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Cancer Letters xxx (2007) xxx–xxx

www.elsevier.com/locate/canlet

Mini-review

The Biology of Ewing sarcoma

Nicolo Riggi, Ivan Stamenkovic *

Division of Experimental Pathology, Institute of Pathology, University of Lausanne, Switzerland

Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland

Received 22 August 2006; received in revised form 5 December 2006; accepted 12 December 2006

Abstract

Sarcomas account for less than 10% of all human malignancies that are believed to originate from as yet poorly definedmesenchymal progenitor cells. They constitute some of the most aggressive adult and childhood cancers in that they have ahigh metastatic proclivity and are typically refractory to conventional chemo- and radiation therapy. Ewing’s sarcoma is amember of Ewing’s family tumors (ESFT) and the second most common solid bone and soft tissue malignancy of childrenand young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generatesfusion of the 5 0 segment of the EWS gene with the 3 0 segment of the ETS family gene FLI-1. The resulting EWS-FLI-1fusion protein is believed to behave as an aberrant transcriptional activator that contributes to ESFT development byaltering the expression of its target genes in a permissive cellular environment. Although ESFTs are among the best studiedsarcomas, the mechanisms involved in EWS-FLI-1-induced transformation require further elucidation and the primarycells from which ESFTs originate need to be identified. This review will highlight some of the most recent discoveriesin the field of Ewing sarcoma biology and origins.� 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ewing sarcoma; EWS-FLI-1; Transformation; Mesenchymal progenitor cells

1. Introduction

Ewing sarcoma, often referred to as Ewing’s sar-coma family tumors (ESFT) is the second mostcommon bone malignancy after osteosarcoma, aris-ing in children and young adults with a peak inci-dence at age 15. The frequency of Ewing sarcomais 1–3 per million per year in the Western hemi-sphere, with a slight predominance in males.Although most Ewing sarcomas occur in bone and

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* Corresponding author. Tel.: +41 21 314 7136; fax: +41 21 3147110.

E-mail address: Ivan.Stamenkovic@chuv.ch (I. Stamenkovic).

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especially in the pelvis, the diaphyseal regions ofthe long bones and bones of the chest wall, 15%of primary ESFT may arise in a variety of extraos-seous sites, including deep soft paravertebral, tho-racic and proximal limb tissues, kidney, bladder,lung, prostate and the meninges [1]. Similar to sev-eral other sarcomas, ESFT displays an aggressivebehavior with a tendency toward recurrence follow-ing resection and pronounced proclivity towardearly hematogenous metastasis primarily to thelung, bone and bone marrow. Lymph node, liverand brain metastases are typically rare. Currently,Ewing sarcomas are treated with a combination ofsurgery, radiation and chemotherapy, but despite

reserved.

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these multimodal approaches the survival rateremains poor: 50% at 5 years (25% when metastasisare present at diagnosis) and less than 30% at10 years.

2. Histology

Morphologically, Ewing sarcoma is composed ofsheets of small round cells with a high nuclear tocytoplasmic ratio and is often classified by patholo-gists into a group of small round blue cell tumorsthat include neuroblastoma, alveolar rhabdomyo-sarcoma and lymphoblastic lymphoma. The cellstypically have scant, weakly eosinophilic cytoplasmthat usually contains glycogen in the form ofperiodic-acid-Schiff-positive, diastase degradablegranules, and round nuclei with evenly distributedchromatin and little mitotic activity (Fig. 1). Immu-nohistochemical analysis has shown that in morethan 90% of cases Ewing sarcoma cells express theadhesion receptor CD99, commonly associated withlymphoid cells and believed to play a role inleukocyte transmigration of the endothelium [2].Depending on the degree of neuroectodermal differ-entiation, Ewing sarcoma cells may also expressneural cell markers, including neural-specific eno-lase (NSE), S-100, synaptophysin and CD57 [1].Ewing sarcoma cells are reactive with anti-vimentinantibodies and, in about 20% of cases, with anti-cytokeratin antibodies. Some of these tumors mayexpress neurofilaments as well.

Immunohistochemistry is frequently required forthe differential diagnosis of small blue round cell

Fig. 1. Histology of Ewing sarcoma, showing the typical small,poorly differentiated, round cell phenotype.

