Over the last decades, the knowledge of the cellular and molecu-lar biology of ESFT has increased remarkably and has led to the development of multidisciplinary therapeutic strategies. However, the prognosis and survival of patients with metastatic or recur-rent disease remains still very poor. Here, we focus on the current state-of-the-art in the research of ESFT and review carefully the main critical points that will highlight the most promising bio-logical targets amenable to future therapies.
Chromosomal Translocations in ESFT: Specificity, Gene and Protein Fusions, Functions
The ESFT family comprises morphologically heterogeneous tumors that are characterized by nonrandom chromosomal trans-locations involving the EWS gene and one of several members of the ETS family of transcription factor genes.1 EWS-FLI1 fusions
*Correspondence to: David Herrero Martín; Email: [email protected]: 01/20/10; Revised: 02/10/10; Accepted: 02/12/10Previously published online:www.landesbioscience.com/journals/cbt/article/11511
are detected in roughly 90% of ESFT; EWS-ERG are present in 5% of cases, while 3% represent some other type of fusion of EWS with a member of the ETS family of transcription factor genes (Table 1). These fusions are specific to this neoplasia, as PCR studies of other tumors that could be included in a differen-tial diagnosis, such as central primitive neuroectodermic tumors, neuroblastomas, rhabdomyosarcomas, adamantinomas and giant cell tumors, have repeatedly yielded negative results.2 The two main sources of variability in the translocations are, on the one hand, the EWS fusion “companion” (one member of the ETS gene family, such as FLI1, ERG, ETV1, E1A or FEV ), and on the other, the location of the translocation break point within each gene involved. In addition to the usual prognostic factors of this neoplasia (stage, localization, volume of the primary tumor, age and response to treatment), recent studies suggest the contribu-tion of molecular heterogeneity toward the prognosis in Ewing sarcoma (ES).
The translocation t(11;22)(q24;q12) is the most common in ES, associated with 90% of cases, and this high specificity sug-gests that the product of this rearrangement is involved in the formation of these malignancies. This translocation leads to an in-frame fusion of EWS at 22q12 to FLI1 at 11q24 and the forma-tion of the EWS-FLI1 fusion protein. The EWS protein is pre-dicted to be an RNA-binding protein containing a transcriptional activation domain(s) in the N-terminus and an RNA recognition motif in its C-terminus.3,4 It belongs to a subgroup of RNA-binding proteins called the TET family, which also includes the liposarcoma/fusion protein (TLS/FUS), and the human TATA binding protein-associated factor (hTAFII68).5,6 Three key features indicate that the EWS protein is encoded by a house-keeping gene: it is expressed ubiquitously, its expression is stable throughout the cell cycle, and its mRNA has a long half-life.5 The transcriptional potency of the N-terminal domain of EWS observed in its various tumorigenic fusion proteins suggests that
The molecular pathogenesis of Ewing sarcomaCarlos Mackintosh,1,† Juan Madoz-Gúrpide,2,† Jose Luis Ordóñez,1,† Daniel Osuna3,† and David Herrero-Martín4,*
1Laboratory of Molecular Pathology of Sarcomas; Centro de Investigación del Cáncer-Instituto de Biología Molecular y Celular del Cáncer; Universidad de Salamanca-CSIC; Salamanca, Spain; 2Group of Cancer Research; Fundación Jiménez Díaz; Madrid, Spain; 3Departamento de Fisiología Vegetal (CIALE); Facultad de Biología;
Universidad de Salamanca; Villamayor (Salamanca), Spain; 4Department of Oncology; University Children’s Hospital; Zurich, Switzerland
Ewing sarcoma family tumors (ESFT) are a group of aggressive solid bone and soft tissue malignancies of children and young adults characterized by specific chromosomal translocations that give rise to EWS-ETS aberrant transcription factors. Identification of EWS-ETS target genes and their role in tumor signaling networks together with the unravelling of the cell of origin will facilitate the translation into new treatment modalities for these neoplasms.
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manner, and the N-terminal EWS domain can act as a potent transcriptional activation domain; several fold stronger than the corresponding native FLI1 domain displaced by the chromo-somal translocation. Several molecular reports have provided experimental evidence to confirm this view. Recent studies have revealed that EWS-ETS chimeric proteins also require the col-laboration of other proteins to perform their functions, such as AP-1 proteins for transformation,12 or RNA helicase A, which acts as a transcriptional cofactor.13 Additionally, although most of the studies regarding the molecular function of the chime-ric proteins in ES have focused on the altered role of the ETS portion, there is data suggesting that EWS modifies its activity when fused to ETS proteins, so that EWS-FLI1 may function both in transcriptional and post-transcriptional processes. In fact, it seems to behave as an aberrant RNA splicing factor in in vitro assays, blocking U1C-, TASR- and YB1-mediated splicing. Consequently, EWS-FLI1 may contribute to malignant transfor-mation through disruption of RNA splicing, uncoupling gene transcription from RNA splicing in the pathogenesis of ES.
Targets of the Chimeric Proteins
Experimental evidence suggests two mechanisms that promote EWS-ETS fusion-mediated oncogenesis: (i) by acting as aberrant ETS transcription factors for a collection of genes, which can vary in their levels of expression and in their identity with respect to those originally regulated by the parental ETS proteins; and (ii) by modulating gene expression at the RNA processing stages of transcription control, acting as aberrant TET factors as well. Consequently, a major strategy towards understanding the mech-anism by which EWS-FLI1 contributes to cell transformation has been to identify its putative target genes and proteins.
