FUS-CHOP Fusion Protein Expression Coupled to p53 Deficiency Induces Liposarcoma in Mouse but Not in...

CANCER STEM CELLS

FUS-CHOP Fusion Protein Expression Coupled to p53

Deficiency Induces Liposarcoma in Mouse but Not in Human

Adipose-Derived Mesenchymal Stem/Stromal Cells

RENE RODRIGUEZ,a RUTH RUBIO,a IVAN GUTIERREZ-ARANDA,a GUSTAVO J. MELEN,a

CAROLINA ELOSUA,aJAVIER GARCIA-CASTRO,

a,bPABLO MENENDEZ

a

aAndalusian Stem Cell Bank, Centro de Investigacion Biomedica, Consejerıa de Salud-Universidad de Granada,

Granada, Spain; bInstituto de Salud Carlos III, Area Biologıa Celular y Desarrollo, Madrid, Spain

Key Words. Mesenchymal stem cells • Adipose-derived mesenchymal stem/stromal cells • Liposarcoma • Fusion genes • FUS-CHOP • p53

• Sarcomagenesis

ABSTRACT

Human sarcomas have been modeled in mice by expres-

sion of specific fusion genes in mesenchymal stem cells(MSCs). However, sarcoma models based on human MSCsare still missing. We attempted to develop a model of lipo-

sarcoma by expressing FUS (FUsed in Sarcoma; alsotermed TLS, Translocated in LipoSarcoma)-CHOP (C/

EBP HOmologous Protein; also termed DDIT3, DNADamage-Inducible Transcript 3), a hallmark mixoid lipo-sarcoma-associated fusion oncogene, in wild-type and p53-

deficient mouse and human adipose-derived mesenchymalstem/stromal cells (ASCs). FUS-CHOP induced lipo-

sarcoma-like tumors when expressed in p532/2but not in

wild-type (wt) mouse ASCs (mASCs). In the absence ofFUS-CHOP, p532/2 mASCs forms leiomyosarcoma,

indicating that the expression of FUS-CHOP redirects thetumor genesis/phenotype. FUS-CHOP expression in wtmASCs does not initiate sarcomagenesis, indicating that

p53 deficiency is required to induce FUS-CHOP-mediated

liposarcoma in fat-derived mASCs. In a human setting,p53-deficient human ASCs (hASCs) displayed a higher invitro growth rate and a more extended lifespan than wt

hASCs. However, FUS-CHOP expression did not inducefurther changes in culture homeostasis nor initiated lipo-

sarcoma in either wt or p53-depleted hASCs. These resultsindicate that FUS-CHOP expression in a p53-deficientbackground is sufficient to initiate liposarcoma in mouse

but not in hASCs, suggesting the need of additionalcooperating mutations in hASCs. A microarray gene

expression profiling has shed light into the potentialderegulated pathways in liposarcoma formation from p53-deficient mASCs expressing FUS-CHOP, which might

also function as potential cooperating mutations in thetransformation process from hASCs. STEM CELLS 2011;29:179–192

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

It is becoming evident that mesenchymal stem cells (MSCs)may be the target cell in which transforming mutations re-sponsible for sarcoma development arise [1]. Several types ofsarcomas have been reproduced in vivo on overexpression ofspecific fusion oncoproteins in bone marrow (BM)-derivedmouse MSCs (mMSCs). These models include the recapitula-tion of Ewing’s sarcoma by expression in BM-mMSCs ofEWS-FLI-1 [2, 3], mixoid liposarcoma (MLS) by expressionof FUS (FUsed in Sarcoma; also termed TLS, Translocated inLipoSarcoma)-CHOP (C/EBP HOmologous Protein; alsotermed DDIT3, DNA Damage-Inducible Transcript 3) [4], and

alveolar rhabdomyosarcoma by expression of PAX-FKHR [5].Furthermore, cancer-initiating cells displaying MSC propertieshave been recently identified in Ewing’s Sarcoma [6]. How-ever, the success in reproducing these tumors using humanMSCs (hMSCs) has not yet been achieved. The expression ofEWS-FLI-1 in hMSCs does not transform but induces agene expression profile similar to that observed in Ewing’ssarcomas [7].

A liposarcoma classification based on the differentiationstage of the MSCs has been suggested [8]. Among liposarco-mas, MLS is the most common, representing 35% of all cases[9]. At the molecular level, MLS is characterized by the pres-ence of the chromosomal translocation t(12;16) (q13;p11)which generates the FUS-CHOP fusion oncogene [10]. This

Author contributions: R. Rodriguez: designed the study, performed experiments, analyzed the data and interpreted the results, and wrotethe paper; R. Rubio, I.G.-A., and G.J.M: performed experiments; C.E.: analyzed data; J.G.-C.: conceived the study and analyzed data;P.M.: conceived the study, analyzed the data and interpreted the results and wrote the paper; P.M. and R. Rodriguez: financiallysupported the study.

Correspondence: Pablo Menendez, Ph.D., Andalusian Stem Cell Bank, Instituto de Investigacion Biomedica, Parque Tecnologico de laSalud, Avda del Conocimiento, Armilla, Granada 18100, Spain. Telephone: 34-958-894-672; Fax: 34-958-894-652; e-mail: [email protected]; or Rene Rodriguez, Ph.D., Andalusian Stem Cell Bank, Instituto de Investigacion Biomedica, ParqueTecnologico de la Salud, Avda del Conocimiento, Armilla, Granada 18100, Spain. Telephone: 34-958-894-672; Fax: 34-958-894-652;e-mail: [email protected] Received April 21, 2010; accepted for publication November 12, 2010; first published onlinein STEM CELLS EXPRESS November 23, 2010. VC AlphaMed Press 1066-5099/2009/$30.00/0 doi:10.1002/stem.571

STEM CELLS 2011;29:179–192 www.StemCells.com

oncogene encompasses the NH2-terminal domain of FUSfused in-frame to the entire coding sequence of CHOP. TheNH2-terminal domain of FUS confers the transactivationdomain to the fusion protein [11]. CHOP is a member of theC/EBP family of transcription factors, capable of formingheterodimers with and inactivate other (CCAAT/enhancerbinding protein) members [12].

Previous in vitro approaches have shown the transformingeffects of FUS-CHOP in fibroblasts [13], but not in 3T3-L1 prea-dipocytes, suggesting that the activity of FUS-CHOP may becell-dependent. The generation of transgenic mice expressingFUS-CHOP under the control of EF1a promoter gave rise to lip-osarcomas, which resembled their human counterparts [14]. Asaforementioned, the expression of FUS-CHOP in BM-mMSCsgave rise to MLS [4] supporting the idea that liposarcoma devel-ops in mesenchymal progenitors, at least in murine models.

The origin and pathogenesis of many tumors has exten-sively been studied in vivo using animal models and in vitrousing murine cells. However, differences between the mouseand human have left gaps in our understanding. Unfortunately,a human model to reproduce FUS-CHOP-mediated liposarcomausing hMSCs is still missing. Such a human model would becrucial to further investigate not only many aspects associatedwith the pathogenesis of liposarcoma but also to undertakehigh-throughput small compounds screening, thus, eliminatingconfounding in vitro and in vivo effects owing to the differen-ces between murine- and human-based disease models [15].

