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[Cancer Research 65, 8698-8705, October 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Expression of the EWS/FLI-1 Oncogene in Murine Primary Bone-Derived Cells Results in EWS/FLI-1–Dependent, Ewing Sarcoma–Like Tumors

Yeny Castillero-Trejo1, Susan Eliazer1, Lilin Xiang1, James A. Richardson2 and Robert L. Ilaria, Jr.1,3

1 Hamon Center for Therapeutic Oncology Research, 2 Department of Pathology, 3 Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, Texas

Requests for reprints: Robert L. Ilaria, Jr., Lilly Research Laboratories, Lilly Corporate Center, DC 2133, Indianapolis, IN 46285. Phone: 317-433-4759; Fax: 317-276-9666; E-mail: ilariaro{at}lilly.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ewing sarcoma is the second most common malignant pediatric bone tumor. Over 80% of Ewing sarcoma contain the oncogene EWS/FLI-1, which encodes the EWS/FLI-1 oncoprotein, a hybrid transcription factor comprised of NH2-terminal sequences from the RNA-binding protein EWS and the DNA-binding and COOH-terminal regions of the Ets transcription factor FLI-1. Although numerous genes are dysregulated by EWS/FLI-1, advances in Ewing sarcoma cancer biology have been hindered by the lack of an animal model because of EWS/FLI-1–mediated cytotoxicity. In this study, we have developed conditions for the isolation and propagation of murine primary bone-derived cells (mPBDC) that stably express EWS/FLI-1. Early-passage EWS/FLI-1 mPBDCs were immortalized in culture but inefficient at tumor induction, whereas later-passage cells formed sarcomatous tumors in immunocompetent syngeneic mice. Murine EWS/FLI-1 tumors contained morphologically primitive cells that lacked definitive lineage markers. Molecular characterization of murine EWS/FLI-1 tumors revealed that some but not all had acquired a novel, clonal in-frame p53 mutation associated with a constitutive loss of p21 expression. Despite indications that secondary events facilitated EWS/FLI-1 mPBDC tumorigenesis, cells remained highly dependent on EWS/FLI-1 for efficient transformation in clonogenic assays. This Ewing sarcoma animal model will be a useful tool for dissecting the molecular pathogenesis of Ewing sarcoma and provides rationale for the broader use of organ-specific progenitor cell populations for the study of human sarcoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ewing sarcoma is the second most common malignant bone tumor of childhood. Approximately 80% of patients present with a skeletal site of disease, whereas the remainder of cases occur in virtually any soft tissue. The fusion gene EWS/FLI-1, found in the majority of Ewing sarcoma tumors, is the result of a genetic fusion of sequences from the EWS gene, a RNA-binding protein, and FLI-1, a gene that encodes a protein in the Ets family of transcription factors (reviewed in ref. 1). Various forms of the EWS/FLI-1 fusion have been found in over 80% of cases of Ewing sarcoma family of tumors (ESFT), whereas the other ESFT cases contain EWS fused to another Ets family member. According to a generally accepted working model, EWS/FLI-1 plays a central role in the pathophysiology of ESFT by disturbing the normal balance of gene expression in the affected cells. Traditionally, ESFT have been included in the group of "small round blue cell tumors," reflecting their somewhat undifferentiated morphologic appearance. Although expression of the cell surface molecule CD99 (MIC2) has been useful in the immunohistochemical diagnosis of ESFT, tumors in this family are equally notable for their general lack of definitive lineage markers (2).

Given the presence of EWS/FLI-1 or one of the related fusions in virtually all Ewing sarcoma cases, a considerable effort has been made to study the biological consequences of EWS/FLI-1 expression. Apart from its obvious association with human Ewing sarcoma tumors, EWS/FLI-1 was identified as an oncogene by its ability to transform NIH 3T3 cells to growth in semisolid medium and to form tumors in immunodeficient mice (3, 4). Interference with EWS/FLI-1 expression has been shown to interfere with the growth and tumorigenicity of EWS/FLI-1-expressing cells (5, 6). These and other studies suggest a pivotal role for EWS/FLI-1 in Ewing sarcoma; however, whether EWS/FLI-1 is capable of transforming primary cells, or is necessary and sufficient for tumorigenesis, is unknown. To gain insight into these questions, we hypothesized the following: (a) The predilection of Ewing sarcoma for skeletal sites indicates that a cellular target of the EWS/FLI-1 fusion resides in bone. (b) Because Ewing sarcoma is usually a pediatric tumor, the EWS/FLI-1 cellular target is more prevalent in younger individuals. To test these hypotheses, we isolated primary bone-derived cells (PBDC) from young mice and stably introduced EWS/FLI-1 by retroviral gene transfer. Compared with control cells, which were difficult to propagate, EWS/FLI-1 murine PBDCs (mPBDC) were immortalized, and with serial passage formed sarcomatous tumors in syngeneic, immunocompetent animals. Whereas some murine EWS/FLI-1 tumors had acquired a novel mutation of p53, mPBDCs were highly dependent on EWS/FLI-1 for transformation. Our findings support the existence of organ-specific progenitor cell populations that may be used to model the cellular and molecular origins of bone and soft tissue sarcomas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and transduction of murine primary bone-derived cells. PBDCs were obtained from 3- to 4-week-old BALB/c mice according to an established animal protocol approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center. The long bones, rib cage, and vertebral bones were carefully dissected away from adjacent soft tissue and gently crushed using a mortar and pestle. After removal of debris, cells were placed in growth medium containing DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT), 2 mmol/L glutamine, nonessential amino acids, and penicillin/streptomycin at 37°C and 5% CO2. After 48 to 72 hours, the nonadherent population, comprised mainly of hematopoietic cells, was removed and the remaining adherent population was subjected to retroviral transduction with a replication-defective EWS/FLI-1 type 1 retrovirus or control vector in the absence of exogenous growth factors, as previously described (7). Stably transduced cells were selected based on resistance to the selectable marker neomycin (0.8 mg/mL) and propagated for further study.

