Histone Chaperone CHAF1A Inhibits Differentiation and ...ting.Anti-H3K9me3antibodies(22-442;Millipore)wereusedat a dilution 1:1,000 after acid extraction of the histones. Xenograft

Molecular and Cellular Pathobiology

Histone Chaperone CHAF1A Inhibits Differentiation andPromotes Aggressive Neuroblastoma

Eveline Barbieri1, Katleen De Preter3, Mario Capasso4, Zaowen Chen1, Danielle M. Hsu2,Gian Paolo Tonini5, Steve Lefever3, John Hicks1, Rogier Versteeg7, Andrea Pession6,Frank Speleman3, Eugene S. Kim2, and Jason M. Shohet1

AbstractNeuroblastoma arises from the embryonal neural crest secondary to a block in differentiation. Long-term

patient survival correlates inversely with the extent of differentiation, and treatment with retinoic acid or otherprodifferentiation agents improves survival modestly. In this study, we show the histone chaperone andepigenetic regulator CHAF1A functions in maintaining the highly dedifferentiated state of this aggressivemalignancy. CHAF1A is a subunit of the chromatin modifier chromatin assembly factor 1 and it regulatesH3K9 trimethylation of key target genes regulating proliferation, survival, and differentiation. Elevated CHAF1Aexpression strongly correlated with poor prognosis. Conversely, CHAF1A loss-of-function was sufficient to driveneuronal differentiation in vitro and in vivo. Transcriptome analysis of cells lacking CHAF1A revealed repressionof oncogenic signaling pathways and a normalization of glycolytic metabolism. Our findings demonstrate thatCHAF1A restricts neural crest differentiation and contributes to the pathogenesis of high-risk neuroblastoma.Cancer Res; 74(3); 765–74. �2013 AACR.

IntroductionNeuroblastoma arises from residual immature neural crest

cells within the peripheral sympathetic ganglia of very youngchildren (1). The clinical and biologic behavior of this tumor isextremely heterogeneous, encompassing fatal tumor progres-sion, as well as spontaneous regression and differentiation intomature ganglioneuroma. Furthermore, the degree of neuronaltumor differentiation strongly affects patient outcome. Studiesfrom transgenicmousemodels of neuroblastomawith targetedoverexpression of the MYCN oncogene also demonstrate thatblocked neural crest differentiation leads to the malignanttransformation of neuroectodermal precursors into neuroblas-toma (2). Efforts to define the mechanisms for this blockage inneuroblast differentiationhavebeen the focusofmajor researchefforts over the past years, and have led to the incorporation ofseveral differentiation strategies into neuroblastoma treatment.Retinoic acid therapy is an important component of treatment

of residual disease of stage IV neuroblastoma after multimodaltherapy (3). Nevertheless, rising resistance and treatment tox-icity represent relevant limiting factors, and the overallresponse rate to retinoic acid in patients with neuroblastomais low, suggesting that only a subgroup of patients benefits fromthe treatment. Therefore, a better understanding of the molec-ular mechanisms that restrict neuroblastoma differentiationcould lead to improved therapeutic approaches for this highlyaggressive malignancy.

Alterations in components of the transcriptional machineryand chromatin modifier genes are now associated with initia-tion and differentiation of multiple cancers (4), including neu-roblastoma (5). A role for epigenetics in tumorigenesis is furthersupported by recent genome-wide sequencing studies revealingrecurrent cancer-associated mutations in key epigenetic regu-lator genes, including histone modifiers, histone chaperones,and DNA methylation modifiers (6). In particular, methylationof histone H3 at position lysine 9 (H3K9) has been extensivelystudied as a major factor regulating transition between tran-scriptionally active euchromatin and inactive heterochromatin(7). In addition, H3K9 histone methyltransferases interact withDNA methyltransferases (e.g., DNMT1/3b) to indirectly modu-late gene silencing through DNA methylation (8). The histonemodifiers EZH2 (9) and LSD1 (10) are deregulated in neuro-blastoma with high expression conferring worse prognosis. Inaddition, repression of the tumor suppressor and chromatinmodifier CHD5 through loss of heterozygosity and DNA meth-ylation negatively correlates with long-term survival (11).