Human cells do not transform as easily as mouse cells,suggesting that in addition to the initiating fusion oncogene,secondary transforming hits may be necessary to transformhuman cells. We and others have demonstrated the relevantrole that the deficiency of different cell cycle regulators,especially p53, has on the transformation of mMSCs [16–20].In fact, p53 mutations and p53 deregulated expression arefrequent in liposarcomas [21, 22], supporting p53 as a poten-tial secondary hit for hMSC transformation.

Here, we attempted to model liposarcoma by ectopicexpression of FUS-CHOP in both wild-type (wt) and p53-defi-cient mouse and human adipose-derived mesenchymal stem/stromal cells (ASCs). In mouse, we found that FUS-CHOPexpression does not transform mouse ASCs (mASCs) on itsown. However, the expression of FUS-CHOP in p53�/�

mASCs induces liposarcoma. On the other hand, FUS-CHOP-was unable to transform either wt or p53-deficient humanASCs (hASCs), therefore, emphasizing a further need forunknown cooperating mutations to induce liposarcoma fromhASCs. A microarray gene expression profiling has revealedpotential deregulated pathways in liposarcoma formation fromp53-deficient mASCs expressing FUS-CHOP, which mightalso function as potential cooperating mutations in the trans-formation process from hASCs.

MATERIALS AND METHODS

MSC Sourcing and Culture

Mouse ASC cultures were established from adipose tissuefrom gonadal, retroperitoneal, and subcutaneous depots ofFVB mice as previously described [19]. p53�/� mASCs on aFVB background were previously described [20]. BM-mMSCswere obtained by flushing the femurs and tibias from 8- to12-week-old FVB mice with phosphate-buffered saline (PBS)supplemented with 2% fetal bovine serum (FBS). Mononu-clear cells were then plated at a density of 106 cells per centi-meter square in murine mesenchymal medium and supple-ments (http://www.stemcell.com; Vancouver, Canada, Stem

Cells Technologies) and incubated at 37�C in a 5% humidi-fied CO2 atmosphere. After 48 hours, nonadherent cells werediscarded and fresh medium was added. When cell cultureachieved >85% of density, adherent cells were trypsinized,washed, and replated at a concentration of 5,000 cells percentimeter square.

Human ASCs were obtained from Inbiobank (www.inbioban-k.org; San Sebastian, Spain). Two independent cultures establishedfrom lipoaspirates from two donors (39- and 45-year-old whitefemales with 29.5 and 32 body mass index, respectively) wereused. hASCs were cultured in advanced-Dulbecco’s modifiedEagle’s medium (http://www.invitrogen.com; Carlsbad, CA,Gibco) plus 10% FBS. When cell cultures achieved over 85% ofdensity, adherent cells were trypsinized, washed in PBS, andreplated at a concentration of 5� 103 cells per centimeter square.

Lentiviral Vectors, Viral Production,and ASC Transduction

FUS-CHOP cDNA (exon 7 of FUS is fused to exon 2 ofCHOP; kindly provided by Dr. I Sanchez-Garcia, Salamanca,Spain) was inserted into the pRRL-EF1a-PGK-GFP lentiviralexpression vector. Viral particles pseudotyped with VSV-Gwere generated on 293T cells using a standard calcium-phos-phate transfection protocol and were concentrated by ultra-centrifugation. Murine and human ASCs were infected over-night with concentrated viral particles expressing eitherpRRL-EF1a-PGK-GFP or pRRL-EF1a-FUS-CHOP-PGK-GFP.The next day, the viral supernatant was removed and transducedcells were washed with MSC media and allowed to expand [23].In those experiments where the transduction efficiency resultedlower than 85%, the transduced (Green Fluorescent Protein)GFPþ fraction was enriched using a FACSAria cell sorter(http://www.bd.com; Franklin Lakes, NJ, Becton Dickinson;supporting information Fig. 1 and Fig. 5A). Depletion of p53was achieved by transduction with lentiviral particles carrying ap53-shRNA expression vector (pLVUH-shp53; Addgene plas-mid 11,653 [24]). In a typical experiment, the efficiency oftransduction using these lentiviral particles was of approxi-mately 80% (supporting information Fig. 2).

In Vitro Culture Homeostasis andDifferentiation Analyses

Morphology of the different ASC cultures was recorded daily.As for the growth kinetics of the different ASC cultures, cellswere counted every 4 days and replated at a density of 3 �103 cells per centimeter square. Cumulative population dou-blings were calculated as previously described [20]. Colony-forming assays and replicative senescence was determined aspreviously described [25]. MSCs differentiation studies wereperformed by plating either mouse or human ASCs in specificdifferentiation inductive media for 2 weeks as described [19].G-banding karyotype analysis was performed as previouslydescribed [26, 27]. Fifty metaphases were consistently ana-lyzed for each ASC genotype.

Flow Cytometry and Cell Sorting

The immunophenotype of cultured ASCs was determined byflow cytometry using fluorochrome-conjugated monoclonalantibodies anti-Sca-1, CD11b, CD14, CD29, CD44, and CD45for mASCs or anti-CD90, CD73, CD105, CD166, CD106,CD45, CD34, HLA-DR, CD19, and CD14 (BD Bioscience,http://www.bdbiosciences.com; Franklin Lakes, NJ) forhASCs as described [19, 23]. 5-bromo-2-deoxyuridine incor-poration and total DNA content were measured as described[25]. Cell sorting of GFPþ ASCs was conducted under BSL-2 conditions using a FACSAria cell sorter Becton Dickinson(http://www.bd.com; Franklin Lakes, NJ).

180 Role of FUS-CHOP in Mouse and Human ASCs

Reverse Transcription Polymerase Chain Reaction

Total RNA extraction and reverse transcription polymerasechain reaction (RT-PCR) were done as previously described[28]. RT-PCR conditions were as follows: cDNA synthesis at37�C for 2 hours, pre-PCR denaturation at 94�C for 2 minutesfollowed by 35 cycles of denaturation at 94�C for 30 seconds,annealing at 64�C for 30 seconds, and extension at 72�C for30 seconds. FUS-CHOP primers (forward: 50-GACAGCAGAACCAGTACAACAG-30 and backward: 50-TGAGTCATTGCCTTTCTCCTTC-30) amplified a 443 bp sequence span-ning the FUS-CHOP fusion breakpoint. GAPDH primers (for-ward: 50-GAAGGTGAAGGTCGGAGTC-30 and backward:50-GAAGATGGTGATGGGATTTC -30) were used as loadingcontrol and amplified a sequence of 228 bp.

Western Blot

Whole cell extracts were prepared as previously described[29]. Thirty micrograms of protein was resolved on 10% (so-dium dodecyl sulfate polyacrylamide gel electrophoresis) gelsand blotted onto nitrocellulose membrane (BioRad, http://www.bio-rad.com; Hercules, CA). Proteins were detectedusing a chemiluminescence detection system (BioRad, http://www.bio-rad.com; Hercules, CA) according to the manufac-turer’s instructions. Antibodies used were as follows: anti-p53([sc-6243 and sc-126], 1:500 dilution), anti-GADD153/CHOP([sc-7351], 1:200; used for the detection of FUS-CHOP), anti-PPARc ([sc-7196], 1:500) and anti-C/EBPa ([sc-61], 1:500)from Santa Cruz Biotechnology (http://www.scbt.com; SantaCruz, CA); anti-FUS/TLS ([A300-292A], 1:7,000) fromBethyl Laboratories (http://www.bethyl.com; Montgomery,TX); anti-p21 ([554262], 1:750) from BD-Pharmingen (http://www.bdbiosciences.com; Franklin Lakes, NJ); and anti-b-actin ([A-1978], 1:20,000) from Sigma (http://www.sigmaal-drich.com; St. Louis, MO).