Retrovirus preparation. The retroviral vectors MINV-EWS/FLI-1 type I and MINV-EWS/FLI-1 R2L2 have been described previously (8). The vectors pBabe-loxP/puro and pBabe-Cre/hygro were kindly provided by Jerry Shay and Woodring Wright (Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX). Helper-free, replication-defective retroviral supernatants were generated by transient transfection of 293T cells with a retroviral construct and an ecotropic retroviral packing construct, as previously described (9).

Cell lines. All cell lines were propagated at 37°C at 5% CO2. NIH 3T3 cells were grown in DMEM supplemented with 10% bovine calf serum. Human 293T cells, the Ewing sarcoma cell lines TC-32 and TC-71, and the human rhabdomyosarcoma cell line RD were grown in DMEM with 10% heat-inactivated FCS. The breast cancer cell line HCC1143 and the leukemia cell line K562 were grown in RPMI (JRH Biosciences, Lenexa, KS) supplemented with 10% FCS and 2 mmol/L glutamine.

Tumorigenesis and transformation assays. Populations of EWS/FLI-1 or control mPBDCs were introduced into syngeneic BALB/c mice by s.c., i.p., or i.v. injection at a dose of 5 x 106 cells per animal. S.c. and bone tumors were harvested for analysis when tumor volumes reached 1 to 2 cm3, or when animals showed signs of morbidity. In the case of i.p. tumors, animals were sacrificed when they showed signs of increased abdominal girth or unusual weight gain. Tumors were analyzed by immunohistochemistry, fluorescence-activated cell sorting (FACS), and molecular studies as described elsewhere in this report. In some cases, tumor cell lines were derived from EWS/FLI-1 mPBDC tumors by incubating small tumor fragments in growth medium and processed for further analysis. For soft agar transformation assays, LoxP-EWS/FLI-1 mPBDCs were transduced with a Cre or control vector retrovirus, selected for resistance to the antibiotic hygromycin, and plated in triplicate at a density of 1 x 104 to 5 x 105 cells per 3.5-cm dish. The number of colonies of >150 µm in diameter was scored on day 14 (SMZ1500, Nikon Instruments, Melville, NY). Statistical analysis was calculated using an unpaired t test using Prism software (GraphPad Software, Inc., San Diego, CA).

Western immunoblot, immunohistochemistry, and fluorescence-activated cell sorting. Protein lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer containing a panel of protease and phosphatase inhibitors (10). Tumor cell lysates were prepared by directly adding RIPA lysis buffer to minced tissue followed by homogenization using a polytron homogenizer (Brinkman Instruments, Westbury, NY). Cellular debris was removed by centrifugation at 14,000 rpm at 4°C for 10 minutes. Lysates were normalized by A595 (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA), resolved by SDS-PAGE, and electrophoretically transferred overnight to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). Immunoreactivity was detected by enhanced chemiluminescence (Amersham, Piscataway, NJ) using the following antibodies: anti-cyclin D1 (Biomol Research labs, Plymouth Meeting, PA), anti-actin, anti-p53, anti-p21, anti-c-myc, and anti-FLI-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA). The preparation, sectioning, and immunohistochemical staining of tumor tissue were done using standard procedures using antibodies against the following markers: CD68, muscle-specific actin, myosin, neural-specific enolase, mast cell tryptase, pankeratin, and epithelial membrane antigen (all from DakoCytomation, Carpinteria, CA); desmin and myoglobin (both from Signet laboratories, Dedham, MA); smooth muscle actin and p75 (both from Chemicon International, Temecula, CA); MAC-3 (PharMingen, San Diego, CA) and S100 (Novocastra Laboratories, Newcastle upon Tyne, United Kingdom). For FACS, EWS/FLI-1 tumor cells were briefly expanded in adherent culture and then analyzed by murine anti-CD117, anti-VEGFR2, anti-CXCR4, anti-MAC-1, anti-CD41 (all from PharMingen), anti-Sca-1 (Caltag Laboratories, Burlingame, CA), or isotype control antibodies using a FACScan flow cytometer and CellQuest software (Becton Dickinson, Franklin Lakes, NJ).