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tumors. Ewing sarcoma and lymphoblastic lympho-ma both express CD99, but the latter also expressesCD45 whereas Ewing sarcoma does not. Neuroblas-toma cells are NSE and S-100 positive but unliketheir ESFT counterparts, they are vimentin-negativeand neurofilament-positive [1]. Alveolar rhabdo-myosarcoma cells may express CD99, but they alsoexpress desmin, myogenin and MyoD1, whichESFT cells typically lack. Perhaps the most difficultdifferential diagnosis is between ES and poorlydifferentiated small cell synovial sarcoma, since thelatter can express CD99 and unlike its more differ-entiated forms may lack cytokeratins. Because ofthe lack of specificity of these markers, moleculargenetic approaches are required to make the defini-tive distinction.

3. Molecular genetics

Based on the genetic mutations associated withtheir development, sarcomas are subdivided intotwo distinct classes. One class is composed of tumorsbearing complex karyotypic abnormalities with noparticular pattern. The second class, which includesEwing sarcoma, encompasses tumors associatedwith unique chromosomal translocations that giverise to specific fusion genes. Ewing’s sarcoma is in85% of cases associated with the translocationt(11;22)(q24;q12), which leads to the formation ofthe EWS-FLI-1 fusion gene (Fig. 2) [3]. In another10–15% of cases the translocation t(21;12)(22;12)generates the EWS-ERG fusion, whereas the remain-ing 1–5% of cases may harbor one of several possibletranslocations, each resulting in a fusion gene con-taining a portion of the EWS gene and a member

Fig. 2. Schematic represenation of the EWS-FLI-1 fusionresulting from the t(22;11) translocation. The TET familyassociated RRM domain, the ETS DNA binding domain (ETS-DBD) and the amino terminal transactivation domain (ATA) areindicated. The fusion gene can vary depending on whether exons5–9 or 6–9 of FLI-1 are included.

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Table 1Summary of the different fusions and their frequency in Ewingsarcoma

Ewing’s sarcoma translocation

EWS member ETS member Frequency (%)

EWS FLI1 85EWS ERG 10EWS ETV1 <1EWS ETV4 <1EWS FEV <1TLS ERG <1

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of the ets family of transcription factors (Table 1). Inaddition to providing the key to understanding thebiology of Ewing sarcoma, these translocations con-stitute its most reliable diagnostic criterion.

3.1. The EWS gene

In sarcomas, the most studied fusion gene, largelydue to its predominance in ESFT, has been EWS-

FLI-1. EWS is related to TLS/FUS, a gene that isfused to the CHOP transcription factor by chromo-somal translocation in myxoid liposarcoma [4].Together with TAFII68, a TBP-associated factorfound in a subset of transcription complexes, thesegenes form the TET family, sharing a characteristic87-amino acid RRM/RNP-CS domain that isthought to be implicated in protein–RNA binding[5]. TET proteins have a variable number of RGG(arginine–glycine–glycine) repeats that are believedto promote binding to RNA, and a glutamine richN-terminal region that becomes fused to ETS genesin ESFT and a variety of genes encoding transcrip-tion factors in other human cancers [6].

Based on their structure and their ability to bindRNA, TET proteins are thought to participate intranscription and RNA processing. EWS has beenobserved to bind RNA in vitro [7] and together withTAFII68, EWS can form complexes with the basaltranscription factor TFIID, RNA polymerase IIand the coactivators CBP/p300 [8]. In addition totheir association with transcription factors and tran-scriptional regulators, both EWS and TLS/FUSinteract with splicing proteins [9,10] and to modu-late splicing. One hypothesis that has been put forthis that TET proteins may provide a link betweentranscription and mRNA processing by bindingcomponents of both the transcription and splicingmachinery.

EWS and TLS/FUS are ubiquitously expressedin mammalian cells, with a primarily nuclear locali-

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sation. However, when serum-starved cells are stim-ulated with fresh serum, EWS is observed totranslocate from the cytoplasm to the nucleus [11].Both EWS and TLS/FUS are substrates of proteinkinase C (PKC) and may undergo post translationalmodifications. PKC-mediated phosphorylation ofTLS/FUS results in its increased degradation bythe proteosome [12].

