Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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[Cancer Research 63, 3464-3468, July 1, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Ewing Tumor Fusion Proteins Block the Differentiation of Pluripotent Marrow Stromal Cells1

Enrique C. Torchia, Sobha Jaishankar2 and Suzanne J. Baker3

Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The Ewing family of tumors are poorly differentiated pediatric solid tumorsarising in bone and soft tissues from an unknown cell of origin. Ewingtumors are molecularly defined by in-frame genomic fusions that combine EWS with one of several ETS genes, most commonly FLI-1. We considered pluripotent marrow-derived stromal cells a likely candidate for the origin of Ewing tumors and assessed the effects of EWS/ETS proteins in this cell background. EWS/ETS expression in marrow-derived stromal cells caused a dramatic change in cellular morphology that was dependent on the presence of the ETS domain and unique to the fusion proteins. EWS/ETS fusion proteins blocked differentiation along osteogenic and adipogenic lineages, consistent with the undifferentiated appearance of Ewing tumors. Inhibition of differentiation may be an important function of EWS/ETS proteins in the genesis of Ewing tumors.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The Ewing family of tumors are pediatric tumors characterized by a lack of distinguishing morphological features and the presence of balanced chromosomal translocations. In 85% of cases, the resulting fusion gene comprises the 5' end of the Ewing sarcoma (EWS) gene and the 3' end of FLI-1, which belongs to the ETS family of transcription factors (1) . Other ETS genes such as ERG, FEV, ETV1, and E1AF are less frequent fusion partners for EWS in Ewing tumors (2) . ETS proteins recognize a common DNA-binding motif (GGAA/T) and are implicated in various cellular processes, including differentiation, apoptosis, development, and cell growth (3) . The oncogenic mechanism of EWS/ETS fusion proteins in Ewing tumors may involve aberrant transcription regulation of ETS target genes, as well as perturbations in mRNA splicing (2 , 4) .

A complete understanding of transformation by the EWS/ETS proteins will require knowledge of the cell of origin for these tumors. Ewing tumors are classified as primitive neuroectodermal tumors because a subset of these cancers express neural markers, and some Ewing tumor-derived cell lines can undergo neural differentiation (5) . However, most Ewing tumors lack all markers of differentiation, and 85% of cases arise in skeletal sites. Ewing tumors that arise in soft tissue are morphologically and molecularly indistinguishable from those that arise in bone (2) . Therefore, the cell of origin for Ewing tumors is likely to be a primitive, multipotent cell that can give rise to bone or cell types found in soft tissues. MSCs4 are pluripotent mesenchymal cells that reside in the bone marrow and can differentiate into a variety of cell types, including bone, fat, cartilage, skeletal muscle, smooth muscle, neurons, and astrocytes (6, 7, 8) . MSCs can be mobilized from the marrow compartment to populate extraskeletal sites (9) , making these cells a good candidate for the cell of origin for Ewing tumors.

In this study, we analyzed the consequence of expressing EWS/ETS fusion proteins in MSCs. EWS/FLI-1 and EWS/ERG induced a distinct morphological change and blocked osteogenic and adipogenic differentiation of MSCs. The presence of the ETS domain was required for some, but not all, of the EWS/ETS activities in MSCs. EWS/ETS activity may play a causative role in the poorly differentiated state of the Ewing family of tumors.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Isolation and Cell Culture of MSCs.
MSCs were isolated and cultured from bone aspirates as described previously (10) . Briefly, marrow aspirates of femurs and tibias of 3-week-old Arf-/- mice, a gift from Drs. C. Sherr and M. Roussel (St. Jude Children’s Research Hospital, Memphis, TN), were isolated in PBS containing 2% heat-inactivated FBS. Aspirated cells were plated and cultured for 5–7 days in {alpha}-MEM (Clonetics/Biowhittaker, Walkersville, MD) containing 20% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Adherent cells were then expanded twice and either frozen or used directly for subsequent experiments.