CHAF1A (CAF p150) is a primary component of the chro-matin assembly factor 1 (CAF-1), composed of p150, p60, andp48 subunits, which promotes rapid assembly of nucleosomeson newly replicated DNA (12). The importance of CHAF1A incancer pathogenesis is supported by the finding that its

Authors' Affiliations: 1Texas Children's Cancer Center and Center for Celland Gene Therapy; 2Department of Surgery, Baylor College of Medicine,Houston, Texas; 3Center for Medical Genetics, Ghent University, Ghent,Belgium; 4CEINGEBiotecnologie Avanzate, Department of Biochemistry andMedical Biotechnology, University of Naples Federico II, Naples; 5PediatricResearch Institute, University of Padua, Padua; 6Paediatric Oncology andHaematology Unit "Lalla Ser�agnoli," Sant'Orsola-Malpighi Hospital, Univer-sityofBologna,Bologna, Italy; and7DepartmentofOncogenomics,AcademicMedical Center, University of Amsterdam, Amsterdam, the Netherlands

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

Corresponding Author: Jason M. Shohet, Department of Pediatrics,Section of Hematology-Oncology, Baylor College of Medicine, FeiginBuilding, Room 750.01, 1102 Bates Street, Houston, TX 77030. Phone:832-824-4735; Fax: 832-825-1604; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1315

�2013 American Association for Cancer Research.

CancerResearch

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on March 24, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 12, 2013; DOI: 10.1158/0008-5472.CAN-13-1315

http://cancerres.aacrjournals.org/

overexpression has been linked to tumor progression (13),cancer susceptibility (14), andmore recently, epigenetic silenc-ing (15). In addition, CHAF1A participates in a complex withMBD1 and SETDB1 during initiation of a gene-silencing pro-gram by promoting H3K9 trimethylation, heterochromatinformation, and DNA methylation (16).

We show here that CHAF1A restricts neuroblastoma differ-entiation using both in vitro and in vivo orthotopic models.Elevated expression of CHAF1A indeed strongly correlatesclinically with an undifferentiated neuroblastoma phenotypeand poor overall survival. We also demonstrate that CHAF1Apromotes oncogenic signaling pathways (including RAS, AKT,BMI1, and WNT) as well as alters glycolytic metabolism path-ways. Together, these data support a novel function for thehistone modifier CHAF1A restricting neural crest differentia-tion and promoting neuroblastoma tumorigenesis.

Patients and MethodsClinical patient cohort groups

Discovery set 1. Versteeg (n¼ 88). Thisdataset of 88uniquetumors was profiled on the Affymetrix HGU133 plus2.0 platformand normalized using the MASS5.0 algorithm. Expression datawere freely downloaded from the R2 website (http://r2.amc.nl).

Validation set 2. Vermeulen (n¼ 348). This cohort includ-ed 348 patients with neuroblastoma taken from the Interna-tional Society of Pediatric Oncology, European NeuroblastomaGroup (SIOPEN) and from the Gesellschaft fuer PaediatrischeOnkologie und Haematologie (GPOH). Patients were onlyincluded if primary untreated neuroblastoma tumor RNA (atleast 60% tumor cells and confirmed histologic diagnosis ofneuroblastoma) was available and of sufficient quality (17).Almost all patients were treated according to the SIOPENprotocols. The median follow-up was 63 months and greaterthan 24 months for most patients (91%). In this cohort, 32% ofthe patients had stage I disease, 18% stage II, 18% stage III, 23%stage IV, and 9% stage IVS.MYCN amplification was present in17% of all patients, and in 45% of stage IV patients. Median ageat diagnosis was 7.4 months for stage I and II, and 23.5 monthsfor stage III and IV. Expression of CHAF1Awas evaluated usingquantitative real-time PCR (qRT-PCR).

Discovery set 3. Delattre (n¼ 64). This public dataset of 64neuroblastic tumors (11 ganglioneuroblastoma and 53 neuro-blastoma) was profiled on Affymetrix chips HGU133 plus 2.0. Itwas freely downloaded from the Gene Expression Omnibusdataset, accession number GSE12460 (18).

qRT-PCR in primary samplesA quantitative PCR assay was designed for CHAF1A and five

reference genes by PrimerDesign and went through an exten-sive in silico validated analysis using basic local alignmentsearch tool (BLAST) and BiSearch specificity, amplicon sec-ondary structure, single-nucleotide polymorphism presence,and splice variant analysis. The mean amplification efficiencywas 98%. Primer design and qRT-PCR analysis were performedas described previously (17). Primer sequences are available inRTPrimerDB (2): CHAF1A (ID ¼ 8273) and reference genes:HPRT1 (ID¼ 5), SDHA (ID¼ 7), UBC (ID¼ 8), andHMBS (ID¼4). Data handling and calculations (normalization, rescaling,

inter-run calibration, and error propagation) were performedin qBasePlus version 1.1 (19, 20).