TET family members are highly conserved acrossspecies, suggesting important physiological roles.Inbred TLS/FUS�/� mice die at birth and displaya developmental block in B lymphocyte develop-ment and marked chromosomal instability [13].Outbred TLS/FUS-deficient mice survive into adult-hood but display impaired spermatogenesis in addi-tion to genomic instability and sensitivity toionizing radiation [14]. These observations suggestthat TLS/FUS plays a significant role in genomicstability. It is noteworthy that TET family membersconstitute a portion of fusion genes associated withnearly half of sarcomas that bear single chromo-somal translocations, including ESFT, clear cell sar-coma, desmoplastic small round cell tumor, myxoidchondrosarcoma, and myxoid liposarcoma [4]. Withone exception, namely, the expression of FUS-ERG

in acute lymphoblastic leukemia [15], EWS andTLS/FUS fusion genes are found only in sarcomas.

3.2. The FLI-1 gene

The FLI-1 gene was identified as the site of inser-tion of Friend’s murine leukemia virus [16] and wassubsequently shown to be proximal to the insertionsite of several other viruses [17]. It is composed of a5 0 and 3 0 ets domain, both of which have a helix–loop–helix structure, separated by a FLI-1-specificdomain (FLS) [18,19]. The 5 0 ets and FLS domainsform the amino terminal transcriptional activation(ATA) domain whereas the 3 0 ets domain containsthe DNA binding sequences. The 3 0 region encodesan 89-amino acid carboxyterminal transactivation(CTA) domain [18].

During development, FLI-1 is expressed in hema-topietic and endothelial cells and in the mesenchymederived from neural crest cells [20]. Similarly, in avi-an embryogenesis, the FLI-1 gene is expressed inneural crest cells that give rise to the developingmesenchyme, whereas in zebrafish embryos FLI-1expression is detected at sites of vasculogenesis[21]. In adult mammalian tissues, FLI-1 expressionis detected principally in hematopietic cells, lower

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expression levels being detected in non-hematopietictissues including the heart, lung and ovaries [16].

Knock-out studies in mice have shown thathomozygous germline deletions within the FLI-1locus result in embryonic lethality with intracranialhemorrhage, attesting in part to vascular abnorma-lites, and absence of megakaryocytes, indicatingdefective megakaryopoiesis [22]. Overexpression ofFLI-1 in transgenic mice resulted in immunedysregulation characterized by augmented B cellproliferation, hypergammaglobulinemia and anautoimmune-type, immune complex-mediated dis-order [23]. Together, these studies provide strongevidence that FLI-1 plays an important physiologi-cal role in hematopoiesis and vasculogenesis. Eluci-dation of the FLI-1 target gene repertoire thatmediates its physiological functions is still incom-plete, but several observations have helped providepotentially important clues as to the mechanismswhereby FLI-1 might participate in the regulationof hematopoiesis. Thus, FLI-1 has been shown tobind promoter sequences of glycoprotein IX, glyco-protein IIb (GpIIb) and the thrombopoietin receptor(MPL) genes, all of which are megakaryocyte-specif-ic [24,25]. In human erythroleukemic cells, transcrip-tion from these promoters is induced by FLI-1.

Because FLI-1 is a target of proviral integrationin F-MuLV-induced erythroleukemia and is trans-located in Ewing’s sarcoma to form a potentiallyoncogenic fusion gene, its putative role in transfor-mation has been the focus of numerous studies.Development of F-MuLV-induced leukemiasrequires insertional activation of FLI-1 as the initialgenetic event, followed by mutations in the TP53

gene [26]. Overexpression of FLI-1 has beenobserved to promote self renewal of erythroid pro-genitor cells at the expense of Epo-induced differen-tiation, consistent with a role in malignanttransformation [27].

Several additional properties of FLI-1 support itsimplication in transformation and oncogenesis.FLI-1 can repress retinoblastoma (Rb) proteinexpression at the transcriptional level, thereby pro-moting the G1 to S transition of the cell cycle [28].Its activation in erythroid cells is accompanied byinduction of Bcl2 expression and a correspondingenhancement of cell survival [27]. There are there-fore at least three, possibly inter-related mechanismswhereby FLI-1 might promote transformation andcarcinogenesis: promotion of cell survival, inductionof the cell cycle and aberrant transcription in asso-ciation with EWS.

