prc2-mediated transcriptomic alterations at the embryonic ...culture is a powerful in vitro...

Molecular and Cellular Pathobiology

PRC2-Mediated Transcriptomic Alterations at theEmbryonic Stage Govern Tumorigenesis andClinical Outcome in MYCN-Driven NeuroblastomaShoma Tsubota1, Satoshi Kishida1, Teppei Shimamura2, Miki Ohira3, Satoshi Yamashita4,Dongliang Cao1, Shinichi Kiyonari1, Toshikazu Ushijima4, and Kenji Kadomatsu1

Abstract

Pediatric cancers such as neuroblastoma are thought to involvea dysregulation of embryonic development. However, it has beendifficult to identify the critical events that trigger tumorigenesisand differentiate them from normal development. In this study,we report the establishment of a spheroid culture method thatenriches early-stage tumor cells from TH-MYCN mice, a preclin-ical model of neuroblastoma. Using this method, we found thattumorigenic cells were evident as early as day E13.5 duringembryo development, when the MYC and PRC2 transcriptomeswere significantly altered. Ezh2, an essential component of

PRC2, was expressed in embryonic and postnatal tumor lesionsand physically associated with N-MYC and we observed thatH3K27me3 was increased at PRC2 target genes. PRC2 inhibitionsuppressed in vitro sphere formation, derepressed its target genes,and suppressed in situ tumor growth. In clinical specimens,expression of MYC and PRC2 target genes correlated strongly andpredicted survival outcomes. Together, our findings highlightedPRC2-mediated transcriptional control during embryogenesis as acritical step in the development and clinical outcome of neuro-blastoma. Cancer Res; 77(19); 5259–71. �2017 AACR.

IntroductionNeuroblastoma is an embryonal childhood malignancy that

originates in sympathoadrenal progenitors derived from migrat-ing neural crest stem cells. Thus, this disease occurs predominant-ly in the adrenal medulla and the sympathetic ganglia (1). Unlikein adult tumors, somaticmutations are rare inneuroblastoma andother childhood cancers (2, 3). Besides, intensive genetic analyseshave identified diverse genetic variations in neuroblastoma,includingMYCN amplification, aberrant copy number alterations(17q gain, 11q loss, etc.), single nucleotide variants (ALK, ATRX,etc.), chromothripsis, TERT rearrangements (3–5), and the dis-ruption of the let-7 miRNA family (6). However, the precisemechanisms regulating causative genomic alterations and epige-netic deregulation in human neuroblastoma are poorly under-stood. Because neuroblastoma is a developmental disorder, itstumorigenic eventsmay be accompanied by normal developmen-tal programs of the sympathoadrenal cell lineage. Therefore, wemay not be able to elucidate the mechanisms of neuroblastoma

tumorigenesis without investigating the early stage of this disease,particularly during embryogenesis. In terms of causative events,the exogenous expression of several genes, such as MYCN (7),LIN28B (8), andmutantALK (9), in genetically engineeredmousemodels, results in thedevelopment of neuroblastoma thatmimicsthe clinical presentation of this disease (10). However, the onco-genic roles of these changes in early pathogenesis, especiallyduring the embryonic stages, remain poorly understood, partlydue to the lack of a proper experimental method that selectivelyisolates transformed tumor cells obtained during the early stage oftumorigenesis.

To address the abovementioned challenges, we utilized theMYCN transgenic (TH-MYCN) mouse neuroblastoma model(7), wherein the expression of humanMYCN by the rat tyrosinehydroxylase promoter results in neuroblastoma formation thatis observable in the postnatal sympathetic ganglia. Spheroidculture is a powerful in vitro experimental tool to isolateproliferative cells, such as cancer cells, and this system providesexperimental opportunities to address cellular and molecularmechanisms in cancer (11). We and others have reportedspheroid culture conditions for neuroblastoma cells fromTH-MYCN mice or human neuroblastoma patients; however,these methods are only applicable to the culture of later-stageneuroblastoma cells (11, 12).

In this study, we report a spheroid culture condition to enrichearly-stage tumor cells from TH-MYCN mice and transcriptomic,epigenomic, and genomic analyses of the early pathogenesis inneuroblastoma. Using this model, we identified the cellular andmolecular events on embryonic day 13.5 (E13.5) in TH-MYCNmice, including the upregulation ofMYC targets and deregulationof polycomb repressive complex 2 (PRC2) targets, which wereessential for neuroblastoma tumorigenesis and strongly impactedlater-stage human neuroblastoma.

1Department of Biochemistry, Nagoya University Graduate School of Medicine,Nagoya, Japan. 2Division of Systems Biology, Nagoya University GraduateSchool of Medicine, Nagoya, Japan. 3Research Institute for Clinical Oncology,Saitama Cancer Center, Saitama, Japan. 4Division of Epigenomics, NationalCancer Center Research Institute, Tokyo, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Kenji Kadomatsu, Nagoya University Graduate Schoolof Medicine, 65 Tsurumai-Cho, Nagoya, Aichi 466-8550, Japan. Phone: 815-2744-2059; Fax: 815-2744-2065; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-3144

�2017 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst August 14, 2017; DOI: 10.1158/0008-5472.CAN-16-3144

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Materials and MethodsAnimals

TH-MYCNmice (7) on a 129þTer/SvJclmice background (CLEAJapan, Inc.) were maintained in a pathogen-free, temperature-controlled environment with a 12-hour light/dark cycle and fedmouse feeder pellets andwater ad libitum at our animal facility. Allanimal experiments were approved by the Animal Care and UseCommittee of the Nagoya University Graduate School of Medi-cine (Nagoya, Japan).

Spheroid cultureThe components of RA (þ) and RA (�) media are summarized

in Supplementary Fig. S1A. Homemade chick embryo extract wasprepared as described previously (13) and described in detail inSupplementary Materials and Methods. For primary culture, theembryonic superior mesenteric ganglion (SMG) was enzymati-cally digested with TrypLE (12563011, Thermo Fisher Scientific)containing 0.25 mg/mL Collagenase Type IV (C5138, Sigma-Aldrich) for 15 minutes at 37�C. The postnatal SMG was enzy-matically digestedfirst with 2.5mg/mLCollagenase Type IV for 20minutes at 37�C, followed by TrypLE for 20 minutes at 37�C.These digested tissues were further dissociated by pipetting withfire-polished Pasteur pipettes in quenching solution [L-15 Medi-um (11415064, Thermo Fisher Scientific) containing 1% BSAFraction V Solution (15260037, Thermo Fisher Scientific),10 mmol/L HEPES (15630106, Thermo Fisher Scientific), andpenicillin–streptomycin (15140148, Thermo Fisher Scientific)]containing 0.1 mg/mL DNase I (D4527, Sigma-Aldrich) and10 mmol/L MgCl2 (20908-65, Nacalai Tesque). The dissociatedcells were filtered by passing them through 35-mm cell strainers(352235, Corning). Single cells were cultured on either low-attachment PrimeSurface dishes (Sumitomo Bakelite) or non-treated culture dishes (Iwaki) at 37�C in a humidified incubatorcontaining 5% CO2.

