n-myc and noncoding rnas in neuroblastoma...n-myc induces mirna expression the mir-17-92 cluster the...

Review

N-myc and Noncoding RNAs in Neuroblastoma

Jochen Buechner and Christer Einvik

AbstractNeuroblastoma is a pediatric tumor of the sympathetic nervous system. Amplification and overexpression of the

MYCN proto-oncogene occurs in approximately 20% of neuroblastomas and is associated with advanced stagedisease, rapid tumor progression, and poor prognosis.MYCN encodes the transcriptional regulator N-myc, whichhas been shown to both up- and downregulate many target genes involved in cell cycle, DNA damage,differentiation, and apoptosis in neuroblastoma. During the last years, it has become clear that N-myc alsomodulates the expression of several classes of noncoding RNAs, in particular microRNAs.MicroRNAs are themostwidely studied noncoding RNA molecules in neuroblastoma. They function as negative regulators of geneexpression at the posttranscriptional level in diverse cellular processes. Aberrant regulation ofmiRNA expression hasbeen implicated in the pathogenesis of neuroblastoma. While the N-myc protein is established as an importantregulator of several miRNAs involved in neuroblastoma tumorigenesis, tumor suppressor miRNAs have also beendocumented to repress MYCN expression and inhibit cell proliferation of MYCN-amplified neuroblastoma cells.It is now becoming increasingly evident that N-myc also regulates the expression of long noncoding RNAs suchas T-UCRs and ncRAN. This review summarizes the current knowledge about the interplay between N-mycand noncoding RNAs in neuroblastoma and how this contributes to neuroblastoma tumorigenesis.Mol Cancer Res;10(10); 1243–53. �2012 AACR.

IntroductionNeuroblastoma is a malignant embryonic childhood

tumor arising from primitive cells of the neural crest. Itaccounts for more than 7% of childhood malignancies andaround 15% of cancer-related deaths in childhood (1). Thehuman proto-oncogeneMYCN is amplified in about 20%ofneuroblastoma tumors (2). MYCN amplification is closelyrelated to poor survival of the patients, despite all modernmultimodal treatment efforts. In contrast, MYCN nonam-plified, low-stage neuroblastoma, and tumors in infants,even when metastasized, have the propensity to differentiateinto benign subtypes, or regress spontaneously. Because of itsprofound effect on clinical outcome,MYCN amplification isroutinely used as a biomarker for treatment stratification.Beside MYCN amplification, hemizygous loss of large

segments on chromosome 11q defines anothermajor geneticsubtype of high-risk neuroblastoma. MYCN amplificationand 11q are inversely correlated and can be found in about70% of all metastatic tumors. Typically, both genetic sub-

types occur with additional genetic alteration. Loss of chro-mosome 1p is frequently found inMYCN-amplified tumors,whereas 11q loss is significantly associated with gain of 7qand 3p, and 4p loss (3). Gain of 17q material is frequent inboth 11q- andMYCN-amplified tumors, most often causedby unbalanced t(11q;17q) and t(1p;17q) translocations,respectively (2).The transcription factor N-myc, which is encoded by

MYCN on chromosome 2p24, belongs to the Myc family ofDNA binding basic region/helix-loop-helix/leucine zipper(bHLHZip) proteins, in which c-MYC, L-Myc, and N-mycare the best characterized members (4). The genomicsequences of MYCN and c-MYC share wide structuralhomology. Both genes consist of 3 exons, where the firstexon is untranslated and exons 2 and 3 encode the translatedregions (5). N-myc and c-MYC proteins are of similar sizes(464 and 454 amino acids, respectively). However, theMYCN mRNA is longer, mainly because of a larger 30-untranslated region (30UTR). In addition to structural andsequence homologies within the Myc family, the functionand biochemical properties of these proteins are closelyrelated. Myc-proteins heterodimerize with the bHLHZip-protein Max to a transcription factor complex that binds tospecific E-box DNA motifs (50-CANNTG-30) and activatestranscription of genes involved in diverse cellular functions,including cell growth and proliferation, metabolism, apo-ptosis and differentiation (6–9). N-myc preferentially bindsto the E-box motifs CATGTG and CAACTG. UnderMYCN-amplified conditions, however, N-myc becomes lessspecific and binds additionally to CATTTG and CATCTG

Authors' Affiliation: Department of Pediatrics, University Hospital ofNorthNorway, Tromsø, Norway

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Christer Einvik, University Hospital of NorthernNorway, Department of Pediatrics, 9038 Tromsoe, Norway. Phone: þ47776 44735; Fax: þ47 776 45350, E-mail: [email protected]: 10.1158/1541-7786.MCR-12-0244

�2012 American Association for Cancer Research.

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(10). In addition to Myc, Max also dimerizes with thebHLHZip-proteins Mad/Mnt. These complexes also bindto E-box elements, but repress transcription through therecruitment of corepressors (11). Through interaction withSp1 and Miz-1 at promoters, N-myc has been shown tosilence gene expression by recruitment of the histone dea-cetylase HDAC1 (12, 13).While c-MYC is expressed during all developmental stages

and in a distinct pattern throughout the cell cycle of dividingcells (14, 15), N-myc expression is restricted mainly to thenervous system and mesenchymal tissues during particularembryonal stages (16).Several individual N-myc downstream targets have been

identified, including p53 (17), TERT (18), ODC1 (19),MCM7 (20), and MDM2 (21). The current knowledgeand models on how these and other protein-coding N-myctargets upon deregulation may contribute to neuroblas-toma formation have recently been reviewed by others(6, 9).During the last 20 years, it has become increasingly

apparent that the expressed non–protein-coding portionof the genome, the noncoding RNA (ncRNA), plays impor-tant infrastructural and regulatory roles in many cellularprocesses. The regulatory ncRNAs are commonly groupedinto 2 classes based on practical reasons related to RNApurification protocols; small ncRNAs (i.e., miRNAs,siRNAs, piRNAs, etc. 200 nt). Dysregulation of ncRNAs hasbeen found to have relevance in several diseases, includingcancer, neurologic, cardiovascular, developmental, and otherdisorders (22, 23).MicroRNAs (or miRNAs) are the most widely studied

ncRNA and constitute an abundant class of endogenoussmall ncRNAs that negatively regulate protein expression incells (24). The biogenesis and action of miRNAs have beencomprehensively reviewed elsewhere (25, 26). The firstmiRNA, lin-4, was discovered in 1993 in the nematodeCaenorhabditis elegans (27, 28). Since that time, miRNAshave been found in nearly every organism, from plants andsimple multicellular organisms to flies, vertebrates, andhumans. MiRNAs are annotated and catalogued in thepublic-accessible database miRBase (www.mirbase.org;refs. 29–33), which was founded at the Sanger Institute inEngland and is now managed by the University of Man-chester. The current miRBase release 19 (August 2012)annotates 25,141 mature miRNAs in 193 species, including2042 unique mature human miRNAs.Expressional changes of even single miRNAs have pro-

found effects on the protein composition in cells (34, 35).The degree of complementarity between thematuremiRNAsequence and the target mRNAs determines the mechanismresponsible for blocking gene expression. Near-perfect pair-ing, as commonly seen in plants, causes mRNA destructionthrough Ago-catalyzed mRNA cleavage (36, 37). In verte-brates, miRNA-mRNA interactions are most often throughimperfect base pairing (24). Here, the precise mechanismsbehind miRNA-mediated gene silencing are still scientifi-cally debated. A current model argues that the process begins

with initiation-targeted translational repression followed bya general mRNA-destabilization scenario ultimately leadingto mRNA decay (38).As miRNAs tend to target many different mRNAs, and

each mRNA may contain several to hundreds of differentmiRNA binding sites, it is obvious that the miRNA-mRNAregulatory network is extremely complex. It has been esti-mated that 30% to 60% of all human genes are regulated bymiRNAs (39, 40); others suggest that small RNAs, includingmiRNAs, will have the potential to regulate all human genes(41). Established roles for miRNAs are their involvement inthe development of organisms and organs, in cellular pro-cesses such as proliferation, differentiation, signal transduc-tion, and apoptosis, in cell fate decisions and immunologicdefense of viral attacks (reviewed in refs. 42 and 43). As aconsequence of this broad function, miRNA biogenesis hasto be tightly controlled. Deregulated miRNA expression hasbeen associated with a diversity of diseases, including cancer;a fact attributed in the term "oncomirs" for cancer-relatedmiRNAs. MiRNA transcription is regulated by severaltranscription factors, including oncogenes such as c-MYC(44, 45) and MYCN, and tumor suppressor genes such asTP53 (46).