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4. The effect of EWS-FLI-1 expression in tumor

development

A major impediment toward understanding sar-coma biology in general and in ESFT biology inparticular, is the lack of adequate transgenic animalmodels. Thus far, development of a transgenicEwing’s sarcoma model in mice has failed, probablybecause of the toxicity of EWS-FLI-1 and otherEWS-associated fusions in most primary cells.However, recent work using a conditional lym-phoid-specific EWS-ERG model of tumorigenesishas demonstrated that EWS-ERG expression inlineage-committed haematopoietic cells can initiateT-cell lymphomas [29]. The invertor knock-in strat-egy used to generate these tumors offers hope for thedevelopment of transgenic mouse models for boneand soft tissue tumors by circumventing transgenetoxicity [30]. There are currently two animal modelsof sarcoma associated with specific chromosomaltranslocations that recapitulate many of the featuresof their human counterparts. They include the con-ditional PAX3-FKHR knock-in model of alveolarrhabdomyosarcoma, where the fusion gene isexpressed in terminally differentiated skeletal musclecells [31], and the TLS/FUS-CHOP transgenicmodel of myxoid liposarcoma, where the ubiquitousexpression of the TLS/FUS-CHOP transgeneresulted in the exclusive generation of myxoid lipo-sarcoma-like tumors in their classical anatomicallocations [32].

In the absence of adequate transgenic mousemodels, two major approaches have been used toaddress the potential role of EWS-FLI-1 in thepathogenesis of Ewing sarcoma: exogenous expres-sion of the translocation in different cell types anddownregulation of EWS-FLI-1 in Ewing sarcomacell lines. Expression of EWS-FLI-1 in murineNIH-3T3 cells resulted in anchorage independentgrowth and accelerated tumorigenesis in immuno-compromized mice with a tumor phenotype reminis-cent of that of human Ewing sarcoma [33,34]. Theseobservations are consistent with the notion thatEWS-FLI-1 can enhance oncogenesis and that it islargely responsible for the histological characteris-tics associated with ESFT. Moreover, expressionof EWS-FLI-1 in non-ESFT tumor cells, includingneuroblastoma and alveolar rhabdomyosarcomacells, resulted in transdifferentiation with theappearance of Ewing sarcoma features, includingneural marker expression [35–37]. By contrast, thesame approach using Rat-1 cells [33], mouse

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embryonic fibroblasts (MEFs) [38] and human pri-mary fibroblasts [39] not only failed to induce trans-formation but resulted in growth arrest andapoptosis, underscoring the importance of the cellu-lar environment for EWS-FLI-1-mediatedoncogenesis.

Growth inhibitory effects of dominant negativeFLI-1 on Ewing sarcoma cell lines support thenotion that EWS-FLI-1 is implicated in ESFTdevelopment [40]. Studies by several groups havealso shown that antisense EWS-FLI-1 and EWS-FLI-1 siRNA expression in human Ewing sarcomacell lines result in decreased cell growth in vitro

and tumorigenicity in vivo [41].A possible explanation for these observations has

been provided by a recent study showing that EWS-FLI-1 increases the Skp2-mediated 26S proteasomedegradation, decreasing p27 protein stability andpreventing cell senescence [42].

5. Mechanism of action of EWS-FLI-1

Current opinion holds that EWS-FLI-1 as well asthe other EWS-ETS fusion proteins function as aber-rant transcription factors. This view is supported byobservations that EWS-ETS proteins localize to thenucleus, bind DNA in site-specific manner and pos-sess, in the EWS N-terminal domain, a powerful tran-scriptional activator that is severalfold more potentthan the corresponding native FLI-1 domain dis-placed as a result of the chromosomal translocation.

Molecular analysis has revealed that several EWS-ETS target gene promoters contain tandem bindingsites for Ets and AP-1 proteins. Ets family membersthat form fusion proteins with EWS, includingFLI-1, ERG and ETV1, were found to cooperativelybind these tandem elements with Fos-Jun whereasother Ets family members were not. C-terminaldomain mutants of EWS-FLI-1 that cannot bindDNA together with Fos-Jun were reported to losethe ability to transform 3T3 fibroblasts. These obser-vations suggest that the cooperation between EWS-FLI-1 and Fos-Jun is essential for at least some ofthe biological activities of the fusion protein [43].