Up to now, only a few genes have been demonstrated to be direct target genes of EWS-FLI1 (Table 2): hsRPB7,14 UPP1,15 tenascin-C,16 Id2,17 p21WAF1/CIP1,18 PTPL1,19 phospholipase D2,20 TGFBR2,21 IGFBP3,22 MK-STYX,23 TERT,24 GLI1,25 and Aurora A and B.26 The rest of the genes described as interacting with EWS-FLI1 have not yet demonstrated to be direct targets, although
EWS may function as a transcription factor. The RNA-binding motif containing the C-terminal half of EWS is replaced, in the EWS-FLI1 fusion protein, by the DNA-binding domain of the FLI1 protein. Although EWS-ETS fusion proteins function as sequence-specific transcription factors, the role of native EWS protein and the regulatory mechanism controlling the coactiva-tor function of EWS are largely unknown.
FLI1, on the other hand, is a member of the ETS family of transcription factors which activate specific target genes by bind-ing to their cognate DNA sequences through their DNA-binding regions, usually located at their carboxy termini.7,8 The ETS family of transcription factors is defined by a conserved ETS domain that recognizes a core DNA motif. This family of approx-imately 30 genes, including FLI1, ERG, ETV1, E1AF, FEV and ZSG; controls a variety of cellular functions in cooperation with other transcription factors and cofactors. Target genes include oncogenes, tumor suppressor genes and genes related to apopto-sis, differentiation, angiogenesis and invasion.9,10
The (11;22) translocation in ESFT leads to a fusion gene that encodes an oncoprotein consisting of the N-terminal domain of EWS and the C-terminal DNA-binding domain of transcrip-tion factors such as ETS family, activating transcription factor-1, Wilms’ tumor 1 and nuclear orphan receptors.11 The replace-ment of the native transcription activation domain(s) of FLI1 by the N-terminal region of EWS converts the non-transforming activator, FLI1, into a transforming protein with new transcrip-tional activation potential. Sequence analysis of the translocated products define at least eight different types of fusion transcripts, depending on the specific breakpoints in the FLI1 and EWS exons included within the chimeric genes. Of them, the most common fusion (63%) is EWS exon 7-FLI1 exon 6, which is referred to as the type 1 fusion.
This singularity of EWS-FLI1 as the main protein actor in ES genesis raises the question of what are the specific functions of these chimeric proteins. Currently, it is widely assumed that EWS-ETS fusion proteins function as either aberrant transcrip-tion factors or potent repressors. In support of this hypoth-esis, they localize to the nucleus, bind DNA in a site-specific
Table 1. Different fusion genes found in ESFT (and ESFT-like)
Chromosomal translocation Fusion protein Tumor type Prevalence factor (%) Reference
t(11;22)(q24;q12) EWS-FLI1 Ewing family of tumors 90 57
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to the overall effects elicited by IGF1 in ES.51 They appeared to be constitutively activated in ES, likely due to the presence of the IGF1R-mediated autocrine loop. This is consistent with results from gene expression studies of pediatric sarcomas, in which sev-eral tyrosine kinases or receptor tyrosine kinases genes have been found to be highly associated with half of the tumor groups: KIT, PDGFRB, JAK1, FLT1, EGFR, PDGFRA and several FGFRs.52
Recent evidence has shown that Wnt/Frizzled signaling is functional in ES cell lines.53-55 Canonical Wnt/β-catenin signal-ing enhances ESFT motility, contributing to metastasis, probably through either autocrine or paracrine modes of Wnt glycopro-teins, since they are expressed in bone, muscle and soft tissues. Wnt proteins activate a canonical pathway that is characterized by accumulation of β-catenin in the cytoplasm. In the absence of Wnt signaling, cytoplasmic β-catenin forms a complex with axin, APC, GSK-3b and casein kinase I-a. Mutations in the axin, APC and β-catenin genes have been characterized in numerous human malignancies including colon cancer, malignant mela-noma, hepatocellular carcinoma, endometrial carcinoma, ovar-ian carcinoma and prostate cancer.56 A common outcome of these mutations is the accumulation of free β-catenin, mimicking con-stitutive Wnt activation. Both canonical and non-canonical Wnt pathways have been shown to modulate cell motility and tumor metastasis, but non-canonical Wnt pathways are pending dem-onstration in ESFT.
Secondary Alterations in ESFT
Although EWS-ETS chimeric proteins are considered the ini-tiating molecular events in ESFT, other alterations (that will be considered here as “secondary alterations”) have been also detected. Their significance and contribution to the pathogen-esis of ES are not completely understood yet. Henceforth we will discuss the main discoveries in the field. p53 mutations, which are frequent in ESFT cell lines,70 confer worse outcome but are rare in ESFT (10–20%). The same can be said for MDM2 and RAS mutations and other genes commonly altered in cancer.71-75 An exception can be made with CDKN2A locus, an alteration in which (a deletion in the vast majority of cases) has been reported as the most frequent secondary molecular aberration in ESFT with a 15–30% overall frequency.72,76-79 It
they are involved in signaling pathways and gene regulation net-works initiated by EWS-FLI1. The EWS-FLI1 target genes listed below are either indirect targets or need verification that they are direct targets (Table 2): EAT-2,27 MFNG,28 mE2-C,29 PIM3,30 IGF1/IGF1R,31 c-Myc and p57KIP2,32 MAPT, PP1R1A, NEK2 and cyclin D1,33 Skp2,34 caveolin-1,35 CD99,36 zyxin,37 DAX1/NR0B1,38 NKX2.2,39 cholecystokinin,40 VEGF-A,41 NOTCH-p53,42 thrombo-spondins 1 and 2,43 EZH244 and TOPK.45 Based on a stable EWS-FLI1 shRNAi model constructed in TC71 (ES type 1 cell line), Herrero-Martín et al.45 revealed a set of potential new targets for the ES fusion protein, including LSM1, BEX2, TK1 and EIF4E.