Anchorage-Independent Cell Growth

Soft agar colony formation assay was carried out using theCytoSelect 96-well cell transformation assay kit (Cell BiolabsInc., http://www.cellbiolabs.com; San Francisco, CA) asdescribed [20]. For each genotype, two independent experi-ments were performed in triplicate. Hela cells and the trans-formed line T-AD-p53-FC #4 were used as positive controls.

In Vivo Tumorogenesis Assays

Nonobese diabetic/severe combined immunodeficient NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NOD/SCID-IL2R�/�) micewere obtained from Jackson Laboratories (http://jaxmice.jax.org; Bar Harbor, ME). All mice were housed under specificpathogen-free conditions, fed ad libitum, and maintainedunder veterinary care according to animal welfare facilitiesguidelines. Eight- to twelve-week-old animals were used [30].Mice were inoculated subcutaneously with 5 � 106 ASCs(between passage 5 and 10 for mASCs and 5 and 15 forhASCs) or 1 � 106 tumor-derived cells. Animals were sacri-ficed when tumors reached approximately 10 mm or 4 monthsafter infusion. All animal research protocols were approvedby the Animal Research Ethical Committee of the Universityof Granada prior to the study. On tumor removal, half tumorwas mechanically disaggregated to establish ex vivo tumorcell lines as described [20]. The remaining portion of the tu-mor was used for immunohistopathology analysis.

Histological Analysis

Tumor samples were fixed in formol, embedded in paraffin,cut into 4-lm sections, and stained with hematoxylin and eo-sin (H&E). Multiple tumor sections were stained with specific

antibodies against S-100 (1:500 dilution), smooth-muscle b-actin (1:50 dilution), desmin (1:100 dilution), myogenin (1:50dilution; all from DakoCytomation, http://www.dako.com;Glostrup, Denmark), and GFP (1:400 dilution) from Invitro-gen (http://www.invitrogen.com; Paisley, UK) as previouslydescribed [31].

Gene Expression Microarray Analysis

ASCs were collected during the exponential cell growth andstabilized in RNA later (Ambion, http://www.ambion.com;Austin, TX) solution until RNA extraction. RNA was isolatedusing the Agilent Total RNA Isolation Kit (Agilent Technolo-gies, http://www.agilent.com; Palo Alto, CA) and its qualitychecked in the Agilent 2100 Bioanalyzer. Total RNA sampleswere labeled with Cy3 using the Quick-Amp Labeling Kitand hybridized with the Gene Expression Hybridization Kit tothe Whole Human Genome Microarray (G4112F) or WholeMouse Genome Microarray (G4122F), following Manufac-turer’s instructions (Agilent Technologies, CA). Each samplewas labeled and hybridized as independent triplicates [32].Primary data was examined using GeneSpring 11.0 software(Silicon Genetics, Redwood City, CA). Gene expression inthe control and experimental groups were compared usingStudent’s t test and a Benjamini Hochberg multitesting cor-rection. Only genes satisfying the threshold of p value < .05and a fold change expression >2 were included and assignedas significant. Pools of genes that were differentiallyexpressed were clustered according to their expression patterndynamics into hierarchical tree clustering algorithms using thePearson’s centered correlation distance definition as similaritymeasure and Centroid’s as the linkage rule. Analysis of path-ways significantly altered by FUS-CHOP was performedusing the Ingenuity Pathway software 8.0 (Ingenuity Systems,Inc., http://www.ingenuity.com; Redwood City, CA). Microar-ray data has been deposited and is available at Gene Expres-sion Omnibus (http://www.ncbi.nlm.nih.gov/geo/).

RESULTS

FUS-CHOP Induces Liposarcoma When Expressedin p53-Deficient Mouse ASCs

FUS-CHOP cDNA was subcloned into a pRRL-EF1a-PGK-GFP lentivector and GFP- and FUS-CHOP-expressing lentivi-ral particles were prepared and used to transduce both wt andp53�/� mASC. Transduction efficiency measured as GFPexpression was determined by fluorescence microscopy andflow cytometry (supporting information Fig. 1). To ensure, weworked with homogeneous mASC cultures, those cultures dis-playing transduction efficiencies <85% underwent cell sortingto enrich the GFPþ cell fraction (supporting informationFig. 1). The expression of FUS-CHOP transcript and proteinwas verified by RT-PCR (Fig. 1A) and Western blot using ananti-GADD153/CHOP antibody, which recognizes a 74-kDaband corresponding to FUS-CHOP and a unspecific band ofhigher molecular weight (Fig. 1B). p53 absence in p53�/�

mASCs was also confirmed by Western blot (Fig. 1B).Phenotypically, both wt and p53�/� mASCs displayed typ-

ical MSC phenotype: CD44þ, CD29þ, Sca1þ (Sca1low inp53�/� mASCs as reported [19, 20]) and absence of the he-matopoietic markers CD45, CD14, and CD11b (Fig. 1C).Similarly, both wt and p53�/� mASCs showed identical osteo-genic differentiation potential, whereas their adipogenicpotential differed as reported [20], with p53�/� mASCs dis-playing smaller oil droplets in the cytoplasm indicating a

Rodriguez, Rubio, Gutierrez-Aranda et al. 181

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poorer adipogenic differentiation (Fig. 1D). Importantly, theexpression of FUS-CHOP did not influence either the pheno-type or the differentiation properties of wt or p53�/� mASCs.

To assay the in vivo tumorogenic potential of the differentmASCs, NOD/SCID IL2Rc�/� mice were inoculated subcuta-neously with 5 � 106 wt GFP-expressing mASCs (wt-GFP),wt FUS-CHOP (FC)-expressing mASCs (mASC-wt-FC),p53�/� GFP-expressing (mASC-p53-GFP), or p53�/� FC-expressing mASCs (mASC-p53-FC). Opposite to wt-GFP orwt-FC mASCs, both p53-GFP and p53-FC mASCs generatedtumors in vivo (Fig. 2A, 2B). Similar to our previous observa-tions [20], p53�/� mASCs (mASC-p53-GFP) gave rise toleiomyosarcoma with a tumor incidence of 50% and a latencyperiod of 70 days. Interestingly, the expression of FC inp53�/� mASCs (mASC-p53-FC) induced a marked aggres-siveness of these cells. On inoculation of mASC-53-FC cells,tumor incidence reached 100% and the latency period was

significantly shortened to 37 days (Fig. 2A). Tumors inducedby mASC-p53-GFP or mASC-p53-FC displayed relevant dif-ferences. Macroscopically, tumors from mASC-p53-FC had asofter texture and displayed large nodules of differentiated fatcells (Fig. 2B). The entire tumors showed GFP fluorescenceconfirming their ASC origin (Fig. 2B). Likewise, immunohis-tochemical analysis of serial sections showed a strong GFPstaining of tumor cells (supporting information Fig. 3). As wepreviously described [20], histology analyses revealed thatmASC-p53-GFP-induced leiomyosarcoma is composed ofinterlacing fascicles of spindle cells positive for desmin andsmooth-muscle actin, partially positive for S-100 and negativefor myogenin (Fig. 2C). However, tumors derived frommASC-p53-FC cells showed large areas composed of lipo-genic cells surrounded by stroma comprising both oval andround-shaped cells (Fig. 2D, panel i). These tumors displayedfeatures of human MLSs including the presence of irregularly