In addition, recent evidence suggests that EWS-FLI-1 associates with other proteins that may influ-ence its function. Thus, a phage display library screenuncovered a EWS-FLI-1 binding peptide containinghomology to RNA helicase A (RHA). Subsequentexperiments identified RHA in a protein complexwith EWS-FLI-1 in ESFT cell lines and showed that

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RHA binds to known EWS-FLI-1 target gene pro-moters, enhancing EWS-FLI-1 function [44].

5.1. EWS-FLI-1 targets

A major goal toward understanding the mecha-nism whereby EWS-FLI-1 contributes to cell trans-formation has been to identify its putative targetgenes. Initial efforts using subtractive hybridizationidentified three genes that were induced by EWS-FLI-1 but not by FLI-1 alone: EAT-2, (an SH-2domain-containing protein), mE2-C (a cyclin-selec-tive ubiquitin ligase) and MFNG (manic fringe geneencoding a glycosyltransferase involved in somaticdevelopment) [45–47]. Forced expression of MFNG

enhanced tumorigenesis of NIH3T3 cells in immu-nodeficient mice but did not induce the small roundcell phenotype typical of ESFT. Several other stud-ies identified potential EWS-FLI-1 target geneswhose induction may be implicated in transforma-tion and/or tumor progression, including MYC

[48], ID2 [49,50], CCND1 [51] and PDGFC [52]. Inaddition, several potentially relevant genes werefound to be repressed by EWS-FLI-1, includingthose encoding p21 [53], p57kip [54], TGF-bRII[55,56] and IGFBP-3 [57]. Among these, only TGF-

BRII and IGFBP3 have been shown to be directEWS-FLI-1 targets and repression of TGFBRIIhas been convincingly associated with Ewing’s sar-coma cell proliferation [55]. Downregulation ofIGFBP3 in a Ewing’s sarcoma cell line, on the otherhand, has been shown to be associated with cell sur-vival. For most of the other candidate EWS-FLI-1target genes, it remains unclear whether they aredirectly or indirectly regulated by the fusion protein.It is also apparent that EWS-FLI-1 induces andrepresses genes in cell type-specific fashion, such thatit has been difficult to determine which of the candi-date targets are implicated in EWS-FLI-1-mediatedtransformation of permissive primary cells.

More recently identified putative EWS-FLI-1target genes include the orphan nuclear receptorDAX1, protein tyrosine phosphatase 1 (PTPL1),phospholipase D2 (PLD2) and the homeobox con-taining protein NKX2-2. DAX1 is upregulatedupon EWS-FLI-1 expression in different cell typesand is selectively expressed in Ewing’s sarcoma cells[58], where it may be implicated in generating ormaintaining the transformed phenotype [59]. Pro-tein tyrosine phosphatase 1 was found to be a directtarget of EWS-FLI-1 and to be highly expressed inESFT cells. Its potential implication in ESFT

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growth and the association between its suppressionand increased ESFT cell sensitivity to cytotoxicdrugs render it a candidate therapeutic target inESFT [60]. A siRNA approach using an ESFT cellline uncovered PLD2 as a potential EWS-FLI-1 tar-get. Down regulation of PLD2 as a result of EWS-FLI-1 siRNA expression resulted in reducedPDGF-mediated signaling and corresponding cellgrowth [61]. Induction of PLD2 but not PLD1 byEWS-FLI-1 was recently confirmed in a differentESFT cell line, and shown to be dependent on thedirect binding of the fusion protein to the PLD2promoter [62].

An elegant approach, consisting of the stableknock-down of EWS-FLI-1 in a Ewing sarcoma cellline, followed by introduction of exogenous EWS-FLI-1 cDNA, thereby providing an «inducible res-cue» system, revealed induction and repression ofnumerous genes, several of which have been identi-fied in expression profiling studies of ESFT [63].Several genes related to neural differentiation wereobserved to be upregulated by EWS-FLI-1 in thismodel, including NKX2-2, which plays a role in neu-ral development. Although it remains to be deter-mined whether the NKX2-2 gene is a direct orindirect target of EWS-FLI-1, its expression appearsto be required for EWS-FLI-1-mediated tumorgrowth promotion in vivo [63,64].