Short hairpin RNA constructs and antibodiesFor p53 short hairpin RNA (shRNA), second-generation

lentiviruses expressing shp53 and shLuc control were used asdescribed (21). To knock down CHAF1A expression, a TRIPZlentiviral inducible shRNAmir with Tet-inducible promoterwas used (Open Biosystems). The tetracycline response ele-ment (TRE) promoter also drives the expression of a TurboRFPreporter. To repress CHAF1A expression, doxycycline wasadded at a final concentration of 1 mg/mL. Control lines usingscrambled shRNAmir were also generated. A GIPZ lentiviralstable shRNA (Open Biosystems) was instead used to trans-duce neuroblastoma lines for in vivo studies. Briefly, 293T cellswere transfected with pLSLPw, TRIPZ, and GIPZ constructsalong with packaging plasmids, pVSVG, and pLV-CMV-delta8.2 by using lipofectamine. Virus-containing supernatantswere collected at 48 and 72 hours and neuroblastoma cellswere transduced in the presence of 8 mg/mL polybrene(Sigma). CHAF1A rabbit monoclonal antibodies (Epitomics;#5464-1; 1:500 dilution) and p53 mousemonoclonal antibodies(Sigma; #P6874; 1:1,000 dilution) were used for Western blot-ting. Anti-H3K9me3 antibodies (22-442;Millipore) were used ata dilution 1:1,000 after acid extraction of the histones.

Xenograft modelOrthotopic xenografts of human neuroblastoma were gen-

erated as described previously (23) by injection under the renalcapsule of an inoculum of 106 tumor cells in 0.1 mL of PBS.Tumors were evaluated at necropsy 5 weeks after inoculation.

Oligonucleotide microarray data analysisTotal RNA was isolated using the RNAeasy Kit (Quiagen)

from IMR32 cells transduced with inducible CHAF1A shRNA.Gene expression profiling using Affymetrix U133þ2.0 arrayswas performed in neuroblastoma cells upon CHAF1A silencingover time course (0, 5, and 10 days) in triplicate. Differentiallyexpressed genes were identified by MAS5 detection P values�0.05, ANOVA P value�0.05, and absolute fold change�2. Foreach time point, genes were ranked with respect to the averageexpression change upon CHAF1A knockdown. Gene SetEnrichment Analysis (GSEA) was then performed for each ofthe three time points using gene permutation alternative (24).GSEA software v2.0.1 was used for the analysis. Default para-meters were used and gene sets that met the false discoveryrate (FDR) �0.25 criterion were ranked by nominal P value.GeneOntology (GO) analysiswas performed as described usingthe DAVID bioinformatic database (25).

Details about cell lines, tissue culture, and qRT-PCR assaysand primers are found in the Supplementary Methods section.

ResultsCHAF1A is repressed by p53 and highly expressed inundifferentiated neuroblastoma

Neuroblastoma is primarily a p53 wild-typemalignancy, andas part of previous efforts to profile the p53 transcriptionalresponse of neuroblastoma, we observed that increased p53

Barbieri et al.

Cancer Res; 74(3) February 1, 2014 Cancer Research766




levels correlated with decreased CHAF1A expression. As therepression of p53 functions is critical to neuroblastoma tumor-igenesis, and CHAF1A expression is altered in other malig-nancies, we proceeded to further analyze CHAF1A function inneuroblastoma.Prognostic factors for neuroblastoma include age, stage at

diagnosis, histology, and specific genetic alterations, includingMYCN amplification/overexpression. We first analyzed theprognostic value of CHAF1A in a clinical cohort of 88 patientswith neuroblastoma (discovery set 1) using the R2 microarraydatabase and showed that increased CHAF1A expressionstrongly correlates with poor overall survival (OS; P <0.0001; Fig. 1A) and higher stage of disease (P < 0.0001;Supplementary Fig. S1). We further confirmed CHAF1A prog-nostic value in a large independent cohort of patients withneuroblastoma. qRT-PCR analysis of CHAF1A expression intumor samples from 348 patients enrolled in SIOPEN andGPOH clinical trials (validation set 2) identified patients withneuroblastoma with poor overall survival (P < 0.0001) andprogression-free survival (PFS; P < 0.001; Fig. 1B). In addition,multivariate logistic regression analysis showed that CHAF1A

expression level is able to predict survival, independent of theMYCN status (amplified vs. nonamplified), age at diagnosis (<or >12 months), and stage (stage IV vs. other stages) with ahazard ratio of 2.37 and 2.22 for OS and event-free survival(EFS), respectively (P < 0.05 and P < 0.005; Table 1).