The medium was changed every 3 to 4 days. When thespheres reached 200–300 mm in diameter, they were passagedby digesting them with StemPro Accutase (A1110501, ThermoFisher Scientific) and subsequently dissociated by pipettingthem with fine-tipped pipettes. To evaluate sphere sizes, singlecells were plated at low density (1–2.5 cells/mL) and allowed togrow clonally. An inverted microscope (IX81, Olympus) wasused to capture images. The sphere sizes were measured, andthe sphere numbers were counted manually using the cellSenssoftware (Olympus).

Subcutaneous transplantationSingle-cell suspensions of E13.5 and 3-week-old TH-MYCNþ/�

sphereswere prepared as described earlier. Single-cell suspensionsfrom TH-MYCNþ/� tumor tissues were isolated as previouslydescribed (14). The dorsal flanks of wild-type mice (7–8 weeksof age, male and female mixed evenly) were subcutaneouslytransplanted with 1 � 105 cells in 50% Matrigel (356234, Corn-ing). The mice were monitored for tumor growth every week, andtumor size was measured using digital calipers. The tumorvolumes were calculated with the following formula: volume(mm3) ¼ (length � (width)2)/2.

Genomic, epigenomic, and transcriptomic analysisArray comparative genomic hybridization (arrayCGH),

methyl-CpG binding domain (MBD) protein-enriched genome

sequencing (MBD-seq), and microarray analysis are describedin detail in Supplementary Materials and Methods. A geneontology analysis was conducted against gene ontology data-base with Bonferroni correction using the PANTHER overrep-resentation test on the PANTHER classification system (15). Agene set enrichment analysis was conducted using GSEA soft-ware with default settings (16, 17). Publicly available data forhuman neuroblastoma analysis were analyzed using R. Geneexpression matrix and annotation information of 498 neuro-blastoma samples obtained from GSE49710 and GSE62564. Aprinciple component analysis (Bioconductor: pcaMethods) wasused to calculate the first principle component for a set ofgenes, which was considered a signature score. Graphs weredrawn using the beeswarm package and survival package.

Chromatin immunoprecipitation sequencingChromatin immunoprecipitation for H3K27me3 was per-

formed as described previously (18) and described in detail inSupplementaryMaterials andMethods. The genomic regionswithrespect to transcription start site (TSS) and transcription termi-nation site (TTS; regions from TSS-3K to TTSþ3K) for each genewere partitioned into nonoverlapping subregions (bins) of 300bps, and the raw reads were assigned to each bin. The number ofreads was further quantile-normalized to adjust sample varia-tions. The average H3K27me3 peak profiles were calculated andplotted using ngs.plot package.

Inhibition of Ezh2 functionKnockdown of Ezh2 using short hairpin RNA and Ezh2 inhib-

itor treatment are described in Supplementary Materials andMethods.

qPCR,Western blotting, immunoprecipitation, overexpressionof MYCN, histologic analysis, and statistical analysis

qPCR, Western blotting, immunoprecipitation, overexpres-sion of MYCN, hematoxylin and eosin (H&E) staining, immu-nofluorescence analysis, IHC, in situ hybridization, and statis-tical analysis are described in Supplementary Materials andMethods.

Accession numberData obtained in this study have been deposited in NCBI's

Gene Expression Omnibus (Edgar et al., 2002) and are acces-sible through GEO Series accession number GSE87784 andGSE89741.

ResultsRetinoic acid-free spheroid culture is suitable for the growth ofMYCN-transformed neuroblasts

The postnatal sympathetic ganglia in TH-MYCN mice, f.e.,the SMG (Supplementary Fig. S1B), contain undifferentiated/proliferating neuroblasts positive for Phox2b (a sympatheticlineage marker), Ki67 (a proliferation marker), and N-Myc(Fig. 1A; Supplementary Fig. S1C; refs. 19, 20). The initial stepof this study consisted of establishing suitable spheroid cultureconditions that enrich and expand these early-stage neuroblastsin vitro. We attempted to use a culture condition that was usedto maintain derivatives of neural crest stem cells, such assympathetic and enteric neural progenitors (21). The mediumcontained retinoic acid (RA), which is known to induce

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Cancer Res; 77(19) October 1, 2017 Cancer Research5260




neuroblastoma differentiation and is used to treat high-riskpatients in the clinic (22). Thus, we prepared medium contain-ing RA [RA (þ) medium] and RA-free medium [RA (�) medi-um; Supplementary Fig. S1A].

Cells from the SMG were cultured in either RA (þ) or RA (�)medium to evaluate their sphere-forming ability. Primary sphereswere formed in both conditions (Fig. 1B). Spheres in RA (þ)medium exhibited neurite-like structures, an indication of differ-entiation, whereas those in RA (�) medium exhibited a distinctround shape (Fig. 1B). The mean sphere size that emerged in RA(�) mediumwas significantly larger than those that developed inRA (þ) medium, and RA (�) spheres were maintained even afterseveral passages (Fig. 1B and C; Supplementary Fig. S1D). We did

not observe differences in the expression levels of the humanMYCN transgene between the two media (Fig. 1D). Neuroblastspositive for Phox2bwere successfully isolated from TH-MYCNþ/�

SMG in both media, but the population of neuroblasts positivefor Ki67 was significantly lower in RA (þ) spheres than in RA (�)spheres (Fig. 1E). We further performed a microarray analysis ofRA (þ) and RA (�) spheres and identified significantly differen-tially expressed genes (Supplementary Fig. S1E and S1F; Supple-mentary Table S1). The data collectively indicated that treatingundifferentiated neuroblasts from TH-MYCNmice with RA accel-erates transcriptomic changes that promote cell differentiation;thus, RA (�) medium is suitable for maintaining proliferativeneuroblasts in vitro.