Southern blot. Genomic DNA was isolated from EWS/FLI-1 mPBDC cells and tumors by Tris/SDS/proteinase K lysis followed by serial phenol-chloroform-isoamyl alcohol extraction. To assess the clonality of EWS/FLI-1-induced tumors, genomic DNA was digested with the restriction enzyme BglII (New England Biolabs, Beverly, MA), which cuts once in the provirus, and at the next BglII site encountered in genomic DNA. The size integrity of the provirus was confirmed by digestion of genomic DNA with XbaI, which cuts once in the multiple cloning site 5' of the EWS/FLI-1 cDNA and again at the 3' retroviral long terminal repeat. Samples were resolved on a 0.8% Tris-Borate-EDTA gel and transferred to a nylon membrane (Zetaprobe, Bio-Rad) by capillary transfer overnight. Membranes were hybridized with a 881-bp 32P-labeled probe (Radprime DNA labeling system; Invitrogen, Carlsbad, CA) derived from the neomycin resistance gene and analyzed by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stable expression of EWS/FLI-1 in primary cells derived from mouse bone. Because Ewing sarcoma tumors generally arise in the bone of children and young adults, murine mPBDCs from young mice were studied as potential EWS/FLI-1 cellular targets. Cells were isolated from 3- to 4-week-old BALB/c mice by gently crushing the axial and long bones, and the adherent population was transduced with a replication-defective EWS/FLI-1 type 1 or control retrovirus. Stably transduced cells were selected based on neomycin resistance and further characterized. At first, EWS/FLI-1-transduced cells seemed somewhat morphologically heterogeneous, comprising a mixture of spindle-like cells, a few larger fibroblast-like cells, and some cells with a more stellate appearance (Fig. 1A, top right). EWS/FLI-1 mPBDCs initially proliferated slowly in culture, but during serial passage, the population became more uniform, comprised of immature-appearing cells with a slightly spindle-like morphology and nuclei with numerous punctate nucleoli (Fig. 1A, bottom left). Cytospins of the EWS/FLI-1-transduced population revealed mitotically active cells comprised of cells with slightly eccentrically placed nuclei with a relatively high nucleus-to-cytoplasmic ratio (Fig. 1A, bottom right). Some cells also possessed faint cytoplasmic granules. In contrast, murine bone-derived cells transduced with control retroviral vector (Fig. 1A, top left) or EWS/FLI-1 R2L2, a point mutant defective in nuclear localization, DNA binding (11), and transformation (ref. 8; data not shown) were comprised of large sheet-like cells with abundant cytoplasm that were difficult to propagate.



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  		Figure 1. Stable expression of EWS/FLI-1 in mPBDCs leads to Ewing sarcoma–like tumors in immunocompetent mice. A, photomicrographs of EWS/FLI-1-transduced mPBDC (top right and bottom) reveal cells with a higher nuclear-to-cytoplasmic ratio than vector control cells (top left) and prominent finger-like cytoplasmic projections, resulting in a stellate morphology. Stable expression of EWS/FLI-1 is confirmed by anti-FLI-1 immunoblot (bottom). The human Ewing sarcoma (TC-71) and rhabdomyosarcoma (RD) cell lines serve as positive and negative controls, respectively. Magnification: 10x (top), 40x (bottom left), 100x (cytospin bottom right). B, multiple, large intra-abdominal tumors (top left, arrows) and numerous pulmonary parenchymal and chest wall tumors (top right) formed after EWS/FLI-1 mPBDC were introduced into syngeneic BALB/c mice by i.p. and i.v. injection, respectively. Positions of esophagus (E), lung (L), and heart (H) are shown for orientation. A large, expansile tibial bone mass arising in an i.v. injected mouse (bottom). Note tumor-associated neovascularization in the affected limb. C, photomicrographs of H&E-stained EWS/FLI-1 mPBDC tumors from two different mice (left and middle). Note how the cells (left) resemble a "small round blue cell tumor"; cells in the middle panel have more abundant cytoplasm and an elongated, spindle-like morphology. For comparison, a metastatic lesion from a patient with Ewing sarcoma (right). D, H&E-stained histologic section of a tibia from a mouse with a bone tumor showing bone marrow infiltration by EWS/FLI mPBDC (left, arrows) and cortical destruction (vertical arrow). Right, a higher magnification of EWS/FLI mPBDC showing numerous neoplastic cells with multiple finely punctate nucleoli (arrows). Bar, 20 µm.

 
To confirm stable expression of EWS/FLI-1, early-passage cells (EF-1) were analyzed by Western immunoblot using an anti-FLI-1 antibody that also recognizes the EWS/FLI-1 fusion protein. EWS/FLI-1 was readily detected in this PBDC population and was expressed at levels similar to the well-characterized human Ewing sarcoma tumor cell line TC-71 (Fig. 1A, bottom). Cells transduced with retroviral vector alone (neo), or a human rhabdomyosarcoma tumor cell line (Rd) showed no evidence of EWS/FLI-1 expression. These results show that primary cells isolated from murine bone can tolerate stable expression of EWS/FLI-1 and become immortalized for serial passage in culture.