To confirm the regulation of CHAF1A by p53 activity,CHAF1A gene expression was assessed in multiple p53 wild-type neuroblastoma lines treated with the MDM2 inhibitor,Nutlin-3a. qRT-PCR demonstrated significant repression ofCHAF1A expression upon treatment (P < 0.005). However, thiseffect is totally abrogated in neuroblastoma p53 mutant(LAN1) or p53 knockdown (LAN5 si p53) cells, confirming thatCHAF1A repression is indeed p53-mediated (Fig. 1C and D).

CHAF1A silencing promotes neuroblastomadifferentiation in vitro

To assess the biologic function of CHAF1A in neuroblastoma,we first generated multiple neuroblastoma cell lines withinducible shRNA-mediated CHAF1A knockdown using a Tet-On conditional system that coexpressed red fluorescent protein(RFP). We found that silencing of CHAF1A leads to a distinct

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Figure 1. CHAF1A expression accurately predicts neuroblastoma outcome and is regulated by p53. A, Kaplan–Meier and log-rank analysis for OSof discoveryset 1 (88 patients with neuroblastoma) based on CHAF1A expression. B, independent validation of CHAF1A expression in a large cohort of patients withneuroblastoma (SIOPEN/GPOH). CHAF1A expression as measured by qRT-PCR versus OS and PFS in validation set 2; survival of 348 patients withneuroblastoma in the 4 quartiles of the signature score is shown. C, p53 activation represses CHAF1A expression. qRT-PCR demonstratessignificant decrease of CHAF1A mRNA levels after Nutlin-3a treatment (10 mmol/L for 8 hours) in multiple p53 wild-type neuroblastoma lines (shownhere LAN5, IMR32, and SY5Y). However, this effect is completely abrogated when the effect of Nutlin-3a is tested in a p53 mutant neuroblastoma cell line(LAN1) or in a p53 wild-type line LAN5 transduced with a shp53 lentivirus (D). Each error bar represents two biologic replicates.

CHAF1A Blocks Neuroblastoma Differentiation

www.aacrjournals.org Cancer Res; 74(3) February 1, 2014 767




morphologic change consistent with the morphologic changeobserved upon retinoic acid-induced differentiation in sensitiveneuroblastoma cell lines (26). Silencing of CHAF1A in twoneuroblastoma cell lines, LAN5 and IMR32 (Fig. 2A), graduallyinduces the development of long dense neurite-like processesover a 7 to 14 day time span. In contrast, no apparent change inmorphology was observed in scramble-control–transducedcells (Fig. 2B). To define the observed morphology change asneuronal differentiation, we measured the gene expression ofseveral well-characterized markers of terminal neuronal differ-entiation: b3 tubulin (TUBB3), nerve growth factor receptor(NGFR), tyrosine hydroxylase (TH), and growth-associatedprotein (GAP43). Silencing of CHAF1A is associated with sig-nificantly increased expression levels of these neuronalmarkerscomparedwithnoninduced andnontargeting siRNAcontrols (P< 0.005; Fig. 3A). In addition, as CHAF1A is known to promotetrimethylation of histone H3 at position lysine 9, we examinedthe global H3K9me3 levels and found that silencing of CHAF1Asignificantly reduces the level of global H3K9me3 in IMR32 cells(Fig. 3B).

To further determine the role of CHAF1A as an inhibitor ofdifferentiation, we evaluated CHAF1A expression levels inneuroblastoma cells treated with retinoic acid. In all threelines tested (LAN5, CHLA255, and NGP), the morphologicdifferentiation (Supplementary Fig. S2) is associated with asignificant (P < 0.005) downregulation of CHAF1A expressionlevels after 7 to 10 days of retinoic acid treatment (Fig. 3C).Finally, we compared the expression of CHAF1A in a smallcohort of less aggressive ganglioneuroblastoma (n ¼ 11) withmore aggressive, undifferentiated neuroblastoma samples (n¼53; discovery set 3; ref. 18). As shown in Fig. 3D, CHAF1Aexpression is significantly (P < 0.05) higher in the undifferen-tiated neuroblastoma group.