Much of the understanding about the nature of the targets of EWS-ETS is derived from studies of oncogenic fusions that arise from gene rearrangements after chromosomal translocation. Even more research has been devoted to understanding the mecha-nisms of action of the ES chimeric proteins themselves. However, although extensive work has been performed using cellular, molecular and genomic lines of attack, little knowledge has been reported using a proteomic approach. To assess the contribution of the N-terminal domain of the EWS protein to the formation of human solid tumors, it is important to understand the normal function(s) of EWS, but this point is not well characterized yet. Nonetheless, a handful of proteins have been suggested as inter-acting with EWS-FLI1, either directly or indirectly: hsRPB7,14 TERT,46 heat shock proteins,47 HSP90,48 IGF1,49 or Interferon (IFN)α/β receptor.50
Taken all these observations together, we can conclude that the fusion protein EWS-FLI1 exerts a certain degree of control in different routes of tumor development, maintenance and pro-gression in ES: cell proliferation and survival (by activation of targets IGF1, MYC and NKX2.2, among others); escape from growth inhibition, senescence and apoptosis (by inhibition of p21, p57kip, TGFBR2 and IGFBP3); and upregulation of criti-cal genes involved in neural tube and neural crest development (NKX2.2, cholecystokinin).
Cellular Signaling Pathways in ES
An inquisitive look at the cell signaling pathways altered in ES, and more specifically at the changes in particular molecules involved in them, such as cell surface adhesion molecules, receptor tyrosine kinases, growth factors and transcription factors would constitute a valuable strategy for identifying potential candidates for therapeutic intervention and diagnostic development (Fig. 1). The most relevant signaling routes reported to be altered in ES are tyrosine kinase pathways and the Wnt signaling pathway.
ESFT proliferation and maintenance is determined by auto-crine and paracrine activation of growth factor receptors and their corresponding ligands, such as insulin-like growth factor 1 (IGF1) and its receptor insulin-like growth factor 1 recep-tor (IGF1R).31 EWS-FLI1 shRNA interference in ES cell lines affected the IGF1/IGF1R survival pathway and its downstream targets.45 An analysis of the contribution of the two major path-ways of the intracellular IGF1R signaling cascade suggested that both the mitogen-activated protein kinase (MAPK) and phos-phatidylinositol-3-kinase (PI3K) signaling pathways contributed
Table 2. A summary of EWS/FLI1 genetic targets identified to date (references found in the text)
On the other hand, the absence of p53 mutations in most ESFT tumors was not consistent with in vitro results: this protein and/or its pathway was expected to be altered because in vitro experiments showed how cell types with different origins cannot be transformed by EWS-ETS because of p53 activation and subsequent cell cul-ture growth arrest and induction of senescence.80 Moreover, forced expression of p53 in vitro demonstrated that p53 is not mutated in tumors and its pathway is functional in ESFT cell lines.81
Another gene involved in the G1 to S cell cycle checkpoint,
p27Kip1, has been shown to be absent in ESFT. Low expression lev-els were detected in most ES patients (76%) with a remarkably worse survival. EWS-FLI1 promotes the cell cycle progression through suppression of the expression of this cyclin-dependent
has been proven that the loss of the CDKN2A locus has a marked clinical outcome as the event-free survival (EFS) is worse in ES patients with p16INK4 and p14ARF mutation/deletion than in those without the mutation/deletion.72,77,78 But also it has been related with the stage of the ES patient and a poor chemoresponse.77,79 Thus, the loss of CDKN2A locus has a clin-ical usefulness in identifying a subset of ES patients with poor prognosis. It is tempting to propose that the loss of products from this locus, p16INK4 and p14ARF, could cooperate with one of the best characterized EWS-ETS downstream targets, cyclin D1,33 to release cell cycle initiation from its negative regulators, rendering it independent from extracellular signals and other controls.
Figure 1. Regulation and role of EWS/FLI1 direct targets in the most important signaling pathways described in ESFT. Signaling pathways involved in ES proliferation-survival and phenotype are delimited in dashed areas. Canonical pathway interactions are depicted by continuous lines, whereas dashed lines represent possible pathway interactions. Red and green colors mean up-regulation and down-regulation, respectively. EWS/FLI1 direct targets are represented in boxes: kinases are shown as hexagons, phosphatases as ovals, transcription factors as octagons and the remaining targets are shown as rectangles.