EWS/FLI-1–transduced murine primary bone-derived cells form sarcomatous tumors in immunocompetent mice. To determine if EWS/FLI-1 mPBDCs were transformed, cells were introduced into syngeneic animals (BALB/c) by s.c., i.p., or i.v. injection. Interestingly, EWS/FLI-1 mPBDCs were fully tumorigenic in immunocompetent syngeneic mice. Upon i.p. injection, EWS/FLI-1 mPBDCs formed large, multifocal intra-abdominal masses (Fig. 1B, top). I.v. injection of EWS/FLI-1 mPBDCs produced intrapulmonary, pleural, and chest wall tumors in some animals (Fig. 1B, top right), whereas other animals developed proximal appendicular bone tumors reminiscent of skeletal Ewing sarcoma (Fig. 1B, bottom).

The efficiency of tumor induction correlated with the number of cell passages in culture. EWS/FLI-1 mPBDCs rarely formed tumors before passage 15. By passages 17 to 18, ~38% (5 of 13) of mice injected with EWS/FLI-1 mPBDCs developed tumors, reaching a size of 2 cm3 by days 17 to 18 after s.c. injection. I.p. tumors from cell passages 17 to 18 took 3 to 6 months to be clinically manifest and were generally multifocal and quite bulky (Fig. 1B, top left). From cell passage 28 to 45, tumor induction was 100% (14 of 14), regardless of the route of administration. Intra-abdominal tumors from passages 28 to 45 also grew more rapidly, being clinically evident by an average of 43 days after inoculation.

EWS/FLI-1 tumors were comprised of morphologically immature cells with a high nuclear to cytoplasmic ratio (Fig. 1C, left), reminiscent of the "small round blue cell tumors" of human Ewing sarcoma (Fig. 1C, right). There were scattered mitotic figures. The tumors were well vascularized, and in some cases, contained large areas of intratumoral necrosis and hemorrhage (data not shown). Most EWS/FLI-1-induced murine tumors were comprised of cells that morphologically resembled human Ewing sarcoma cells; however, some EWS/FLI-1-induced tumors consisted of cells possessing more cytoplasm and assuming a more spindle cell morphology (Fig. 1C, middle). Upon i.v. injection, some animals developed bone tumors that resembled skeletal Ewing sarcoma. These tumors occurred at an average of 49 days after injection and were comprised of a diffuse intramedullary infiltration with undifferentiated cells with numerous punctate nucleoli (Fig. 1D, right). Besides diffuse infiltration of the bone marrow, there was also evidence of focal cortical destruction and invasion by tumor cells (Fig. 1D, vertical arrow).

Characterization of murine EWS/FLI-1 tumors. Tumors arising in mice inoculated with EWS/FLI-1-transduced mPBDCs were analyzed by anti-FLI-I immunoblot to confirm preserved expression of the EWS/FLI-1 oncoprotein. Although there was some intertumor variability in level of expression, all tumors stably expressed EWS/FLI-1 protein (Fig. 2A). Because EWS/FLI-1 mPBDC-induced tumors were undifferentiated by conventional microscopy (Fig. 1A), EWS/FLI-1 mPBDCs and tumors were analyzed by immunohistochemistry for lineage and tumor marker expression (12). Interestingly, EWS/FLI-1 mPBDCs were negative for most markers in the panel but did consistently stain positive for smooth muscle actin (SMA). EWS/FLI-1 mPBDCs were negative for the neural-associated markers S100, p75 nerve growth factor receptor, and neural-specific enolase, and the muscle-related markers desmin, muscle-specific actin, myoglobin, and myosin (Table 1). EWS/FLI-1 mPBDCs were also negative for MAC-3 and CD68, markers of macrophage and dendritic lineage cells. EWS/FLI-1 mPBDCs were generally negative for the epithelial marker pan-keratin, but some murine EWS/FLI-1 tumors had some cells with focal cytoplasmic staining, a pattern also observed in some human Ewing sarcoma tumors (2).



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  		Figure 2. Murine sarcomatous tumors express EWS/FLI-1 protein and are oligoclonal for the EWS/FLI-1 provirus. A, protein lysates from EWS/FLI-1 mPBDC tumors were analyzed by anti-FLI-1 Western immunoblot. Individual mice are represented by letter; numbers denote different tumor samples from the same mouse. EWS/FLI-1 (+/–) expression controls (left). Positions of EWS/FLI-1 and the endogenous FLI-1 protein recognized by this antibody (arrows). B, genomic DNA from early-passage (EP, passage 6) or late-passage (LP, passage 20) EWS/FLI-1 (EF-1) mPBDC, or tumors from four different late-passage cell-injected mice were analyzed by Southern blot to evaluate the clonality of EWS/FLI-1 proviral integration. A dominant clone found in late passage cells and derived tumors (arrow). Representative tumors from two independent injection experiments. A clonal EWS/FLI-1 (EC) and a polyclonal vector control (N) 3T3 cell line as controls (far left). A clonal EWS/FLI-1 line (E3) obtained from an independent mPBDC retroviral transduction (middle).