Figure 1. Characterization of FUS-CHOP expressing mouse ASCs. (A): Reverse transcription polymerase chain reaction confirming the expression ofFUS-CHOP in wt and p53�/� ASCs at mRNA level. GAPDH was used as housekeeping gene. (B): Western blotting showing the expression of FUS-CHOP using an anti-GADD153/CHOP antibody and the p53 status of the indicated mASCs. b-actin was used as loading control. (C): Immunophenotypicprofile of the indicated ASC genotypes analyzed by flow cytometry. Representative dot plots are shown for Sca-1, CD29, CD44, CD14, CD11b, andCD45. Filled lines represent the irrelevant isotypes. (D): Adipogenic (oil red staining, upper panels) and osteogenic (Alizarin red staining, bottom panels)differentiation potential of ASCs with the distinct genotypes indicated. Inset images represent negative controls of differentiation. Abbreviations: FC,FUS-CHOP; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, mASC, mouse adipose-derived mesenchymal stem/stromal cell; wt, wild type.


Figure 2. Characterization of tumors developed by mASC-p53-GFP and mASC-p53-FC. (A): Summary table indicating tumor incidence, la-tency, and histological analysis of tumors developed in immunedeficient Nonobese diabetic/severe combined immunodeficient NOD.Cc-Prkdcscid

IL2rctm1Wjl/SzJ mice inoculated with the indicated ASCs. (*) The average number of days needed to observe an approximate tumor diameter of 8mm. Mice carrying tumors were sacrificed at this point. (**) NT detected. Mice were sacrificed 150 days after injection. (§) previously reportedby [20]. (B): Macroscopic appearance of tumors developed from the indicated ASCs (upper panel). Images demonstrating GFP fluorescence posi-tivity of tumors exposed to UV light (lower panel). (C, D): Histological analysis of tumors arising in mice inoculated with either mASC-p53-GFP (C) or mASC-p53-FC (D) cells. Staining is shown for H&E, S-100, desmin, SM-actin, and myogenin as indicated. Red arrows show preadi-pocytes/lipoblasts and black arrows indicate the presence of capillaries. Inset shows �20 original magnification. Abbreviations: FC, FUS-CHOP;GFP, green fluorescent protein; mASC, mouse adipose-derived mesenchymal stem/stromal cell; NT, no tumors; SM-actin, smooth muscle-actin;wt, wild type.


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sized adipocytes and lipoblasts (Fig. 2D, panel ii; red arrows)and a certain level of capillary vascularization (Fig. 2D, paneliii; black arrows). Tumoral lipogenic areas stained positivefor S-100, whereas the larger host-derived mature adipocyteswere S-100 negative (Fig. 2D, panel iv). Interestingly,although the expression is weaker than that observed inmASC-p53-GFP tumors, mASC-p53-FC tumors were alsopartially positive for desmin and SM-actin (Fig. 2D, panels v,vi). Together, the expression of FC in p53�/� mASCs redi-rects the tumor genesis/phenotype and fate from leiomyosar-coma to a liposarcoma histologically mixed with areas ofleiomyosarcoma.

Notably, previous studies reported the development of amouse model of MLS induced by the sole ectopic expressionof FUS-CHOP in wt BM-mMSCs [4]. However, ectopic FUS-CHOP expression in wt mASCs did not generate sarcomagen-esis, indicating that different genetic insults (i.e., p53 defi-ciency) may be required to induce FC-mediated liposarcomain mASCs but not in BM-mMSCs. To confirm this idea, weexpressed FUS-CHOP in BM-mMSCs derived from wt mice,and we addressed their in vivo tumorogenic potential as wepreviously did for mASCs (supporting information Fig. 4).Opposite to control cells (GFP), FUS-CHOP-expressing BM-mMSCs generated tumors in all injected mice (n ¼ 5). Mac-roscopically, tumors displayed fat nodules like the tumorsfrom FUS-CHOP-expressing mASCs, although they weremuch more vascularized. Overall, the tumors are highly vas-cularized undifferentiated sarcomas; although they also dis-play characteristics typical of MLSs as shown by the presenceof atypical multivacoulated lipoblasts and the presence of aplexiform vascular pattern (see supporting information Fig. 4for a detailed description). These results suggest that BM-mMSCs are more susceptible to FUS-CHOP-induced transfor-mation and sarcomagenesis than mASCs.

Ex Vivo-Established Cell Lines fromFUS-CHOP-Expressing Tumors Show MSCProperties In Vitro and Initiate Liposarcoma In Vivo

To gain further insights into these experimentally inducedliposarcoma-like tumors, five lines were derived from p53�/�

FC-expressing tumors (T-AD-p53-FC #1 to T-AD-p53-FC #5;characterization of two lines is shown in supporting informa-tion Fig. 5). These ex vivo-established T-AD-p53-FC-trans-formed mASC lines retained FUS-CHOP expression as con-firmed by RT-PCR (Fig. 3A) and Western blot (Fig. 3B).

To investigate their tumorogenic potential, two T-AD-p53-FC lines were reinoculated into immunodeficient mice.These transformed ASCs forms liposarcoma-like tumors in alltransplanted mice (100% tumor penetrance) within a 2- to 4-week period (Fig. 3C). The histology of these secondarytumors was similar to that of the primary tumors (Fig. 3D).Furthermore, cultures derived from these tumors showed thepresence of small fat vacuoles in the cytoplasm even in theabsence of adipogenic differentiation factors, suggestingthe partial commitment to fat lineage of these T-AD-p53-FCtumor cells (Fig. 3D).

FUS-CHOP Expression in mASCs Alters SignalingPathways Relevant to Liposarcoma Formation

To gain further insights into the potential molecular and cellu-lar mechanisms underlying the tumor genesis/phenotype redi-rection induced by FUS-CHOP expression in p53-deficientmASCs, we compared the gene expression profile of mASC-p53-GFP (which form leiomyosarcoma-like tumors) andmASC-p53-FC cells (which form liposarcoma-like tumors) bymicroarray analysis. We initially generated the lists of genesdifferentially modulated (p value <0.01; expression more thantwo fold up or down) between p53-GFP or p53-FC and wt-GFP mASC cultures (supporting information Table 1). A totalof 7,603 and 7,170 genes were altered in mASC-p53-GFP andmASC-p53-FC cells, respectively. Most of these genes(5,766) were common in both lists, whereas 1,837 or 1,404genes were specifically altered in mASC-p53-GFP andmASC-p53-FC cells, respectively (Fig. 4A). Because bothmASC-p53-GFP and mASC-p53-FC cells are transformed, thegenes commonly altered in both mASC genotypes (5,766)would likely affect important signaling pathways responsiblefor the tumoral transformation of mASCs. Meanwhile, thosegenes specifically altered in mASC-p53-FC cells would reflectthe genetic program induced by FUS-CHOP in p53-deficient