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EWS-ETS fusions into diverse cellular systems gave rise to differ-ent responses like induction of apoptosis, growth arrest or dedif-ferentiation.80,96-98 Considering that most ESFT occurs in bone and soft tissue, bone marrow-human mesenchymal stem cells (MSCs) may serve as one cellular source susceptible to EWS-FLI1 transformation. Bone marrow-human MSCs, which are classic mesodermal derivatives, express an extensive assortment of neural genes, and therefore, are predisposed to differentiate to neural and glial lineages.99 The EWS-FLI1 fusion has been shown to impose a neural crest parasympathetic lineage direc-tion to the cells but inhibits terminal differentiation and could act through the Wnt signalling pathway, a possible common mechanism for vertebrate neural crest induction.100,101 EWS-FLI1 upregulates critical genes in neural crest development, acting as a cell lineage determinant rather than a pure “oncogene” and EWS-ETS expression is considered to be the initiating malignant event of ESFT tumorigenesis.101-103
In fact, the development of ES-like tumors from MSCs has been already described in mice.103,104 Gene expression profile analysis of tumor-derived cells revealed upregulation of known EWS-FLI1 gene targets, including MYC and ID-2, and repres-sion of p21 and TGFβRII, both of which have been shown to be downregulated in ES cells. Furthermore, these tumors were highly sensitive to IGF1R inhibition, a hallmark of ES, and expressed ES-associated markers, including NSE and CD99. Importantly, bone marrow MSCs used in this study expressed a functional p53 in addition to retaining the p16INK4A/p19ARF locus.104
In immortalized human MSCs, the phenotype and ontogen-esis of human MSC-TERT20 tumors were consistent with the hypothesis that sarcomas may arise from MSCs.105 Riggi et al.44 have shown that expression of EWS-FLI1 in human MSCs is able to induce a gene expression profile highly similar to that of ESFT although the cooperation of additional genetic alterations is required for a full tumoral transformation. Recent evidence suggests that five events are required to transform human MSCs. In this study, retroviruses encoding for the catalytic subunit of human telomerase (hTERT), HPV-16 E6 and E7, SV40 small T antigen (ST), and an oncogenic allele of H-Ras (H-RasV12) were used to induce transformation.106 Functional IGF1R expression has been shown to be a pre-requisite for EWS-FLI1-mediated transformation, underscoring the importance of the IGF1/IGF1R pathway in the initial phase of ESFT develop-ment.45,107 Furthermore, the DNA binding ability of EWS-FLI1 is required for IGF1 induction in bone marrow-human MSCs.44 IGF1/IGF1R signaling has been observed to be critical during the initiating phases of ES development, and its block-ade induces growth arrest and apoptosis in ESFT cell lines.108 The polycomb group gene EZH2 is another EWS-FLI1 can-didate target gene that was induced in human MSCEWS-FLI-1. EZH2, implicated in the maintenance of glioblastoma stem cells,109 may help to achieve the tumorigenic phenotype of ES by preventing cell senescence while promoting survival and proliferation and hindering differentiation.110 This is supported by a study of EZH2 repression, which resulted in the reduction of ESFT cell proliferation and tumorigenicity.44 In a complemen-tary approach, the profiles of different EWS-FLI1-silenced ES
kinase inhibitor via activation of the proteasome-mediated degra-dation pathway. Forced p27Kip1 expression in ES cell lines proved its ability to reduce cell growth and promote apoptosis in vitro and in vivo.32,34,82
The tumor suppressor, p21WAF1 shows a similar situation, with a 55% of ES cases showing loss of expression.32,83 The activity of the p21WAF1 is negatively regulated by EWS-FLI1 fusion protein through at least two ETS-binding sites in its promoter.84 Silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p21WAF1.42 As the fusion protein interacts with the p300 cotransactivator and suppresses its histone acetyltransferase activity, the use of a histone deacetylase inhibitor results in the induction of p21WAF1.84,85 Thus p21WAF1 could be a target for a molecularly based therapy for ESFT. As can be easily concluded, abrogation of G
1-S transition repressors
accounts for all the secondary alterations identified in ESFT sug-gesting that molecular events targeting these regulators are needed for tumor development.
Another set of studies has enhanced our understanding of the genomic imbalances in ESFT, making use of diverse tech-niques such as comparative genomic hybridization on metaphase spreads (CGH), CGH array, fluorescent in situ hybridization (FISH) or multiplex ligation-dependent probe amplification (MLPA).70,77,78,86-95 Results from these works gave similar esti-mations of the abundance of genomic aberrations, showing an overall incidence of 80% of cases bearing copy number altera-tions (CNA). Specifically, gains of chromosomes 1q, 8 and 12 and losses of 9p and 16q are the main alterations reported by CNA.
The prognostic impact of these aberrations is still unclear although studies support an adverse survival for the most frequent alterations (1q, 12 and 16q), especially in those studies with larger sample sizes. Remarkably, the gain of chromosome 8, present in 50–60% of ES, has never demonstrated prognostic value in clinical correlations. However, it seems clear that the global amount of CNA correlates with poor survival. Data has been divided by two types of thresholds for this sort of analysis: overall chromosome number above 50, based on karyotyping, and CNA above three, by CGH studies. Patients above these thresholds clearly demonstrated worse survival. Besides large chromosomal aberrations, CGH array stud-ies using oligonucleotide platforms have unveiled the existence of very short CNA (less than 100 kbs) not detectable with other tech-nologies.93 The loss of the CDKN2A locus was the only one found to be recurrent; reinforcing its role as a major secondary alteration.
Finally, a few studies have contrasted the impact of copy number changes on the transcriptome.91,93 The first of these studies deter-mined that those samples showing genomic instability (more than three CNA) displayed overexpression of genes implicated in cell cycle regulation, chromosomal segregation and mitosis.91 Other authors have selected a list of candidate genes based on gene copy-expression level correlation studies, and tried to validate one of them, HDGF, as a prognostic marker, although they did not find it statistically relevant.93
ESFT Cell Type of Origin
The cellular context plays a fundamental role in ES pheno-type, as proven by the fact that the experimental insertion of
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The Pediatric Preclinical Testing Program supported by the National Cancer Institute aims to identify new drugs active against childhood cancers. The program makes use of a panel of 61 childhood tumor xenografts (four of them are ES xeno-grafts) in which tumor-type selectivity of novel treatments is tested. The ES xenografts were obtained by using CB17SC-M SCID-/- mice implanted with ES cell lines (SK-NEP-1; EW5, EW8, TC-71).121
Xenograft models have been used to gain knowledge about the pathogenesis and histogenesis of ES. Tumors with charac-teristics of ES were developed in mice implanted with murine primary bone-derived cells and mesenchymal progenitor cells expressing EWS-FLI1.103,104 Recently, we identified TOPK, a kinase involved in cell proliferation and motility, as a target of EWS-FLI1 by using an RNA interference system to knockdown EWS-FLI1 in an ES cell line. NOD/SCID mice implanted with the interfered cells developed smaller tumors than controls.45
Moreover, several specific ES metastatic models have been also published. In these models, ES cells were injected through the mouse tail vein. These models resemble the pattern of metastatic diffusion of ES in humans, including skeletal and extraskeletal locations.122,123 Gonzalez et al.118 reported a different strategy to induce metastasis formation in mice. They injected cells trans-duced with the EWS-FLI1 fusion types 1, 2 and 3 into the left cardiac ventricle of athymic mice. The aim of this approach was to overcome the limitation imposed by the fact that lung metastasis formation, after injection of cells via tail vein, may not necessarily reflect metastatic activity, as lungs bear the first capillary bed that injected cells face after tail vein injection. This model could be useful to test the effects of new inhibitors of tumor growth.