CHAF1A promotes tumorigenesis and opposesdifferentiation in vivo

We then generated neuroblastoma lines with stableCHAF1A knockdown and found that CHAF1A silencingmarkedly inhibited in vitro proliferation of LAN5 and IMR32neuroblastoma cell lines by day 4 (MTT assay; Supplemen-

tary Fig. S3). To assess the role of CHAF1A in promotingtumorigenesis, loss-of-function studies using three distinctneuroblastoma cell lines (LAN-5, IMR-32, and NGP) withstable CHAF1A knockdown were performed in vivo in anorthotopic neuroblastoma model (subrenal capsule injec-tion). This model closely recapitulates the highly angiogenicand invasive growth characteristics of undifferentiatedhuman neuroblastoma (27). We found that CHAF1A silenc-ing significantly reduces tumor growth in all the three celllines tested (Fig. 4A and B). Tumors with CHAF1A knock-down grossly seemed less vascular and Western blot analysissuggested that tumor growth is proportional to CHAF1Alevels (Supplementary Fig. S4).

Detailed histologic analysis shows that the control tumorshave a more undifferentiated phenotype with closely apposedneuroblasts, decreased neuropil as highlighted on S100 proteinimmunostaining, and high mitotic karyorrhectic index (MKI;525 þ 37 per 5,000 tumor cells). In contrast, CHAF1A knock-down tumors display more neuronal differentiation withincreased well-developed neuropil-separating neuroblasts,and a much lower MKI (193 þ 43 per 5,000 tumor cells).Finally, electron microscopy confirmed the presence of fre-quent cell processes with a well-developed neuropil andincreased dense-core neurosecretory granules (Fig. 4C) inCHAF1A knockdown tumors compared with controls. Overall,the histologic differences are consistent with a change in gradefrom "undifferentiated" to "poorly differentiated," which cor-relates with the reduced growth observed in vivo.

CHAF1A silencing induces neuronal differentiationpathways and inhibits major oncogenic pathways

To unveil changes in gene expression associated withchanges in neuroblastoma phenotype induced by CHAF1A,we performed gene expression profiling (Affymetrix U133þ2.0arrays) in IMR32 cells 5 and 10 days after CHAF1A silencing.Clustered heat map of the differentially expressed genes isshown in Fig. 5A (GEO series accession number GSE51978).Wethen examined the occurrence of GO terms of genes associatedwith changes in CHAF1A expression using the DAVID onlineanalysis platform (28). Notably, themost significantly enriched

Table 1. Multivariate logistic regression analysis in the SIOPEN/GPOH cohort

OS P Risk factor 0.95 Confidence interval

Stage (IV vs. others) 5.46E�08 10.33 4.45 23.96Age (< or >1 year) 1.68E�01 1.9 0.76 4.75MYCN status (� amplification) 4.20E�04 3.28 1.69 6.34CHAF1A expression (< or > median) 3.79E�02 2.37 1.05 5.35

EFS P Risk factor 0.95 Confidence interval

Stage (IV vs. others) 1.06E�08 4.46 2.67 7.44Age (< or >1 year) 4.77E�01 0.83 0.5 1.38MYCN status (� amplification) 9.67E�02 1.56 0.92 2.65CHAF1A expression (< or > median) 1.12E�03 2.22 1.37 3.59

NOTE: P values, risk factors, and 95% confidence interval are shown for disease stage (stage IV vs. others), age (< or >1 year),MYCNstatus (amplified vs. nonamplified), and CHAF1A expression. Significant values for CHAF1A expression are shown in bold.

Barbieri et al.





functional categories (P < 0.05) upon CHAF1A silencing areassociated with multiple processes involved in neuronal dif-ferentiation (axonogenesis, synaptic transmission, cell-cellsignaling, catecholamine biosynthesis, and nervous systemdevelopment; Table 2), validating CHAF1A as a potentialcritical regulator of neuronal differentiation. Furthermore,these functional categories were distinct from the onesdescribed to be enriched in neuroblastoma-cell differentiationupon Cyclin D1 and Cdk4 silencing (29) or retinoic acidtreatment (30), suggesting a distinctive mechanism forCHAF1A in inducing cell differentiation (SupplementaryTables S1 and S2).In addition, GSEA revealed that genes regulated by CHAF1A

were associated with major metabolic and oncogenic path-ways. CHAF1A silencing significantly enriches for cell metab-olism pathways (valine, leucine, and isoleucine degradation,

glutamate metabolism, and insulin pathways; Fig. 5B) andsuppresses pathways with known oncogenic function in neu-roblastoma (KRAS, ALK, AKT, and BMI1; nominal P < 0.05 andFDR q < 0.25; Fig. 5C). A complete list of the significantpathways is shown in Supplementary Table S3. qRT-PCRconfirmed that CHAF1A affects the expression of selectedmetabolic genes with important roles in insulin, type 2 dia-betes, valine, leucine, and isoleucine degradation pathwaysboth in IMR32 and LAN5 cells. Notably, DHRS2, an enzymewith crucial oxidoreductase activity, is markedly upregulatedupon CHAF1A silencing (Fig. 5D).

