EWS WT1 Oncoprotein Activates Neuronal Reprogramming ... · Tumor and Stem Cell Biology EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation

Tumor and Stem Cell Biology

EWS–WT1 Oncoprotein Activates Neuronal ReprogrammingFactor ASCL1 and Promotes Neural Differentiation

Hong-Jun Kang1, Jun Hong Park1, WeiPing Chen2, Soo Im Kang1, Krzysztof Moroz1, Marc Ladanyi3, andSean Bong Lee1

AbstractThe oncogenic fusion gene EWS–WT1 is the defining chromosomal translocation in desmoplastic small round-

cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS–WT1 functionsas an aberrant transcription factor that drives tumorigenesis, but themechanistic basis for its pathogenic activityis not well understood. To address this question, we created a transgenic mouse strain that permits physiologicexpression of EWS–WT1 under the native murine Ews promoter. EWS–WT1 expression led to a dramaticinduction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neuralreprogramming factor ASCL1. Mechanistic analyses demonstrated that EWS–WT1 directly bound the proximalpromoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS–WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells toneuronal induction media increased neural gene expression and induced neurite-like projections, both of whichwere abrogated by silencing EWS–WT1. Taken together, our findings reveal that EWS–WT1 can activate neuralgene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neuraldifferentiationmight offer a novel therapeutic approach to treatDSRCT.Cancer Res; 74(16); 4526–35.�2014AACR.

IntroductionDesmoplastic small round-cell tumor (DSRCT) is a rare but

aggressive tumor occurring predominantly in adolescents andyoung adults (1). DSRCT is poorly understood and highly lethal,resulting in 85% mortality within 5 years despite aggressivemultimodal therapy (2, 3). The majority of tumors are found inserous membrane of abdominal or pelvic cavity without anapparent involvement of any organ systems and form multiplenests of malignant cells embedded in dense desmoplasticstroma (1). A distinct immunophenotypic feature of DSRCT isa multilineage expression of epithelial, mesenchymal, neuronal,and muscle markers (3). Despite the presence of these multi-lineage markers, the tumor cells seem poorly differentiated. Todate, there is nomolecular rationale for themultilineage expres-sion and the tumor cell of origin remains undefined.

DSRCT is caused by a balanced chromosomal transloca-tion t(11;22)(p13;q12) that results in a fusion of the N-terminaldomain (NTD)of theEwing sarcomagene,EWSR1 (termedEWS)to the C-terminal domain of the Wilms tumor gene,WT1 (4, 5).EWS encodes an RNA/ssDNA–binding protein that plays mul-tiple roles in diverse cellular processes, such as maturation ofpre-B lymphocytes, meiosis, hypersensitivity to DNA damage,prevention of premature senescence of fibroblasts (6), andhematopoietic stem cells (7), mitosis (8), cell-fate determinationof classical brown fat (9), regulation of microRNAs (10), andregulationof genotoxic-induced alternative splicing (11, 12). TheNTD of EWS contains multiple degenerate repeats of theSYGQQS motif that functions as a potent transcriptional acti-vationdomain(13, 14).WT1 is inactivated in10%to15%ofWilmstumors, a childhood kidney cancer (15). WT1 encodes a tran-scription factor with four Cys2–His2 zinc fingers at theC-terminus, andundergoes an alternative splicing involvingonlythree amino acids (Lys, Thr, and Ser, termed KTS) between thezinc fingers three and four (16). This splicing produces twoisoforms that either lacks (�) or contains (þ) the KTS, whichalters the DNA-binding specificity. In DSRCT, only the last threezinc fingers of WT1 are fused to EWS and the KTS splicing ispreserved (5), resulting in the production of two isoforms: EWS–WT1–KTS (herein termed E�KTS) and EWS–WT1þKTS(EþKTS).

The presence of EWS–WT1 translocation in all DSRCTpoints to the fusion products as the initiator of this tumor.Interestingly, the two isoforms differ in their oncogenic activ-ities as only the E�KTS, but not EþKTS, has the potential totransform NIH3T3 cells in vitro (17). Therefore, the majority ofstudies have focused on identifying the target genes of E�KTS

1Tulane University School of Medicine, Department of Pathology andLaboratory Medicine, New Orleans, Louisiana. 2Genomics Core Facility,National Institute of Diabetes and Digestive and Kidney Diseases, NIH,Bethesda,Maryland. 3Department of Pathology,Memorial Sloan-KetteringCancer Center, New York, New York.

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

Current address for S.I. Kang: Department of Pathology and Cell Biology,Institute for Cancer Genetics, Columbia University Medical Center, NewYork, NY.

Corresponding Author: Sean Bong Lee, Tulane University School ofMedicine, Department of Pathology and LaboratoryMedicine, 1700 TulaneAvenue Room 808, New Orleans, LA 70112. Phone: 504-988-1331; Fax:504-988-7389; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3663

�2014 American Association for Cancer Research.

CancerResearch

Cancer Res; 74(16) August 15, 20144526

on February 13, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663

http://cancerres.aacrjournals.org/

(18–22), whereas only two genes have been identified as directtargets of EþKTS (22, 23). These studies also revealed differentDNA recognition sequences of each isoform: E�KTS binds toeither a GC-rich, 50-GXG(T/G)GGGXG-30 (X is any base;refs. 17, 20), or TCCn repeats (n > 3; ref. 18), whereas EþKTSrecognizes 50-GGAGG(A/G)-30 (23). Despite these findings,how EWS–WT1 drives oncogenesis remains poorly under-stood; consequently, the prognosis of DSRCT remains grimand the development of effective therapy is urgently needed.