However, despite the great amount of information that these xenograft models provide, they also face many drawbacks. For instance, the normal architecture present in the patient tumor specimens is altered in xenografts and the genetic heteroge-neity is diminished. The tumor heterogeneity characteristic of the original ES is lost as a result of the selective pressure of cell culture or tissue explantation. Moreover, they do not accurately reflect the course of human disease. Therefore, the data obtained by using these models should be examined care-fully, especially when a new compound is tested. For all these reasons, we believe that xenograft models should be considered as an intermediate step between cell culture and murine cancer models, and therefore they should be named more properly as “animal cultures.”124
Genetically engineered models in ESFT. So far, no murine genetically engineered model (GEM) of ES has been created and importantly, there has been no report indicating any attempt to achieve it until recently.125,126 The embryonal lethality, due to the expression of the EWS-ETS fusions, is probably the main explanation for the lack of a GEM of ES to date.126,127 To avoid embryonic lethality, a key approach could be to drive the expres-sion of EWS-FLI1 exclusively in MSCs, as they seem to be the cells of origin for ES.128 In this regard, it would be important to identify more specific mesenchymal genes by which the expres-sion of EWS-FLI1 could be directed, thus avoiding the expres-sion in hematological tissues, as it induces leukemia.126 Another
cell lines converge on that of mesenchymal stem cells,111 and the “core” EWS/FLI1 transcriptional signature has been correlated with that of human MSC.112,113
Bone marrow-human MSCs may be the right cells in the right place for EWS-FLI1-mediated pathogenesis, and their capability to migrate from the bone marrow niche would help to explain the extraosseous locations of ESFT.102,103,114 It is important to study these progenitors or even earlier precursor cells, whose devel-opmental program seems to be deranged and blocked early in differentiation, in the search for specific molecular markers that would help to describe the ontogeny of the disease, and would allow the early detection of cells involved in sarcomagenesis. Because of the lack of knowledge about the first molecular events involved in translocation, the characterization of bone marrow-human MSCs from ESFT patients and healthy donors and the comparison at several levels (transcriptomic, epigenetic and proteomic ones), would be a key factor in highlighting the earli-est tumorigenic processes. Recently, a subpopulation of cancer stem cells in ES have been identified and characterized. These CD133+ tumor cells are able to initiate and maintain tumoral growth through the course of transplantations in immunodefi-cient mice and show a high degree of plasticity as MSCs.115
Taken together, all these data support the notion that MSCs would be suitable candidates as ES precursor cells as has been proposed already for other sarcomas. Furthermore, these data point towards MSCs as putative targets for more effective thera-peutic strategies.116,117
Animal Models in ESFT
From cell culture to mouse models. The information obtained from tissue culture studies using human and animal cell lines has provided great and inestimable information to the scientific community working on sarcomas and particularly on ESFT. The research done with cell lines has made it possible to infer the gene fusion role in the pathobiology of ES and also to discover new genes involved in this neoplasia.32,118 Cell culture models also pro-vide the primary platform for the testing of new drugs.49 However, in vitro conditions exert selective pressure on ES tumor cells and research has shown that only those tumor cells harboring particu-lar molecular alterations are prone to growth in cell culture.119
Moreover, cancer and particularly ES is a complex disease in which microenvironment and surrounding cells (normal, stromal and immune cells) play a crucial role. In fact, some aspects of ES development, for instance angiogenesis or metastases, can not be assessed by cell culture studies alone. Therefore, the informa-tion obtained by in vitro studies needs to be confirmed and there-fore should be taken into account carefully. For these and other reasons, the development of animal models in ES is an issue of paramount relevance. As reviewed by Beltinger and Debatin,120 few animal models of ES have been developed by implantation of tissue of murine origin into immunocompetent mice (synge-neic model). At present, several models created by implantation of tissue of human origin (xenogeneic or xenograft model) have been reported.48,103 These models have been used mainly to test the effects of novel therapeutic compounds.
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tissues. Interestingly, induction of FUS-CHOP is not lethal for the mouse, as seen with other sarcoma fusion genes such as EWS-FLI1 and EWS-ERG.126,127,130
In some transgenic mice, the expression of gene fusions is controlled with exogenous substances such as tetracycline or its analog, doxycycline. This approach has identified the role played by BCR-ABL1 in both the induction and maintenance of acute B-cell leukemia.131 More recently, Lin et al.125 created a condi-tional transgenic GEM by inducing the expression of EWS-FLI1 in the MSCs of embryonic limb buds using a Cre-Lox system, which did not induce tumors. Only in a setting of p53 deletion did mice develop poorly differentiated sarcomas but not ES.