Expression Vectors.
Flag epitope-tagged EWS, FLI-1, EWS/FLI-1, EWS/ERG, and EWS/FLI-1{Delta}ETS, a mutant EWS/FLI-1 lacking 54 amino acids from the ETS domain (11) , were subcloned into the retroviral expression vector MSCV-IRES-GFP (12) . This vector also encodes the GFP directly downstream of an internal ribosomal entry site. High titer ecotropic viruses were made in 293-T cells by cotransfection of retroviral vectors with a plasmid encoding a packaging defective helper virus (13) .

Differentiation Assays.
MSCs were transduced with retrovirus and selected 72-h post-transduction for equivalent levels of GFP expression by FACS. Only GFP-positive cells were used in subsequent experiments. Cells were plated for differentiation assays 24 h after sorting at a density of 1 x 105 cells/35-mm wells and allowed to reach confluency, usually an additional 24 h, before induction of differentiation. Osteogenic differentiation was induced by maintaining cells in DMEM (Clonetics/Biowhittaker) containing 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml L-ascorbic acid, 100 nM dexamethasone, and 10 mM ß-glycerophosphate for 8 days, after which 8 nM 1{alpha}, 25{alpha}-dihydroxy vitamin D3 was included in the culture media for an additional 2 days. Cells were then incubated in DMEM with 10% FBS without supplements for 10 days. Calcification in differentiated cultures was detected by staining with Alizarin red as described previously (10) . Adipocyte differentiation was induced by culturing MSCs in DMEM containing 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, and 1 µM dexamethasone for 21 days. Accumulation of neutral lipids in cells was detected by Oil-Red-O staining (10) .

RNA and Immunoblot Analysis.
Total RNA was isolated from MSCs using the TRIzol reagent (Invitrogen, Carlsbad, CA), and RNA blot analysis was performed as described previously (14) . Expression of introduced genes was detected using a cDNA probe for EWS/FLI-1. Osteocalcin mRNA was detected using a cDNA probe encoding the first 92 amino acids of the protein. ß-actin was detected using a full-length cDNA probe, and Ppar{gamma} was detected using a cDNA probe encoding the first 50 amino acids of the protein. For Western blotting, cells were sorted by FACS 72 h after retroviral transduction, plated, and lysed 24 h later. SDS-PAGE and immunoblot detection of Flag-tagged proteins expressed in MSCs were performed as described previously (15) , using 10 µg/ml M2 anti-FLAG antibody (Fisher Scientific, Pittsburgh, PA).

Immunocytochemical and Cytochemical Staining.
MSCs or murine medulloblastoma cells SJMM1 (a gift from Tom Curran, St. Jude Children’s Research Hospital) were grown on poly-L-lysine glass slides (Clontech/BD Science, Palo Alto, CA), fixed in 4% paraformaldehyde in PBS, and permeabilized in 0.1% Triton X-100 in PBS. Slides were then blocked in 10% goat serum and incubated with antisera against mouse glial fibrillary acidic protein (1:200; DAKO, Carpinteria, CA), S100-ß (1:500; Sigma Corp., St. Louis, MO), or Mr 145,000 neurofilament M (1:500; Chemicon, Temecula, CA) before detection by appropriate Cy3-conjugated secondary antibody. Detection of senescence-associated ß-galactosidase activity was done as described previously (16) . Vector and EWS/FLI-1-transduced cells were sorted by FACS 72 h after transduction, plated, and stained for ß-galactosidase activity 48 h later.