Materials and MethodsCell culture and reagentsHEK293 (CRL-1573) and U2OS (HTB-96) cells were pur-

chased from the American Type Culture Collection andmouseembryonic fibroblasts (MEF) were generated as describedpreviously (6). These cells were grown in DMEM with 10%FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Invi-trogen). JN–DSRCT-1 cells (24), authenticated by the presenceof EWS–WT1 translocation and free of mycoplasma, weregrown in DMEM/F-12 media with 10% FBS. Tamoxifen, 4-hydroxytamoxifen (4-HT) and G418 (Sigma), and N2 supple-ment (Invitrogen) were purchased.

Generation of EWS–WT1 knockin mice and animal careTo generate a conditional EWS–WT1 mouse, we followed

the strategy that was used to generate a conditional EWS–FLI1mouse (25). Briefly, we inserted a loxP-flanked transcriptional"STOP" cassette (26) in an antisense direction into Ews intron 6(Fig. 1A). A human WT1(�KTS) cDNA (exons 8–10) was fusedto Ews exon 7 to create an EWS/WT1(�KTS) "knockin" allele.The targeting construct was used to generate correctly tar-geted mouse ES cells (TC-1; ref. 27) as determined by Southernblot analysis (25). Positive ES clones were injected into C57BL6

blastocysts (NIDDK Mouse Knockout Core) and the result-ing chimeras were crossed to C57BL6 females to achievegermline transmission. A conditional EWS–WT1(þKTS)knockin mouse was generated previously (6). A transgenicmouse constitutively expressing the CreER allele (B6.Cg-Tg(CAG-cre/Esr1)5Amc/J) was purchased (The Jackson Labo-ratory). The primers for genotyping EWS–WT1 or CreERmiceare described in the Supplementary Information. All animalprocedures were approved and handled according to theguidelines provided by the Tulane Institutional Animal Careand Use Committee and by the NIH Animal ResearchAdvisory Committee.

Microarray analysisMEFs harboring E�KTS/CreERþ, EþKTS/CreERþ or

CreERþ were either untreated or treated with 1 mmol/L 4-HT.At 24 hours, total RNA was prepared using the RNeasy Kit(Qiagen). Gene-expression profiling with or without (refer-ence) 4-HT was performed using Affymetrix Mouse Genome430 2.0 arrays. Three biologic replicates were analyzed for eachsample. The data were analyzed using an Affymetrix RMAalgorithm. Genes with greater than 1.5-fold difference and a Pvalue of <0.05 were selected by ANOVA using Partek Pro(Partek). The heatmaps were generated by using either RMAraw signal values or fold change values from ANOVA lists byPartek. Microarray data have been submitted to GEO database(GSE53301). Gene ontology (GO) analysis of primary tumorswas performed using DAVID v6.7 (The Database for Annota-tion, Visualization and Integrated Discovery; refs. 28, 29).

Real-time qRT-PCRTotal RNA was converted to cDNA using SuperScript III

Reverse Transcriptase (Invitrogen). Each sample was analyzed

Figure 1. Generation of inducible EWS–WT1 MEFs. A, schematics of conditional EWS/WT1 alleles; pA, poly-adenylation signal; NEO, neomycin resistancegene. Arrows, PCRprimers. B, CreER-mediated recombination analyzed by PCRof genomicDNA isolated fromMEFs culturedwith or without 1 mmol/L 4-HT.C, inducible expression of EWS/WT1 transcripts was analyzed by RT-PCR inMEFs treated for the indicated times with 4-HT. EWS–STOP–WT1 indicates theaberrantly spliced transcript. Gapdh was used as a control. D, MEFs treated as in C were immunoblotted with anti-WT1 or anti-actin.

EWS–WT1 Activates ASCL1 and Induces Neural Differentiation

www.aacrjournals.org Cancer Res; 74(16) August 15, 2014 4527




by real-time quantitative RT-PCR (qRT-PCR) using a TaqManprobe for Ascl1 (Mm03058063_m1; Applied Biosystems). Therelative transcript quantity was calculated by the comparativeCt method normalized against Gapdh.

Promoter–reporter assaysA human ASCL1 promoter (�1,043 to þ233) luciferase

reporter construct was purchased (GeneCopoeia) and sub-cloned into pGL3Basic vector (Promega). The ASCL1 promot-er–luciferase construct, along with Renilla luciferase plasmid,was transfected into JN–DSRCT-1 and HEK293 cells by Lipo-fectamine 2000 (Invitrogen). At 48 hours posttransfection,luciferase activities were measured using the Dual LuciferaseAssay Kit (Promega). Site-specific mutations (M1-M3) in theASCL1 promoterwere generated byDNA synthesis (BlueHeronBiotechnology) and confirmed by sequencing.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) assay was per-

formed as described (30) using anti-WT1 (C19; Santa CruzBiotechnology) or anti-RNA Pol II (Millipore) antibodies. Theprimers used to amplify the ASCL1 promoter are listed inSupplementary Information.

Colony formation assayCells were transduced with lentiviruses expressing shWT1

(shWT1-2 or shWT1-3), shASCL1 (shASCL1-1 or shASCL1-2) orscrambled control and cultured in presence of puromycin for 2weeks. After staining with crystal violet, colonies were countedand photographed. Three independent experiments were per-formed in triplicates. The shWT1 and shASCL1 sequences(Sigma) are listed in Supplementary Information.

ImmunofluorescenceCells were fixed with 4% paraformaldehyde for 15 minutes,

permeabilized with 0.5% Triton X-100 for 10 minutes, blockedwith 5% goat serum (Sigma), and incubated with the followingprimary antibodies: mouse Tuj1 (1:1,000) or rabbit anti-MAP2(1:1,000). Alexa Fluor 488 and 594 (Invitrogen) were used assecondary antibodies. Confocal microscopy was performedusing an inverted laser scanning confocal microscope (ZeissAxiovert 200 M) and analyzed with the LSM 510 confocalsoftware (Zeiss).