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Retinoic acid-free spheroid culture is suitable for the growth of MYCN-transformed neuroblasts. A, H&E staining of the SMG from 3-week-old WT or TH-MYCNþ/�

mice. Arrows, differentiated ganglion cells. Dotted line, enclosed areas are clustered neuroblasts. B, Primary and passaged spheres from 3-week-oldTH-MYCNþ/� SMG cultured in either RA (þ) or RA (�) medium. C, Quantification of the distribution and mean sphere sizes. D, Relative expression levelsof MYCN transgene in RA (þ) and RA (�) primary spheres evaluated by quantitative PCR. The data shown represent the average � SD from four biologicalreplicates. E, Immunofluorescence of RA (þ) spheres (n ¼ 23) and RA (�) spheres (n ¼ 22) for Phox2b and Ki67, with nuclear staining by DAPI. The ratio ofPhox2b- or Ki67-positive cells to DAPI-positive cells was determined. �� , P < 0.001. NA, not available.

Essential Role of PRC2 in Early Neuroblastoma Tumorigenesis

www.aacrjournals.org Cancer Res; 77(19) October 1, 2017 5261




MYCN-transformed neuroblasts are enriched and maintainedas spheres in vitro

Sphere formation was observed in primary culture and sub-sequent passages from the SMG of 3-week-old TH-MYCNþ/�

mice but not that from WT mice in RA (�) medium (Fig. 2A).We performed a microarray analysis to investigate the transcrip-tomic differences among 3-week-old WT SMG, TH-MYCNþ/�

SMG, and TH-MYCNþ/� spheres. A principal component anal-ysis revealed that the PC1 component (85.6% contribution)clearly separated three sample groups, wherein TH-MYCNþ/�

primary and passage spheres were clustered together (Fig. 2B).This clear separation was also evidenced by an unsupervisedhierarchical clustering analysis according to the differentiallyexpressed probes among the four groups (Fig. 2C; Supplemen-tary Table S2). It clustered the four sample groups except for oneTH-MYCNþ/� SMG, which was clustered with WT SMG, likelydue to the lower number of neuroblasts in the original SMGtissue (Fig. 2C). In addition, the clustering grouped genes intothree major groups, groups A (2,049 genes), B (3,713 genes),and C (195 genes; Fig. 2C; Supplementary Fig. S2A). The genes

in group A were upregulated in the TH-MYCNþ/� SMG andspheres, including genes that positively regulate neuroblastomadevelopment, such as Lin28b (8), Alk (9), Bmi1 (23), Mybl2(24), Lmo3 (25), Bdnf (26), Aurka (27), FACT (facilitates chro-matin transcription, composed of Supt16 and Ssrp1; ref. 28),Mdk (20), andNeurod1 (29). The genes in group B were stronglydownregulated in TH-MYCNþ/� spheres, including genes thatare negatively associated with neuroblastoma development,such as Ntrk1/2/3 (26), Ngfr (30), Casz1 (30), and Clu (30).The genes in group C were only upregulated in the TH-MYCNþ/�

SMG, including several immune-related genes, such as Ccl3/4,Cxcl1, Ifna11, Il1a/b, and Tlr1; this pattern was likely due toinfiltrating immune cells, such as myeloid cells (31) and T cells(32). A gene ontology analysis showed enrichment in cell pro-liferation–related GO terms in group A, extracellular molecule-related GO terms in group B, and cytokine activity in group C(Supplementary Fig. S2B; Supplementary Table S2), which wereconsistent with the functions of the genes listed above. Together,these results suggest that undifferentiated neuroblasts from the3-week-old TH-MYCN SMG were selectively enriched and almost

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Group B (3713 genes):strongly downregulated in TH-MYCN+/- SphereNB negatively related genes: Ntrk1/2/3, Ngfr, Casz1, Clu

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MYCN-transformed neuroblasts are enriched and maintained as spheres in vitro. A, Primary and passaged spheres from the SMG of 3-week-oldWT or TH-MYCNþ/�

mice. B, A microarray analysis was performed for SMG from eight independent 3-week-old WT and TH-MYCNþ/� mice as well as TH-MYCNþ/� primary spheresand passaged spheres (passage 5). A principal component analysis revealed that principal component 1 (85.6%) predominantly contributed to separatesample groups; WT SMG (green dots), TH-MYCNþ/� SMG (orange dots), and TH-MYCNþ/� spheres (red dots are primary spheres, and red circles are passagespheres). C, An unsupervised hierarchical clustering of all samples according to differentially expressed probes among the four groups.

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stably maintained as spheres in vitro, while expressing moleculescharacteristic of neuroblastoma. Thus, spheroid culture in RA (�)medium is an ideal experimental tool to address the molecularmechanisms of early neuroblastoma tumorigenesis.

MYCN-driven tumorigenesis is observable on as early asembryonic day 13.5 in TH-MYCN mice

We next investigated the initial timing of MYCN-driventumorigenesis and the critical molecular events in its earlypathogenesis in TH-MYCN mice. We examined the transverseplane of the E13.5 SMG region, where Phox2b-positive sym-pathoadrenal progenitors were clustered around the dorsalaorta in both WT and TH-MYCNþ/� mice (Fig. 3A). A specificantisense RNA probe for humanMYCNmRNA (SupplementaryFig. S3A) detected human MYCN mRNA expression in a smallsubset of neuroblasts in the E13.5 TH-MYCNþ/� SMG region(Fig. 3B; Supplementary Fig. S3B). The number of MYCN-positive cells increased on postnatal day 0 SMG and furtherincreased in 2-week-old TH-MYCNþ/� SMG (Fig. 3B).