Another large group of models expresses the fusion genes from their native promoters through knock-in technology. These models use homologous recombination in ESCs to induce gene fusions and therefore transfer them into the mouse germline. The conditional knock-in models make use of specific recombinases, allowing a spatio-temporal control of the chimeric gene expres-sion. The P1 bacteriophage Cre-lox system is the most widely employed. To generate Cre-lox mice expressing the fusion gene, it is necessary to engineer two transgenic murine lines: a Cre mouse and a loxP mouse. The Cre mouse harbors the Cre recom-binase gene under the control of a tissue specific promoter. Cre recombinase mediates the recombination between loxP sites, a pair of inverted, repeated DNA elements. The loxP mouse can be engineered to harbor the chimeric fusion gene, plus a stop cas-sette upstream of the fusion site flanked by loxP sites in the same orientation. Depending on the orientation and location of the loxP sites, recombination mediated by Cre induces deletion, inversion or translocation of the sequence of interest. After breeding the two mouse lines, expression of Cre induces the excision of the stop cassette and consequently the expression of the fusion gene.
Higuchi et al.132 generated a transgenic mouse harboring a conditional knock-in allele containing a loxP-STOP cassette placed upstream of the AML1-ETO fusion site. The fusion gene was induced after crossing the AML1-ETO murine line
important aspect that could help to explain the absence of a GEM ES model is the different genetic background of mice as compared to humans. As has been pointed out earlier, Hancock and Lessnick,112 compared the gene expression profiles of dif-ferent ES models with human ES samples. They found strong correlations between human-based EWS-FLI1 models and ES. However, none of the murine-based model systems showed any correlation to ES samples. The data demonstrated that models using murine cellular backgrounds perform poorly in general, as compared to human-based ones. One possibility is that EWS-FLI1-mediated pathways in mouse cells could be different than those in the original human disease. Another possibility is that crucial EWS-FLI1 binding sites may not be present in the mouse genome. Alternatively, coregulators of the EWS-ETS chime-ric proteins (such as coactivators or cooperating transcription factors) might interact differently in the mouse.112
Overcoming the problem of the embryonic lethality induced by gene fusions, different genetically engineered models of sarcomas, such as liposarcoma, alveolar rhabdomyosarcoma and synovial sarcoma, as well as of some malignant hematological disorders have been generated (reviewed in Ordóñez et al. submit-ted). Chromosomal translocations and their corresponding gene fusions are common events in all of these malignancies, playing a crucial role in the initial steps of sarcomagenesis.129 A general view of the strategies used and their advantages and disadvan-tages could be of help to understand why all attempts to generate an ES mouse model have failed (Table 3).
Some transgenic mice have been engineered to induce the expression of fusion genes in a non-physiological manner under the control of exogenous promoters or enhancer elements through the injection of cDNA constructs into fertilized oocytes or through gene targeting in embryonic stem cells (ESCs).124,130 Pérez-Losada et al.130 generated a model of liposarcoma by pronuclear injection into fertilized eggs, of a cDNA construct including the human FUS-CHOP gene under the control of the elongation factor 1α promoter to direct expression to all mice
Table 3. Genetically engineered models of common fusion genes found in sarcomas
Ref. Model Fusion Promoters Expression place Phenotype
130, 136 Transgenic FUS-CHOP EF1-α All tissues Liposarcoma
MX1-creBone marrow liver, spleen, and hematopoietic tissues
Myeloid/erytroid leukemia
127 Invertor EWS-ERGEWS endogenous
(loxP mice) Rag1 (Cre-mouse)
Lymphocyte Leukemia
125Conditional transgenic (Cre-loxP)
deletion of p53EWS-FLI1 Prx1 (Cre-mouse)
Primitive mesenchymal tissues of the embryonic
limb bud
Poorly differentiated sarcomas
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cassette flanked by loxP sites was knocked-in to the EWS gene but in the reversed orientation with respect to the direction of transcription. Later, Cre recombinase, under the lymphocyte-specific Rag1 promoter, mediated inversion of the ERG gene and produced the EWS-ERG gene fusion, subsequently generating leukemia.127 The invertor model was later used for a model of B-cell lymphomas by expression of MLL-AF4.135
Immune System Role in ESFT: Significance of Immunotherapy
One of the mechanisms of tumor relapse is escape from immu-nity. Understanding the relevant molecular connections between tumor development and the immune system will help to develop more effective therapies. ES is not able to induce an effective antitumor response, which has been attributed to low level major histocompatibility complex (MHC) expression and lack of co-stimulatory surface molecules. Immunotherapy is an emer-gent and exciting treatment field in ESFT and several different approaches have been already tested.