ResultsGeneration of mice harboring conditional EWS–WT1knockin alleles

To determine the physiologicmechanisms underlying EWS–WT1-driven tumorigenesis, we decided to express EWS–WT1under the control of native Ews transcriptional network. Wefused the WT1(�KTS) cDNA encoding the last three zincfingers of WT1 lacking the KTS to the mouse Ews exon 7 (Fig.1A), recreating the exact fusion transcript found in DSRCT.Similar to EWS–FLI1 mice (25), the loxP-flanked transcrip-tional "STOP" was inserted in the antisense direction, whichwas essential for the successful targeting of EWS–WT1(�KTS).The STOP inserted in the sense direction completely preventedhomologous recombination (data not shown), suggesting that,

similar to EWS–FLI1, a leaky EWS–WT1(�KTS) expressionimpedes ES cell growth. Successfully targeted ES cells wereused to generate a conditional EWS–WT1(�KTS) knockinmouse. We have previously generated a conditional EWS–WT1(þKTS) knockin mouse (6). We, herein, designateheterozygous mice carrying EWS–WT1(�KTS) as E�KTS andEWS–WT1(þKTS) as EþKTS. E�KTS and EþKTSmice seemedhealthy and were backcrossed to C57BL6 mice for more thaneight generations.

Inducible expression of E�KTS and EþKTSWe first crossed E�KTS or EþKTS mice with EIIa-Cre

transgenic mice (general deleter line), but we only obtainedEþKTS;Creþ mice (data not shown), suggesting that consti-tutive expression of E�KTS caused lethality. Therefore, wecrossed E�KTS or EþKTS mice with a transgenic mouseconstitutively expressing Cre recombinase fused to mutatedestrogen receptor (CreER), allowing an inducible expression ofEWS–WT1 with tamoxifen (Supplementary Fig. S1A). To testthe efficiency, we intraperitoneally (i.p.) administered 3 dosesof tamoxifen (3 mg/40 g body weight, given every 3 days) andexamined CreER-mediated excision of STOP by PCR. Weobserved varying degrees of recombination in different tissues,with the highest recombination observed in the kidney andpancreas, intermediate recombination in the brain, heart, andlung, and very little recombination in the liver and spleen(Supplementary Fig. S1B). Tamoxifen did not induce recom-bination in any of the tissues in E�KTSmicewithout CreER. Todetermine the cytotoxicity of E�KTS or EþKTS, we adminis-tered three doses of tamoxifen and monitored the mice daily.Notably, all E�KTS;CreERþmice (n¼ 4, age 6–8weeks) died by10 to 11 days from the first tamoxifen injection, whereastamoxifen-treated EþKTS;CreERþ (n ¼ 4) or E�KTS;CreER�

(n ¼ 4) mice lived more than 16 months. These observationsindicate that global expression of E�KTS, but not EþKTS,causes lethality in mice.

Expression of E�KTS, but not EþKTS, leads to inhibitionof cell growth

To examine the cytotoxic effects of E�KTS in detail, wederived MEFs from E�KTS;CreERþ and EþKTS;CreERþ

embryos. We observed near complete excision of STOP in bothMEFs following 4-HT treatment (Fig. 1B). There was norecombination in the absence of 4-HT or without CreERexpression. Similar to EWS–FLI1 MEFs (25), an aberrantlyspliced transcript (EWS–STOP–WT1) containing 147 bpderived from the antisense STOP is present in either MEFs inthe absence of 4-HT (Fig. 1C). Upon 4-HT–mediated excision ofSTOP, expression of the correctly spliced E�KTS and EþKTStranscripts (EWS–WT1) emerged at 6 hours and reachedmaximal levels at 24 hours (Fig. 1C). Expression of E�KTSand EþKTS proteins was detectable at 12 hours and reachedmaximal levels at 24 hours, but the expression of E�KTS wassubstantially lower than EþKTS (Fig. 1D). As previously dem-onstrated for EWS–FLI1, no detectable E�KTS protein wasgenerated in the absence of 4-HT (Fig. 1D), likely due to a rapiddegradation of the proteins generated from the aberrantlyspliced transcripts (25). Strikingly, the expression of E�KTS

Kang et al.

Cancer Res; 74(16) August 15, 2014 Cancer Research4528




resulted in a rapid cessation of cell growth whereas EþKTS–expressing cells grew normally (Fig. 2A). To gain insights intothe E�KTS–induced growth arrest, we examined expression ofvarious cell-cycle proteins by immunoblotting. Expression ofkey cell-cycle regulators, cyclin A and cyclin D1, was markedlyreduced in cells expressing E�KTS at 48 hours compared withCreER MEFs (Fig. 2B). Notably, phosphorylated AKT wasabsent in E�KTS–expressing cells even though total AKTlevels were comparable. However, expression of other cell-cycle proteins and various cyclin-dependent kinase inhibitors(Fig. 2B, right panels) was unaltered. MEFs expressing E�KTSdid not undergo apoptosis as determined by PARP cleavage(data not shown).

Genome-wide gene-expression profilingTo gain insights into the E�KTS– and EþKTS–mediated

transcriptional regulation, we performed whole-genomeexpression analysis. Interrogation of more than 39,000 probesets with RNA isolated from E�KTS– or EþKTS–expressingMEFs revealed that 3,228 transcripts (2,051 induced and 1,177repressed) showed significant expression changes (P< 0.05 and>1.5-fold change) following E�KTS expression whereas 1,557transcripts (780 induced and 777 repressed) showed significantalterations upon EþKTS expression (Supplementary Fig. S2A).About 300 transcripts were significantly altered by 4-HT treat-

ment in CreERMEFs, demonstrating a relatively small effect of4-HT. Notably, there was a very little overlap of genes regulatedby the two isoforms, suggesting that each isoform is recruitedto distinct promoter/enhancer regions and regulate differentgenes. GO analysis revealed that a large number of neural-related genes were induced by E�KTS but not by EþKTS(Supplementary Table S1 and Fig. 3A). This was surprisingbecause EWS–WT1 has never been shown to regulate neuralgenes. However, it is well established that most primaryDSRCTs express neural markers such as neuron-specific eno-lase and S100 protein (3). GO analysis revealed that theexpression of EþKTS resulted in a repression of genes involvedin DNA replication and repair pathways (SupplementaryTable S1).