Studying N-myc and miRNA Expression—General AspectsBasically, 3 different approaches have been used to

study the role of N-myc on miRNA expression in neu-roblastoma: (i) comparison of miRNA expression profilesbefore and after experimental MYCN-knockdown orN-myc overexpression in MYCN-amplified or nonampli-fied neuroblastoma cell lines, respectively; (ii) comparisonof miRNA expression profiles in MYCN-amplified andnonamplified primary tumors; and (iii) analysis of directN-myc binding to miRNA promoters or promoters ofthe host genes, for example, by chromatin immunopre-cipitation (ChIP).The 2 very first studies investigating the role of N-myc on

miRNA expression in neuroblastoma tumors were publishedby Chen and Stallings (47) and Schulte and colleagues (48)almost 5 years ago. Both studies profiled the miRNAexpression in a smaller set of primary tumors (18 and 24tumors including 6 and 7 with MYCN amplification,respectively) to define differentially expressed miRNAsbetween the MYCN-amplified and nonamplified groups.Using miRNA-specific real-time RT-PCR, Chen and Stal-lings profiled 157 known miRNAs, whereas Schulte andcolleagues used a microarray approach, supplemented byreal-time RT-PCR validation, to profile 384 miRNAs inboth neuroblastoma tumors and cell lines. Both studiesfound subsets of miRNAs that were differentially expressedbetween MYCN-amplified and nonamplified tumors, indi-cating for the first time that miRNAs play a role in neuro-blastoma pathogenesis.In the following years, a rapidly growing number of

subsequent studies extended and refined the knowledge onN-myc–regulated miRNAs, taking general methodologicconsiderations into account:

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Tumor sample sizeThe heterogeneous genetic background of neuroblastoma

tumors requires large tumor sets to delineate miRNA expres-sion signatures for complex genetic subgroups. In one of thelargestmiRNAprofiling studies inneuroblastoma so far,Brayand colleagues (49) profiled 430 miRNAs in a total of 145primary neuroblastoma tumors, including 36 with MYCNamplification. They found both up- and downregulatedmiRNAs (14 and 23, respectively) when MYCN-amplifiedtumors were compared with nonamplified tumors. Impor-tantly, they also determined large-scale genomic gainsand losses in each tumor by array-comparative genomichybridization and correlated the genomic localization ofdifferentially expressed miRNAs to chromosomal gains andlosses. About 15% of all detectablemiRNAs changed expres-sion as a result of chromosomal imbalances in the tumors,highlighting that gains or losses of miRNA encodingregions contribute significantly to miRNA deregulation inneuroblastoma, in addition to N-myc overexpression.

MYCN expressionExperimental systems using MYCN induction or knock-

down do not reflect 2 sides of the same coin, but initiate 2distinct biologic processes, where the former results in cell-cycle progression and proliferation, whereas the latter indifferentiation and apoptosis.

Profiling platformsThe nature of miRNAs (small size and base-paired

structure) poses a challenge for miRNA-detection techni-ques. Different technical platforms (e.g., northern blot-ting, high-throughput RT PCR techniques, microarrayanalyses, next-generation sequencing) may, therefore, gen-erate partially diverging expression profiles, mandatingconfirmation between the platforms. Moreover, differentnormalization methods for miRNA expression data canaffect the calculation of expressional changes, which canresult in a nonuniform interpretation of differentiallyexpressed miRNAs (50).

Number of miRNAsThe number of investigated individual miRNAs varies

between studies, especially over time, not least because theoverall number of identified miRNAs (and other smallRNA molecules) in the human genome is still increasing.Profiling studies based on ultradeep next-generationsequencing of the total small RNA transcriptome inneuroblastoma (51) have the potential to provide ultra-specific and absolute miRNA expression data in futurestudies.

Functional confirmationDifferential miRNA expression data should be supported

by functional studies in vitro and in vivo to prove biologicrelevance of each individual miRNA.In the following sections of the review, functional

studies on N-myc–regulated miRNAs will be summarizedin detail.

N-myc Induces miRNA ExpressionThe mir-17-92 clusterThe mir-17-92 cluster, which is transcribed as a polycis-

tronic unit from chromosome 13, comprises 7 individualmiRNAs (mir-17, mir-18a, mir-19a, mir-19b-1, mir-20a,and mir-92a-1; ref. 52). The transcription of mir-17-92 isdirectly activated by both c-MYC (53) and N-myc (48)oncoproteins.Fontana and colleagues (54) published the first compre-

hensive functional study on theMYCN-regulatedmir-17-92cluster in neuroblastoma. They confirmed the observationmade by Schulte and colleagues (48), showing that miRNAsof the mir-17-92 cluster are higher expressed in tumors andneuroblastoma cell lines with high N-myc expression. Bythe use of ChIP, they validated direct binding of N-mycto several E-box motifs in the mir-17-92 promoter andshowed transcriptional activation in luciferase reporter geneassays. Moreover, Fontana and colleagues shed light on thefunctional consequences of mir-17-92 overexpression inMYCN-amplified neuroblastoma cells: the tumor suppres-sor p21 (CDKN1A) was shown to be targeted bymir-17, andoverexpression of mir-17 in nonamplified cells increasedproliferation, colony formation and in vivo tumor growth.Vice versa, inhibition of mir-17 by antagomirs in MYCN-amplified cells decreased proliferation and tumorigenesis,and increased p21 expression. Surprisingly, antagomir-17increased apoptosis in neuroblastoma cells; an effect notattributable to increased p21. Instead, mir-17 was found toadditionally target BIM (BCL2 interacting mediator of celldeath, or BCL2L11), a pro-apoptotic BH3-only member ofthe BCL2 (B-cell lymphoma 2) family. In conclusion,Fontana and colleagues proposed that mir-17 functions asa major effector of MYCN-mediated tumorigenesis, by tar-geting p21, whereas at the same time protecting MYCN-amplified cells from N-myc induced apoptosis throughtranslational inhibition of BIM.Other studies have confirmed direct binding of N-myc to

the mir-17-92 promoter (50, 55), as well as a positivecorrelation between expression of MYCN and members ofthe mir-17-92 cluster in primary tumors and/or neuroblas-toma cell lines (49, 51, 55–61). As miRNAs simultaneouslytarget a variety of different mRNAs, it became clear thatactivation of the mir-17-92 cluster enables N-myc to turnmultiple cellular processes toward malignant transforma-tion. Beveridge and colleagues (62) showed that mir-17 andmir-20a target 3 differentiation-associated genes in neuro-blastoma cells; BCL2, MEF2D (myocyte enhancer factor-2D) and MAP3K12. Another differentiation-associatedprotein, the estrogen receptor-a (ER-alpha), was alsoreported to be a target for miRNAs of themir-17-92 cluster.ER-alpha is expressed in fetal sympathetic ganglia duringhuman neuronal development and has been shown to beinversely correlated to MYCN expression in neuroblastomatumors (55). Loven and colleagues showed that mir-18aand 19a target ER-alpha, providing a mechanism on howN-myc regulates ER-alpha expression. Stable knockdown ofmir-18a inhibited proliferation and induced differentiationof MYCN-amplified neuroblastoma cells. Notably, Loven