 

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  		Table 1. Immunohistochemistry profile of EWS/FLI-1 mPBDC and tumors

 
Because the general lack of differentiation markers suggested the possibility of a mesenchymal or other primitive stem cell, cells were obtained from two different murine EWS/FLI-1 tumors, expanded briefly in adherent culture, and harvested for analysis by FACS. Interesting, the vast majority of EWS/FLI-1 tumor cells (95-99%) were Sca-1 positive but lacked expression of the hematopoietic stem cell marker c-Kit (Table 2). EWS/FLI-1 cells were also negative for vascular endothelial growth factor receptor 2 (VEGFR2), CXCR4, and MAC-1, whereas a minority of cells (9% in one tumor) were positive for CD41 (integrin-{alpha} IIB). Thus, EWS/FLI-1 mPBDC tumors showed little evidence of definitive lineage commitment by morphology, immunohistochemistry, or FACS, consistent with the general lack of definitive lineage marker expression seen in human Ewing sarcoma tumors (2).


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  		Table 2. Tumor mesenchymal stem cell marker profile (FACS)

 
Because EWS/FLI-1 was introduced into mPBDCs by retroviral transduction, the clonality of murine EWS/FLI-1-induced tumors was assessable by Southern blot. Early-passage (passage 6) EWS/FLI-1 mPBDCs in culture contained four or five distinct clones by Southern blot, whereas later-passage (passage 20) cells were even more oligoclonal, with one clone predominating (Fig. 2B, arrow). This EWS/FLI-1 clone, alone or in association with a couple of other minor clones, was found in all tumors arising in animals inoculated with this EWS/FLI-1 mPBDC population (Fig. 2B, tumors 1-4). EWS/FLI-1 cells from another independent transduction experiment showed predominance of a different unique proviral integration site, indicating that tumorigenesis was not dependent on a particular retroviral genomic integration site (Fig. 2B, E3). None of the EWS/FLI-1 mPBDC lines or tumors showed any abnormality in proviral size by Southern blot (data not shown). Because early-passage EWS/FLI-1 mPBDCs were inefficient in tumorigenesis compared with later-passage cells, these findings suggested that passage in culture selected for EWS/FLI-1 mPBDC clones with greater tumorigenic potential.

Progressive cell cycle dysregulation in EWS/FLI-1 murine primary bone-derived cells. Altered regulation of cell cycle regulatory factors has been reported in EWS/FLI-1-expressing cells and primary Ewing sarcoma tumors, but their role in Ewing sarcoma tumorigenesis has not been firmly established (7, 13–17). In early-passage EWS/FLI-1 mPBDCs, p53 protein expression was constitutively increased compared with control mPBDCs (Fig. 3A), consistent with an EWS/FLI-1-dependent increase in p53 gene expression (14). Increased expression of p53 in early-passage EWS/FLI-1 mPBDC was associated with a marked increase in the expression of the cell cycle regulatory protein p21, an established p53 target gene (18). However, in late-passage EWS/FLI-1 mPBDC, p21 expression was not increased, despite even higher constitutive levels of p53. Furthermore, in one EWS/FLI-1 mPBDC tumor (Fig. 3A, TC), there was no detectable expression of p21 by Western immunoblot despite high-level p53 protein expression. This progressive alteration in p53 and p21 expression was not attributable to differences in EWS/FLI-1 protein expression, which remained constant from early-passage EWS/FLI-1 mPBDCs to established tumors (Fig. 3A, large arrow). Expression of other cell cycle–related genes, such as cyclin D1 and c-myc, was generally increased in EWS/FLI-1 mPBDC cell lines and tumors, consistent with previous reports (7, 11, 13).



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  		Figure 3. P53 and cell cycle-related gene expression analysis reveals a novel p53 mutation in some murine EWS/FLI-1 tumors. A, early-passage (EP) or late-passage (LP) EWS/FLI-1 mPBDC, a EWS/FLI-1 tumor (TC), or cells transduced with vector alone (N) were analyzed by immunoblot using the indicated antibodies. Right, positions of EWS/FLI-1 (large arrow) and endogenous FLI-1 (double arrows). NIH 3T3 cells (–), a human Ewing sarcoma (ES, TC-71) and a rhabdomyosarcoma (RS, RD) cell line as controls (far left). Anti-actin immunoblot serves as a loading control. B, a schematic of murine p53 depicting the approximate amino acid positions of the transcriptional activation (TA) domain, src-homology 3-like domain with PXXP motif, DNA-binding domain, and COOH (C)-terminal region. An expanded view of a highly conserved region encoded by exon eight (E8) of the murine p53 gene depicts the novel deletion mutation (dashed lines) found in some EWS/FLI-1 mPBDC tumors. Note that the remaining bases from the two serine (S) codons flanking the deleted region form a single serine and preserve the reading frame. C, Western immunoblot analysis of six different murine EWS/FLI-1 (EF-1) tumors. In tumors 1 to 3 and vector control mPBDC (N), p53 is wild type and is constitutively expressed at modest levels (top arrow), whereas tumors 5 and 6 constitutively express the mutant form of p53 depicted in (B), which exhibits a slightly faster mobility on gradient gel electrophoresis (asterisk). Tumor 4 expresses a mixture of wild-type and mutant p53. Note that p21 is constitutively expressed in cells with wild-type p53 and lost in cells expressing mutant p53. Right, for comparison, a human breast cancer cell line (BC, HCC1143) overexpressing mutant P53 (47) and a leukemia cell line (K5, K562) lacking p53 expression (48). Primary murine myoblasts in growth (–) or myogenic differentiation conditions (+) are shown as controls for p21 induction (7, 49, 50). EWS/FLI-1 (human ES cell line TC-32, single arrow) and FLI-1 (F1, double arrows) expression controls (far right). Anti-actin immunoblot (bottom) serves as a loading control.