Neural genes are overexpressed in primary DSRCTTo determine whether neural genes are enriched in primary

DSRCT, we analyzed the expression profile of 28 primaryDSRCT performed previously in comparison with four othertumor types (31). GO analysis of genes that are upregulated(>2-fold) in DSRCT compared with either Ewing sarcoma (ES),alveolar rhabdomyosarcoma (ARMS), or alveolar soft partsarcoma (ASPS) revealed an enrichment of neural pathways(Supplementary Table S2), but not when compared withsynovial sarcoma (SS). The enrichment of neural pathways

Figure 2. E�KTS induces cellgrowth arrest in MEFs. A, E�KTSand EþKTSMEFs were grownwithor without 1 mmol/L 4-HT and cellnumber was counted daily. Threeindependent experiments wereperformed in triplicates. B, whole-cell lysates fromCreERandE�KTSMEFs grown with or without 4-HTwere analyzed by immunoblottingwith the indicated antibodies.






was not evident by genes that were repressed in DSRCT (datanot shown). We next examined the expression of all the geneslisted in the identified GO neural pathways in 137 primarytumors. Remarkably, a large number of these neural geneswereuniquely overexpressed in 28 DSRCT compared with othertumors (Supplementary Fig. S3 and Supplementary Table S2).A smaller subset of neural genes was also uniquely overex-pressed in SS, which might explain the failure of our GOanalysis to reveal neural pathway enrichment when DSRCTwas compared with SS. Notably, a subset of neural genes thatwere highly activated by E�KTS in MEFs was also enriched inprimary DSRCT, including ASCL1, PLXNB1 (Plexin B1), andNTRK3 (neurotrophic tyrosine kinase, receptor 3; Fig. 3B).These results suggest that neural gene expression in DSRCTmight be directly regulated by E�KTS.

ASCL1 is directly activated by E�KTSASCL1 is one of the neural reprogramming factors that

directly converts fibroblasts into neurons (32, 33). Given theenrichment of neural genes in DSRCT, we next determinedwhether E�KTS directly activated ASCL1 transcription. Ofnote, 4-HT–mediated the expressionof E�KTS, but not EþKTSor CreER, resulted in amassive increase inASCL1mRNA (>150-fold) and protein expression in MEFs (Fig. 4A and B). Theexpression of E�KTS in a heterologous osteosarcoma cells(U2OS) also resulted in a robust induction of ASCL1 comparedwith EþKTS or empty vector control (Supplementary Fig. S2B).ASCL1 expressionwasmost abundant in a DSRCT cell line, JN–

DSRCT-1, as compared with U2OS or three ES cell lines (Fig.4C). Depletion of EWS–WT1 in JN–DSRCT-1 cells with lenti-viruses containing shRNAs against the 30 region of WT1(shWT1-2 or shWT1-3 but not with shWT1-1) resulted in asuccessful knockdown of EWS–WT1 (Fig. 4D). Concomitantly,silencing EWS–WT1 led to almost complete inhibition ofASCL1 expression, but not in controls. We note that endoge-nous WT1 is not expressed at detectable levels in JN–DSRCT-1cells by immunoblotting (data not shown). Collectively, theseresults demonstrate that E�KTS is directly responsible for theASCL1 transcription in MEFs and JN–DSRCT-1 cells.

EWS–WT1 or ASCL1 expression is critical for DSRCT cellsurvival

EWS–WT1 is the defining oncogene in DSRCT but it is notknown whether continued expression of EWS–WT1 is neces-sary to sustain tumor cell growth. Depletion of EWS–WT1 orASCL1 by two independent shRNAs in JN–DSRCT-1 cellsresulted in a complete loss of tumor cell growth as revealedby colony formation assay (Fig. 4E–G). These results suggestthat persistent expression of EWS–WT1 is required for tumorcell growth. Interestingly, the expression of ASCL1 was alsoessential for DSRCT tumor cell growth.

Identification of E�KTS–responsive elements in theASCL1 promoter

To identify the regulatory sequences responsive to E�KTS,we tested a proximal (1.2 kb) human ASCL1 promoter in a

Figure 3. E�KTS activates neural genes in MEFs and in primary tumors. A, a heatmap of significantly altered genes in neural pathways in E�KTS–expressingMEFs. B, a heatmap of some neural genes that are enriched in primary DSRCT (n ¼ 28) compared with other tumors (31). ARMS, (n ¼ 23); ES, (n ¼ 28); SS,(n ¼ 46); and ASPS, (n ¼ 12).

Kang et al.





luciferase reporter assay. Expression of E�KTS, but notEþKTS, in HEK293 cells resulted in a concomitant increasein the ASCL1 promoter–driven luciferase activity in a dose-dependent manner (Fig. 5A). Transfection of the ASCL1 pro-moter–reporter in JN–DSRCT-1 cells also resulted in a modestbut significant induction of luciferase reporter compared withU2OS or A4573 cells, whereas a small (1.5-fold) increase wasobserved in CHP100 cells (Supplementary Fig. S4A). Inspectionof human ASCL1 proximal promoter sequences (�1,043 toþ233) revealed a presence of 12 potential GC-rich E�KTS–binding sites (E�KTS–BS): two upstream (between �951 and�820) and 10within 400 bp of the transcriptional start site (Fig.5B). ChIP analysis of JN–DSRCT-1 cells with an antibodyagainst the C-terminus of WT1 demonstrated that EWS–WT1is mostly bound to the proximal (�408 to �56) ASCL1 pro-moter but not in the region (�765 to �384) harboring noE�KTS–BS. A weak recruitment was detected in the upstreamregion (�1,078 to �743; Fig. 5B). Consistent with this, the D2promoter (�406 toþ233) containing the 10 potential E�KTS–BS was fully responsive to E�KTS as the 1.2-kb promoter

(Fig. 5C). Alignment of the human and mouse ASCL1 proximalpromoter regions (�400 toþ1) revealed a high conservation ofthe entire region (86% identity), including the multiple poten-tial E�KTS–BS (Supplementary Fig. S4B).