We next investigated the sphere-forming ability of E13.5 WTand TH-MYCNþ/� neuroblasts. Because normal sympathoadre-nal progenitors can proliferate at E13.5, spheres formed fromboth E13.5WT and TH-MYCNþ/� SMG, and these spheres did notdiffer in cellular morphology and mean sphere size (Fig. 3C andD). However, E13.5 TH-MYCNþ/� spheres, but not E13.5 WTspheres, were maintained after several passages (Fig. 3E). All cellsin E13.5 WT primary and TH-MYCNþ/� passaged spheres werepositive for Phox2b, and thus they are sympathoadrenal lineage(Fig. 3F). All TH-MYCNþ/� sphere cells were positive for N-Myc,and the ratio of Ki-67 positive cells were higher than that of E13.5WT sphere cells, suggesting thatMYCN-positive transformed cellswere selected by subculture. Accordingly, human MYCN expres-sion was increased by passages (Supplementary Fig. S3C). WTprimary spheres formed from the E13.5 and postnatal day 0 SMG,but these spheres could not be passaged (Fig. 3G). In contrast,TH-MYCNþ/� spheres formed from all different stages and weremaintained even after several passages (Fig. 3G). To evaluate thetumorigenicity of these spheres, E13.5 and 3-week-oldTH-MYCNþ/� spheres were subcutaneously transplanted intoWTmice. Allografts of TH-MYCNþ/� tumor cells (1 � 105 cells)developed into subcutaneous tumors within 4 weeks of trans-plantation (Fig. 3H). Surprisingly, E13.5 and 3-week-old spherecells (1 � 105 cells) also formed subcutaneous tumors thatprogressed slower than tumor cell allografts (Fig. 3H). All trans-planted tumors were undifferentiated neuroblastoma, andwe didnot observe obvious histologic differences among these tumors(Supplementary Fig. S3D). These results collectively demonstratethat MYCN-driven tumorigenesis is observable on as early asE13.5 in the TH-MYCN SMG; thus, critical molecular events inthe early pathogenesis of neuroblastoma should be captured byanalyzing E13.5 TH-MYCN spheres.

Transcriptomic alterations in MYC and PRC2 targets areprominent events during early neuroblastomatumorigenesis

We compared the transcriptomes of E13.5 WT andTH-MYCNþ/� spheres using a microarray analysis. Genes posi-tively associated with neuroblastoma, such as Neurod1, Lmo3,Bdnf, Bmi1, Lin28b, Mybl2, Lmo1, and FACT (Ssrp1 and Supt16),were significantly upregulated, and genes negatively associatedwith neuroblastoma, such as Ngfr, Clu, and Ntrk3, were signifi-

cantly downregulated in E13.5 TH-MYCNþ/� spheres (Fig. 4A). Inaddition, most of the well-defined 51 MYC core targets (28, 33)were significantly upregulated in E13.5 TH-MYCNþ/� spheres(Fig. 4A). A GO analysis revealed the enrichment of cell prolif-eration-related GO terms in upregulated genes and the enrich-ment of extracellular compartment- and development-relatedGOterms in downregulated genes (Fig. 4B; Supplementary Table S3).To reveal the upstream regulatory mechanisms, we focused onwell-definedmolecular target gene sets created by Ben-Porath andcolleagues, including targets of MYC, PRC2, and NOS (NANOG,OCT4, and SOX2), which are associated with human embryonicstem cell identity and certain types of cancers (34). A gene setenrichment analysis revealed the significant enrichment of MYCtarget gene sets in upregulated genes (Fig. 4C; SupplementaryTable S4). Remarkably, PRC2 target gene sets were significantlyenriched in downregulated gene sets (Fig. 4C; SupplementaryTable S4). The OCT4 target gene set was also enriched in down-regulated genes, but the NANOG and SOX2 target gene sets werenot enriched (Supplementary Table S4). The absolute values ofthe normalized enrichment scores of PRC2 target gene sets(approximately 1.7–1.8) were higher than those of the MYCtarget gene set (approximately 1.4) and OCT4 target gene set(approximately 1.6). These results suggest that PRC2 target genesmore significantly contribute to the differential expression pat-terns between E13.5 WT and TH-MYCNþ/� spheres. Indeed, themajority of differentially expressed PRC2 targetswere significantlydownregulated in E13.5 TH-MYCNþ/� spheres (Fig. 4D; Supple-mentary Table S5). The PRC2 major components Ezh2, Eed, andSuz12 were slightly upregulated, whereas the Ezh2 family geneEzh1was downregulated in E13.5 TH-MYCNþ/� spheres, suggest-ing that Ezh2 is the enzyme primarily responsible for PRC2 in thiscellular context (Supplementary Fig. S4A). The expressions ofPRC2 components and a set of differentially expressed genes werevalidated by qPCR (Supplementary Fig. S4B). But, Ezh2 proteinonly showed a tendency to increase (Supplementary Fig. S4C).These results suggest that the differential expression of PRC2componentsmaynot directly contribute to the differential expres-sion of PRC2 target genes. On the other hand, exogenous expres-sion of MYCN in E13.5 WT spheres resulted in increase in Ezh2expression (Supplementary Fig. S4D), suggesting that N-Mycpromotes the transcription of Ezh2.

We performed chromatin immunoprecipitation sequencing(ChIP-seq) for the tri-methylation of Histone H3 at lysine 27(H3K27me3), which is a histone mark modified by PRC2 andresults in gene silencing (35).H3K27me3was strongly enriched atthe promoter region of PRC2 targets compared with non-PRC2targets (Fig 4E). Importantly, the H3K27me3 enrichment wasincreased in E13.5 TH-MYCNþ/� spheres and further elevated inTH-MYCNþ/� tumor spheres, suggesting that the PRC2 targetgenes were transcriptionally suppressed by Ezh2-mediatedH3K27me3 modification. Physical association between N-Mycand Ezh2, and the transcriptional suppression of PRC2 targetgenes by the complex were previously reported (36). Indeed, N-Myc, Ezh2, and Suz12 proteins were physically associated inE13.5 TH-MYCNþ/� spheres evidenced by coimmunoprecipita-tion (Fig. 4F), suggesting that N-Myc regulates the function ofPRC2 for example by recruiting PRC2 on certain genomic regions.

We also performed array comparative genomic hybridization.As previously reported (37), segmental chromosomal gains orlosses were identified in TH-MYCNþ/� tumor tissues and spheres;however, they were not observed in E13.5 and 3-week-old






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TH-MYCNþ/� spheres (Supplementary Table S6), suggesting thatcopy number alterations were not a major cause of neuroblasto-ma tumorigenesis in TH-MYCN mice. DNA methylation of pro-moter CpG islands is a gene-silencing mechanism, and weassumed that promoter-associated DNA methylations mightaffect the differential expression pattern between E13.5 WT andTH-MYCNþ/� spheres. Thus, we performed methyl-CpG bind-ing domain protein-enriched genome sequencing (MBD-seq).We considered � 500-bp regions of TSSs as promoter regionsand identified 23 and 73 specifically promoter-methylatedgenes in E13.5 WT and TH-MYCNþ/� spheres, respectively(Supplementary Fig. S5A and S5B). We then assessed differ-ences in the expression levels of promoter-methylated genesbetween E13.5 WT and TH-MYCNþ/� spheres; however, theexpression levels of most genes remained unchanged (Supple-mentary Fig. S5C–S5E). Thus, we concluded that the DNAmethylation of promoter CpG islands did not contribute totranscriptomic changes. Collectively, these results highlight thecontribution of PRC2 in the transcriptome to early neuroblas-toma pathogenesis in TH-MYCN mice.