Real-time PCR Analysis for Ppar{gamma}.
We designed a specific set of amplification primers and Taqman probe to detect the expression of murine Ppar{gamma} in MSC: 5'-CCCACCAACTTCGGAATCAG-3' and 5'-CCATTGGGTCAGCTCTTGTGA-3', Taqman probe: 6FAM-CTCCGTGATGGAAGACCACTCGCA-TAMRA. Relative quantitation of Ppar{gamma} message was performed from 25 ng of reverse transcribed total RNA using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). Levels of Ppar{gamma} were normalized to ribosomal 18S RNA abundance (Taqman probe Cat: 4310893E; Applied Biosystems).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
EWS/ETS Proteins Alter the Morphology of MSCs Isolated from Arf-/- Mice.
We established primary cultures of MSC from Arf-/- mice to evaluate the effect of expressing EWS/ETS fusion proteins in this cell background. We chose Arf-/- mice because mutations in the INK4A/ARF locus are important secondary genetic alterations found in 25% of Ewing tumors (17, 18, 19, 20) . In addition, stable expression of EWS/FLI-1 is tolerated in cultures of Arf-null fibroblasts (21) . MSC cultures from Arf-/- mice retained the ability to differentiate into bone and fat cells, indicating that the absence of Arf did not alter the pluripotency of MSCs in culture (data not shown). To determine whether EWS/ETS proteins could alter the differentiation potential of Arf-/- MSCs, we used a retroviral system (Fig. 1A)Citation to express EWS/FLI-1 and EWS/ERG fusion proteins. In addition, we also expressed empty vector, EWS, FLI-1, and a mutated EWS/FLI-1 lacking the ETS domain (EWS/FLI-1{Delta}ETS) to determine the activities that are unique to the fusion proteins and the relative contribution of the ETS domains in the function of EWS/ETS fusion proteins. EWS/FLI-1{Delta}ETS does not bind DNA or promote anchorage-independent growth of NIH 3T3 cells (11 , 14) . All constructs also expressed GFP, so transduced cells were FACS sorted for GFP expression to eliminate interference of subsequent differentiation assays by untransduced cells. All retroviral vectors gave rise to proteins of expected molecular weights as shown by Western blot analysis (Fig. 1B)Citation .



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  		Fig. 1. Expression of EWS/FLI-1 and EWS/ERG alters the morphology of MSCs. A, diagram of retroviral vectors used to express bicistronic messages encoding Flag epitope-tagged EWS, FLI-1, EWS/FLI-1{Delta}ETS, EWS/FLI-1, or EWS/ERG proteins, along with GFP, which was used to select transduced cells by FACS and verify expression of transgenes. In B, expression of introduced proteins in MSCs was detected by Western blot analysis in 20 µg of cell lysates using anti-Flag antibody. In C, MSCs expressing EWS/FLI-1 or EWS/ERG showed similar dramatic changes in cellular morphology in comparison with cells expressing vector, full-length EWS, FLI-1, or EWS/FLI-1{Delta}ETS. In D, vector and EWS/FLI-1-transduced MSCs were stained for neutral pH ß-galactosidase activity, a marker associated with senescent cells. As a positive control for senescence in this cell background, wild-type MSCs were maintained in culture for 2 weeks. Senescent cells stain blue. Both vector and EWS/FLI-1-transduced MSCs showed very few S-ß-gal-expressing cells. Scale bar, 100 µm.

 
Expression of either EWS/FLI-1 or EWS/ERG in MSCs resulted in a dramatic change in cellular morphology within 48 h after transduction, with cells appearing round with a centrally located nucleus. This pronounced change in morphology was evident regardless of the density of the cells and was not attributable to cell cycle arrest (data not shown). In contrast, cells transduced with vector, EWS, FLI-1, or EWS/FLI-1{Delta}ETS showed mesenchymal cell morphology similar to untransduced MSCs (Fig. 1C)Citation . Given the enlarged morphology of MSCs expressing the fusion proteins, we also evaluated whether EWS/FLI-1 induced cellular senescence in MSC cultures by assaying for the presence of senescence-associated ß-galactosidase activity (S-ß-gal; Ref. 16 ). As shown in Fig. 1DCitation , very few cells transduced with empty vector or EWS/FLI-1 showed S-ß-gal staining as compared with widespread staining of MSCs isolated from wild-type mice that were allowed to senesce, indicating that EWS/ETS fusion proteins do not induce cellular senescence in the MSC background. Furthermore, the morphology of senescent MSCs did not resemble the morphology induced by expression of EWS/ETS proteins.