To determine the precise E�KTS–responsive elements in theASCL1 promoter, we introduced substitution mutations at fourdistal (M1), sixproximal (M2),orall10 (M3)potentialE�KTS–BSin theD2 promoter (Supplementary Fig. S4C) and tested them inreporter assays. Destroying the four distal sites (M1) resulted inabout 88% reduction in the luciferase activity, whereas abolish-ing the six proximal sites (M2) resulted in 95% inhibition ofE�KTS transcription (Fig. 5D). The M3 promoter, whichdestroyed all E�KTS–BS, completely (99%) lost the ability tomediate transcription by E�KTS, demonstrating that multipleE�KTS–BS are necessary for the full activation by E�KTS.

Partial reprogramming of fibroblasts to neuron-likecells by E�KTS

ASCL1, along with POU3F2 (POU domain, class 3, transcrip-tion factor 2, also called BRN2) and MYT1L (Myelin

Figure 4. E�KTS activates ASCL1expression. A, MEFs cultured withor without 1 mmol/L 4-HT for 24hourswere analyzed for expressionof Ascl1 by qRT-PCR. Threeindependent experiments wereperformed in triplicates; �, P < 0.05;Student t test. B, whole-cell lysatesfromCreERor E�KTSMEFs grownwith or without 4-HT wereimmunoblotted with anti-ASCL1 oranti-actin. C, U2OS, CHP100,A4573, RD–ES, and JN–DSRCT-1cells were immunoblotted withanti-WT1, anti-ASCL1, or anti-actin. Arrow, EWS/WT1; �,nonspecific band. Note that EWS–WT1 migrates higher (62 kDa) inJN–DSRCT-1 cells due to adifferent EWS translocationbreakpoint (24). D, JN–DSRCT-1cells were transduced withlentivirus carrying threeindependent shWT1 or control andanalyzed by immunoblotting withanti-EWS, anti-ASCL1, or anti-actin. E, JN–DSRCT-1 cells weretransduced with lentivirusescarrying control, two independentshWT1, or two independentshASCL1 and the total numberof colonies was counted andcompared with the control(set to 100%). Three independentexperiments were performed intriplicate; �,P < 0.05; Student t test.F, representative images fromEareshown. G, JN–DSRCT-1 cells weretransduced with lentivirusescarrying scrambled or twoindependent shASCL1 andanalyzed by immunoblotting withanti-ASCL1 or anti-actin.






transcription factor 1-like), has been shown to directly repro-gram fibroblasts into excitatory neurons (32). Direct repro-gramming of fibroblasts into other types of neurons, such asdopaminergic or motor neuron, can be achieved by a combi-nation of different neural transcription factors, but ASCL1 isrequired for nearly all direct neural reprogramming (33).Furthermore, ASCL1, when expressed alone, has been shownto induce a partial reprogramming of fibroblasts into neuron-like cells (32). Thus, we tested whether E�KTS, which directlyactivates ASCL1 expression, could lead to a partial neuralreprogramming of MEFs. Following induction of E�KTS orEþKTS with 4-HT for 2 days, cells were cultured in neuralinduction media [DMEM/F12 þ N2 supplement (1�)] for 10days (fresh media given every 2 days). Remarkably, immatureneuron-like cells appeared in the E�KTS–expressing MEFs

displaying elongated bi- or tripolar projections that resem-bled neurites (Fig. 6A). Immunofluorescence with antibodiesagainst neuron-specific b-III-tubulin (TUBB3 recognized byTuj1 antibody) and microtubule-associated protein 2(MAP2) revealed that about 20% of cells expressed theseneural markers in E�KTS–expressing cells (Fig. 6B andSupplementary Fig. S5A). There were less than 5% ofTuj1-positive cells in E�KTS MEFs without 4-HT and noTuj1-positive cells appeared in EþKTS MEFs regardless of 4-HT. Extended neural induction period (up to 18 days) did notincrease the number of neuron-like cells nor result in morecomplex neuronal morphology in E�KTS MEFs (data notshown). These results demonstrate that E�KTS expressionleads to a partial neural reprogramming of MEFs, likely viaASCL1.

Figure 5. E�KTS activates ASCL1 via multiple E�KTS–responsive elements. A, HEK293 T cells were transfected with empty vector or increasing amounts(0.1, 0.2, and 0.6 mg) of E�KTS or EþKTS along with the 1.2-kb ASCL1 promoter construct and luciferase activity was measured. Three independentexperiments were performed in triplicates; ��, P < 0.01; ��, P < 0.001; Student t test. B, ChIP analysis of the ASCL1 promoter in JN–DSRCT-1 cells.Cross-linked chromatin was immunoprecipitated with rabbit IgG or anti-WT1 (C19) antibody and was amplified by PCR with the indicated primers (arrows).RNA Pol II antibody was used as a positive control. Circles, putative E�KTS–binding sites. TSS, transcription start site (þ1). C, ASCL1 promoter–reporterdeletion constructs or a pGL3-Basic (control) were transfected in JN–DSRCT-1 cells and luciferase activity was measured. D, the D2 promoter ormutated (M1,M2, andM3) promoter–reporter constructswere transfected in JN–DSRCT-1 cells and luciferase activitywasmeasured. TheX-circles representmutated E�KTS–binding sites. Three independent experiments were performed in triplicates; ��, P < 0.001.