 
The disconnection between p53 and expression of its transcriptional target p21 in late-passage EWS/FLI-1 cells and a murine EWS/FLI-1 tumor suggested the presence of a mutation in p53. To explore this possibility, total RNA was prepared from an EWS/FLI-1 mPBDC tumor (TC), and the entire p53 transcript was amplified by reverse transcriptase-PCR (RT-PCR) and sequenced. The only abnormality found was a 24-nucleotide deletion in exon 8, located within a region of the P53 DNA-binding domain highly conserved between mouse and man (Fig. 3B). Interestingly, the mutation occurred within the nucleotides spanning Ser264 and Ser272 (Fig. 3B, mouse sequence in bold), resulting in deletion of the eight amino acid sequence GNLLGRDS. Because the S264 codon was recreated, there was preservation of the reading frame, resulting in a virtual full-length mutant p53 protein still detectable by a COOH-terminal anti-p53 antibody (Fig. 3A). Sequence analysis of early-passage EWS/FLI-1 and control mPBDCs revealed no evidence of p53 mutation (data not shown), consistent with the relatively low levels of constitutive p53 protein in these cells (Fig. 3A, N and early passage compared with TC). Thus, some EWS/FLI-1 cells had acquired a novel p53 mutation containing an in-frame eight amino acid deletion in the p53 DNA-binding domain, associated with a constitutive loss of p21 protein expression in vivo.

Although loss or mutation of p53 is frequent in human Ewing sarcoma tumor cell lines, it is less common in primary Ewing sarcoma tumor specimens (19–21). To determine if p53 mutation was a required step in EWS/FLI-1-mediated transformation of primary murine bone-derived progenitors, additional murine EWS/FLI-1 tumors were analyzed for p53 expression and mutational status. Of a total of six murine EWS/FLI-1 tumors analyzed, two showed constitutive p53 overexpression and no detectable expression of p21 (Fig. 3C, lanes 5-6). Both tumors had the same in-frame p53 deletion on sequence analysis (data not shown). Three EWS/FLI-1 murine tumors exhibited low-level p53 expression, associated with preserved expression of p21 (lanes 1-3). No mutation of p53 was detected in these tumors (data not shown). One tumor expressed two different p53-immunoreactive proteins (lane 4), one migrating as wild type, and the other with a slightly faster electrophoretic mobility that comigrated with the mutant p53 band seen in the tumors with the small in-frame deletion (Fig. 3C, asterisk). Comprised of p53 mutant and p53 wild-type cells, this tumor expressed significantly less p21 protein than p53 wild-type tumors. As expected, all EWS/FLI-1 tumors expressing the p53 deletion mutation arose in animals injected with the same EWS/FLI-1 mPBDC population. No other type of p53 mutation was detected in tumors or cells from three independently generated EWS/FLI-1 mPBDC populations (data not shown). As seen earlier (Fig. 3A), all tumors constitutively expressed a range of EWS/FLI-1 protein. Interestingly, expression of endogenous FLI-1 (double arrows) was absent in one mouse EWS/FLI-1 tumor (lane 5) and barely detectable in another (lane 6). These results show that EWS/FLI-1-mediated transformation of mPBDCs did not require mutation of p53.

EWS/FLI-1 is required for the efficient transformation of murine primary bone-derived cells. With successive passage in culture, EWS/FLI-1 mPBDCs became more efficient in tumor induction. This finding suggested that EWS/FLI-1 was an initiating event in tumorigenesis but that secondary events were required for the full tumorigenic potential of EWS/FLI-1 mPBDC. To gauge the relative contribution of EWS/FLI-1 in the transformation of primary cells, a mPBDC cell line was established that stably expressed EWS/FLI-1 flanked by loxP sites (22–24). After propagation in culture, EWS/FLI-1 loxP mPBDCs were transduced with a replication-defective Cre retrovirus, and the effect of cre-mediated EWS/FLI-1 excision was quantitated by soft agar clonogenic assay. Passage 11 EWS/FLI-1 loxP mPBDCs transduced with vector alone formed a mean of 184 ± 17 colonies per plate, compared with 65 ± 4 colonies for Cre-transduced cells, a 2.8-fold reduction (P = 0.002; Fig. 4A). Importantly, the reduction in clonogenicity paralleled the Cre-mediated decrease in EWS/FLI-1 protein expression by immunoblot (Fig. 4A, bottom left). Later-passage (p24) EWS/FLI-1 LoxP mPBDCs also showed a Cre-mediated decease in soft agar transformation and EWS/FLI-1 expression (Fig. 4A, right). Control-transduced cells formed an average of 116 ± 8 colonies per plate, compared with 48 ± 7 colonies by Cre-transduced cells, a reduction of 2.4-fold (P = 0.003). There was no difference in constitutive EWS/FLI-1 protein expression between early- and later-passage cells (Fig. 4A).