Figure 3. Characterization of ex vivo-established transformed mASC lines from FUS-CHOP-expressing tumors. (A): Reverse transcription poly-merase chain reaction showing the expression of FUS-CHOP at mRNA level in the indicated tumor-derived cell lines (T-AD-p53-FC #1 to #5).GAPDH was used as a housekeeping gene. (B): Western blotting confirming the expression of FUS-CHOP protein using an anti-GADD153/CHOPantibody. b-actin was used as loading control. (C): Summary table indicating tumor incidence, latency, and histological analysis of tumors developedin NOD/SCID IL2Rc�/� mice inoculated with the indicated ASC lines. (*) The average number of days needed to observe a tumor diameter of �8mm. (D): H&E and oil red O staining of tumors arising in mice inoculated with the indicated ASC lines. Abbreviations: FC, FUS-CHOP; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; mASC, mouse adipose-derived mesenchymal stem/stromal cells.


mASCs responsible for tumor phenotype redirection. There-fore, we searched for the signaling pathways associated tothese groups of genes using the Ingenuity Pathway Analysis(IPA) software (Fig. 4B, 4C). Many relevant signaling path-ways including routes involved in the control of proliferationand/or differentiation of MSCs/ASCs were commonly alteredin mASC-p53-GFP and mASC-p53-FC cells. Thus, the dis-rupted p53 signaling may cooperate with pathways controlled

by G-protein-coupled receptors, Wnt, phosphatase and tensinhomolog (PTEN), fibroblast growth factor (FGF), and phos-phoinositide-3-kinase/v-akt murine thymoma viral oncogenehomolog (PI3K/AKT) among others, contributing to tumoraltransformation in mASCs (Fig. 4B and supporting informationTable 2). On the other hand, the expression of FUS-CHOPinduced changes in signaling pathways related with lipid me-tabolism and/or sarcoma genesis such as the inhibition of the

Figure 4. Signaling pathways altered by the expression of FUS-CHOP in mASCs. (A–C): After gene expression microarray analysis, the groupsof genes differentially expressed (p value <0.01; expression more than two fold up or down) in mASC-p53-GFP or mASC-p53-FC versusmASC-wt-GFP were compared and the lists of pathways significantly altered were generated using the Ingenuity Pathways Analysis 8 software.(A): Venn diagram showing the number of genes commonly and specifically altered in mASC-p53-GFP and/or mASC-p53-FC cells. (B): List ofsignificantly modulated pathways (p < 0.05) generated with the genes commonly altered in both mASC-p53-GFP and mASC-p53-FC cells. Onlya selection of relevant pathways is shown (see Supporting Information Table 2 for the complete list of pathways). (C): List of significantlyaltered pathways (p < 0.05) generated with the genes specifically altered in mASC-p53-FC cells. Arrowheads point to key pathways that arelikely to contribute to liposarcoma formation. (D): Expression of adipogenic regulators in mASC-wt-FC and mASC-p53-FC cells relative tomASC-wt-GFP cells. Differentially expressed genes were obtained by microarray analysis (p value <0.01; expression more than twofold up ordown). þ/� indicates genes upregulated/dowregulated up to four times; þþ/�� indicates genes upregulated/dowregulated more than four times.‘‘o’’ represents genes not significantly altered. (E): Western blotting showing the level of expression of PPARc and C/EBPa in cell lines derivedfrom tumors arisen from mASC-p53-FC cells (T-AD-p53-FC-3) and mASC-p53-GFP cells (T-AD-p53-GFP-1). Total extracts from murine whiteadipose tissue was included as a positive control and b-actin was used as loading control. Abbreviations: AKT, v-akt murine thymoma viral onco-gene homolog; CNTF, ciliary neurotrophic factor; C/EBPa, CCAAT/enhancer binding protein a; FC, FUS-CHOP; FGF, fibroblast growth factor;GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; mASC, mouse adipose-derived mes-enchymal stem/stromal cells; PAK, p21 protein (Cdc42/Rac)-activated kinase; PDGF, platelet-derived growth factor; PPARc, peroxisome prolifer-ator-activated receptor c; PTEN, phosphatase and tensin homolog; WAT, white adipose tissue.


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retinoid X receptor (RXR), sphingolipid and fatty acid metab-olism, prolactin signaling, and platelet-derived growth factor(PDGF) pathway (Fig. 4C). These changes could explain, atleast in part, the FUS-CHOP-induced tumorogenesis redirec-tion toward the development of a liposarcoma phenotype.

FUS-CHOP-mediated deregulation of genes involved inadipogenic differentiation seems to be key for liposarcomadevelopment. We thus analyzed the effect of FUS-CHOPexpression on relevant adipogenic regulators [33, 34] byassessing the gene expression changes observed in the micro-rarray analysis of wt-FC and p53-FC versus wt-GFP mASCs(Fig. 4D). In wt-FC mASCs, FUS-CHOP seems to induce a

proadipogenic gene expression profile by upregulating severalpositive regulators of adipogenesis while inhibiting the nega-tive regulator FoxA2. However, in p53-FC mASCs, there wasa more complex regulation of adipogenesis, with several posi-tive and negative regulators being either upregulated or down-regulated (Fig. 4D), suggesting an impairment of the adipo-genic pathways in these p53-FC-mASCs. Among theseadipogenic regulators, the most relevant transcription factorscontrolling the final stages of adipogenesis, peroxisome prolif-erator-activated receptor c (PPARc) and CCAAT/enhancerbinding protein a (C/EBPa), are repressed in liposarcomas ofFC-expressing transgenic mice as well as in MLS cell lines

Figure 5. Generation of FUS-CHOP-expressing wt or p53-deficient human adipose-derived mesenchymal stem/stromal cells (hASCs). (A): hASCswere transduced with lentiviral particles expressing FUS-CHOP or GFP. The GFPþ population was enriched to purify FUS-CHOP-expressing hASCs.Subsequently, p53 was depleted from some of the cultures to obtain the following genotypes: GFP, FC, p53-GFP, and p53-FC. Representative imagesof GFPþ hASCs and GFP analysis by flow cytometry are shown. (B): RT-PCR (upper panel) and Western blot (lower panel) showing FC expressionat RNA and protein levels in FC-transduced hASCs. GAPDH and FUS were respectively used as loading controls. (C): Western blotting showingp53 and p21 protein levels after transduction of GFP- and FC-expressing hASCs with lentiviral particles-expressing p53 shRNA. Cells were treatedwith or without 1 lM CPT for 24 hours. b-actin was used as loading control. (D): Phase-contrast morphology, adipogenic (oil red staining), andosteogenic (Alizarin red staining) differentiation potential of the distinct hASCs. Abbreviations: CPT, campthotecin; FC, FUS-CHOP; GAPDH, glyc-eraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; RT-PCR, reverse transcription polymerase chain reaction; WB, western blot.


[35]. We thus analyzed the protein levels of PPARc and C/EBPa in cell lines derived from tumors induced by p53-wt(TAD-53-1) and p53-FC mASCs (TAD-FC53-3; Fig. 4E).Similar to previous reports [35], the expression of PPARc andespecially of C/EBPa was found heavily repressed in the FC-expressing tumors (TAD-FC53) as compared with TAD-53tumor or white adipose tissue protein extract.