In summary, we demonstrate that the expression of CHAF1Ais regulated by p53 and positively correlates with a moreundifferentiated aggressive neuroblastoma phenotype in vitroand in vivo. In addition, silencing of CHAF1A leads to upre-gulation of genes controlling neuronal differentiation,

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Figure 2. CHAF1A silencing induces a differentiated neuronal phenotype. A, knockdown of CHAF1A expression by CHAF1A siRNA but not siRNAcontrol in LAN5 and IMR32 cells. CHAF1A mRNA and protein level after knockdown was determined by SYBR Green qRT-PCR and Western blotting.B, conditional siRNA-mediated knockdown of CHAF1A is compared with conditional siRNA control and retinoic acid (RA) treatment over time(Tet-on cells visualizedby fluorescentmicroscopy for RFP). CHAF1Asilencing induces longneurite extension comparablewith retinoic acid treatment in LAN5cells. By contrast, IMR32 cells did not show the same morphologic changes and underwent marked apoptosis after 5 to 7 days of retinoic acidtreatment. However, silencing CHAF1A strongly promotes neurite extension in this same cell type.






normalized glucose metabolism, as well as downregulation ofmajor oncogenic pathways. As discussed below, these datasuggest that CHAF1A or downstream pathways may representnovel therapeutic targets, which could sensitize neuroblasto-ma to differentiation in vivo.

DiscussionCurrently, the predictive risk factors used for neuroblastoma

risk stratification are age, stage, tumor histology, and MYCNgene amplification status. We observed that elevated expres-sion of one such chromatin chaperone, CHAF1A, significantlycorrelateswith poor survival in several large cohorts of patientswith neuroblastoma independently of these clinical features.CHAF1A expression is also much lower in spontaneouslyregressing infant neuroblastomas and in ganglioneuroblasto-mas (a highly differentiated form of neuroblastoma), andmarkedly elevated in the most undifferentiated aggressivemetastatic cases.

These clinical observations suggest that CHAF1A plays animportant role in neuroblastoma biology. Oncogenic func-tions of deregulated CHAF1A and the CAF-1 histone chap-erone complex continue to be defined. Expression of CAF-1has been associated with cell proliferation in breast cancer

(31), deregulation of DNA repair in squamous cell carcinoma(32), and genomic instability and cancer susceptibility in therecessively inherited Bloom syndrome (14). Furthermore,activating single nucleotide polymorphisms within theCHAF1A gene strongly correlate with glioma tumorigenesis(33).

Mirroring the clinical observations, using an orthotopicxenograft model of neuroblastoma, we show that CHAF1Aexpression drives a more undifferentiated neuroblastoma phe-notype in vivo. Suppression of CHAF1A strongly induces neu-roblastoma to differentiate, suggesting that CHAF1A restrictsinnate differentiation pathways and may modulate resistanceto differentiation-based therapies. Of note, we present geneexpression data suggesting that CHAF1A may act throughmechanisms independent of previously characterized CyclinD1/Cdk4 or retinoic acid-driven pathways (SupplementaryTables S1 and S2). However, neuroblastoma differentiation isa complex and poorly understood process involving multiplenetworks of genetic and epigenetic pathways. Understandingthe regulatorymechanisms of neuroblastoma differentiation isimportant for obtaining insight into basic biology and fordeveloping novel therapies that may overcome the resistanceto retinoids.

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Figure 3. CHAF1A silencing promotes molecular neuronal differentiation in vitro. A, CHAF1A silencing upregulates molecular markers of neuronaldifferentiation. qRT-PCR confirms induction of neuron-specific marker genes (NGFR, TH, GAP43, and TUBB3) 10 to 14 days after doxycycline-inducibleCHAF1A siRNA expression. All changes in expression were normalized to non-induced siRNA. B, CHAF1A knockdown reduces the level of globalH3K9me3 in IMR32cells 10daysafterCHAF1AsiRNAexpression.Western blot datawere analyzedbydensitometry.C, qRT-PCR revealsdecreasedCHAF1AmRNA levels after 7 to 10 days of treatment with retinoic acid (10 mmol/L) in LAN5, CHLA255, and NGP neuroblastoma cells. D, CHAF1A expressionin ganglioneuroblastoma (GNB; n ¼ 11) versus undifferentiated neuroblastoma (NB; n ¼ 53) in discovery set 3.