EWS/ETS Proteins Blocked Osteogenic Differentiation.
To determine whether EWS/FLI-1 or EWS/ERG could modulate the osteogenic potential of MSCs, we cultured transduced MSCs with inducers of bone differentiation and assayed for the presence of mature osteoblasts. As shown in Fig. 2ACitation , when vector, EWS-, and EWS/FLI-1{Delta}ETS-transduced cells were cultured with osteogenic inducers, strong mineral deposition was detected by Alizarin red staining. In contrast, mineral deposition was absent in EWS/FLI-1- and EWS/ERG-transduced cells. Interestingly, FLI-1-transduced cells also showed a similar lack of mineral deposition. All transduced cells retained expression of the introduced constructs as shown by GFP fluorescence of cells immediately before Alizarin red staining (data not shown). To evaluate the block in osteogenic differentiation at the molecular level, we used RNA blotting to detect the expression of osteocalcin, an osteoblast marker. As expected, osteocalcin was undetectable in transduced MSCs not cultured with osteogenic inducers (Fig. 2BCitation , Day 0) and induced at levels proportional to the degree of Alizarin red staining in vector, EWS-, and EWS/FLI-1{Delta}ETS-transduced cells (Fig. 2BCitation , Day 10). Osteocalcin was not detectable in EWS/FLI-1-, EWS/ERG-, or FLI-1-transduced cells. Expression of the introduced transgenes was maintained in cells induced to differentiate as shown by Northern blot (Fig. 2B)Citation . The inability of EWS/FLI-1{Delta}ETS to prevent osteogenic differentiation indicates that a functional ETS domain is important for the inhibition of osteogenesis by EWS/ETS fusion proteins.



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  		Fig. 2. EWS/ETS proteins block osteogenic differentiation of MSCs. In A, transduced MSCs were cultured with osteogenic inducers for 21 days, fixed, and stained with Alizarin red. MSCs expressing empty vector, EWS, and EWS/FLI-1{Delta}ETS stained heavily for calcification, whereas FLI-1-, EWS/FLI-1-, and EWS/ERG-transduced cells showed no evidence of calcification. Scale bar, 300 µm. In B, detection of osteocalcin, transgenes, and ß-actin mRNA in 10 µg of total RNA isolated from transduced MSCs at the start of differentiation (Day 0) or 10 days after induction of differentiation. Induction of osteocalcin was consistent with calcification observed in A. Expression of all introduced transgenes, detected with a probe for EWS/FLI-1, was maintained. ß-actin expression is shown as a loading control.

 
EWS/ETS Proteins Blocked Adipocyte Differentiation.
We cultured transduced MSCs with inducers of adipocyte differentiation to determine whether the effect of EWS/ETS proteins was specific to bone or a general effect on differentiation. Similar to osteogenic induction, vector and EWS-transduced MSCs were not perturbed in adipocyte differentiation and showed strong accumulation of neutral lipids as indicated by Oil-Red-O staining (Fig. 3A)Citation . In contrast, EWS/FLI-1-, EWS/ERG-, and FLI-1-transduced cells did not accumulate neutral lipids. In EWS/FLI-1{Delta}ETS-transduced cells, only a few lipid droplets could be observed. To evaluate adipogenic differentiation at the molecular level, we performed Northern blot analysis. Expression of the transgenes was maintained throughout the induction of differentiation (data not shown). Ppar{gamma}, a marker of adipocyte differentiation, was detectable in vector and EWS-transduced cells at day 21 of differentiation but undetectable in EWS/FLI-1-, EWS/ERG-, or EWS/FLI-1{Delta}ETS-transduced cells, consistent with Oil-Red-O staining (Fig. 3B)Citation . Surprisingly, Ppar{gamma} was highly expressed in FLI-1-transduced cells after adipogenic induction, despite the absence of neutral lipid accumulation (Fig. 3A)Citation . We quantitated Ppar{gamma} expression in transduced cells by real-time PCR. Consistent with Northern blot analysis, Ppar{gamma} was nearly undetectable in EWS/FLI-1-, EWS/ERG-, and EWS/FLI-1{Delta}ETS-transduced cells (Fig. 3C)Citation . FLI-1-transduced cells showed a 4-fold increase in Ppar{gamma} levels as compared with vector and a 105-fold increase as compared with EWS/FLI-1 cells. Therefore, FLI-1 inhibition of adipogenesis occurred at a later stage of differentiation than the block induced by EWS/ETS proteins, because FLI-1-expressing cells responded to adipogenic signals to induce Ppar{gamma} expression. The EWS/ETS inhibition of adipocyte differentiation also did not require the presence of a functional ETS domain because EWS/FLI-1{Delta}ETS-transduced cells also showed impaired adipogenesis.