FUS-CHOP Expression Neither Induces Changes inCulture Homeostasis nor Initiates Liposarcomain Either wt or p53-Depleted hASCs

According to the above liposarcoma-like model based onFUS-CHOP expression coupled to p53 deficiency in mASCs,we next wanted to reproduce this model using hASCs. Thesefat-derived ASCs were transduced with either GFP- or FC-expressing lentiviruses (Fig. 5A). To ensure, we work withhomogeneous hASC cultures, those cultures displaying trans-duction efficiencies <85% underwent cell sorting to enrichthe GFPþ cell fraction (Fig. 5A). Then, p53 expression wasdepleted in both GFP- and FC-expressing hASCs using lenti-viruses expressing a p53-specific shRNA. Four genotypes

were generated for downstream experiments: GFP-expressinghASCs (GFP), FUS-CHOP-expressing hASCs (FC), p53-defi-cient GFP-expressing hASCs (p53-GFP), and p53-deficientFUS-CHOP-expressing hASCs (p53-FC; Fig. 5A). The expres-sion of the FUS-CHOP was confirmed by RT-PCR and WB(Fig. 5B). We also confirmed by western blot (WB) the reduc-tion of p53 protein levels in p53-GFP and p53-FC cells both inunstressed conditions or after the induction of DNA damage bytreatment with the topoisomerase I inhibitor campthotecin (CPT;Fig. 5C). The activation of p21 after CPT treatment was alsoprevented, indicating that p53 depletion is effectively blockingthe downstream signaling of this protein (Fig. 5C).

All hASCs genotypes displayed a typical hMSC immuno-phenotype (supporting information Fig. 6). Likewise, all hASCsgenotypes showed similar morphology and adipogenic differen-tiation potential (Fig. 5D). However, FC-expressing hASCsshowed a much robust osteogenic differentiation potential ascompared with GFP hASCs (Fig. 5D). Intriguingly, thisenhanced osteogenic differentiation potential observed in FChASCs is lost in either p53-GFP or p53-FC hASCs, suggestingthat FUS-CHOP expression boosts osteogenic differentiation ina p53-dependent manner.

Figure 6. In vitro growth properties of FUS-CHOP-expressing wt or p53-deficient hASCs. (A): Cumulative population doublings of the indicatedcultures. The time of p53 depletion (dashed arrow), senescence entry (blue arrows), and a representative image of a senescence-associated b-galacto-sidase activity staining are shown. (B): Cell cycle analysis including BrdU labeling of S-phase. (C): Colony-forming ability. (D): Soft agar assayshowing no anchorage-independent growth of either wt or p53-deficient hASCs-expressing FUS-CHOP. HeLa cells and T-AD-p53-FC #4 were usedas a positive control. (E): Summary table indicating the inability of hASC-p53-GFP and hASC-p53-FC cells inoculated in NOD/SCID IL2Rc�/�

mice to form tumors. Data from mouse ASCs represented in Figure 2A are reproduced in gray color text. Abbreviations: ASCs, adipose-derived mes-enchymal stem/stromal cells; BrdU, 5-bromo-2-deoxyuridine; FC, FUS-CHOP; GFP, green fluorescent protein; NT, no tumors.


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Long-term analysis of hASC growth kinetics revealed thatp53 depletion robustly enhanced the growth rates of p53-GFPand p53-FC hASC cultures (Fig. 6A), and consequently, thesecultures showed a higher proportion of cells in S-phase(Fig. 6B) and an increased clonogenic capacity (Fig. 6C). Theexpression of FUS-CHOP, however, did not impact these cul-ture homeostasis properties in wt or p53-depleted hASCs. Thelifespan of p53-GFP and p53-FC hASC cultures was longeras compared with wt GFP- or FC-expressing hASCs, whichreached senescence (measured as b-galactosidaseþ staining)about 30 days earlier (Fig. 6A). Additionally, all hASCsremained euploid after long-term in vitro expansion (support-ing information Fig. 7)

Finally, we assayed the in vitro and in vivo transformationcapacity for the different hASCs genotypes. In contrast to ourpositive controls, none of the hASCs cultures were capable ofgenerating colonies in the anchorage-independent growth-based in vitro transformation assays (Fig. 6D). Furthermore,none of the hASCs cultures formed tumors on inoculation inNOD/SCID IL2Rc�/� mice (Fig. 6E). Altogether, these results

indicate a differential outcome of FUS-CHOP overexpressionin ASC of mouse versus human origin.

Differences in Gene Expression Profiling BetweenGFP- and FC-Expressing hASCs Suggest thatFUS-CHOPActs As anOverall Transcriptional Repressor

To identify patterns of gene expression, which could help toelucidate the biological effect of FUS-CHOP expression inhASCs, we compared the transcriptional profiles of GFP- ver-sus FC-expressing hASCs by microarray analysis. Data analy-sis identified 183 genes differentially expressed (p value <.05; expression more than twofold up or down) between GFP-and FC-expressing hASCs (supporting information Table 1).Overall, FUS-CHOP expression functioned as a transcriptionalrepressor in hASCs because its expression induced the down-regulation of 127 differentially expressed genes (70%) andthe upregulation of 56 genes (30%; Fig. 7A). This trend wasvery similar to that observed after FUS-CHOP expression inmASCs (70.5% and 29.5% of downregulated and upregulatedgenes, respectively).

Figure 7. Signaling pathways altered by the expression of FUS-CHOP in hASCs. (A): Heat map diagram summarizing the microarray geneexpression changes (p value < .05; expression more than twofold up or down) in hASC-wt-FC compared with hASC-wt-GFP cells. Genesinvolved in Wnt/bcatenin (red), endothelin-1 (blue), G-protein-coupled receptor (green), and other relevant signaling pathways (black) are indi-cated. (B–D): Lists containing the significantly (p < .05) altered pathways generated using Ingenuity Pathway Analysis software with the genesdifferentially expressed in hASC-wt-FC versus hASC-wt-GFP cells (B), hASC-p53-FC versus hASC-wt-GFP cells (C), and mASC-p53-FC versusmASC-wt-GFP cells (D). Only a selection of relevant pathways is shown in ([D]; see Supporting Information Table 2 for the complete list ofpathways). Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; ATM, ataxia telangiectasia mutated; BRCA1, breast cancer 1,early onset; CHK, CHK checkpoint homolog; FC, FUS-CHOP; FGF, fibroblast growth factor; GFP, green fluorescent protein; GNRH, gonadotro-pin-releasing hormone; hASC, human adipose-derived mesenchymal stem/stromal cell; HER-2, Human Epidermal growth factor Receptor; IL-1,interleukin-1; IRF, interferon regulatory factor; LPS, lipopolysaccharide; PDGF, platelet-derived growth factor; PKR, protein kinase R; PTEN,phosphatase and tensin homolog; RXR, retinoid X receptor; THOP1, thimet oligopeptidase 1; TNFR2, tumor necrosis factor receptor 2; TREM1,triggering receptor expressed on myeloid cells 1; VDR, vitamin D receptor; wt, wild type.


Analysis of the altered genes using the IPA softwarerevealed that not many signaling pathways and gene familiesdisplayed a significantly altered gene expression profile inFC-expressing hASCs (Fig. 7A, 7B). Among the altered path-ways endothelin-1, Wnt/b-catenin, and G-protein-coupledreceptors signaling were previously reported to be deregulatedin cancer [36–38]. Indeed, among the biological/pathologicalfunctions identified by IPA software, cancer becomes themost significantly altered after the expression of FUS-CHOPboth in human and mouse ASCs (supporting informationTable 3). Thus, 46 of the 183 genes (25.1%) modulated byFC in hASCs were previously involved in tumorogenesis(supporting information Table 3).