Kang et al.





Partial differentiation of JN–DSRCT-1 cells to neuron-like cellsWe next examined whether JN–DSRCT-1 cells could be

differentiated towardneural lineage by the endogenous expres-sion of ASCL1. When JN–DSRCT-1 cells were cultured with theneuronal induction media for 3 days, about 30% of cellsdisplayed short, but occasionally long, bi- or tripolar Tuj1-positive neurite-like projections (Fig. 6C andD). TUBB3 expres-sionwas only observed inN2-treated JN–DSRCT-1 cells but notin the absence of N2 or in U2OS cells with or without N2 (Fig.6C). Induction of TUBB3 was observed as early as 24 hours andcontinued to increase to 72 hours (Fig. 6E). Notably, siRNA-mediated acute depletion of EWS–WT1 led to nearly completeinhibition of Tuj1-positive neurite projections and to markeddecrease in TUBB3 expression in JN–DSRCT-1 cells (Supple-mentary Fig. S5B–S5D), demonstrating that EWS–WT1 isresponsible for the partial neural differentiation.

DiscussionIn this study, we report the generation of two conditional

EWS–WT1 knockin mice, each expressing a specific isoformunder the control of endogenous EWS transcriptional network.A previous study has shown that the expression of E�KTS, butnot EþKTS, was sufficient to transform NIH3T3 cells (17).Consistent with this, the expression of EþKTS in MEFs had noeffects on cell growth and mice expressing EþKTS did notdevelop any spontaneous tumor (data not shown). However,our study does not preclude a supporting role for EþKTS inDSRCT tumorigenesis. In contrast, the expression of E�KTSresulted in a rapid cessation of cell growth and rapid death ofmice. Though the exact cause of death is unknown, Ki67immunostaining showed that the expression of E�KTS causeda significant reduction in the number of proliferating cells incrypts of intestinal epithelium (Supplementary Fig. S6), sug-gesting a block in proliferation of gastrointestinal epithelium

Figure 6. E�KTS induces partialneural reprogramming. A, E�KTSand EþKTS MEFs cultured withor without 1 mmol/L 4-HT werecultured for 10 days in N2-containing media. Cells wereimmunostained with Tuj1 antibodyand DAPI; scale bar, 20 mm. B, thetotal number of Tuj1-positive cellswas counted from 26 randomlyselected fields (n > 790). C,immunostaining of JN–DSRCT-1and U2OS cells with Tuj1 antibodyfollowing 3 days of culture with orwithout N2 media; scale bar, 20mm. D, the total number of Tuj1-positive JN–DSRCT-1 cells wascounted from 26 randomlyselected fields (n > 290). E, the N2-treated JN–DSRCT-1 cells wereimmunoblotted with Tuj1 or anti-Lamin A/C.






and malabsorption might be responsible. In support ofthis, mice expressing E�KTS lost weight by an average of3.9 g (n ¼ 4) in 4 days, whereas the control mice gained anaverage of 0.8 g (n ¼ 3).

Othermousemodels expressing oncogenic fusion genes alsoshowed lethal effects (25, 34–39), suggesting that expression ofchimeric oncogenes in primary cells often leads to negativecellular effects. Therefore, we postulate that in DSRCT, EWS–WT1 translocationmust occur in a permissive cell type(s) or inthe context of permissive microenvironment that will allowpersistent EWS–WT1 expression. These cells will likely be thetumor cells of origin inDSRCT. An analogous situation exists inES in which the expression of EWS–FLI1 is toxic to most celltypes (25, 40, 41) but its expression is permitted in mesenchy-mal stem cells (42, 43) or in neural crest stem cells (44).Therefore, identifying a cell type that tolerates E�KTS expres-sion is critical to understanding DSRCT tumorigenesis and forthe development of a mouse model.

One of the hallmarks of DSRCT is that tumor cells expressneural and other lineage markers (3), but the rationale for themultilineage expression has not been provided. Our findingsdemonstrate that E�KTS induces a number of neural genes,including ASCL1, which will likely induce additional neural-related genes. Thus, this study provides a molecular rationalefor the neural gene expression in DSRCT.

Before this study, it was not known whether continuedexpression of EWS–WT1 is required for tumor cell growth.Our findings showed that persistent EWS–WT1 expression isessential for tumor cell growth, making it an ideal target fortherapy. Surprisingly, depletion of ASCL1 also led to inhibitionof JN–DSRCT-1 cell growth, revealing another potential ther-apeutic target. When JN–DSRCT-1 cells were stimulated toundergo neural differentiation, however, ASCL1 expressionwas further induced along with its target genes (Supplemen-tary Fig. S5E) and partial neural differentiation ensued. Thus,ASCL1 seems to have dual functions: (i) maintaining tumor cellgrowth under a proliferative signal and (ii) promoting neuraldifferentiation under a neural differentiation signal.

Direct reprogramming of fibroblasts into neurons requiresthe expression of three factors: ASCL1, POU3F2, and MYT1L

(32). When we attempted to induce full neural reprogram-ming in JN–DSRCT-1 cells by expressing POU3F2, MYT1L, orNEUROD1, either alone or in combination, expression of anyof these factors under neural differentiation conditionresulted in a rapid cell death (data not shown), demonstrat-ing that JN–DSRCT-1 cells are incompatible with full neuralreprogramming. However, a partial neural differentiationwas achieved in JN–DSRCT-1 cells, likely due to E�KTS–activated ASCL1. These findings suggest a possibility thatinduction of partial neural differentiation in DSRCT patientsmight lead to an arrest or delay of aggressive tumor cellgrowth, providing a potentially novel and effective therapyagainst this incurable disease.