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  		Fig. 3. EWS/ETS proteins block adipogenic differentiation of MSCs. In A, transduced MSCs were incubated with adipogenic inducers, fixed, and stained with Oil-Red-O. Only empty vector and EWS-transduced cells showed significant accumulation of neutral lipids seen as red droplets. In contrast, only a few cells with fat droplets were evident in EWS/FLI-1{Delta}ETS-expressing cells, whereas no fat droplets were present in FLI-1-, EWS/FLI-1-, and EWS/ERG-transduced MSCs. Scale bar, 300 µm. B, Northern blot detection of the adipocyte marker Ppar{gamma} and ß-actin mRNA in 10 µg of total RNA isolated from transduced MSCs at the start of differentiation (Day 0) and at day 21 of differentiation. EWS/FLI-1-, EWS/ERG-, or EWS/FLI-1{Delta}ETS-transduced cells failed to induce Ppar{gamma} consistent with the absence of neutral lipids in these cells. Unexpectedly, FLI-1-transduced cells showed a significant induction of Ppar{gamma} but failed to accumulate lipid droplets. In C, real-time PCR was used to quantitate the levels of Ppar{gamma} in differentiated MSCs. Ppar{gamma} levels were normalized to 18S RNA, and all samples were assayed in quadruplicate. Reverse transcribed RNA from 293T cells was used as a negative control for Ppar{gamma} expression, and all values of Ppar{gamma} shown are relative to Ppar{gamma} levels in 293T cells.

 
MSCs Express Neural Markers.
The cell of origin of Ewing tumors may be a progenitor cell with the capacity to express neural markers. We therefore characterized MSCs for the presence of neural markers in comparison with a murine medulloblastoma-derived cell line used as a control. As shown in Fig. 4Citation , MSCs showed strong expression of S100-ß, a glial marker enriched in brain tissue. Only few cells showed strong expression of neurofilament M (Mr 145,000), a neural specific protein, whereas Gfap, an astrocyte marker, was undetectable in MSCs. Expression of EWS/FLI-1 did not alter the proportion of MSCs expressing these neural markers (data not shown). Historically, Ewing tumors are classified as neuroectodermal tumors because of the subset of tumors that expresses neural markers. MSCs can differentiate along the neural lineage (22 , 23) , and we show that a subset of MSCs expresses neural markers in the absence of specific induction stimuli. Thus, Ewing tumors may arise from mesenchymal progenitor cells rather than primitive neuroectodermal cells. It is possible that the subset of Ewing tumors with neural expression patterns arises from the population of MSCs that express these markers. Alternatively, another event during tumorigenesis may stimulate this differentiation program in some Ewing tumors.



