1. The snoRNP signature significantly and independently predicted prognosis when compared to the established clinical risk factors. Association of snoRNP protein expression and prognosis was confirmed using tissue micro-arrays. Knockdown of snoRNP expression in NB cell lines resulted in reduced telomerase activity and an increase in anaphase bridge frequency. In addition, in patient material, expression of the snoRNP complex was significantly associated with telomerase activity, occurrence of segmental aberrations, and expression-based measurements of chromosomal instability. Together, these results underscore the prognostic value of snoRNP complex expression in NB and suggest a role for snoRNPs in telomere maintenance and genomic stability.
The management of patients with International Neuroblastoma Staging System (INSS) stage 3 neuroblastoma (NB) is not consistent worldwide. We describe a single centre approach at Memorial Sloan-Kettering Cancer Centre (MSKCC) from 1991 to 2007 that minimizes therapy except for those patients with MYCN-amplified NB.
In this retrospective analysis of 69 patients, tumour MYCN was not amplified in 53 and amplified in 16. Event-free survival (EFS) and overall survival (OS) were determined by Kaplan–Meier analysis.
Fourteen patients with non-MYCN-amplified tumours were treated with surgery alone (group A) and the remaining 39 (group B) with surgery following chemotherapy that was initiated and administered at non-MSKCC institutions. Chemotherapy was discontinued after surgery in 38/39 of the latter. The 10-year EFS and OS for all patients with MYCN-non-amplified NB were 74.9 ± 16.9% and 92.6 ± 5.5%, respectively. There was no difference in OS between groups A and B (p = 0.2; 10-year OS for groups A and B was 84.6 ± 14% and 97.1 ± 2.9%, respectively). Patients with MYCN-amplified disease (group C) underwent dose-intensive induction, tumour resection and local radiotherapy: 13 achieved complete or very good partial remission, and 10 received myeloablative chemotherapy. 11/16 patients also received 3F8-based immunotherapy: 10 remain free of disease. The 10-year EFS and OS for patients with MYCN-amplified neuroblastoma treated with immunotherapy were both 90.9 ± 8.7%.
Patients with MYCN-non-amplified stage 3 NB can be successfully treated with surgery without the need for radiotherapy or continuation of chemotherapy. Combination of dose-intensive chemotherapy, surgery, radiotherapy and immunotherapy was associated with a favourable outcome for most patients with MYCN-amplified stage 3 NB.
Stage 3 neuroblastoma; Immunotherapy; Prognosis
Neuroblastoma tumour cells show complex combinations of acquired genetic aberrations, including ploidy changes, deletions of chromosome arms 1p and 11q, amplification of the MYCN oncogene, and—most frequently—gains of chromosome arm 17q. Despite intensive investigation, the fundamental role of these features in neuroblastoma initiation and progression remains to be understood. Nonetheless, great progress has been made in relating tumour genetic abnormalities to tumour behaviour and to clinical outcome; indeed, neuroblastoma provides a paradigm for the clinical importance of tumour genetic abnormalities. Knowledge of MYCN status is increasingly being used in treatment decisions for individual children, and the clinical value of 1p and 17q data as adjuncts or refinements in risk stratification is under active investigation. Reliable detection of these molecular cytogenetic features should be regarded as mandatory for all new cases at presentation.
Key Words: neuroblastoma genetics • 17q gain in neuroblastoma • neuroblastoma: 1p and MYCN • tumour genetics and prognosis