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

Authors' ContributionsConception and design: H.-J. Kang, S.B. LeeDevelopment of methodology: H.-J. Kang, S.B. LeeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.-J. KangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.-J. Kang, J.H. Park, W. Chen, S.I. Kang, K. Moroz,M. Ladanyi, S.B. LeeWriting, review, and/or revision of the manuscript: H.-J. Kang, M. Ladanyi,S.B. LeeAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H.-J. Kang, J.H. Park, S.B. LeeStudy supervision: H.-J. Kang, S.B. Lee

AcknowledgmentsThe authors thank Cuiling Li and Chuxia Deng (NIDDK Mouse Knockout

Core) for the mouse ES cell injection and Chithra Keembiyehetty (NIDDKGenomics Core) for performing the microarray analysis and Yun-Ping Wu(NIDDK) for confocal microscopy.

Grant SupportThis research was supported in part by the Intramural Research Program of

the NIH, NIDDK (S.B. Lee), and by the Tulane Startup Fund (S.B. Lee).The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 26, 2013; revised May 5, 2014; accepted May 19, 2014;published OnlineFirst June 16, 2014.

References1. Gerald WL, Miller HK, Battifora H, Miettinen M, Silva EG, Rosai J.

Intra-abdominal desmoplastic small round-cell tumor. Report of 19cases of a distinctive type of high-grade polyphenotypic malig-nancy affecting young individuals. Am J Surg Pathol 1991;15:499–513.

2. Gerald WL, Haber DA. The EWS–WT1 gene fusion in desmoplasticsmall round-cell tumor. Semin Cancer Biol 2005;15:197–205.

3. Gerald WL, Ladanyi M, de Alava E, Cuatrecasas M, Kushner BH,LaQuaglia MP, et al. Clinical, pathologic, and molecular spectrum oftumors associated with t(11;22)(p13;q12): desmoplastic smallround-cell tumor and its variants. J Clin Oncol 1998;16:3028–36.

4. Ladanyi M, Gerald W. Fusion of the EWS and WT1 genes in thedesmoplastic small round-cell tumor. Cancer Res 1994;54:2837–40.

5. Gerald WL, Rosai J, Ladanyi M. Characterization of the genomicbreakpoint and chimeric transcripts in the EWS–WT1 gene fusion ofdesmoplastic small round-cell tumor. Proc Natl Acad Sci U S A1995;92:1028–32.

6. Li H, Watford W, Li C, Parmelee A, Bryant MA, Deng C, et al. Ewingsarcoma gene EWS is essential for meiosis and B lymphocyte devel-opment. J Clin Invest 2007;117:1314–23.

7. Cho J, Shen H, YuH, Li H, Cheng T, Lee SB, et al. Ewing sarcoma geneEws regulates hematopoietic stem cell senescence. Blood 2011;117:1156–66.

8. Azuma M, Embree LJ, Sabaawy H, Hickstein DD. Ewing sarcomaprotein ewsr1 maintains mitotic integrity and proneural cell survival inthe zebrafish embryo. PloS ONE 2007;2:e979.

9. Park JH, Kang HJ, Kang SI, Lee JE, Hur J, Ge K, et al. A multifunctionalprotein, EWS, is essential for early brown fat lineage determination.Dev Cell 2013;26:393–404.

10. Kim KY, Hwang YJ, Jung MK, Choe J, Kim Y, Kim S, et al. Amultifunctional protein EWS regulates the expression of Drosha andmicroRNAs. Cell Death Differ 2014;21:136–45.

11. Dutertre M, Sanchez G, De Cian MC, Barbier J, Dardenne E, GratadouL, et al. Cotranscriptional exon skipping in the genotoxic stressresponse. Nat Struct Mol Biol 2010;17:1358–66.

Kang et al.





12. Paronetto MP, Minana B, Valcarcel J. The Ewing sarcoma proteinregulates DNA damage-induced alternative splicing. Mol Cell2011;43:353–68.

13. Ng KP, Potikyan G, Savene RO, Denny CT, Uversky VN, Lee KA.Multiple aromatic side chains within a disordered structure are criticalfor transcription and transforming activity of EWS family oncoproteins.Proc Natl Acad Sci U S A 2007;104:479–84.

14. May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB,et al. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a morepotent transcriptional activator and is a more powerful transforminggene than FLI-1. Mol Cell Biol 1993;13:7393–8.

15. Lee SB, Haber DA. Wilms tumor and the WT1 gene. Exp Cell Res2001;264:74–99.

16. Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE.Alternative splicing and genomic structure of the Wilms tumor geneWT1. Proc Natl Acad Sci U S A 1991;88:9618–22.

17. Kim J, Lee K, Pelletier J. The desmoplastic small round-cell tumort(11;22) translocation produces EWS/WT1 isoforms with differingoncogenic properties. Oncogene 1998;16:1973–9.

18. Lee SB, Kolquist KA, Nichols K, Englert C, Maheswaran S, Ladanyi M,et al. The EWS–WT1 translocation product induces PDGFA in desmo-plastic small round-cell tumour. Nat Genet 1997;17:309–13.

19. Wong JC, Lee SB, Bell MD, Reynolds PA, Fiore E, Stamenkovic I, et al.Induction of the interleukin-2/15 receptor beta-chain by the EWS–WT1translocation product. Oncogene 2002;21:2009–19.

20. Palmer RE, Lee SB, Wong JC, Reynolds PA, Zhang H, Truong V,et al. Induction of BAIAP3 by the EWS–WT1 chimeric fusion impli-cates regulated exocytosis in tumorigenesis. Cancer Cell 2002;2:497–505.

21. Ito E, Honma R, Imai J, Azuma S, Kanno T, Mori S, et al. A tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated anti-gen 1, is induced by the Ewing's sarcoma-Wilms' tumor 1 fusionprotein of desmoplastic small round-cell tumor. Am J Pathol2003;163:2165–72.