Fas-FasL interactions and the death-receptor pathway play a central role in the regulation of the immune response. ESFT are resistant to the cytolytic death-receptor pathway but main-tain their sensitivity to the perforin-granzyme pathway, which could be exploited for inducing apoptosis; for example through restoration of caspase-8 expression.137,138 Despite some ES cell lines expressing a non-functional FasL, it is possible to induce Fas-mediated apoptosis after IFNγ preincubation and/or cycloheximide treatment through the generation of cytotoxic T-cell lymphocytes (CTLs).139
Survival in ESFT is not improved by allogeneic stem cell transplants (SCT),140 although some patients benefit from high-dose chemotherapy plus autologous SCT.141,142 The cytokine interleukin-2 (IL-2) has been used with SCT patients to reinforce antitumor immunity.143,144 Side effects have been circumvented by the use of IL-2 transgenic ES cells showing an increase in the number of T and natural killer (NK) cells and a reduction of tumoral cell growth in vitro and in vivo.145 Thus immunomod-ulation using cytokine-induced cells as tumor-reactive T cells and/or NK cells is another treatment option for advanced-stage ESFT.146 NK cell cytotoxicity depends on the combination of NKG2D and DNAM-1 signaling pathways and is increased by activation with interleukin-15. ESFT cells are potentially susceptible to NK cell lysis due to the expression of activating NK cell receptor ligands.147 Dendritic cell (DCs)-based immu-notherapy may be a promising complementary strategy as these antigen-presenting cells are capable of stimulating T-cells.148,149 DC-based vaccines have already been shown to be effective in ESFT as tumoral growth reduction is achieved in vitro and in vivo through stimulated CTLs.150
A wide search has been done in ESFT for tumor-associated antigens (TAA) in order to identify targets for therapeutic cancer vaccines. Due to their specific expression, EWS-ETS chimeric proteins are perfect candidates for TAA targeting. Peptides span-ning the breakpoint of these fusion transcription factors with MHC I and II binding motifs could be susceptible to CTLs
with a transgenic murine line expressing Cre recombinase under the control of the IFNα/β-inducible Mx1 promoter. This promoter is activated in vivo after treatment with IFNα/β (or polyinosinic-polycytidylic acid, pI-pC), which induces Cre expression exclusively in those cells harboring IFNα/β receptors. After expression of Cre recombinase under the IFNα/β inducible Mx1 promoter, the stop cassette was eliminated in hematopoietic and non-hematopoietic tissues. However, the mice retained the fusion gene that was expressed exclusively in myeloid progenitors under the endogenous AML1 regulatory sequences.
In a recent report, Torchia et al.126 circumvented the embry-onic lethality induced by the chimeric protein, generated a transgenic mouse of EWS-FLI1 by using a knock-in condi-tional approach, using a loxP-flanked (floxed) transcription stop cassette (loxP-STOP-loxP). As described by Higuchi et al.132 a Cre-inducible recombinase under the control of the Mx1 promoter was used. However, in contrast to the aforementioned study, EWS-FLI1 was not under the control of the fusion promoter but rather, was under the control of the murine ubiqui-tous promoter Rosa 26. This is the first GEM mouse of EWS-FLI1 published to date. However, EWS-FLI1 expression following Cre induction after a single administration of pI-pC did not produce ES tumors, but rather myeloid/erythroid leukemia, which caused animal death about 2 w after administration. Even a group of animals that were not administered pI-pC developed a similar pathology as compared with pI-pC treated animals, indicative of a basal expression of Cre, and consequently of EWS-FLI1 protein.
Keller et al.133 have used the conditional knock-in strategy to generate an alveolar rhabdomyosarcoma model, by induc-ing the expression of the PAX3-FKHR fusion in skeletal muscle cells of a Myf6-Cre murine line. However, the expres-sion of PAX3-FKHR did not produce an alveolar rhabdomyo-sarcoma phenotype by itself, so it was necessary to inactivate the Ink4a/Arf and Trp53 genes. Recently, a conditional knock-in model of synovial sarcoma has been described in which the SYT-SSX2 fusion was expressed in Myf5 expressing myoblasts. When the expression of the chimeric gene occurred in cells of skeletal muscle lineage other than myoblasts, animals suffered myopathy or embryonic lethality but not synovial sarcoma.134 This fact clearly indicates the crucial relevance of an accurate selection of the cellular context for fusion gene expression in order to avoid lethality and other side effects. However, some-times it is not possible to avoid minimal but meaningful tran-scription levels of fusion genes, which provoke lethality. Such minimal transcription occurs with EWS-FLI1 in ES and MLL-Af4 in hematological malignancies.126,127
A different strategy used to generate GEM mouse is the “invertor model,” a new version of knock-in strategy using loxP sites. In this model a floxed cassette is introduced into the intron of a target gene by homologous recombination in ESCs but the target gene is oriented in the reverse direction for transcription. Only after Cre recombinase expression is the cassette correctly orientated so that the fusion gene can be generated. This strat-egy was used for generation of T-cell lymphomas by conditional expression of EWS-ERG in hematopoietic cells. An ERG invertor
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GGAA-containing elements are enriched close to EWS-FLI1-upregulated genes but not downregulated ones.184,185 Even micro-satellite polymorphisms may confer differences in susceptibility to ES.185 It has been shown that the ability of the EWS-FLI1 to bind DNA and the strength of the transcriptional activa-tion depend on the number of consecutive GGAA motifs.184,186 Recently glutathione S-transferase M4 (GSTM4) has been identi-fied as a direct EWS-FLI1 target gene in ES. EWS-FLI1 binds the GSTM4 promoter and regulates its expression through a GGAA-microsatellite motif. GSTM4 inhibition reduces onco-genic transformation and increased sensitivity of ES cells to che-motherapeutic agents and high levels of GSTM4 expression has been associated to a worse outcome in ES patients. This study defines a wider role for GGAA-microsatellites in EWS/FLI tran-scriptional regulation than previously appreciated.187