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Epigenetic changes, including histone modifications, playa central role in controlling differentiation and defining thepluripotent state of embryonic and cancer stem cells (34).Recent comprehensive genome-wide studies define distinctpatterns of histone modifications and DNA methylationduring multilineage differentiation of stem cells (35, 36).These and other studies point to a complex interaction ofDNA histone modifications and DNA methylation control-ling cellular differentiation barriers (37), and disruption ofthese epigenetic mechanisms is strongly implicated intumorigenesis and survival of cancer stem populations(38). H3K9 trimethylation has been characterized as a majorfactor regulating transitions between transcriptionallyactive euchromatin and inactive heterochromatin (39). Bind-ing of UHRF1 to methylated H3K9 is required for DNAmethylation maintenance (8). Methylated H3K9 serves as

a binding platform for heterochromatin protein 1, whichdirects DNMT1-dependent DNA methylation (40). Of note,previous studies demonstrate that CHAF1A acts indepen-dently of CAF-1 as an epigenetic-silencing factor (15), reg-ulating H3K9me3 epigenetic marking of heterochromatindomains in pluripotent embryonic cells (41). CHAF1A alsomodulates DNA methylation, forming a complex with MBD1and SETDB1, and modulating DNA methylation (16).

As with other aggressive embryonal malignancies, histonemodification and DNA methylation alterations are implicatedin the pathogenesis of neuroblastoma. High expression of theclass II HDAC SIRT1 stabilizes MYCN and promotes tumori-genesis (42), whereas aberrant DNMT3B transcripts areexpressed in high-risk neuroblastoma with globally alteredDNA methylation (43). In addition, altered EZH2 expression(polycomb histone methyltransferase) leads to repression of

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Figure 4. CHAF1A silencing opposes tumor growth and promotes differentiation in vivo. A, Western blotting confirms knockdown of CHAF1A expression inLAN5, IMR32, and NGP cell lines. B, average tumor weight for each cohort � SEM. Tumors with CHAF1A knockdown are significantly smaller than control(LAN5 xenografts: �, Kruskal–Wallis method P ¼ 0.0033, mean � SEM, n ¼ 8 in each group; IMR32 xenografts: �, Kruskal–Wallis method P ¼ 0.035,mean � SEM, n ¼ 10 in each group; NGP xenograft: �, Kruskal–Wallis method P ¼ 0.028, mean � SEM, n ¼ 5 in control group, n ¼ 8 in siRNA group).C, representative tumor samples in control and CHAF1A shRNA group for histologic comparison. Hematoxylin and eosin (H&E) staining, S100 proteinimmunostaining, electron microscopy (EM), and MKI quantification are shown.






multiple tumor suppressor genes in neuroblastoma (9), andgenome-wide DNA methylation studies identified candidateDNA methylation markers with important prognostic value in

neuroblastoma (44). Finally, the definition of the roles of novelchromatin regulators in neuroblastoma, such as CHD5, high-lights the importance of these histone posttranslational

Metabolic pathways Oncogenic pathways

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isoleucine degradationKRAS.600_UP

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Figure 5. Gene expression profiling and GSEA in IMR32 cells upon CHAF1A silencing. A, clustered heat map of the differentially expressed genes uponCHAF1A silencing in IMR32 cells over time course (0, 5, and 10 days). GSEA analysis identifies differentially expressed pathways upon CHAF1A silencing.Pathways related tocellmetabolism(B) andoncogenicsignatures (C) areamong the topdifferentiallyexpressedpathways.D,change inexpressionbyqRT-PCRofselected transcripts involved in cell metabolism upon CHAF1A silencing in IMR32 and LAN5 cells. Each error bar represents two biologic replicates.

Table 2. CHAF1A silencing modulates genes associated with neuronal differentiation

GO ID Name Size P

0007399 Nervous system development 32 0.00420007267 Cell–cell signaling 27 0.00420007268 Synaptic transmission 16 0.01120019226 Transmission of nerve impulse 17 0.01120042423 Catecholamine biosynthetic process 4 0.04010007409 Axonogenesis 12 0.04250007154 Cell communication 71 0.04250010243 Response to organic nitrogen 5 0.04250048856 Anatomical structure development 57 0.04250007417 Central nervous system development 15 0.04250007274 Neuromuscular synaptic transmission 4 0.04250006836 Neurotransmitter transport 7 0.04290000904 Cell morphogenesis involved in differentiation 15 0.04290051050 Positive regulation of transport 10 0.04290048667 Cell morphogenesis involved in neuron differentiation 12 0.0490

NOTE: GO enrichment analysis. The significant enriched GO terms based on biologic processes are shown. Functional categoriesterms, number of genes within each functional category, and corrected P values are indicated.