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and colleagues showed that N-myc also binds to E-boxes ofthe mir-17-92 paralogous miRNA clusters mir-106b-25(chromosome 7) and mir-106a-363 (chromosome X),enabling a concerted action of N-myc–activated miRNAsto synergize N-myc functions. In a genome-wide proteomeanalysis, Mestdagh and colleagues (63) used a tetracycline-inducible mir-17-92 expression system in nonamplifiedneuroblastoma cells (SHEP-TR-mir-17-92) to show that144 proteins were downregulated upon mir-17-92 induc-tion, including multiple key effectors along the TGF-bsignaling cascade. Both TGFBR2 and Smad2/Smad4 wereshown to be direct targets of mir-17/20 and mir-18a,respectively (63). Interestingly, TGF-b responsive genesinclude CDKN1A and BCL2L11 in gastric cancer (64),both direct targets of mir-17-92 in neuroblastoma (54).Recently, our research group reported that mir-92a is

positively correlated to MYCN expression both in neuro-blastoma cell lines and tumors. Mir-92a was further shownto directly target the tumor suppressor DKK3 (Dickkopf-3)mRNA, resulting in decreased secretion of DKK3 fromneuroblastoma cell (65). This observation has been con-firmed by De Brouwer and colleagues (66).These studies illustrate how N-myc is able to regulate

multiple steps of oncogenic processes through the activationof the mir-17-92 cluster (Fig. 1).

Mir-9Another functionally characterized miRNA positively

correlated to MYCN expression is mir-9. This miRNA ishighly expressed in the brain and other neural tissues andcoordinates the proliferation andmigration of human neural

progenitor cells (67). Ma and colleagues (57) used aninducible MYCN expression system and genome-wideChIP-on-chip analyses to confirm that mir-9 (at the mir-9-3 locus) is directly activated by N-myc and that mir-9targets the tumor suppressor E-cadherin (CDH1). E-cad-herin is an ubiquitously expressed transmembrane glyco-protein on the surface of epithelial cells, with a pivotal role forcell–cell adhesion of adjacent cells. E-cadherin function isfrequently lost in epithelial cancers and associated withinvasion andmetastasis. In neural crest development, duringthe process of neurulation, downregulation of E-cadherinallows the neural crest cell to detach from the neural tube andmigrate along the migratory pathway (68). Ma and collea-gues found that mir-9 was significantly higher expressed in23metastasized neuroblastoma tumors (stage IV, allMYCN-amplified), compared with 22 nonamplified tumors withoutmetastases. They showed that mir-9 promotes motility andinvasiveness of both human mammary epithelial and breastcarcinoma cell lines through the suppression of E-cadherin.Moreover, the decrease in E-cadherin increased expression ofthe proangiogenic factor VEGFA through activated b-cate-nin signaling in the cells. The study by Ma and colleaguespropose for the first time a model on how N-myc might beable to contribute to metastasis formation through theactivation of a single microRNA (Fig. 2).

Mir-421A link between disturbed double-strand break (DSB)-

induced DNA damage response and an N-myc–activatedmiRNA has been reported by Hu and colleagues (69). Theauthors reported increased expression of mir-421 covarying

N-myc

mir-17 mir-18a mir-19a mir-19b mir-92a

mir-17-92 cluster

p21

Proliferation Inhibition of apoptosis Inhibition of differentiation

mir-20a

BIM ESR1 BCL2 DKK3MEF2 MAP3-K12TGFBR2 SMAD2/4

Figure 1. N-myc induces theexpression of the mir-17-92cluster. Several miRNAs in thecluster have been confirmed totarget genes involved inproliferation, inhibition ofapoptosis and inhibition ofdifferentiation.

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with reduced levels of ATM (ataxia-telangiectasia mutatedkinase) in MYCN-amplified neuroblastoma cell lines.With the use of a luciferase reporter assay, mir-421 wasshown to directly target the 30UTR sequence of ATM.They further showed that N-myc binds to the promoterregion of mir-421 to enhance its expression. This estab-lishes a linear signaling pathway (N-myc – mir-421 –ATM) explaining how N-myc negatively regulates ATMexpression. ATM is a tumor suppressor that transduces theDSB damage signals to downstream effectors of the DNArepair machinery during cell cycle checkpoints at G1-S andintra-S phase. Impaired ATM activity leads, most oftenthrough gene mutations, to genomic instability and pre-disposes for cancer transformation, especially in the con-text of radiation exposure (70).

In conclusion, this study reported a new mechanism forATMdysregulation related to neuroblastoma tumorigenesis.

N-myc is Predominantly a Repressor of miRNAExpressionAlthough the expression of several miRNAs have been

documented to correlate with MYCN expression, there isnow growing evidence that N-myc predominantly actsrepressive on the overall miRNA composition in MYCN-amplified neuroblastoma cells (47, 49, 59–61, 71) and uponN-myc induction in nonamplified neuroblastoma cells (55).Lin and colleagues (71) used real-timeRT-PCR to profile theexpression of 162 miRNAs in 66 primary neuroblastomatumors (including 13 withMYCN-amplification) and founda nearly global downregulation of miRNAs in high-risktumors, especially in those with MYCN amplification. Theauthors hypothesized that dysregulation in Dicer and/orDrosha, key enzymes in the miRNA-processing pathway,may contribute to the widespread miRNA downregulation.Indeed, bothDicer andDrosha were lower expressed in stage4 tumors compared with other stages, with the most strik-ingly differential expression between stage IV and stage IVS.This suggests that repression of miRNAs may be involved intumor progression.

N-myc–Regulated Tumor Suppressor miRNAs inNeuroblastomaIn the study by Chen and Stallings (47), mir-184 was

significantly downregulated in MYCN-amplified tumorsand upregulated upon siRNA-mediated MYCN-knock-down in aMYCN-amplified neuroblastoma cell line. Over-expression of mir-184 reduced cell viability of bothMYCN-amplified and nonamplified cell lines through the inductionof apoptosis and G1 cell-cycle arrest. A follow-up study byFoley and colleagues (72) confirmed the inverse correlationbetween N-myc and mir-184 expression in primary tumorsand showed that inhibition of mir-184 by antagomir treat-ment increased proliferation of neuroblastoma cells. More-over, they showed that mir-184 directly targets AKT2(protein kinase B beta). AKT2 is a downstream effector ofthe phosphatidylinositol 3-kinase (PI3K) pathway, one ofthe most potent prosurvival pathways in cancer. Activationof AKT is associated with poor prognosis in neuroblastoma(73). Finally, Tivnan and colleagues (74) used an in vivomurine xenograft model where mir-184–transfectedMYCN-amplified or MYCN nonamplified neuroblastomacells were orthotopically injected into CB-17/SCID mice.Tumors arising from mir-184–transfected cells were smallerthan the controls, and mice survived longer. In summary,these comprehensive studies clearly established mir-184 as atumor suppressor in neuroblastoma.Another tumor suppressor miRNA repressed by N-myc is

mir-542-5p. Several studies have shown an inverse correla-tion between mir-542-5p and MYCN amplification inprimary tumors (49, 51, 60, 75). In a large-scale profilingstudy of 430 miRNAs in 69 primary tumors, Schulte andcolleagues (60) found increased expression of 4 miRNAs in

N-myc

mir-9mir-9-3 locus

E-cad

Angiogenesis

Metastasis

β-cat

VEGF

Figure 2. Model for an N-myc–mir-9–E-cadherin pathway involved inmetastasis formation. N-myc activates mir-9, which suppresses theexpression of E-cadherin and promotes cell motility and invasiveness. Inaddition,b-catenin signaling is activated, leading to increasedexpressionof VEGF and induction of angiogenesis.