The latest therapeutic approaches in ESFT are quite diverse and pointed toward several different pathways as manipula-tion of oxidative stress through targeting specific cellular anti-oxidants,188 disruption of EWS-FLI1 interactions—for example, with RNA helicase A in order to promote apoptosis,189 use of antagonists of the receptors of the neuroendocrine peptide chole-cystokinin,190 proteasome blockers as bortezomib utilized either alone or in combination with other drugs to overcome chemo-resistance,191,192 and new classes of kinase inhibitors.193-196 The availability of a well-defined preclinical model, the pediatric preclinical testing program, is helping to develop novel treatment modalities.121,196,197
Blocking of angiogenesis and vasculogenesis seems to be one of the most promising therapeutic alternatives in ESFT treat-ment. Both processes are critical in the growth and expansion of ES. Bone marrow cells participate in the generation of new vessels that supports the growth of ES and vascular endothe-lial growth factor 165 (VEGF165) plays a crucial role in this process. Blocking VEGF receptor 2 with the antibody, DC101, suppresses tumor growth, reduces tumor vessel density, inhibits the migration of vessel endothelial cells and increases apopto-sis.198 VEGF165 is also involved in the ES-induced bone lysis as it modulates the expression of a critical osteoclastogenic factor, the receptor activator NFkB, thus contributing to the meta-static process.199 Silencing of VEGF165 achieves a reduction in metastases,199 as also happens in an ES orthotopic xenograft model after platelet-derived growth factor receptor beta inhi-bition.200 Delta-like ligand 4 and the MEK kinase, MEKK3, have been recently described as important actors in ES vascu-logenesis.201,202 Delta-like ligand 4 is necessary for the forma-tion of bone marrow-derived pericytes/vascular smooth muscle cells,201 and MEKK3 is required for tumor vessel generation.202 Molecular-targeted therapies against these pro-angiogenic and pro-vascular factors will hopefully impair ESFT development and spreading as preclinical evaluation studies confirm.203,204
Therefore the new treatment strategies with respect to ESFT are not only focused on targeting the chimeric fusions EWS-ETS or their target genes. The influence of tumor microenvironment through key pathways for ES proliferation and dissemination including angiogenesis or vasculogenesis or the increasing impor-tance of the role of microsatellites point towards novel therapeutic
or DCs killing.151-153 Several other TAA have been proposed: cancer-germline genes (CGGs),154 papillomavirus binding factor (PBF),155 human leukocyte antigen (HLA),156 membrane-associ-ated phospholipase A1beta (LIPI),157 or cofactors such as 4-1BBL that could help to stimulate the immune response.158
Recently, a clinical study has been conducted in which patients with metastatic or recurrent ESFT receive autologous T cells and DCs pulsed with peptides derived from sarcoma-specific trans-location breakpoints and E7, a peptide known to bind HLA-A2, plus IL-2. Patients experienced minimal toxicity and favorable survival.159 Thus, several trials are being conducted with the purpose of integrating immunotherapy into multimodal treat-ment regimens in ESFT, thereby leading to more effective and specific therapies.
Novel Treatment Modalities for ESFT
Knowledge of the mechanisms of malignancy of the EWS-ETS fusion proteins and their target genes have allowed the devel-opment of directed molecular targeted therapies in ESFT. An IGF1/IGF1R signaling pathway blockade based on the use of neutralizing antibodies and/or small-molecule compounds repre-sents the best example of an ES molecular-based treatment.160-163
The relevance of some EWS-FLI1 target genes, such as DAX1/NR0B1,164,165 GLI1,166 NKX-2.2,167 or caveolin-1,168 in the ESFT pathogenesis has been confirmed by several studies thereby making them attractive treatment objectives. Some other novel actors, with putative tumorigenic roles and thus susceptible to therapeutic intervention, have been identified in ES. For example, polo-like kinase 1, which is highly expressed in other sarcomas,169 or Src family tyrosine kinases such as Lyn, which when inhibited, diminishes tumor growth and reduces lung metastases of ES.170 BMI-1 is a polycomb group gene that promotes anchorage-in-dependent growth in vitro and tumorigenicity in vivo, indepen-dent of CDKN2A repression, through modulation of adhesion pathways.171 Caspase 3 has been revealed as another direct target gene of EWS-FLI1.172 Furthermore, proangiogenic factors such as stromal cell-derived factor-1, which stimulates vasculogenesis,173 may also turn out to be suitable targets.
During the past few years not only have some well-known EWS-ETS targets been confirmed and novel ones been identified but additionally, some new useful molecular markers of progno-sis,174-178 recurrence,179 metastasis or drug resistance have been discovered.176,180
Post-translational modifications of the chimeric fusions such as O-GlcNAcylation or phosphorylation in response to DNA damage could affect their transcriptional activity although how these modifications work in ES is an unresolved question.181,182 Methylation could be also an important phenomenon.183
As has been shown recently, microsatellites sequences seem to play an important role in ESFT tumorigenesis.184-187 Microsatellites have been often regarded as useless or “junk” DNA regions but their role in ESFT malignancy has been proven. EWS-FLI1 modulates the expression of some direct target genes such as NR0B1 through binding to GGAA mic-rosatellites present in their promoters. These highly repetitive
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of the secondary genetic alterations and the discovery of more accurate markers of prognosis and relapse are the main tasks to accomplish.
Resolving these questions will shed light on ES pathogenesis and hopefully will help to identify novel therapeutic modalities.
Acknowledgements
CIC-IBMCC belongs to NoE Eurobonet, FP6-2004- Lifescihealth-5, proposal number 018814, European Commission. Work at CIC is also funded by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation-FEDER (PI052524; RD06/0020/0059, CD06/00001). This work has been done with the support of the Fundación Memoria D. Samuel Solórzano Barruso.
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Conclusion
There are still many important unsolved questions concerning the molecular and cellular biology of ESFT. There is an urgent need to discover the ES cell of origin and the first steps of malignant transformation.
Identification of the cellular environment permissive for the action of the gene fusions; the elucidation of the mechanisms giving rise to EWS-ETS rearrangements; the identification of the most significant cellular signaling pathways involved in ESFT initiation, development and spread; the clarification of the role
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