Barbieri et al.





modifications in controlling gene expression during neuronaldifferentiation (45).In further support of an oncogenic role of CHAF1A, our

GSEA analysis demonstrates that RAS as well as AKT, BMI, andALK pathways are strongly repressed upon CHAF1A knock-down (Fig. 5 and Supplementary Table S3). Ras signalingnetworks drive cellular proliferation and restrict differentia-tion, and previous studies have also suggested a role for RAS–MEK signaling in regulation of responses to retinoic acid indifferent cellular systems (46). Activation of NRAS seems to becritical in neuroblastoma tumorigenesis, considering its func-tion in stabilizing MYCN, promoting MYCN-dependent cellcycle progression, and blocking p53-mediated cell cycle checkpoints and proapoptotic effects (22, 47).In addition, bioinformatic analyses of gene expression

changes also suggest that CHAF1A may alter metabolic path-ways to promote the Warburg effect (increased glucose con-sumption and decreased oxidative phosphorylation). Themostenriched KEGG and Biocarta pathways after CHAF1A knock-down were pathways involved in cell metabolism (valine,leucine, and isoleucine degradation, and glutamate metabo-lism, among others, Fig. 5 and Supplementary Table S3). Valine,isoleucine, and leucine are three essential amino acids, whosecatabolism, together with glutamate, supports ATP produc-tion. The upregulation of these Kreb-cycle components,together with the downregulation of the insulin-Akt signalingupon CHAF1A silencing, suggests that suppression of CHAF1Amay have a role in shifting the cell metabolism to oxida-tive phosphorylation. Although these observations suggestCHAF1A overexpression may force neuroblastoma cellstoward the aerobic glycolysis, detailed metabolic studies willbe required to formally link CHAF1A to modulation of neu-roblastoma metabolism.Taken together, our findings in both neuroblastoma patient

cohorts and tumor models implicate this histone chaperonemolecule in multiple oncogenic pathways in neuroblastoma.

CHAF1A primarily restrains differentiation, which helps inexplaining its high expression in the most aggressive neuro-blastoma cases. Loss-of-function studies suggest that targetingCHAF1A or its downstream pathways would provide a noveltherapeutic approach to high-risk neuroblastoma.

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

Authors' ContributionsConception and design: E. Barbieri, K.D. Preter, A. Pession, E.S. Kim, J.M.ShohetDevelopment of methodology: E. Barbieri, E.S. Kim, J.M. ShohetAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):E. Barbieri, K.D. Preter, D.M. Hsu, J. Hicks, R. Versteeg,A. PessionAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Barbieri, K.D. Preter, M. Capasso, Z. Chen, D.M.Hsu, G.P. Tonini, F. Speleman, E.S. Kim, J.M. ShohetWriting, review, and/or revision of themanuscript: E. Barbieri, K.D. Preter,M. Capasso, F. Speleman, E.S. Kim, J.M. ShohetAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Z. Chen, S. Lefever, E.S. Kim, J.M.ShohetStudy supervision: E.S. Kim, J.M. Shohet

AcknowledgmentsThe authors thankOlivier Delattre and the SIOPEN for the datasets and Els De

Smet for technical help with qRT-PCR analysis.

Grant SupportThis work was supported by the Children's Neuroblastoma Cancer Founda-

tion (E. Barbieri), by Alex's Lemonade Stand (E. Barbieri and J.M. Shohet), theChildren's Cancer Research Foundation (E. Barbieri), and a Research ScholarsGrant from the American Cancer Society (J.M. Shohet). K.D. Preter is supportedby the Flemish Fund for Scientific Research. M. Capasso is supported byAssociazione Italiana per la Lotta al Neuroblastoma and MIUR–FIRB Ricercain Futuro.

The costs of publication of this article were defrayed in part 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.

ReceivedMay 7, 2013; revisedNovember 13, 2013; acceptedNovember 22, 2013;published OnlineFirst December 12, 2013.

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2014;74:765-774. Published OnlineFirst December 12, 2013.Cancer Res Eveline Barbieri, Katleen De Preter, Mario Capasso, et al. Aggressive NeuroblastomaHistone Chaperone CHAF1A Inhibits Differentiation and Promotes

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