When the gene expression profile induced by FUS-CHOPwas compared between human and mouse ASCs, we foundthat 17.5% (32 of 183) and 36.8% (174 of 473) of the genesmodulated in hASCs-wt-FC versus hASCs-wt-GFP andhASCs-p53-FC versus hASCs-wt-GFP, respectively, were alsoaltered in the matching mASCs. Targets commonly altered byFUS-CHOP expression in both mouse and human ASCs includekey regulators of the Wnt signaling pathway (Wnt4, Sfrp1,Sfrp2, and Axin2) and other relevant factors involved in MSCsfate control (Bmp6, Fos, etc.; supporting information Table 4).

Opposite to mASC-p53-FC, hASC-p53-FC cells did notpromote tumor formation when injected in immunodeficientmice. In an attempt to elucidate candidate secondary-transform-ing events, which could be important to drive tumor formationin hASCs, we analyzed the signaling pathways differentiallyaltered in the hASC-p53-FC cells (as compared with hASC-wt-GFP) using the IPA software. The signaling pathways modu-lated in hASC-p53-FC cells were then compared with thosealtered in the tumorogenic mASC-p53-FC cells (Fig. 7C, 7Dand supporting information Table 2). Among the pathwaysaltered in hASCs-p53-FC cells, there are several routes relatedto cell cycle checkpoints and apoptosis whose alteration couldbe relevant in the process of tumoral transformation. On theother hand, mASC-p53-FC cells showed significant changes inmany other signaling routes whose deregulation is stronglyassociated with enhanced tumorogenesis, such as G-protein-coupled receptors, cAMP-mediated factors, PTEN, Wnt/b-cate-nin, or PI3K/AKT signaling. This robust microarray analysissuggests that some of these pathways may need to be targetedto promote full transformation of FC-expressing human ASCs.

DISCUSSION

Increasing evidence indicates that MSCs might constitute thetarget cell for transforming mutations responsible for sarcomadevelopment, suggesting that MSCs/ASCs may become aninstrumental tool in studies aimed at dissecting the pathogene-sis and cellular origin of sarcomas [1]. It has been demon-strated that BM-mMSCs provided a permissive environmentfor tumoral transformation and sarcoma development medi-ated by several sarcoma-associated fusion genes [2–5]. Never-theless, a human sarcoma model remains to be developed.Here, we have explored the role of FUS-CHOP overexpres-sion on both wt and p53-deficient mouse and human ASCs.

Recent findings show that MSCs carrying specific muta-tions initiate different types of sarcomas depending on the tis-sue from which those MSCs are sourced. For instance, p53deficiency in BM-mMSCs forms osteosarcoma [17], whereasp53 deficiency in fat-derived mASCs induces leiomyosarcoma[20]. Riggi et al. [4] previously reported the formation of lipo-sarcoma-like tumors when FUS-CHOP was overexpressed inBM-mMSCs. Here, however, we report that the expression of

FUS-CHOP in fat-derived mASCs is not sufficient to transformthese mASCs or initiate liposarcoma in vivo, suggesting a differ-ential impact of FUS-CHOP in MSCs derived from BM or adi-pose tissue.

We recently reported that p53�/� mASCs form leiomyo-sarcoma-like tumors, linking this type of smooth muscle sar-coma to p53 deficiency in ASCs [20]. Intriguingly, expressionof FUS-CHOP in p53�/� mASCs induced higher aggressive-ness in the tumorogenic properties of mASC-p53-FC as com-pared with mASC-p53-GFP: tumor incidence reached 100%and the latency period significantly shortened. Importantly,tumors resulting from mASC-p53-FC displayed a phenotypicswitch from leiomysarcoma to liposarcoma-like tumors. Thesetumors displayed features of human MLSs including irregu-larly sized lipoblasts and numerous dilated capillaries. Ourdata is further supported by a previous study reporting aswitch of tumoral phenotype after FUS-CHOP expression in ahuman fibrosarcoma cell line [39]. Together, these data sug-gest that the expression of FUS-CHOP triggers the formationof liposarcoma-like tumors from mASCs, but, in contrast toBM-mMSCs, secondary cooperating oncogenic hits such asp53 deficiency are required for liposarcoma development.Similar results have been reported in a model of alveolarrhabdomyosarcoma based on the expression of PAX-FKHR inmMSCs in which p53 deficiency is needed for tumor develop-ment [5].

Similar to recent studies, which highlight the role thatFUS-CHOP plays in the control of important adipogenic regu-lators, especially PPARc and C/EBPa [35], we found a simi-lar downregulation of these transcription factors in thosetumors derived from FUS-CHOP-expressing mASC-p53-FCcells. Nevertheless, the gene expression levels of PPARc arenot affected by the expression of FUS-CHOP in mASCs, inline with previous data from BM-mMSCs [4]. Furthermore,the levels of C/EBPa were upregulated by FUS-CHOP inboth wt and p53-deficient mASCs (Fig. 4D). Together, thesedata indicate that FUS-CHOP seems to predispose mASCs toadipogenic differentiation in vitro through the upregulation ofC/EBPa, although the in vivo cellular environment seems toplay a relevant role in blocking adipogenesis and therefore inthe potential development of the malignancy. In line with thepartial commitment of FUS-CHOP-expressing mASCs to theadipogenic lineage, an incomplete spontaneous adipogenicdifferentiation of cells derived from FUS-CHOP-expressingtumors was observed (Fig. 3D). In addition, we observed aconsistent loss of Sca-1 expression in p53-deficient mASCs(Fig. 1C) which has been associated with a reduced adipo-genic potential of MSCs [40]. Nevertheless, as we previouslyreported, the loss of Sca-1 in p53-deficient mASCs is not cor-related with an enhanced tumorogenic potential [20].

The molecular basis underlying how FUS-CHOP redirectstumor genesis/phenotype in p53-deficient mASCs could be, atleast in part, explained by the signaling pathways specificallyaltered by the presence of FUS-CHOP in mASC-p53-FC cells(Fig. 4A–4C). Among these pathways, the inhibition of RXRsignaling is relevant as this nuclear receptor is an obligate het-erodimeric partner for PPARc necessary to modulate theexpression of genes under the control of peroxisome prolifera-tor response elements [41]. Other significantly altered pathwayssuch as prolactin signaling and sphingolipid metabolism werealso reported to modulate PPARc activity and adipogenesis[42, 43]. Notably, other FUS-CHOP-specific altered routes,such as fatty acid metabolism, were also found to be altered inliposarcoma samples [44]. In addition, Riggi et al. [4] haveidentified PDGFA as a new relevant target induced by theexpression of FC in BM-mMSCs. Importantly, although wehave not observed a significant variation in PDGFA, the PDGF


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signaling was also significantly modulated by the presence ofFUS-CHOP in mASC-p53-FC cells (Fig. 4C). Likewise, wealso found a relevant overlap of 39 and 112 commonly deregu-lated genes when the effect of FUS-CHOP expression in BM-mMSCs (data from Riggi et al.) was compared with mASC-wt-FC and mASC-p53-FC cells, respectively (supporting informa-tion Table 5). Moreover, Matushansky et al. [8] have reported4,027 genes differentially expressed between samples of MLSsand normal fat. We found that 229 and 482 of these geneswere also upregulated or downregulated by FUS-CHOP expres-sion in wt and p53-deficient mASCs (supporting informationTable 6). In addition, three genes (PEG3, GPX3, and ADH1B)altered in mASC-wt-FC cells and five genes (TRO, SAA1,SOX11, GPX3, and ADH1B) deregulated in mASC-p53-FCcells, were also among the 11 most informative genes for MLSas described by Singer et al. [45]. These gene expression pro-file comparisons suggest that (a) FUS-CHOP expression inmASCs modify the expression profile toward a liposarcoma-likepattern and (b) that p53 deficiency cooperates in this process.