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  		Figure 4. mPBDCs are highly dependent on EWS/FLI-1 for transformation. A, LoxP-EWS/FLI-1 murine PBDC were transduced with Cre (C)-recombinase or control (H) retroviral vector at the indicated cell passage (p) and plated in semisolid medium. Input cell number per plate for each passage (top right). Bars, SE from triplicate plates. Data were confirmed in at least two independent experiments. The difference in colony number between Cre- and control-transduced p11 and p24 cells was statistically significant (P = 0.002 and 0.003, respectively, unpaired t test). Bottom, input cells were analyzed by anti-FLI-1 immunoblot to assess Cre-mediated reduction in EWS/FLI-1. Sham-transduced (–) EWS/FLI-1 mPBDC are shown for each set. A human rhabdomyosarcoma (RD) and Ewing sarcoma (ES) cell line (TC71) are shown as EWS/FLI-1 expression controls (left). Anti-actin immunoblot serves as a loading control. B, a working model of Ewing sarcoma depicting the EWS/FLI-1 fusion as an early but not solitary event in the ultimate transformation of a primitive bone-derived cell.

 
Consistent with the results of the tumorigenicity assays, soft agar transformation efficiency correlated with the length of time EWS/FLI-1 loxP mPBDC cells were propagated in culture. At passage 11, the clonogenicity of EWS/FLI-1 loxP mPBDC was 0.03% (colony number per cell input), compared with 1.2% for passage 24 cells, a difference of 40-fold. Thus, although the transformation efficiency of EWS/FLI-1 mPBDCs increased with time in culture, cellular transformation remained dependent on the EWS/FLI-1 fusion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EWS/FLI-1 seems to play a central role in ESFT pathogenesis, but recent studies have suggested that EWS/FLI-1 is associated with considerable cytotoxicity when expressed in primary cells (14, 15). Nonetheless, the fact that Ewing sarcoma patients develop tumors containing the EWS/FLI-1 fusion is compelling evidence that certain primary cells thrive "despite" EWS/FLI-1. This implies that secondary genetic or epigenetic events play a role in lessening EWS/FLI-1-mediated cytotoxicity, or that EWS/FLI-1 toxicity is cell context dependent. In this study, we have tested the hypothesis that intramedullary bone contains a cellular target for EWS/FLI-1 and have developed cell culture conditions that biologically select for cells tolerant of EWS/FLI-1 expression. Cells immortalized and transformed by EWS/FLI-1 in our model seem to comprise a fairly rare subpopulation of intramedullary bone. Even early-passage EWS/FLI-1 mPBDCs were markedly oligoclonal by Southern blot, and in one experiment, the early population was monoclonal (Fig. 2B, E3). Furthermore, the cellular target in our mouse Ewing sarcoma model was more abundant in regions of cancerous or compact bone accessible by mechanical disruption than in bone marrow per se, because aggressive flushing of the intramedullary compartment alone did not yield a cell population capable of stable EWS/FLI-1 expression (data not shown).

EWS/FLI-1 mPBDCs and tumors were notable for their lack of definitive lineage markers, which has been the hallmark of human ESFT (2), and is consistent with studies implicating EWS/FLI-1 in suppressing cellular differentiation (7, 25). Expression of the transmembrane glycoprotein CD99 (MIC2) has been a useful marker for ESFT (26), but there is no known murine homologue. EWS/FLI-1 mPBDC tumors differed from human Ewing sarcoma tumors in their lack of neural-specific enolase and c-Kit staining (positive in 24-35% Ewing sarcoma cases; refs. 27, 28) and their consistent staining for SMA. The absence of lineage marker expression and the prominent expression of SMA and Sca-1 suggest that EWS/FLI-1 targeted a primitive mesenchymal stem cell, perhaps with features of a stromal (CFU-F) or pericytic precursor cell (29–33). In these cells, EWS/FLI-1 expression conferred a survival advantage over control vector–transduced cells, which were difficult to propagate. Murine mesenchymal stem cell isolation, characterization, and propagation vary considerably among laboratories (32–36), indicating that the bone is a complex and not yet fully characterized milieu of multipotential progenitors.