Ezh2 function is vital for the growth of MYCN-transformedneuroblastoma cells in vitro

We next investigated the role of Ezh2 in early tumorigenesisbecause it is the enzyme responsible for the function of PRC2(35). We used lentivirus-mediated shRNAs targeting Ezh2 toimpair Ezh2 function. Four of 5 different shRNAs decreased theEzh2 protein levels by over 50% in 3 days after infection (Fig. 5A).The knockdown of Ezh2 by two different shRNAs (#4 and #5)drastically suppressed E13.5TH-MYCNþ/� sphere formation (Fig.5B). We next used a potent and selective Ezh2 inhibitor,EPZ-6438, which inhibits the histone methyltransferase activitiesof EZH2, leading to a decrease in H3K27me3 (38, 39). Treatmentwith EPZ-6438 resulted in dose-dependent decreases in theH3K27me3 levels (Fig. 5C). Accordingly, the mean sphere sizeswere significantly and dose-dependently decreased in EPZ-6438-treated E13.5 TH-MYCNþ/� spheres (Fig. 5D). The average cellgrowth IC50 values of EPZ-6438 treatment were approximately 1–2 mmol/L for the three different sphere types, that is, E13.5 TH-MYCNþ/�, E13.5 TH-MYCN homozygote (TH-MYCNþ/þ), and 3-week-old TH-MYCNþ/� spheres (Supplementary Fig. S6). Theexpressions of PRC2 targets including cyclin-dependent kinaseinhibitors (40) and Ezh2 targets in neuroblastoma (30) werebroadly downregulated in E13.5 TH-MYCNþ/� spheres, andrecovered at the comparable levels to that in E13.5 WT spheresby EPZ-6438 treatment (Fig. 5E). Together, these results demon-strate that the function of Ezh2 maintains the cell identity oftransformed neuroblastoma cells in vitro by epigenetically repres-sing the expression of its target genes.

Ezh2 is overexpressed in postnatal neuroblasts and is apotential target for neuroblastoma treatment

IHC staining showed that Ezh2 was expressed in sympathoa-drenal progenitors in both the E13.5 WT and TH-MYCNþ/� SMG(Fig. 6A). The Ezh2 expression levels in these cells were compa-rable with those in surrounding cells, suggesting that Ezh2 iswidely and similarly expressed in different types of cells in theE13.5 transverse plane around the dorsal aorta (Fig. 6A). Incontrast, Ezh2 expression was minimal in differentiated ganglioncells both in the 3-week-old WT and TH-MYCNþ/� SMG, buthighly expressed in undifferentiated neuroblasts from theTH-MYCNþ/� SMG (Fig. 6A). Higher Ezh2 protein levels in the3-week-old TH-MYCNþ/� SMG were also observed by Westernblotting (Fig. 6B).

We next evaluated the potential of Ezh2 as a target for neuro-blastoma treatment. TH-MYCNþ/þ mice were used in this exper-iment because tumor formation is stable; all mice exhibitedenlarged tumors in the SMG area at three weeks of age and dieddue to the tumor at 7 to 8 weeks of age (Fig. 6C). We used EPZ-6438 because its efficacy has already been demonstrated in certaintypes of cancers, including rhabdoid tumors (38) and non-Hodg-kin lymphoma (39), and it is currently being evaluated in a phaseI/II clinical trial (ClinicalTrials.gov Identifier: NCT01897571).Wefollowed the treatment procedures described by Knutson andcolleagues (39) and selected 300mg/kg per day as a suitable dosefor this study. EPZ-6438 drastically reduced H3K27me3 levels inthe tumor mass (Fig. 6D). Accordingly, we observed significant insitu tumor growth suppression in TH-MYCNþ/þ mice by thetreatment (Fig. 6E; Supplementary Fig. S7A). However, tumorcells were observed without obvious histologic changes (Supple-mentary Fig. S7B), although the drug inhibited the function ofEzh2 also evidenced byH3K27me3 staining (Fig. 6F; Supplemen-tary Fig. S7C), suggesting that the treatment did not lead tocomplete regression. Accordingly, cessation of the EPZ-6438treatment led to tumor regrowth and resulted in no obviousextension of overall survival (Supplementary Fig. S7D). We didnot observe weight loss and other visible adverse effects. Toproperly assess the benefit of EPZ-6438 to the survival, treatmentregimens such as the dose, drug delivery system, combinationtherapies, and treatment schedule should be considered in future.Collectively, these results clearly demonstrated that Ezh2 is highlyexpressed in MYCN-transformed neuroblastoma in vivo and is apotential target for neuroblastoma treatment.

Transcriptomic characteristics during early pathogenesisare strongly associated with the malignant phenotype ofhuman neuroblastoma

We also examined the early transcriptomic changes found inTH-MYCN mice in human neuroblastomas. To this end, weutilized publicly available datasets (gene expression of 498

Figure 3.MYCN-driven tumorigenesis is observable on as early as embryonic day 13.5 in TH-MYCN mice. A, Immunofluorescence for Phox2b. White dotted line, enclosedareas are the SMG, with clusters of Phox2b-positive cells located around the dorsal aorta (DA, white line, enclosed area). Transverse plane: D, dorsal; V, ventral.B, In situ hybridization forMYCNmRNA in the SMG of E13.5, postnatal day 0, and 2-week-old mice (black dotted line, enclosed areas are the SMG). Dark blue signalswere MYCN-positive cells (arrows). C, Primary spheres from the SMG of E13.5 WT or TH-MYCNþ/� mice. D, Comparison of the mean sphere sizes. The datashown represent the average� SD from independent biological replicates (numbers are indicated by n). E, Passaged spheres fromE13.5WT primary or TH-MYCNþ/�

primary spheres. F, Immunofluorescence of E13.5 WT primary and TH-MYCNþ/� passage spheres for Phox2b, Ki67, and N-Myc, with nuclear staining by DAPI.The ratio of Phox2b-, Ki67-, N-Myc-positive cells toDAPI-positive cellswasdetermined. �� ,P<0.001.G,Sphere-forming ratio of E13.5, postnatal day0, or 3-week-oldWT or TH-MYCNþ/� spheres in primary and passaged culture (more than three passages). Actual spheres formed/tested numbers (biological replicates) areindicated. H, Tumor-forming ratios and tumor growth kinetics of E13.5 spheres, 3-week-old spheres, and dissociated tumor cells from TH-MYCNþ/� mice.Tumor formed/tested numbers are indicated.