It has been reported that the expression of FUS-CHOP [46]or EWS1-FLI1 [47] caused cell death in several human celllines although p53 or p16 gene depletion increased cell survival[47]. We thus attempted to create a model of human liposar-coma by expressing human FUS-CHOP in either wt or p53-de-ficient hASCs. Based on the role that the deficiency of p53plays in the transformation of mASCs [20] and in the fact thatthe expression of this cell cycle regulator is frequently alteredin soft tissue sarcomas, we hypothesized that p53 proteindepletion would be a secondary cooperating hit candidate tissuesarcomas [21, 22]. In contrast to previous studies on humancell lines [46], FUS-CHOP expression resulted nontoxic in wtor p53-depleted primary hASCs, suggesting that hASCs pro-vide a permissive environment for the expression of this fusionprotein. p53 depletion resulted on a much higher growth rate,colony-forming ability, and increased lifespan. However, FUS-CHOP expression neither induced further changes in culturehomeostasis nor initiated liposarcoma in either wt or p53-depleted hASCs. Similar to our data, the expression of EWS-FLI-1 was able to transform BM-mMSC [3] but unable totransform BM-hMSCs although it induced a gene expressionprofile resembling features of Ewing’s Sarcomas [7]. Moreover,mMSCs but not hMSCs generated osteosarcoma-like lesions inthe lung following systemic injection in mice [48]. These dataconfirm that mMSCs are much more susceptible to transforma-tion and sarcoma development [49] than hMSCs, which seemto require additional cooperating mutations to constitute a use-ful cellular model for sarcomagenesis. It is plausible that sev-eral cooperating oncogenic hits are needed to induce lineage-specific transformation of hMSCs [50].

Gene expression profiling showed that FUS-CHOP func-tions as a transcriptional repressor in hASCs. This effect waspreviously observed in the human fibrosarcoma cell lineHT1080 [39]. FUS-CHOP expression results in constitutiveexpression of CHOP mediated by the FUS promoter, whereas innormal cells, the transcription of CHOP is tightly regulated andgenerally repressed [51, 52]. Both CHOP and FUS-CHOP formdimmers and inactivate many members of the C/EBP family oftranscription factors [12, 35], explaining, in part, the transcrip-tional repression observed in FUS-CHOP-expressing hASCs.

Among the signaling pathways displaying a significantlyderegulated gene expression profile in FUS-CHOP-expressinghASCs, Wnt signaling functions as an important regulator ofMSC fate. Activation of Wnt signaling stimulates osteogenesisand inhibits adipogenesis [53]. Wnt signaling also controls thegrowth and transformation of MSCs. Thus, the expression ofthe Wnt signaling inhibitor Dkk-1 in hMSCs induced the for-mation of undifferentiated pleiomorphic sarcomas in vivo [38]

and, likewise, this inhibitor is overexpressed in human osteo-sarcoma [54]. In addition, Dkk-1 is required for BM-MSCsentry into the cell cycle [55]. FUS-CHOP expression in hASCsseems to disrupt the Wnt/b-catenin pathway because severalinducers and repressors genes of the pathway were modulatedby FUS-CHOP expression. Importantly, the expression ofmany of these Wnt signaling members (Wnt4, Axin2, Sfrp1,and Sfrp2) is also modulated by FC in mASCs. Other relevantpathways altered by FUS-CHOP in hASCs include the Endo-thelin axis and the G-protein-coupled receptor signaling. TheEndothelin signaling has been implicated in cancer develop-ment and deregulation of this pathway has been found in sev-eral types of sarcomas [36]. Likewise, the G-protein-coupledreceptors play a pivotal role in a wide array of important sig-naling pathways and many of these receptors have been impli-cated in tumorogenesis [37].

Overall, the adipogenic program of hASCs does not seemhighly altered by the presence of FUS-CHOP. Among themost relevant proadipogenic factors only Klf-4 was downre-gulated by FUS-CHOP. Likewise, the overall overlap betweengenes modulated in hASC-wt-FC cells (25 genes) and hASC-p53-FC cells (37 genes) and those genes differentiallyexpressed between MLSs and normal fat [8] is more modestthan that observed for mASCs (supporting information Table6). This reduced gene expression deregulation induced byFUS-CHOP expression in hASCs may explain the failure ofthe hASC-p53-FC cell to produce tumors when injected intoimmunodeficient mice. On careful comparison of the geneexpression profiles of both mASCs and hASCs, several genesspecifically modulated in mASC-p53-FC cells came out aspotential additional oncogenic insults which may cooperatewith FUS-CHOP and p53 deficiency in the transformationprocess of hASCs. For instance, based on its previouslyreported link with cancer development [56], the role of thePTEN/PI3K/AKT pathway in the transformation of hASCsshould be carefully addressed in future studies.

Together, our results suggest a differential outcome ofFUS-CHOP overexpression in mouse versus human ASCs andindicate that FC expression in a p53-deficient background issufficient to induce liposarcoma-like tumors in mouse but notin human ASCs, suggesting the need of still uncharacterizedadditional cooperating mutations in hASCs.

CONCLUSION

We attempted to model liposarcoma by expression of FUS-CHOP in wild type and p53-deficient mouse and human adi-pose-derived mesenchymal stem/stromal cells (m/hASCs). Theexpression of FUS-CHOP triggers the formation of liposarcoma-like tumors from mASCs but, in contrast to bone marrow-derived mMSCs, secondary cooperating hits such as p53 defi-ciency are required for liposarcoma development. In the humansetting, FUS-CHOP was unable to transform either wild type orp53-deficient hASCs, thus emphasizing the need for furthercooperating mutations. A microarray gene expression profilinghas revealed potential deregulated pathways in liposarcoma for-mation from p53-deficient mASCs expressing FUS-CHOP.

ACKNOWLEDGMENTS

We thank Enrique de Alava (CIC, Salamanca), FranciscoNogales, and Francisco O’Valle (University of Granada) fortheir assistance on the pathology diagnosis, Purificacion Catalinaand Paola Leone for assistance with G-banding assays, and Dr.


Patrick Aebischer for the pLVUHshp53 plasmid. This work wassupported by the CSJA (0030/2006 to P.M. and 0108/2007 to R.Rodriguez) and CICE (P08-CTS-3678 to P.M.) de la Junta deAndalucıa, The FIS/FEDER (PI070026 and PI100449 to P.M.),and the MICINN to P.M. (PLE-2009-0111). R. Rodriguez is sup-ported by a Fellowship from the AECC.

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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FUS-CHOP Fusion Protein Expression Coupled to p53 Deficiency Induces Liposarcoma in Mouse but Not in...

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