MYCN-amplified tumors, whereas 35 miRNAs wererepressed, including mir-542-5p. Mir-542-5p expressionwas found to be predictive for outcome, with a significantlyhigher expression in patients with event-free survivalcompared with relapsed patients. Bray and colleagues (75)profiled the expression of 449 miRNAs in 145 neuroblas-toma tumors and correlated mir-542-5p expression toclinical data. Expression of mir-542-5p was nonrandomlydistributed among tumor genetic subtypes, with lowestexpression in MYCN-amplified tumors (77% completelylacking expression) and highest expression in stage 1,2,3 and4S tumors (24% lacking mir-542-5p). Patients with tumorslacking mir-542-5p expression had the poorest prognosisindependently of the MYCN status in the tumors (60, 75).Bray and colleagues further showed that mir-542-5p over-expression in MYCN-amplified and nonamplified neuro-blastoma cells reduced invasiveness in vitro, and restrictedtumor growth and metastasis in vivo when cells were ortho-topically injected into CB-17/SCID mice.

Genome and Small RNA Transcriptome – WideAnalyses of N-myc – Regulated miRNAExpressionTwo studies have recently used deep sequencing

approaches to analyzeMYCN-regulated miRNA expressionin neuroblastoma (51, 61). Schulte and colleagues (51) usedthe SOLiD v3 sequencing platform to compare the totalsmall RNA transcriptome in 5 unfavorable MYCN-ampli-fied tumors with 5 favorable non-amplified tumors. Ana-lyzing the absolute number of miRNA reads, there was atrend toward a higher proportion of mature miRNAs in thefavorable patient group, indicating a possible global sup-pression of miRNA transcription in MYCN-amplifiedtumors. Expression data of 204 miRNAs were validated byreal-time RT-PCR with good correlation between the tech-nical platforms. The SOLiD sequencing data confirmedpreviously data on differential expression in MYCN-ampli-fied versus non-amplified tumors, including the mir-17-92cluster and mir-181 (positive N-myc-correlation) and mir-542-5p (nearly absent inMYCN-amplified tumors). In total,76 miRNAs were differentially expressed between MYCN-amplified and non-amplified tumors (43 upregulated and 33downregulated). This study also allowed the discovery ofseveral newmiRNAs in neuroblastoma and revealed valuableinsight into miRNA editing and distribution of mir-5p/-3pand mir� forms. Cluster analysis was able to exactly separatethe 2 clinical outcome groups based on their differentialmiRNA expression, indicating that the miRNA transcrip-tome reflects tumor aggressiveness (51).Very recently, Shohet and colleagues (61) conducted a

genome-wide search for N-myc binding sites in promotersdriving miRNA expression in neuroblastoma. Using a com-bination of ChIP and the Illumina GA-I sequencing plat-form (ChIP-seq) in a neuroblastoma cell line with inducibleN-myc expression, they identified 20 gene promoters, host-ing a total of 30 miRNAs, to which N-myc specificallybound to E-boxmotifs. The majority of host genes that were

correlated with survival were downregulated by high N-myclevels, suggesting a tumor suppressor function for these hostgenes as well as the coexpressed miRNAs. However, func-tional in vitro and in vivo studies of 2 MYCN-regulatedintronic miRNAs (mir-591 and mir-558) identified tumorsuppressor functions for mir-591 as expected, whereas mir-558 was reported to function as an oncomir.These seemingly contrasting results probably highlight the

complexity of oncogenes and downstream miRNAs inaggressive cancers. Indeed, it is well-established that deregu-latedMYC proteins on one hand exhibit oncogenic activitiesthat contribute to cancer development, but on the otherhand also activate antitumorigenic responses, for example,through activation of p53 (8).

C-MYC/N-myc – Induced miRNAs Repress GeneNetworksIn a large-scale miRNA expression study, Mestdagh and

colleagues. (59) profiled the expression of 430miRNAs in 95neuroblastoma tumors and delineated a signature of 50unique miRNAs differentially expressed between MYCN-amplified and MYCN single-copy tumors (16 upregulatedand 34 downregulated miRNAs). Interestingly, the miRNAsignature further delineated 2 distinct tumor subgroupswithin the MYCN single-copy group: tumors with high orlow c-MYC expression. The 3 tumor groups defined by the50-miRNA signatures correlated well with the clinical stageand prognosis. Mestdagh and colleagues concluded thatMYCN/c-MYC signaling rather than MYCN amplificationalone underlies the differential expression of miRNAs inneuroblastoma. To identify mRNA targets downstream ofthe MYCN/c-MYC–regulated miRNAs, they integratedmRNA and miRNA expression data sets from 40 neuro-blastoma tumors and calculated correlations between each ofthe 50miRNAs and around 15,000mRNAs. In the group ofmRNAswith inversemiRNA correlation, significant 30UTRseed enrichment was only found for the 16N-myc–activatedmiRNAs, indicating that these miRNAs have a widespreadeffect on differential gene expression in high-risk neuroblas-toma. One-third of the mRNAs were predicted targets of 2or more MYCN/c-MYC–activated miRNAs, indicating aconcerted action toward target gene suppression. Lowexpression of predicted mRNA targets in the tumors corre-lated with a particular poor patient prognosis. MYCN/c-MYC—activated miRNAs were predicted to repress severalpathways known to be involved in neuroblastoma, includingintegrin signaling. In summary, the study by Mestdagh andcolleagues comprehensively showed widespread transcrip-tional repression of coding genes by MYCN/c-MYCthrough miRNA induction, serving as an additional mech-anism of MYCN/c-MYC–induced oncogenicity.

MYCN-Driven miRNA Expression in a MurineTransgenic Neuroblastoma ModelThe TH-MYCN mouse is a widely used transgenic neu-

roblastomamodel inwhich tumorigenesis is driven by neuralcrest-specific expression of the human MYCN transgene

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(reviewed in ref. 76). TH-MYCN mice develop aggressiveneuroblastoma tumors that recapitulate many histologic andpathologic features of high-risk MYCN-amplified humantumors. Terrile and colleagues (77) conducted a miRNAexpression study on 22 TH-MYCN neuroblastoma tumorsand compared their expression profile with 12 normalmurine adrenal glands. Of 440 expressed miRNAs, 159miRNAs were found differentially expressed, with 81 and 78miRNAs being over- and underexpressed in tumors, respec-tively. MiRNA expression in tumors with wild-type p53(TH-MYCN) or p53 haploinsufficiency (TH-MYCN/p53ERTAM mice) did not substantially differ. Furthermore,29 of 63miRNAs (49%) that are conserved between humansand mice and shown to be differentially expressed in humanMYCN-amplified versus nonamplified tumors in 2 previousstudies (49, 59), were also differentially expressed betweenmouse tumors and normal adrenals. All but 7 miRNAs werechanged in the same direction when high-N-myc and low-N-myc states were compared in both species. Principalcomponent analysis (PCA) suggested that N-myc regulatessimilar miRNAs in human and mice, including miRNAswith established functions in neuroblastoma, like membersof the mir-17-92 cluster (54, 63), mir-9 (57), and mir-152(78). The miRNA profiles across murine tumors were morehomogeneous than in human tumors, which might reflectthe inbred genetic environment in mouse versus humantumors (77). In conclusion, TH-MYCN tumors recapitulatea pattern ofMYCN-dependent miRNA expression similar tothat in human MYCN-amplified tumors. TH-MYCNproves, therefore, to be a useful mouse model for studyingmiRNA expression in MYCN-amplified neuroblastoma.

N-myc Alters Expression of Other NoncodingRNAsLong noncoding RNAs (lncRNAs) belong to a diverse

group of regulatory ncRNAs. LncRNAs are commonlydefined as 200–100,000 nt long mRNA-like transcriptslacking significant open reading frames (79). The functionsof lncRNAs are far from understood, but it is clear that thispoorly conserved group of ncRNAs is involved in diversemechanisms for widespread regulation of chromatin mod-ifications and gene expression (80).Compared with the 2042 known unique mature human

miRNAs (miRBase v19), the number of lncRNAs has beenestimated to be at least as numerous as mRNAs (81).LncRNAs have been shown to be aberrantly expressed inseveral human diseases, including cancers (79, 82).Transcribed ultraconserved regions (T-UCR) are 200-779

bp lncRNAs expressed from 481 genomic regions that are100% conserved between human, rat, and mouse (83). T-UCRs are widely expressed in neuroblastoma, and a signa-ture based on the expression of 28 T-UCRs has been shownto be associated with good outcome in noninfant patientswith metastatic disease (84). Furthermore, Mestdagh andcolleagues (85) investigated the global T-UCR expression inneuroblastoma and found a signature of 7 T-UCRs signif-icantly upregulated in MYCN-amplified tumors (n ¼ 18)

compared with nonamplified tumors (n¼ 31). Of these 7, 3(uc.350, uc.379, and uc.460) were also upregulated whenN-myc expression was induced in the SHEP-MYCN-ER cellline, supporting that these T-UCRs are N-myc responsive.These data indicate that differential T-UCR expressioncorrelates with clinicogenetic parameters such as MYCNamplification (85).The most frequent genetic abnormality in neuroblasto-

mas is chromosome 17q gain. Gain of 17q has been shownto be linked to advanced stage disease and amplification ofthe MYCN oncogene (86, 87). Several candidate genes of"'17q gain" have been proposed, including a lncRNA calledncRAN (noncoding RNA expressed in aggressive neuro-blastomas). ncRAN consists of 2 transcripts, Nbla10727(2186 nt) and Nbla12061 (2087 nt), which are splicevariants of the same gene mapped to chromosome region17q25.1. Both transcripts are upregulated in advancedneuroblastomas with 17q gain. High or moderate levelsof ncRAN expression were also observed in neuroblastomacell lines with MYCN amplification, most of which had17q gain. Furthermore, suppression of ncRAN expressionsignificantly inhibited cell growth in a neuroblastoma cellline and overexpression of ncRAN in mouse fibroblastsenhanced anchorage-independent growth (88). Recently,expression of ncRAN was found to be upregulated inbladder cancers compared with normal tissue. Overexpres-sion of ncRAN in a superficial low-ncRAN expressingbladder cancer cell line enhanced cell proliferation andinvasion, whereas shRNA knockdown of ncRAN in a high-ncRAN expressing invasive bladder cancer cell line sensi-tized the cells to chemotherapeutic drugs (89). Accordingto these data, ncRAN appears to have oncogenic propertiesin several cancers.Despite its relative small size (�130 nt), NDM29 (Neu-

roblastoma Differentiation Marker 29) is classified as alncRNA. This Alu-like RNA is transcribed by RNA poly-merase III from the first intron of theASCL3 (Achaete Scute-Like homologue 3) gene. Stable overexpression of NDM29in a MYCN-amplified neuroblastoma cell line inducedneuronal differentiation, reduced the proliferation rate,reduced c-kit expression, induced anchorage-dependentgrowth, and decreased anticancer drug resistance throughMDR1 (multidrug resistance 1) downregulation (90).Although the status of N-myc expression was not reportedin these experiments, it is interesting to notice thatNDM29overexpression coincides the effects ofMYCN knockdown inMYCN-amplified neuroblastoma cells (91–93).DEIN (differentially expressed in neuroblastoma) is a

putative lncRNA expressed as 5 isoforms. Isoform A(4186 bp) and isoformB (5278 bp) are the 2major transcriptvariants of DEIN in primary neuroblastomas. DEIN hasbeen shown to be differentially expressed in neuroblastomasubtypes, with higher expression in tumors with goodprognosis and spontaneous regression (children

bidirectional promoter. This suggests a role for DEIN insuppression of cell proliferation and promotion of differen-tiation (95).

N-myc Expression is Regulated by miRNAsFinally, the interaction between N-myc and miRNAs is

mutual, as MYCN itself is targeted by miRNAs. The func-tionally best-characterizedMYCN-targeting miRNA is mir-34a (96, 97), which is located at chromosome 1p36, a regionfrequently deleted in MYCN-amplified neuroblastomatumors (1). Overexpression of mir-34a inMYCN-amplifiedneuroblastoma cell lines decreased N-myc levels, inhibitedproliferation, and induced apoptosis. Interestingly, mir-34ais transcriptionally activated by p53 (46), implicating thatdeletion of mir-34a has similar cellular consequences as p53deficiency (97). In addition to MYCN, mir-34a targetsBCL2 (96) and E2F3 (98), making mir-34a a multifacetedtumor suppressor miRNA in neuroblastoma. Recently, ourresearch group systematically investigated the MYCN30UTR for miRNA binding sites (99). In addition to mir-34a, we validated mir-34c, mir-449, mir-19, mir-29, mir-101, and let-7/mir-202 as MYCN-targeting miRNAs(Fig. 3). Overexpression of mir-101 and let-7e in MYCN-amplified neuroblastoma cells diminished N-myc levels, andreduced proliferation and clonogenic growth. While mir-101 is generally sparsely expressed in neuroblastoma and hasestablished tumor-suppressor functions in other cancers[(100) and references therein], let-7 family members areupregulated during neuroblastoma cell differentiation(47, 56, 78, 101–103), suggesting tumor suppressor func-tions for these miRNAs in neuroblastoma.

SummaryThe proto-oncogenic transcription factor N-myc is

emerging as an important regulator of non–protein-cod-ing gene expression in neuroblastoma. Most studies havefocused on the interplay between MYCN expression and agroup of small regulatory noncoding RNAs, the micro-RNAs (miRNAs). From several studies published duringthe last 5 years, it is evident that N-myc both activates andrepresses the expression of several miRNAs (summarizedin Supplementary Tables S1 and S2), many of themknown to have important roles in cancer progression.MiRNA induction by N-myc (and also c-MYC) is asso-ciated with a widespread repression of coding genes anddisease-relevant pathways in neuroblastoma cells, suggest-ing that miRNA-controlled regulation of certain groups of

mRNAs may function as an additional mechanism ofMYCN/c-MYC-induced oncogenicity. Indeed, amongthe targets of MYCN-responsive miRNAs (mir-17-92cluster, mir-9 and mir-421) are mRNAs of tumor sup-pressors, and genes involved in the metastatic process. Onthe other hand, miRNAs with tumor suppressor functions(mir-184 and mir-542-5p) have been shown to be inverse-ly correlated to MYCN expression. In fact, miRNArepression seems to be the predominant consequence ofMYCN activation, as high MYCN expression is identifiedwith a global repression of miRNAs, possibly throughimpaired Drosha/Dicer expression. Vice versa, some miR-NAs (mir-34a, mir-101, and let-7) have been documentedto target the MYCN mRNA, resulting in reduced N-mycexpression and decreased proliferation of MYCN-ampli-fied neuroblastoma cells.Long noncoding RNAs (lncRNAs) have recently been

implicated in playing important regulatory functions inseveral diseases, including cancers. It is now becoming clearthat N-myc levels also affect the expression of lncRNAs(such as T-UCRs and ncRAN) in neuroblastoma. Under-standing the mechanisms by which N-myc alters theexpression of the diverse class of regulatory lncRNAs, andthrough which targets and pathways deregulated lncRNAsmay subsequently contribute to oncogenic transformationin aggressive neuroblastoma, will possibly explain missingmolecular links in the pathogenesis of this disease, andfurther elucidate how oncogenic N-myc signaling is exe-cuted. A distinct role for noncoding RNAs in neuroblas-toma is further supported by the fact that several noncodingRNAs with functional roles were initially identified becauseof their genomic location in areas of frequent losses or gainsin advanced disease stages.In conclusion, further understanding of the effect of N-

myc on the expression of non–protein-coding genes in thegenome will help to understand the pathogenesis of aggres-sive neuroblastomas, and provides a basis for novel targetedtherapies for neuroblastoma treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Buechner, C. EinvikWriting, review, and/or revision of the manuscript: J. Buechner, C. EinvikAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J. Buechner, C. Einvik

Received April 17, 2012; revised July 10, 2012; accepted August 20, 2012;published OnlineFirst August 30, 2012.

100 200 300 400 500 600 700 800 900

mir-19a/b mir-29a/b/c mir-101 mir-34a/c/mir-449 let-7e/mir-202

MYCN 3’UTR

Figure 3. Schematic overview ofmiRNAs confirmed to target the30UTR of MYCN.

Buechner and Einvik





References1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet

2007;369:2106–20.2. Deyell RJ, Attiyeh EF. Advances in the understanding of constitu-

tional and somatic genomic alterations in neuroblastoma. CancerGenet 2011;204:113–21.

3. Buckley PG, Alcock L, Bryan K, Bray I, Schulte JH, Schramm A, et al.Chromosomal andmicroRNA expression patterns reveal biologicallydistinct subgroups of 11q- neuroblastoma. Clin Cancer Res2010;16:2971–8.

4. Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer2008;8:976–90.

5. Kohl NE, Legouy E, DePinho RA, Nisen PD, Smith RK, Gee CE, et al.Human N-myc is closely related in organization and nucleotidesequence to c-myc. Nature 1986;319:73–7.

6. Bell E, Chen L, Liu T, Marshall GM, Lunec J, Tweddle DA. MYCNoncoprotein targets and their therapeutic potential. Cancer Lett2010;293:144–57.

7. Eilers M, Eisenman RN. Myc's broad reach. Genes Dev 2008;22:2755–66.

8. Larsson LG, Henriksson MA. The Yin and Yang functions of the Myconcoprotein in cancer development and as targets for therapy. ExpCell Res 2010;316:1429–37.

9. Westermark UK, Wilhelm M, Frenzel A, Henriksson MA. The MYCNoncogene and differentiation in neuroblastoma. Semin Cancer Biol2011;21:256–66.

10. Murphy DM, Buckley PG, Bryan K, Das S, Alcock L, Foley NH, et al.Global MYCN transcription factor binding analysis in neuroblastomareveals association with distinct E-box motifs and regions of DNAhypermethylation. PLoS ONE 2009;4:e8154.

11. Nilsson JA, Cleveland JL. Myc pathways provoking cell suicide andcancer. Oncogene 2003;22:9007–21.

12. Iraci N, Diolaiti D, Papa A, Porro A, Valli E, Gherardi S, et al. A SP1/MIZ1/MYCN repression complex recruits HDAC1 at the TRKA andp75NTR promoters and affects neuroblastoma malignancy by inhi-biting the cell response to NGF. Cancer Res 2011;71:404–12.

13. Liu T, Tee AE, Porro A, Smith SA, Dwarte T, Liu PY, et al. Activation oftissue transglutaminase transcription by histone deacetylase inhibi-tion as a therapeutic approach for Myc oncogenesis. Proc Natl AcadSci U S A 2007;104:18682–7.

14. Hooker CW, Hurlin PJ. Of Myc and Mnt. J Cell Sci 2006;119:208–16.15. Rabbitts PH, Watson JV, Lamond A, Forster A, Stinson MA, Evan G,

et al. Metabolism of c-myc gene products: c-myc mRNA and proteinexpression in the cell cycle. EMBO J 1985;4:2009–15.

16. Stanton BR, Perkins AS, Tessarollo L, Sassoon DA, Parada LF. Lossof N-myc function results in embryonic lethality and failure of theepithelial component of the embryo to develop. Genes Dev1992;6:2235–47.

17. Chen L, Iraci N, Gherardi S, Gamble LD,WoodKM, Perini G, et al. p53is a direct transcriptional target of MYCN in neuroblastoma. CancerRes 2010;70:1377–88.

18. Mac SM, D'Cunha CA, Farnham PJ. Direct recruitment of N-myc totarget gene promoters. Mol Carcinog 2000;29:76–86.

19. Lutz W, Stohr M, Schurmann J, Wenzel A, Lohr A, Schwab M.Conditional expression of N-myc in human neuroblastoma cellsincreases expression of alpha-prothymosin and ornithine decar-boxylase and accelerates progression into S-phase early aftermitogenic stimulation of quiescent cells. Oncogene 1996;13:803–12.

20. Shohet JM, Hicks MJ, Plon SE, Burlingame SM, Stuart S, Chen SY,et al. Minichromosome maintenance protein MCM7 is a direct targetof the MYCN transcription factor in neuroblastoma. Cancer Res2002;62:1123–8.

21. Slack A, Chen Z, Tonelli R, Pule M, Hunt L, Pession A, et al. The p53regulatory gene MDM2 is a direct transcriptional target of MYCN inneuroblastoma. Proc Natl Acad Sci U S A 2005;102:731–6.

22. Esteller M. Non-coding RNAs in human disease. Nat Rev Genet2011;12:861–74.

23. Taft RJ, Pang KC, Mercer TR, Dinger M, Mattick JS. Non-codingRNAs: regulators of disease. J Pathol 2010;220:126–39.

24. Bartel DP. MicroRNAs: target recognition and regulatory functions.Cell 2009;136:215–33.

25. Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. NatRev Mol Cell Biol 2009;10:126–39.

26. Krol J, Loedige I, Filipowicz W. The widespread regulation ofmicroRNA biogenesis, function and decay. Nat Rev Genet 2010;11:597–610.

27. Lee RC, FeinbaumRL, Ambros V. The C. elegans heterochronic genelin-4 encodes small RNAs with antisense complementarity to lin-14.Cell 1993;75:843–54.

28. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of theheterochronic gene lin-14 by lin-4 mediates temporal pattern forma-tion in C. elegans. Cell 1993;75:855–62.

29. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res2004;32:D109–11.

30. Griffiths-Jones S. miRBase: microRNA sequences and annotation.Curr Protoc Bioinformatics 2010;9:1–10.

31. Griffiths-JonesS,GrocockRJ, vanDongenS, BatemanA, Enright AJ.miRBase: microRNA sequences, targets and gene nomenclature.Nucleic Acids Res 2006;34:D140–4.

32. Griffiths-JonesS,Saini HK, vanDongenS, Enright AJ.miRBase: toolsfor microRNA genomics. Nucleic Acids Res 2008;36:D154–8.