N-Myc

Inpu

t

IgG

Ant

i-N-M

yc

Ant

i-Ezh

2

Ant

i-Suz

12

IP:

Loading ratio: 1/20 4 4 41

Ezh2

Suz12

Loading ratio: 1/20 4 1 14

E13.5 TH-MYCN+/- sphere

A B

Ribosome biogenesis

Mitotic cell cycle process

DNA Replication

Mitotic nuclear division

Cell division

DNA repair

Regulation of cell migration

0 2 4 6Fold enrichment

GO term (biological process)

Cell adhesion

Blood vessel development

Extracellular matrix organization

Embryonic organ development

403020100P (-log10)

Upr

egul

ated

Dow

nreg

ulat

ed

0.4

ES

0.0

MYC_MAX_TARGETS

NES: 1.423FDR q-val: 0.222

MYC Target gene sets PRC2 Target gene sets

-0.4

0.0

ES

EED_TARGETS

NES: -1.776FDR q-val: 0.016

-0.5

0.0

ES

SUZ12_TARGETS

NES: -1.730FDR q-val: 0.019

-0.4

0.0

ES

ES_WITH_H3K27ME3

NES: -1.711FDR q-val: 0.020

-0.4

0.0

ES

PRC2_TARGETS

NES: -1.657FDR q-val: 0.025

C

D

F

E13.5 TH-MYCN+/- sphere vs E13.5 WT sphereUpregulatedDownregulated

Supt16

0

2

4

6

8<

Wel

ch’s

t-te

st: q

-val

ue (-

log 10

)

Neurod1

Bdnf

Lmo3

Lin28bBmi1

Mybl2Lmo1

CluNtrk3

Ngfr

< -8 -4 0 4 8 <Fold change (log2)

MYC core target

Ssrp1

E13.5 WT sphere E13.5 TH-MYCN+/- sphere

Dow

n(1

54 g

enes

)U

p(5

0 ge

nes)

Differentially expressed PRC2 targets (204/610 genes)Fold change > ×1.5, Welch’s t-test q-val < 0.1

Processed signal (log2 ratio)-2 20

20

40

60

80

Genomic region (5' −> 3')−3000 TSS 33% 66% TTS 3000

Rea

d co

unt p

er m

illio

n m

appe

d re

ads


TH-MYCN+/- tumor sphere

E13.5 WT sphere

PRC2 targets Others

ChIP-seq:H3K27me3

E

Figure 4.

Transcriptomic alterations in MYC and PRC2 targets are prominent events during early neuroblastoma tumorigenesis.A,Amicroarray analysis of E13.5 TH-MYCNþ/�

spheres that could be passaged and WT primary spheres represented by a volcano plot. Pink or light blue dots are significantly differentially expressed genes(probes). Welch t test false discovery rate (FDR) q-value <0.1 was considered significant. Green dots, 51-MYC core targets. B, Statistical overrepresentationtests were performed for up- and downregulated genes against GO terms. Fold enrichment and Bonferroni-corrected P values are shown (dotted lines are 2).C, A gene set enrichment analysis was performed for up- and downregulated genes against gene sets of C2_CPG_v5.1 from MsigDB. An FDR q-value <0.25was considered significant. NES, normalized enrichment score. D, The hierarchical clustering of all samples according to differentially expressed PRC2 targets(204/610 genes). E, ChIP-seq for H3K27me3 in E13.5 WT and TH-MYCNþ/� spheres and TH-MYCNþ/� tumor spheres. Read counts per million mapped readsare reported as the mean� SEM (shadow). F, Coimmunoprecipitation of E13.5 TH-MYCNþ/� spheres by antibodies indicated, followed byWestern blotting analysisfor N-Myc, Ezh2, and Suz12 proteins.

Tsubota et al.





A

0

10

30

Sph

ere

size

(×10

3 μm

2 )

20

Sphere n = 214 237 261 118211

DMSO 0.01 0.1 1 10

EPZ-6438 (μmol/L)

Mean size = 10503 9361 8058 7116 2794

******

***

DM

SO

EP

Z-64

38_1

0 μm

ol/L

E13.5 TH-MYCN+/- sphere: 6-day treatment

500 μm

B

shNT

0

10

20

#4

Sph

ere

size

( ×10

3 μm

2 )

Sphere n = 327 162129Mean size = 6778 8971338

#5

***

shEzh2

***shN

Tsh

Ezh

2_#4

E13.5 TH-MYCN+/- sphere: 7 days after infection

500 μm

Histone H3

Ezh2

Ezh2/H3: 1.00 0.82 0.29 0.45

#1 #2 #3

shEzh2

0.12 0.22

#4 #5shNT

E13.5 TH-MYCN+/- sphere: 3 days after infection

C D

E

E13.5 TH-MYCN+/- sphere: 3-day treatment

Histone H3

H3K27me3

H3K27me3/H3: 1.00 0.52 0.28 0.07

Ezh2

Ezh2/H3: 1.00 1.11 1.20 1.14

DM

SO

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1.12

10

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1

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6

Cdkn1a(p21Cip1)

Cdkn1b(p27Kip1)

Cdkn1c(p57Kip2)

Cdkn2a(p19ARF)

Cdkn2a(p16INK4a)

Cdkn2b(p15INK4b)

Cdkn2c(p18INK4c)

Cdkn2d(p19INK4d)

Cdkn3(KAP) Clu Ngfr Casz1

Rel

ativ

e va

lue

(gen

e/A

ctb)

E13.5 WT sphere (n = 5)

E13.5 TH-MYCN+/- sphere: DMSO (0.1%) (n = 4)

Outlier (Grubbs’ test, α = 0.05)

E13.5 TH-MYCN+/- sphere: EPZ-6438 (1 μmol/L) (n = 4)

*** *****

******

** **

***

ND

***

****

**

****

*

*****

*

*****

**

**** ***

Cyclin dependent kinase inhibitor family Ezh2 targets in Neuroblastoma(Chunxi Wang, et al., Cancer Res., 2012)

Figure 5.

Ezh2 function is vital for the growth of MYCN-transformed neuroblastoma cells in vitro. A,Western blotting for Ezh2 and Histone H3 proteins in E13.5 TH-MYCNþ/�

spheres infected with nontargeting shRNA (shNT) or five different shRNAs targeting Ezh2 (shEzh2, #1–#5). The relative Ezh2/Histone H3 ratio was quantifiedby dividing the measured densities. B,Quantification of the distribution and mean sphere sizes of E13.5 TH-MYCNþ/� spheres infected with shNT or shEzh2 (#4 and#5). C, Western blotting for Ezh2, H3K27me3, and Histone H3 proteins in E13.5 TH-MYCNþ/� spheres treated with DMSO or EPZ-6438. D, Quantificationof the distribution and mean sphere sizes of E13.5 TH-MYCNþ/� spheres treated with DMSO or EPZ-6438. E, Relative expression levels of indicated genes inE13.5 WT spheres, E13.5 TH-MYCNþ/� spheres treated with DMSO or 1 mmol/L EPZ-638 for 7 days evaluated by qPCR. The data shown represents the average� SD from the biological replicates without outliers detected by Grubbs' test (a ¼ 0.05). � , P < 0.05; ��, P < 0.01; ��, P < 0.001.






3-Week-old

TH-M

YC

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+

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WT

TH-MYCN+/+ mice (weeks of age)

3 4 5

Tumor weight & IHC (Fig. 6E & 6F)

Western blotting (Fig. 6D)Western blotting (Fig. 6D)10 days treatment10 days treatment

14 days treatment14 days treatment5 mm5 mm

A

Histone H3

Ezh2

3-Week-oldWT SMG

3-Week-oldTH-MYCN+/- SMG

0.0

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0.1 1.0

Histone H3

H3K27me3

H3K27me3/H3: 1.00 0.91 0.36 0.34

Ezh2

Ezh2/H3: 1.00 1.03 1.16 1.08

Vehicle EPZ-643810 days treatment (IP, once daily)

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C

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-E13.5 SMG 3-Week-old SMG

100 μm100 μm

Ezh2 Expression

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Rat

io (p

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l cel

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EPZ-6438(n = 3)

H3K27me3

Vehi

cle

EP

Z-64

38

Figure 6.

Ezh2 is overexpressed in postnatal neuroblasts and is a potential target for neuroblastoma treatment. A, IHC for Ezh2 proteins in E13.5 and 3-week-old WTand TH-MYCNþ/� SMG. Dotted line, enclosed areas are clustered neuroblasts in the SMG. Arrows, differentiated ganglion cells. DA, dorsal aorta. B, Westernblotting for Ezh2 and Histone H3 proteins in SMG from 3-week-old WT and TH-MYCNþ/� mice. The relative Ezh2/Histone H3 ratios were quantified by dividing themeasured densities and are indicated by the average� SD. C, Tumor formation in TH-MYCNþ/þ mice at 3–5 weeks of age. Treatment schedules for D–F are shown.D, Western blotting for Ezh2, H3K27me3, and Histone H3 proteins in tumor tissues in four independent TH-MYCNþ/þ mice treated with either vehicle orEPZ-6438 (300 mg/kg/day for 10 days). The relative Ezh2/Histone H3 and H3K27me3/Histone H3 ratios are indicated. E, The weights of dissected tumortissues treated with either vehicle or EPZ-6438 (300 mg/kg/day for 14 days). The data shown represent the average � SD. F, IHC for H3K27me3 marks intumor tissues treated with either vehicle or EPZ-6438 (300 mg/kg/day for 14 days). Tumor cells were weakly stained in vehicle-treated tumor sections, but werenot stained in EPZ-6438–treated ones. Infiltrating immune cells were strongly stained both in vehicle- and EPZ-6438–treated tumor sections. H3K27me3-positivecells were masked by red color, while unstained cells were masked by green color, and the ratio of H3K27me3 positive cells was automatically counted andcalculated by TissuemorphDP software. Data shown represents the average � SD. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Tsubota et al.





neuroblastoma samples), and calculated the signature scoresbased on the expression levels of a set of genes using a principlecomponent analysis, and used the first principle componentvalues as the signature scores. The signature scores for genes thatwere significantly (over 2-fold) upregulated (191 genes) anddownregulated (1,563 genes) between E13.5 TH-MYCNþ/� andWT spheres were calculated, and these changes represented thetranscriptomic changes that occur during early neuroblastomatumorigenesis. In addition, the signature scores for MYC coretargets (51 genes) and PRC2 targets (654 genes) were also calcu-

lated. The signature scores of up- and downregulated genes inE13.5 TH-MYCNþ/� spheres positively (r ¼ 0.76) and negatively(r ¼ �0.56) correlated with the signature score of MYC coretargets, respectively (Fig. 7A). Importantly, the signature score ofPRC2 targets was strongly and negatively associated with thesignature score of MYC core targets (r ¼ �0.84; Fig. 7A). Thesecorrelations were stronger than those with the expression levels ofMYCN (Fig. 7A). Moreover, these signature scores were stronglyassociated with several clinical statuses, including the MYCNstatus, RISK factors, and INSS staging categories (Fig. 7B). We

A

E13

.5 T

H-M

YC

N+/

- sph

ere

Upr

egul

ated

(191

gen

es)

E13

.5 T

H-M

YC

N+/

- sph

ere

Dow

nreg

ulat

ed (1

563

gene

s)

PR

C2

Targ

ets

in E

S c

ells

(654

gen

es)

with MYC targets signature

-10

010

20S

igna

ture

sco

re-1

00-5

00

50S

igna

ture

sco

re–4

0–2

00

20S

igna

ture

sco

re

r = 0.76

r = –0.56

r = –0.84

−6 0 6

MYC core targets

(51 genes)

Signature score

with MYCN expressionr = 0.65

r = –0.50

r = –0.75

<2 4 128

MYCN

log2 expression

Pearson correlation coefficient BP = 1.4 × 10–58

Student’s t-test

P = 7.5 × 10–15

P = 5.1 × 10–60

MYCN status

Non-amp

(401)

Amplified

(91)

P = 6.7 × 10–49

Student’s t-test

P = 3.5 × 10–16

P = 2.0 × 10–39

RISK

Low/Intermediate

(322)

High

(176)

P = 4.0 × 10–32

ANOVA

P = 1.6 × 10–13

P = 7.1 × 10–28

INSS staging

1

(121)

2

(78)

3

(63)

4

(183)

4S

(53)

C

Ove

rall

surv

ival

pro

babi

lity

0

0.2

0.4

0.6

0.8

1.0

P = 9.0 × 10–6 P = 2.8 × 10–26 P = 7.8 × 10–22 P = 6.4 × 10–7 P = 4.6 × 10–18

0 5 10 15 Year

High (249)Low (249)

0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15


Downregulated (1563 genes)

Signature score


Upregulated (191 genes)

Signature score

PRC2 targets in ES cells

(654 genes)

Signature score

MYC core targets

(51 genes)

Signature score

MYCN

log2 expression

Figure 7.