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  		Fig. 4. Detection of neural markers in culture of MSCs. MSCs were grown on poly-L-lysine-coated slides, fixed, permeabilized, and incubated with antiserum for S100ß, neurofilament (Mr 145,000), and Gfap. MSCs showed strong S100-ß expression, few cells showed strong expression of neurofilament, and Gfap (a glial cell-specific marker) was undetectable in MSCs. A murine medulloblastoma-derived cell line with strong expression of all three markers is included as a control. Scale bar, 50 µm.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
MSCs are good candidates for the cell of origin for Ewing tumors because they are primitive cells that can express neural markers, reside in bone, and can be mobilized to populate peripheral tissues. Therefore, we expressed EWS/FLI-1 and EWS/ERG fusion proteins in primary cultures of MSCs to determine the effects of the fusion proteins in this cell background. Any activities relevant to Ewing tumors should be shared by these two EWS/ETS fusion proteins because they appear to function interchangeably in the context of human tumors. Indeed, both EWS/ETS fusion proteins had an equivalent ability to induce a pronounced change in cellular morphology, producing rounded cells that were larger and contained more cytoplasm than the typical appearance of a human Ewing tumor cell. Thus, the fusion proteins have unique properties in this cell background but do not necessarily completely recapitulate the Ewing tumor phenotype.

EWS/FLI-1 and EWS/ERG blocked osteogenic and adipogenic differentiation of primary MSCs, an activity that is very relevant to the poorly differentiated appearance of Ewing tumors that often arise in bone. A block in differentiation is also consistent with the ability of EWS/FLI-1 to inhibit myogenesis in the immortalized cell line C2C12 (24) . Disruption of cellular differentiation is a common feature associated with oncogenic transformation, e.g., myogenic differentiation is blocked by expression of oncogenic forms of ras or fos (25 , 26) . A number of well-characterized fusion proteins block differentiation in leukemia (27) , such as acute myelogenous leukemia fusion proteins that interfere with hematopoietic maturation, leading to the accumulation of circulating blast cells in acute myeloid leukemia (28) . In the current study, we showed that FLI-1 blocked osteogenic and adipogenic MSC differentiation. FLI-1 also inhibits erythroid differentiation in hematopoietic cells, an activity that is believed to be relevant to its oncogenic role in Friend virus-induced mouse erythroleukemias (29) .

All EWS fusion proteins found in Ewing tumors include an ETS domain, indicating an important oncogenic function for this motif. In our studies, morphological change and osteogenic inhibition by EWS/ETS proteins required the ETS domain. Interestingly, EWS/ETS inhibition of adipogenic differentiation did not require the ETS domain of the EWS/ETS proteins, and FLI-1 blocked adipogenesis at a different stage of differentiation. Therefore, EWS/ETS exert unique effects distinct from FLI-1 and in some cases independent of the ETS domain in the regulation of differentiation. This is likely to be relevant to tumorigenesis, because a component of the oncogenic activity of EWS/FLI-1 is also independent of ETS-mediated DNA binding (14 , 30 , 31) .

In summary, using a primary pluripotent cell culture system, we developed a unique and relevant model system to study EWS/ETS function. The ability of EWS/FLI-1 and EWS/ERG to inhibit osteogenic and adipogenic differentiation is consistent with the poorly differentiated state of Ewing sarcomas and suggests that one critical function of EWS chimeric proteins in tumorigenesis may be to arrest differentiation.


   ACKNOWLEDGMENTS
 
We thank Gerard Grosveld and Edwin Horwitz for helpful discussions about marrow stromal cells. We also thank Cynthia Wetmore and Tom Curran for murine medulloblastoma cells and Peter McKinnon for comments on this manuscript.


   FOOTNOTES
 
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.

1 Supported in part by an NIH Cancer Center Support CORE grant, NIH Grant CA92117 (to S. J. B.), and the American Lebanese Syrian Associated Charities. Back

2 Present address: The University of Tennessee Health Science Center, 403 Hyman Building, 62 South Dunlap Avenue, Memphis, TN 38163. Back

3 To whom requests for reprints should be addressed, at Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail: suzanne.baker{at}stjude.org Back

4 The abbreviations used are: MSC, marrow-derived stromal cell; GFP, green fluorescent protein; Ppar{gamma}, peroxisome proliferative-activated receptor {gamma}; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorting. Back

Received 3/31/03. Accepted 5/19/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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