Chromatin immunoprecipitation (ChIP) experi-ments have yielded mixed results so far. One studyreported the discovery of numerous potentialEWS-FLI-1 targets using this approach [65] whereasanother study found an unacceptably high level ofbackground immunoprecipitation using commer-cially available anti-EWS-FLI-1 antibodies [63].

From the sum of these observations, it wouldappear that EWS-FLI-1 may participate in Ewingsarcoma pathogenesis by promoting at least twoset of events that synergize in tumor developmentand progression: cell proliferation and survival, byinducing among other candidate genes, PDGFC,

IGF1, MYC, CCND-1 and NKX2-2 and escapefrom apoptosis and growth inhibition, by repressingp21, p57kip, TGFbRII and IGFBP3. In addition,EWS-FLI-1 appears to play a critical role in induc-ing the ESFT small round cell phenotype.

6. The potential origin of Ewing sarcoma

From the preceding discussion, it appears obvi-ous that at least two key issues still remain to beaddressed in order to understand Ewing’s sarcoma

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biology: the identity of the cells from which ESFToriginate, and which presumably display permissive-ness for EWS-FLI-1-mediated transformation, andthe possibility that EWS-FLI-1 is the unique initiat-ing event in ESFT development. Although mutationof p53 and loss of p16INK4A/p14ARF have been doc-umented in ESFT, they occur in a minority oftumors, and in about 15–20% of ESFT, the onlydetected genetic event appears to be the t(11;22)chromosomal translocation leading to EWS-FLI-1expression. These observations are consistent withthe existence of a primary cell that can be trans-formed by EWS-FLI-1. Until recently, there hasbeen no indication as to the possible nature of sucha cell, other than the widely held view that sarcomasin general originate from undefined mesenchymalstem/progenitor cells [66]. The histological featuresof Ewing’s sarcoma suggest a poorly differentiatedtumor that has both mesenchymal and neuroecto-dermal features [1]. This has led to an as yet unre-solved debate as to the neuroectodermal ormesenchymal origin of these tumors.

Numerous efforts have been made to identify pri-mary cells that might undergo transformation as aresult of EWS-FLI-1 expression. However, initialresults were somewhat surprising. Thus, introduc-tion of EWS-FLI-1 into mouse embryonic fibroblasts(MEFs) resulted in cell cycle arrest and cell death,with the surviving MEFs losing EWS-FLI-1 expres-sion [38]. MEFs from p19ARF�/� mice transfectedwith EWS-FLI-1 were observed to maintain EWS-FLI-1 expression but did not form tumors in vivo

[38]. Loss of p53 also failed to induce tumorigenesisby MEFs expressing EWS-FLI-1 [38]. Only upontransformation with SV40-T antigen could MEFslacking p19ARF or p53 and expressing EWS-FLI-1form tumors in vivo with histological features thatresemble the human Ewing sarcoma phenotype [38].

Similar observations were made in hTERT-im-mortalized human primary fibroblasts where EWS-FLI-1 expression induced p53-mediated growtharrest and apoptosis [39]. Together, these studies sug-gest that fibroblasts are unlikely to provide an originof Ewing sarcoma. Moreover, as discussed above,most Ewing’s sarcomas appear to have a functionalp53 pathway and to retain p19ARF expression.

In an effort to identify a candidate primary cellfrom which ESFT originates, we expressed EWS-FLI-1 in murine cells with a variable spectrum ofplasticity, ranging from embryonic stem (ES) cellsand primary mesenchymal progenitor cells (MPC)to embryonic fibroblasts. Whereas ES cells and