EWS/FLI-1 mPBDCs initially proliferated slowly in culture, in association with the induction of p53 and the p21 (Fig. 3), consistent with an EWS/FLI-1-induced, p53-mediated cytotoxic response to EWS/FLI-1 (14). Despite this initial hurdle, EWS/FLI-1 mPBDCs were immortalized in culture and were more readily propagated than control mPBDCs. Although p53 was wild type in early-passage EWS/FLI-1 mPBDCs, p53 mutation occurred in some EWS/FLI-1-expressing cells. Interestingly, the novel p53 mutation, discovered during the course of this study, seems to have indirectly mapped an eight-amino-acid region of p53 that is critical for the induction of p21. Other EWS/FLI-1 murine tumors did not overexpress p53 or contain p53 mutations, however, indicating that loss of p53 was not an essential feature of EWS/FLI-1-mediated tumorigenesis in this murine Ewing sarcoma model. These findings are consistent with the observation that most human Ewing sarcoma tumor cell lines have p53 loss or mutation but that p53 is mutated in only a minority of primary Ewing sarcoma tumor specimens (19–21). Indeed, numerous cell cycle regulator factors are dysregulated in EWS/FLI-1-expressing cells (13, 17, 19–21), but no single cell cycle regulatory factor has been shown to be permissive for the EWS/FLI-1-mediated transformation of primary cells (15, 25). Thus, for Ewing sarcoma tumors, the list of candidate EWS/FLI-1-collaborating factors is heterogeneous and likely extends beyond the category of cell cycle regulatory factors (8, 11, 13, 37, 38).

Despite the likelihood of collaborating factors in EWS/FLI-1-associated malignancy, several studies have shown that interference with EWS/FLI-1 expression inhibits human Ewing sarcoma tumor cell line proliferation and viability (5, 6, 39–41). Given the cytotoxicity of EWS/FLI-1 in primary cells, these findings suggest that the cellular response to EWS/FLI-1 may evolve from cytotoxic to dependency during the course of Ewing sarcoma biology. Consistent with this hypothesis, EWS/FLI-1 mPBDC proliferation was slow initially but grew with subsequent passage in culture, suggesting the possibility of occult secondary genetic and epigenetic changes. Nonetheless, mPBDC transformation strongly correlated with Cre-dependent changes in EWS/FLI-1 expression (Fig. 4A). Thus, like human Ewing sarcoma, EWS/FLI-1 was a central feature in this murine Ewing sarcoma model. Whether EWS/FLI-1 dependency is absolute in human Ewing sarcoma is unknown, but the forces underlying clonal evolution in cancer (42), including Ewing sarcoma (43–46), make it likely that Ewing sarcoma patients harbor some EWS/FLI-1-independent clones. Interestingly, although Cre-transduced loxP-EWS/FLI-1 cells showed a significant reduction in soft agar colony formation, Western immunoblot, and RT-PCR analysis of surviving colonies revealed that some mPBDC clones could still grow in soft agar despite successful "loss" of EWS/FLI-1 (data not shown). This suggests that whereas many clones were dependent on EWS/FLI-1 for transformation, some evolved to EWS/FLI-1 independence, suggesting potential limitations of EWS/FLI-1-targeted therapy as a means to eradicate Ewing sarcoma.

Taken together, our findings suggest that EWS/FLI-1 is an initiating factor in sarcomagenesis. In this working model, an EWS/FLI-1 fusion develops in a bone-resident progenitor cell population that is more active in young individuals (Fig. 4B). The cytotoxicity of EWS/FLI-1 provides a natural barrier to cellular transformation by limiting the time frame in which additional events (p53 mutation or other genetic/epigenetic events) must occur to avoid apoptosis or senescence. In most cases, secondary events do not occur, and no malignancy results. Thus, Ewing sarcoma would be a rare disease, with EWS/FLI-1 as the central feature, associated with a heterogeneous list of potential secondary cooperating factors. Certainly, our study does not rule out other possible scenarios. For example, EWS/FLI-1 alone might be sufficient for transformation, but the cellular target is rare, or that a variety of secondary events occur before the development of the EWS/FLI-1 fusion. Nonetheless, primary cell-based models systems like the one described here should prove valuable tools in testing these hypotheses and provide rationale for using discrete progenitor cell populations as platforms to study the origins of sarcoma. Lastly, this murine Ewing sarcoma model will allow careful dissection of the molecular events that lead to EWS/FLI-1-dependent and EWS/FLI-1-independent tumorigenesis.


   Acknowledgments
 
Grant support: Nearburg Family Fund for Pediatric Cancer Research.

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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jerry Shay and Woodring Wright for the pBabe/loxP and pBabe/Cre vectors; Krista Lladik and the UT Southwestern Immunohistochemistry Core Facility, Frank Marini, and Michael Andreef for assistance in the FACS analysis of EWS/FLI-1 mPBDC; and Eric Olson and Richard Gaynor for critical review of the article before publication.


   Footnotes
 
Note: Y. Castillero-Trejo and S. Eliazer contributed equally to this work.

S. Eliazer is currently at CyThera, Inc., 3550 General Atomics Drive, Building 2, Room 503, San Diego, CA 92121.

Received 5/18/05. Revised 7/15/05. Accepted 7/26/05.


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