The transcriptomic characteristics during early pathogenesis are strongly associatedwithmalignant phenotypeof humanneuroblastoma.A,Plots of signature scoresor log2 expression for indicated signature scores or log2 expression. Pearson correlation coefficients are indicated with regression lines. B, Distribution ofsignature scores among patients with several clinical statuses, MYCN status, RISK factors, and INSS staging categories. The numbers in brackets indicate thenumber of neuroblastoma samples. C, Kaplan–Meier survival curves for the overall survival probability of 498 patients with neuroblastoma grouped bymedian of the log2 expression or signature scores (high, red; low, green). Log-rank t test P values are indicated.






next investigated the ability of these scores to predict patientprognosis. Specifically, a highMYC target signaturewas associatedwith poor prognosis (P¼ 2.8� 10�26), and the prognostic powerof this signature was stronger than that ofMYCN expression (P¼9.0 � 10�6), which is consistent with the findings of previousstudies (Fig. 7C; refs. 28, 41). Notably, the signature scores of up-and downregulated genes in E13.5 TH-MYCNþ/� spheres as wellas the signature score of PRC2 targets were also able to predictpatient prognosis (Fig. 7C). The expression levels of PRC2 com-ponents, that is, EZH2, EED and SUZ12, were not stronglyassociated with the signature score for MYC targets and MYCNexpression (Supplementary Fig. S8A), and did not predict patientprognosis (Supplementary Fig. S8B). These results indicate thatthe transcriptomic characteristics, including the deregulation ofPRC2 targets, during early neuroblastoma pathogenesis inTH-MYCN mice are sustained and are critical determinants ofthe malignant phenotype of neuroblastoma.

DiscussionThe targeted expression of oncogenes in sympathoadrenal

lineage cells in several mouse models results in neuroblastomaformation (7–9). However, the detailed molecular mechanismsof the cellular context-dependent oncogenic events have not yetbeen investigated. We found that tumorigenic cells wereobservable as early as E13.5 in TH-MYCN mice. This cellularevent was accompanied by transcriptomic alterations, that is,the up- and downregulation of gene sets, the activation of MYCtargets, and the deregulation of PRC2 targets. Because PRC2inhibition suppressed the growth of neuroblastoma cells in vitroand markedly reduced tumor mass in vivo, PRC2 should be anessential player in neuroblastoma tumorigenesis in TH-MYCNmice. Notably, the activation of MYC targets and deregulationof PRC2 targets predicted the prognosis of human neuroblas-toma. Overall, our study revealed cellular events that initiatetumorigenesis at the mid-gestation period in TH-MYCN miceand molecular events in which early-stage transcriptionalalterations, including the deregulation of MYC and PRC2targets, govern tumorigenesis and the malignant phenotype ofhuman neuroblastoma.

Changes in the DNA methylome are a characteristic epigeneticderegulation in neuroblastoma. Notably, the presence of a CpGisland methylator phenotype is associated with MYCN amplifi-cation and high-risk neuroblastoma (42, 43), and a recent studysuggested the cooccurrence of DNA hypermethylation and anincrease in H3K27me3 marks on the same genomic locus inhuman neuroblastoma (43). Furthermore, PRC2 has beenreported to physically interact with DNA methyltransferases todirectly regulate DNA methylation (44). A physical interactionhas also been identified between MYCN and PRC2 in neuroblas-toma (36). We also observed it in early neuroblastoma (Fig. 4F).Although these lines of evidence suggest a link between DNA

methylation and PRC2 function, we did not observe a strongassociation among differences in mRNA expression, DNA meth-ylation, and H3K27me3 modifications in E13.5 TH-MYCNspheres (Supplementary Fig. S5D and S5E). Therefore, our resultsparticularly highlight the essential role of PRC2 and H3K27me3among various types of epigenetic regulations during early tumor-igenesis in TH-MYCN mice, although the axis of MYCN–PRC2–DNA methylation in neuroblastoma development warrants fur-ther investigation.

The MYCN and MYC target signatures are strongly associatedwith the malignant phenotype of neuroblastoma (28); however,directly targeting these transcription factors with small-moleculeinhibitors is challenging, aswidely discussed in the literature (45).In this study, we clarified that the specific inhibition of the histonemethyltransferase activity of Ezh2 suppressed tumor growth inTH-MYCN mice, and the MYC target signature and PRC2 targetsignature strongly correlated in human neuroblastoma. There-fore, the disruption of PRC2 function, such as the inhibition ofEzh2 by EPZ-6438, may provide a promising option for thetreatment of human neuroblastoma.

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

Authors' ContributionsConception and design: S. Tsubota, S. Kishida, K. KadomatsuDevelopment of methodology: S. Tsubota, D. CaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Tsubota, M. Ohira, S. Yamashita, T. UshijimaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Tsubota, T. Shimamura, S. YamashitaWriting, review, and/or revision of the manuscript: S. Tsubota, S. Kishida,D. Cao, K. KadomatsuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Ohira, S. KiyonariStudy supervision: K. Kadomatsu

AcknowledgmentsWe thank Dr. Hideki Enomoto in Kobe University Graduate School of

Medicine for providing anti-Phox2b antibody and the experimental procedureof spheroid culture method, and Ayaka Hatano for her technical assistance.

Grant SupportS. Tsubota was supported by JSPS KAKENHI grant JP14J00157. S. Kishida

was supported by JSPS KAKENHI grant JP24590377. T. Ushijima and K.Kadomatsu were supported by a grant for the Practical Research for InnovativeCancer Control from Japan Agency for Medical Research and Development(16ck0106011h0003). K. Kadomatsu was supported by JSPS KAKENHI grantJP15k15079 and CREST, JST (15656320).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 25, 2016; revised May 23, 2017; accepted July 27, 2017;published OnlineFirst August 14, 2017.

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2017;77:5259-5271. Published OnlineFirst August 14, 2017.Cancer Res Shoma Tsubota, Satoshi Kishida, Teppei Shimamura, et al. NeuroblastomaGovern Tumorigenesis and Clinical Outcome in MYCN-Driven PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage

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