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embryonic fibroblasts lost EWS-FLI-1 expression atthe protein level by 14 days following infection witha retrovirus containing the fusion gene, bonemarrow-derived MPCs maintained EWS-FLI-1expression for several months [67]. Mesenchymalprogenitor cells expressing EWS-FLI-1 displayedrobust upregulation of insulin-like growth factor 1(IGF-1) and the corresponding binding proteins 3and 5 (IGFBP-3 and IGFBP-5). Upon injection intomice, these cells formed tumors composedpredominantly of sheets of small round cells. Geneexpression profile analysis of tumor-derived cellsrevealed upregulation of known EWS-FLI-1 tar-gets, including MYC and ID-2 and repression ofp21 and TGFbRII both of which have been shownto be downregulated in Ewing sarcoma cells.Furthermore, these tumors displayed high sensitivi-ty to IGF-1R inhibition, a hallmark of Ewingsarcoma, as well as expression of Ewing sarcoma-as-sociated markers, including NSE and CD99. Impor-tantly, MPC used in this study had not beenimmortalized prior to EWS-FLI-1 introductionand expressed functional p53 in addition to retain-ing the p16INK4A/p19ARF gene [67].

In a study performed simultaneously by anothergroup, EWS-FLI-1 introduction into unsorted mur-ine bone marrow-derived cells resulted in tumorswith various phenotypes including one that is con-sistent with that of Ewing sarcoma [68]. The tumorsexpressed markers associated with Ewing sarcomaand displayed more aggressive behavior upon subse-quent loss of p53 [68]. Prior to these studies, workfrom Suzanne Baker’s laboratory showed that intro-duction of EWS-FLI-1 into p19ARF�/� MPCsresulted in a block in differentiation toward osteo-genic and adipocytic lineages [69]. Together, thesethree independent studies provide solid evidencethat EWS-FLI-1 expression may not only be impli-cated in the pathogenesis of ESFT but may consti-tute its initiating event. They also suggest thatMPCs may provide the necessary permissivenessfor the transforming potential of EWS-FLI-1. Inother words, MPCs may be the right cells in theright place for EWS-FLI-1-mediated oncogenesis.Their capability to migrate from the bone marrowto most organs may help explain the extraosseouslocations of Ewing’s sarcoma.

The discovery that MPC transformed by EWS-FLI-1 upregulate IGF-1 and are dependent onIGF-1R signaling for survival is consistent withthe behavior of Ewing sarcoma cells, which havebeen observed to require IGF-1 for growth [70]

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and to be among the most sensitive tumor cell typesto IGF-1R inhibition [71,72]. Moreover, functionalIGF-1R expression has been shown to be a pre-req-uisite for EWS-FLI-1-mediated transformation [73],underscoring the importance of the IGF-1 pathwayin the initial phase of ESFT development.

Although IGF-1 upregulation was not sufficientto transform MPC in our model of ESFT, it is likelyto be one of several factors directly or indirectlyinduced by EWS-FLI-1 that are implicated inMPC transformation and tumorigenicity. It isnoteworthy that the age associated with the peakincidence of Ewing’s sarcoma coincides with aug-mented IGF-1 secretion in bone as a result of a burstin growth hormone production. IGF-1 inductioncould provide a survival signal that is essentialduring early cell transformation to circumventEWS-FLI-1-induced growth arrest and apoptosis.Consistent with this notion, several studies, includ-ing our EWS-FLI-1-transformed MPC model, havesuggested that IGF-1R blockade may provide apotentially relevant therapeutic avenue for Ewing’ssarcoma [67,74]. These observations are of interestbecause conventional chemotherapeutic approachesfor Ewing’s sarcoma control have failed to signifi-cantly improve its notoriously poor prognosis.

7. Future directions

The model systems used thus far have providedsubstantial insight into the biological properties ofEWS-FLI-1 that may be relevant to transformation.The evidence that EWS-FLI-1 can transform mouseMPC to yield Ewing’s sarcoma-like tumors consti-tutes a quantum step toward understanding the cel-lular environment required for expression of itsoncogenic potential. It also underscores the notionthat a single genetic event in the appropriate cellularcontext may be sufficient for ESFT development.The next phase of Ewing’s sarcoma research shouldaddress the susceptibility of human MPCs to trans-formation by EWS-FLI-1 and elucidate the mecha-nism whereby direct and indirect EWS-FLI-1 targetgenes initiate and promote ESFT development.Hopefully, such endeavors will lead to the discoveryof new therapeutic targets and more effectivetreatment.

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