33. Kozomara A, Griffiths-Jones S. miRBase: integrating microRNAannotation and deep-sequencing data. Nucleic Acids Res 2011;39:D152–7.

34. BaekD, Villen J, Shin C, Camargo FD,Gygi SP, Bartel DP. The impactof microRNAs on protein output. Nature 2008;455:64–71.

35. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R,Rajewsky N. Widespread changes in protein synthesis induced bymicroRNAs. Nature 2008;455:58–63.

36. Jones-Rhoades MW, Bartel DP, Bartel B. MicroRNAS and theirregulatory roles in plants. Annu Rev Plant Biol 2006;57:19–53.

37. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ,et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science2004;305:1437–41.

38. Djuranovic S, Nahvi A, Green R. A parsimonious model for generegulation by miRNAs. Science 2011;331:550–3.

39. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, oftenflanked by adenosines, indicates that thousands of human genesare microRNA targets. Cell 2005;120:15–20.

40. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalianmRNAs are conserved targets of microRNAs. Genome Res 2009;19:92–105.

41. Siomi H, Siomi MC. On the road to reading the RNA-interferencecode. Nature 2009;457:396–404.

42. Huang Y, Shen XJ, Zou Q, Wang SP, Tang SM, Zhang GZ. Biologicalfunctions of microRNAs: a review. J Physiol Biochem 2011;67:129–39.

43. Guarnieri DJ, DiLeone RJ. MicroRNAs: a new class of gene regula-tors. Ann Med 2008;40:197–208.

44. Chang TC, Yu D, Lee YS, Wentzel EA, Arking DE, West KM, et al.Widespread microRNA repression by Myc contributes to tumorigen-esis. Nat Genet 2008;40:43–50.

45. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Good-son S, et al. A microRNA polycistron as a potential human oncogene.Nature 2005;435:828–33.

46. Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M,Lee KH, et al. Transactivation of miR-34a by p53 broadly influencesgene expression and promotes apoptosis. Mol Cell 2007;26:745–52.

47. Chen Y, Stallings RL. Differential patterns of microRNA expression inneuroblastoma are correlated with prognosis, differentiation, andapoptosis. Cancer Res 2007;67:976–83.

48. Schulte JH, Horn S, Otto T, Samans B, Heukamp LC, Eilers UC, et al.MYCN regulates oncogenic MicroRNAs in neuroblastoma. Int JCancer 2008;122:699–704.

49. Bray I, Bryan K, Prenter S, Buckley PG, Foley NH, Murphy DM, et al.Widespread dysregulation of MiRNAs by MYCN amplification andchromosomal imbalances in neuroblastoma: association of miRNAexpression with survival. PLoS ONE 2009;4:e7850.






50. Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F,Speleman F, et al. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 2009;10:R64.

51. Schulte JH,Marschall T,MartinM,Rosenstiel P,MestdaghP, SchlierfS, et al. Deep sequencing reveals differential expression of micro-RNAs in favorable versus unfavorable neuroblastoma. Nucleic AcidsRes 2010;38:5919–28.

52. Olive V, Jiang I, He L. mir-17-92, a cluster of miRNAs in the midst ofthe cancer network. Intl J Biochem Cell Biol 2010;42:1348–54.

53. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005;435:839–43.

54. Fontana L, Fiori ME, Albini S, Cifaldi L, Giovinazzi S, Forloni M, et al.Antagomir-17-5p abolishes the growth of therapy-resistant neuro-blastoma through p21 and BIM. PLoS ONE 2008;3:e2236.

55. Loven J, Zinin N, Wahlstrom T, Muller I, Brodin P, Fredlund E, et al.MYCN-regulated microRNAs repress estrogen receptor-alpha(ESR1) expression and neuronal differentiation in human neuroblas-toma. Proc Natl Acad Sci U S A 2010;107:1553–8.

56. Buechner J, Henriksen JR, Haug BH, Tomte E, Flaegstad T, Einvik C.Inhibition ofmir-21, which is up-regulated duringMYCN knockdown-mediated differentiation, does not prevent differentiation of neuro-blastoma cells. Differentiation 2011;81:25–34.

57. MaL,Young J, PrabhalaH,PanE,MestdaghP,MuthD, et al.miR-9, aMYC/MYCN-activated microRNA, regulates E-cadherin and cancermetastasis. Nat Cell Biol 2010;12:247–56.

58. Mestdagh P, Feys T, Bernard N, Guenther S, Chen C, Speleman F,et al. High-throughput stem-loop RT-qPCR miRNA expression pro-filing using minute amounts of input RNA. Nucleic Acids Res 2008;36:e143.

59. Mestdagh P, Fredlund E, Pattyn F, Schulte JH, Muth D, Vermeulen J,et al. MYCN/c-MYC-induced microRNAs repress coding gene net-works associated with poor outcome in MYCN/c-MYC-activatedtumors. Oncogene 2009;29:1394–404.

60. Schulte JH, SchoweB,MestdaghP, Kaderali L, Kalaghatgi P, SchlierfS, et al. Accurate prediction of neuroblastoma outcome based onmiRNA expression profiles. Int J Cancer 2010;127:2374–85.

61. Shohet JM, Ghosh R, Coarfa C, Ludwig A, Benham AL, Chen Z, et al.A Genome-wide Search for MYCN binding sites reveals both newoncogenic and tumor suppressor MicroRNAs associated withaggressive neuroblastoma. Cancer Res 2011;71:1–11.

62. Beveridge NJ, Tooney PA, Carroll AP, Tran N, Cairns MJ. Down-regulation of miR-17 family expression in response to retinoic acidinduced neuronal differentiation. Cell Signal 2009;21:1837–45.

63. Mestdagh P, Bostrom AK, Impens F, Fredlund E, Van Peer G, DeAntonellis P, et al. The miR-17-92 microRNA cluster regulates mul-tiple components of the TGF-beta pathway in neuroblastoma. MolCell 2010;40:762–73.

64. PetroccaF, VisoneR,OnelliMR,ShahMH,NicolosoMS,deMartino I,et al. E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 2008;13:272–86.

65. Haug BH, Henriksen JR, Buechner J, Geerts D, Tomte E, Kogner P,et al. MYCN-regulated miRNA-92 inhibits secretion of the tumorsuppressor DICKKOPF-3 (DKK3) in neuroblastoma. Carcinogenesis2011;32:1005–12.

66. De Brouwer S, Mestdagh P, Lambertz I, Pattyn F, De Paepe A,Westermann F, et al. Dickkopf-3 is regulated by the MYCN-induced miR-17-92 cluster in neuroblastoma. Int J Cancer2012;130:2591–8.

67. Delaloy C, Liu L, Lee JA, Su H, Shen F, Yang GY, et al. MicroRNA-9coordinates proliferation and migration of human embryonic stemcell-derived neural progenitors. Cell Stem Cell 2010;6:323–35.

68. Taneyhill LA. To adhere or not to adhere: the role of Cadherins inneural crest development. Cell Adh Migr 2008;2:223–30.

69. Hu H, Du L, Nagabayashi G, Seeger RC, Gatti RA. ATM is down-regulated by N-Myc-regulated microRNA-421. Proc Natl Acad SciUSA 2010;107:1506–11.

70. Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigmfor cell signalling and cancer. Nat Rev Mol Cell Biol 2008;9:759–69.

71. Lin RJ, Lin YC, Chen J, Kuo HH, Chen YY, Diccianni MB, et al.microRNA signature and expression of Dicer and Drosha can predictprognosis and delineate risk groups in neuroblastoma. Cancer Res2010;70:7841–50.

72. Foley NH, Bray IM, Tivnan A, Bryan K, Murphy DM, Buckley PG, et al.MicroRNA-184 inhibits neuroblastoma cell survival through targetingthe serine/threonine kinase AKT2. Mol Cancer 2010;9:83.

73. Opel D, Poremba C, Simon T, Debatin KM, Fulda S. Activation of Aktpredicts poor outcome in neuroblastoma. Cancer Res 2007;67:735–45.

74. Tivnan A, Foley NH, Tracey L, Davidoff AM, Stallings RL. MicroRNA-184-mediated inhibition of tumour growth in an orthotopic murinemodel of neuroblastoma. Anticancer Res 2010;30:4391–5.

75. Bray I, Tivnan A, Bryan K, Foley NH, Watters KM, Tracey L, et al.MicroRNA-542-5p as a novel tumor suppressor in neuroblastoma.Cancer Lett 2011;303:56–64.

76. Chesler L, Weiss WA. Genetically engineered murine models–con-tribution to our understanding of the genetics, molecular pathologyand therapeutic targeting of neuroblastoma. Semin Cancer Biol2011;21:245–55.

77. Terrile M, Bryan K, Vaughan L, Hallsworth A, Webber H, Chesler L,et al. miRNA expression profiling of the murine TH-MYCN neuro-blastomamodel reveals similarities with human tumors and identifiesnovel candidate miRNAs. PLoS ONE 2011;6:e28356.

78. Das S, Foley N, Bryan K, Watters KM, Bray I, Murphy DM, et al.MicroRNA mediates DNA demethylation events triggered by retinoicacid during neuroblastoma cell differentiation. Cancer Res 2010;70:7874–81.

79. Wapinski O, Chang HY. Long noncoding RNAs and human disease.Trends Cell Biol 2011;21:354–61.

80. Wang KC, Chang HY. Molecular mechanisms of long noncodingRNAs. Mol Cell 2011;43:904–14.

81. Clark MB, Mattick JS. Long noncoding RNAs in cell biology. SeminCell Dev Biol 2011;22:366–76.

82. GibbEA, VucicEA, EnfieldKS,StewartGL, LonerganKM,Kennett JY,et al. Human cancer long non-coding RNA transcriptomes. PLoSONE 2011;6:e25915.

83. Scaruffi P. The transcribed-ultraconserved regions: a novel class oflong noncoding RNAs involved in cancer susceptibility. Scientific-WorldJournal 2011;11:340–52.

84. ScaruffiP, Stigliani S,Moretti S, CocoS, De Vecchi C, Valdora F, et al.Transcribed-Ultra Conserved Region expression is associated withoutcome in high-risk neuroblastoma. BMC Cancer 2009;9:441.

85. Mestdagh P, Fredlund E, Pattyn F, Rihani A, Van Maerken T,Vermeulen J, et al. An integrative genomics screen uncovers ncRNAT-UCR functions in neuroblastoma tumours. Oncogene 2010;29:3583–92.

86. Bown N, Cotterill S, Lastowska M, O'Neill S, Pearson AD, Plantaz D,et al. Gain of chromosome arm 17q and adverse outcome in patientswith neuroblastoma. N Engl J Med 1999;340:1954–61.

87. Spitz R, Hero B, Ernestus K, Berthold F. Gain of distal chromosomearm 17q is not associatedwith poor prognosis in neuroblastoma.ClinCancer Res 2003;9:4835–40.

88. YuM,OhiraM, LiY,NiizumaH,OoML, ZhuY, et al.High expressionofncRAN, a novel non-coding RNA mapped to chromosome 17q25.1,is associated with poor prognosis in neuroblastoma. Int J Oncol2009;34:931–8.

89. Zhu Y, Yu M, Li Z, Kong C, Bi J, Li J, et al. ncRAN, a newly identifiedlong noncoding RNA, enhances human bladder tumor growth, inva-sion, and survival. Urology 2011;77:510 e1-5.

90. Castelnuovo M, Massone S, Tasso R, Fiorino G, Gatti M, Robello M,et al. An Alu-like RNA promotes cell differentiation and reducesmalignancy of human neuroblastoma cells. FASEB J 2010;24:4033–46.

91. Vitali R, Cesi V, Nicotra MR, McDowell HP, Donfrancesco A, Man-narino O, et al. c-Kit is preferentially expressed in MYCN-amplifiedneuroblastoma and its effect on cell proliferation is inhibited in vitrobySTI-571. Intl J Cancer J intl Du Cancer 2003;106:147–52.

92. Henriksen JR, Haug BH, Buechner J, Tomte E, Lokke C, FlaegstadT, et al. Conditional expression of retrovirally delivered anti-MYCN

Buechner and Einvik





shRNA as an in vitro model system to study neuronal differenti-ation in MYCN-amplified neuroblastoma. BMC Dev Biol 2011;11:1.

93. Blanc E, Goldschneider D, Ferrandis E, BarroisM, LeRouxG, LeonceS, et al. MYCN enhances P-gp/MDR1 gene expression in the humanmetastatic neuroblastoma IGR-N-91 model. Am J Pathol 2003;163:321–31.

94. Voth H, Oberthuer A, Simon T, Kahlert Y, Berthold F, Fischer M.Identification of DEIN, a novel gene with high expression levels instage IVS neuroblastoma. Mol Cancer Res 2007;5:1276–84.

95. Voth H, Oberthuer A, Simon T, Kahlert Y, Berthold F, Fischer M. Co-regulated expression of HAND2 andDEIN by a bidirectional promoterwith asymmetrical activity in neuroblastoma. BMC Mol Biol2009;10:28.

96. Cole KA, Attiyeh EF, Mosse YP, Laquaglia MJ, Diskin SJ, BrodeurGM, et al. A functional screen identifies miR-34a as a candidateneuroblastoma tumor suppressor gene. Mol Cancer Res 2008;6:735–42.

97. Wei JS, Song YK, Durinck S, Chen QR, Cheuk AT, Tsang P, et al. TheMYCN oncogene is a direct target of miR-34a. Oncogene 2008;27:5204–13.

98. Welch C, Chen Y, Stallings RL. MicroRNA-34a functions as a poten-tial tumor suppressor by inducing apoptosis in neuroblastoma cells.Oncogene 2007;26:5017–22.

99. Buechner J, Tømte E, Haug BH, Henriksen JR, Løkke C, Flægstad T,et al. Tumour-suppressor microRNAs let-7 and mir-101 target theproto-oncogene MYCN and inhibit cell proliferation in MYCN-ampli-fied neuroblastoma. Br J Cancer 2011;105:296–303.

100. Wang HJ, Ruan HJ, He XJ, Ma YY, Jiang XT, Xia YJ, et al. MicroRNA-101 is down-regulated in gastric cancer and involved in cell migrationand invasion. Eur J Cancer 2010;46:2295–303.

101. Foley NH, Bray I, Watters KM, Das S, Bryan K, Bernas T, et al.MicroRNAs 10a and 10b are potent inducers of neuroblastoma celldifferentiation through targeting of nuclear receptor corepressor 2.Cell Death Differ 2011;18:1089–98.

102. Fukuda Y, Kawasaki H, Taira K. Exploration of human miRNA targetgenes in neuronal differentiation. Nucleic Acids Symp Ser (Oxf)2005:341–2.

103. Laneve P, DiMarcotullio L, Gioia U, FioriME, Ferretti E, Gulino A, et al.The interplay between microRNAs and the neurotrophin receptortropomyosin-related kinase C controls proliferation of human neu-roblastoma cells. Proc Natl Acad Sci USA 2007;104:7957–62.






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