22. Li H, Smolen GA, Beers LF, Xia L, Gerald W, Wang J, et al. Adenosinetransporter ENT4 is a direct target of EWS/WT1 translocation productand is highly expressed in desmoplastic small round-cell tumor. PLoSONE 2008;3:e2353.

23. Reynolds PA, Smolen GA, Palmer RE, Sgroi D, Yajnik V, Gerald WL,et al. Identification of a DNA-binding site and transcriptional target forthe EWS–WT1(þKTS) oncoprotein. Genes Dev 2003;17:2094–107.

24. Nishio J, Iwasaki H, Ishiguro M, Ohjimi Y, Fujita C, Yanai F, et al.Establishment and characterization of a novel human desmoplasticsmall round-cell tumor cell line, JN–DSRCT-1. Lab Invest 2002;82:1175–82.

25. Sohn EJ, Li H, Reidy K, Beers LF, Christensen BL, Lee SB. EWS/FLI1oncogene activates caspase 3 transcription and triggers apoptosis invivo. Cancer Res 2010;70:1154–63.

26. Sauer B. Manipulation of transgenes by site-specific recombination:use of Cre recombinase. Methods Enzymol 1993;225:890–900.

27. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast growthfactor receptor 3 is a negative regulator of bone growth. Cell1996;84:911–21.

28. Huang da W, Sherman BT, Lempicki RA. Systematic and integrativeanalysis of large gene lists using DAVID bioinformatics resources. NatProtoc 2009;4:44–57.

29. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichmenttools: paths toward the comprehensive functional analysis of largegene lists. Nucleic Acids Res 2009;37:1–13.

30. Kim HS, Kim MS, Hancock AL, Harper JC, Park JY, Poy G, et al.Identification of novel Wilms' tumor suppressor gene target genesimplicated in kidney development. J Biol Chem 2007;282:16278–87.

31. FilionC,Motoi T,OlshenAB, LaeM,Emnett RJ,GutmannDH, et al. TheEWSR1/NR4A3 fusion protein of extraskeletal myxoid chondrosar-coma activates the PPARG nuclear receptor gene. J Pathol2009;217:83–93.

32. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, WernigM. Direct conversion of fibroblasts to functional neurons by definedfactors. Nature 2010;463:1035–41.

33. Yang N, Ng YH, Pang ZP, Sudhof TC, Wernig M. Induced neuronalcells: how tomake and define a neuron. Cell StemCell 2011;9:517–25.

34. CodringtonR,Pannell R, Forster A, DrynanLF, Daser A, LobatoN, et al.The Ews–ERG fusion protein can initiate neoplasia from lineage-committed haematopoietic cells. PLoS Biol 2005;3:e242.

35. Haldar M, Hancock JD, Coffin CM, Lessnick SL, Capecchi MR. Aconditional mouse model of synovial sarcoma: insights into a myo-genic origin. Cancer Cell 2007;11:375–88.

36. Higuchi M, O'Brien D, Kumaravelu P, Lenny N, Yeoh EJ, Downing JR.Expression of a conditional AML1-ETO oncogene bypasses embry-onic lethality and establishes a murine model of human t(8;21) acutemyeloid leukemia. Cancer Cell 2002;1:63–74.

37. Keller C, Hansen MS, Coffin CM, Capecchi MR. Pax3:Fkhr interfereswith embryonic Pax3 and Pax7 function: implications for alveolarrhabdomyosarcoma cell of origin. Genes Dev 2004;18:2608–13.

38. Lin PP, Pandey MK, Jin F, Xiong S, Deavers M, Parant JM, et al. EWS–FLI1 induces developmental abnormalities and accelerates sarcomaformation in a transgenicmousemodel. Cancer Res 2008;68:8968–75.

39. Lagutina I, Conway SJ, Sublett J, Grosveld GC. Pax3-FKHR knock-inmice show developmental aberrations but do not develop tumors. MolCell Biol 2002;22:7204–16.

40. Deneen B, Denny CT. Loss of p16 pathways stabilizes EWS/FLI1expression and complements EWS/FLI1 mediated transformation.Oncogene 2001;20:6731–41.

41. Lessnick SL, Dacwag CS, Golub TR. The Ewing's sarcoma oncopro-tein EWS/FLI induces a p53-dependent growth arrest in primaryhuman fibroblasts. Cancer Cell 2002;1:393–401.

42. Castillero-Trejo Y, Eliazer S, Xiang L, Richardson JA, Ilaria RL Jr.Expression of the EWS/FLI-1 oncogene in murine primary bone-derived cells Results in EWS/FLI-1-dependent, ewing sarcoma-liketumors. Cancer Res 2005;65:8698–705.

43. Riggi N, Cironi L, Provero P, Suva ML, Kaloulis K, Garcia-Echeverria C,et al. Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res 2005;65:11459–68.

44. von LevetzowC, Jiang X, Gwye Y, von LevetzowG, Hung L, Cooper A,et al. Modeling initiation of Ewing sarcoma in human neural crest cells.PLoS ONE 2011;6:e19305.






2014;74:4526-4535. Published OnlineFirst June 16, 2014.Cancer Res Hong-Jun Kang, Jun Hong Park, WeiPing Chen, et al. ASCL1 and Promotes Neural Differentiation

WT1 Oncoprotein Activates Neuronal Reprogramming Factor−EWS

Updated version

10.1158/0008-5472.CAN-13-3663doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/06/16/0008-5472.CAN-13-3663.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/74/16/4526.full#ref-list-1

This article cites 44 articles, 15 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/74/16/4526To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-3663

http://cancerres.aacrjournals.org/content/suppl/2014/06/16/0008-5472.CAN-13-3663.DC1

http://cancerres.aacrjournals.org/content/74/16/4526.full#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/74/16/4526


Documents

EWS WT1 Oncoprotein Activates Neuronal Reprogramming ... · Tumor and Stem Cell Biology EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation