SRC Signaling Is Crucial in the Growth of Synovial Sarcoma ......tured in Dulbeccos' Modiﬁed...

Molecular and Cellular Pathobiology

SRC Signaling Is Crucial in the Growth of Synovial SarcomaCells

Sebastian Michels1,2, Marcel Trautmann1, Elisabeth Sievers1, Dagmar Kindler1, Sebastian Huss1,Marcus Renner4, Nicolaus Friedrichs1, Jutta Kirfel2, Susanne Steiner2, Elmar Endl3, Peter Wurst3,Lukas Heukamp1, Roland Penzel4, Olle Larsson5, Akira Kawai6, Shinya Tanaka7, Hiroshi Sonobe8,Peter Schirmacher4, Gunhild Mechtersheimer4, Eva Wardelmann1, Reinhard B€uttner1, andWolfgang Hartmann1

AbstractSynovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a

chimeric transcriptional modifier. Several receptor tyrosine kinases have been found activated in synovialsarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of theresults of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of theSRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK andPTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence ofSRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcomaxenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC wasdetected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for theactivation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated withincreased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway.Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation,associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure ofcells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasionwere dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA.Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. Insummary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovialsarcoma. Cancer Res; 73(8); 2518–28. �2013 AACR.

IntroductionSynovial sarcomas account for 5% to 10% of all soft-tissue

sarcomas. In the majority, synovial sarcoma arise mainly inadolescents and young adults with predominance in malegender. They are molecularly characterized by a reciprocal

t(X; 18) translocation, which juxtaposes the SS18 gene onchromosome 18 to either the SSX1, the SSX2, or rarely to theSSX4 gene on the X chromosome. The SS18/SSX chimericproteins act as transcriptional coactivators, leading to dereg-ulation of oncogenic pathways (1–3).

Current treatment protocols for synovial sarcoma are basedon radical surgery and standardized chemo- and radiotherapy;however, prognosis is still poor in advanced disease (4). Tar-geted therapeutic approaches, which have significantlyimproved the clinical course of patients with gastrointestinalstromal tumors (GIST) or dermatofibrosarcoma protuberans,are still lacking for synovial sarcomas (5, 6). Several receptortyrosine kinases have been shown to be expressed in synovialsarcomas, including the EGF receptor (EGFR; ref. 7) and theinsulin-like growth factor-I receptor (IGF-IR; ref. 8), leading toan activation of the PI3K/AKT signaling pathway (9). Non–receptor tyrosine kinases, includingmembers of the SRC familykinases (SFK), are important in tumor cell growth, survival, andmotility in different tumor entities, including mesenchymaltumors as osteosarcoma and Ewing sarcoma (10).

The 60-kDa human c-SRC (SRC) tyrosine kinase contains 2phosphorylation sites regulating its enzymatic activity. Phos-phorylation at Tyr527 leads to a reduced activity, whereas

Authors' Affiliations: 1Department of Pathology, University HospitalCologne, Cologne; Departments of 2Pathology and 3Molecular Medicine,University Hospital Bonn, Bonn; 4Department of Pathology, UniversityHospital Heidelberg, Heidelberg, Germany; 5Department of Oncology-Pathology, Karolinska Hospital, Stockholm, Sweden; 6Division of Ortho-paedic Surgery, National Cancer Center Hospital, Tokyo; 7Laboratory ofMolecular and Cellular Pathology, Hokkaido University Graduate School ofMedicine, Sapporo; and 8Department of Laboratory Medicine, ChungokuCentral Hospital, Fukuyama, Hiroshima, Japan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S. Michels and M. Trautmann contributed equally to this work.

Corresponding Author: Wolfgang Hartmann, Department of Pathology,University Hospital Cologne, Kerpener Strasse 62, Cologne D-50937,Germany. Phone 49-221-47898562; Fax: 49-221-4786360; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-3023

�2013 American Association for Cancer Research.

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autophosphorylation at Tyr416 is associated with full kinaseactivity (11, 12). SRC phosphorylation status is modulated bythe c-SRC tyrosine kinase (CSK) and the protein tyrosinephosphatase PTP1B, which modify SRC phosphorylation atTyr527 (12, 13). CSK has been reported to be critical for SRCderegulation in colon cancer cells (14). Among the PTPs,PTP1B has been shown to be of particular importance, beingoverexpressed in breast cancer cell lines with elevatedSRC activity (15–17). Different receptor tyrosine kinasesincluding the IGF-IR and effectors of the PIK3/AKT, RAS/MAPK, and STAT3 pathways represent important interactionpartners of SRC, leading to cellular survival and proliferation.Furthermore, SRC is capable of modulating cell migration andinvasion through interaction with integrins, the focal adhesionkinase (FAK), and regulators of the family of Rho-GTPases(18, 19).The present study was conducted to analyze the functional

relevance of SRC signaling in synovial sarcoma biology andto test its potential as a target for innovative therapeuticapproaches.

Materials and MethodsPatients, tumor samples, and cell linesThirty cases of synovial sarcoma were analyzed compris-

ing 22 monophasic and 8 biphasic tumors. Approval of thestudy by the Ethical Committee of the University of BonnMedical Center (Bonn, Germany) was obtained. FISH or PCRanalyses were used to confirm the diagnosis of synovialsarcoma revealing a t(X; 18) translocation as describedbefore (20). The synovial sarcoma cell lines CME-1, 1273/99, FUJI, SYO-1 (all carrying a SS18/SSX2 translocation), andHS-SY-II (carrying a SS18/SSX1-translocation) have beendescribed earlier (21–25); presence of the SS18/SSX trans-location was confirmed by PCR using primers specific for thetranslocation subtypes.

Phosphokinase arrays1273/99 (SS18/SSX2-translocated) andHS-SY-II (SS18/SSX1-

translocated) cells were grown for 48 hours in medium sup-plemented with 10% FBS. Protein extraction and phosphoki-nase arrays (R&D Systems), comprising spotted antibodies for46 kinase phosphorylation sites, were conducted as indicatedby the manufacturer. Filter development was conducted usingthe ECL Kit (Amersham) as described before (26). Densito-metric analysis was conducted using the ImageJ software(http://rsb.info.nih.gov/ij).

ImmunohistochemistryPTP1B andCSKantibodieswere purchased fromAbcam, the

p-(Ser10)-histone H3 antibody from Merck Millipore, and p-(Tyr416)-SRC, p-(Tyr527)-SRC and cleaved caspase-3 (Asp175)antibodies fromCell Signaling Technologies. Tissue specimens(including xenografts) were fixed in 4% buffered formaldehydeand embedded in paraffin. After antigen retrieval (10 mmol/Lsodium citrate buffer, pH 6.0, microwave 600 W, 10 minutes)PTP1B, CSK, p-(Ser10)-histone H3 and cleaved caspase-3(Asp175) immunohistochemical stainings were conductedon 4-mm sections with an Autostainer (DAKO) or manually

[p-(Tyr416)-SRC and p-(Tyr527)-SRC]. For PTP1B and CSK,the antigen–antibody binding was visualized with the avidin–biotin complex (ABCmethod) using AEC (3-amino-9-ethylcar-bazol) or 3,30-diaminobenzidine (DAB) as chromogen. Forp-(Tyr416)-SRC and p-(Tyr527)-SRC stainings, the CatalyzedSignal Amplification System (CSA II; DAKO) was used accord-ing to the manufacturer's instructions using DAB aschromogen. Positive and negative control stainings using anappropriate rabbit IgG subtype (DCS) were included. For allproteins, cytoplasmic and membranous immunoreactivitywere assessed using a semiquantitative score (negative, weak,moderate, strong) defining the staining intensity in the positivecontrol (invasive ductal breast cancer/intraductal breastcancer) as strong.

SS18/SSX fusion gene overexpression in T-REx-293 cellsT-REx-293 cells were purchased from Invitrogen and cul-

tured in Dulbeccos' Modified Eagles' Media (DMEM) supple-mented with 10% FBS, 2 mmol/L L-glutamine, and 5 mg/mLblasticidin. SS18/SSX1, SS18/SSX2, SS18, SSX1, and SSX2 wereamplified by PCRusing templates expression vectors describedbefore (27, 28). The cDNAs were then cloned into the tetracy-cline-regulated pTREx DEST30 Gateway expression vector(Invitrogen). Using Lipofectamine 2000 reagent, T-REx-293cells were transfected with expression vectors for SS18/SSX1,SS18/SSX2, SS18, SSX1, and SSX2, respectively, and the pT-REx/GW-30/lacZ vector (Invitrogen) expressing b-galactosidasewas included as control. Forty-eight hours after transfection,T–REx–293 cells were selected in culture medium supplemen-ted with 1 mg/mL geneticin (G418), and drug-resistant colo-nies were isolated after 6 weeks of selection. To induce geneexpression, 1mg/mL tetracycline (Sigma-Aldrich) was added tothe T–REx–293 cell lines for 24 hours.

Culture and treatment of human synovial sarcoma cellsand MTT cell proliferation assays

Cell lines were grown inmonolayer cultures andmaintainedat 37�C in a humidified 5% CO2 atmosphere as described (20).Because of their low proliferative rate, HS-SY-II synovial sar-coma cells were not suitable for further functional assays. Forproliferation assays, cells were cultured in medium supple-mented with 2% FBS in 96-well dishes at least in triplicate. Celldensity was 5 � 103 per well. Cells were exposed to increasingconcentrations of dasatinib (0.01–10 mmol/L; Santa Cruz Bio-tech), and appropriate controls were included. For combina-tion treatments with chemotherapeutic drugs, cell lines wereincubated with increasing concentrations of doxorubicin, vin-cristine, and actinomycin D (0.1–100 ng/mL), alone or incombination with dasatinib in a concentration resulting ingrowth inhibition of 20% to 30%. Synergy was evaluated bythe fractional product method (29). A difference of more than10% between the observed and the predicted effect was con-sidered to signify synergistic activity between dasatinib andthe chemotherapeutic drug, a difference of less than 10% wasdefined as additive. All assays were conducted for 72 hours.MTTproliferation assays (Roche)were conducted according tothe manufacturer's instructions. The formazan dye was quan-tified using a scanningmultiwell spectrometer (BGMLabtech).

SRC Signaling in Synovial Sarcoma

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Knockdown of SRC and IGF-IR by RNA interference1273/99 and CME-1 cells were cultured in 25-cm2 flasks in

medium supplemented with 10% FBS as described above. At acell density of 50%, cells were transfected with 60 pmol StealthRNAi (SRC: HSS186080, HSS186081, HSS186082; IGF-IR:HSS105253, HSS105254, HSS179797; Invitrogen) or nontarget-ing control siRNA (Invitrogen) using Lipofectamine RNAiMAX(Invitrogen) according to the manufacturer's instructions.After 24 hours, cells were trypsinized, reseeded, and MTTassays were conducted as described above (2% FBS). Todocument SRC knockdown, 5 � 104 siRNA-transfected cellswere plated in 12-well dishes in medium supplemented with2% FBS and cultured for 72 hours.

Western blot analysisA total of 2� 105 to 4� 105 cells were seeded in 6-well dishes

(Greiner) in medium supplemented with 2% FBS for 48 hoursbefore treatment with increasing doses of dasatinib for 60minutes. To document SRC activation via the IGF-IR pathway,1273/99, FUJI, and CME-1 cells were starved for 4 hours andtreated with 200 ng/mL recombinant IGF-II (R&D Systems).Cell lysis and Western blots were conducted as describedbefore (20). Following primary antibodies were used accordingto the manufacturer's instructions: b-actin (Sigma-Aldrich),c-SRC (Santa Cruz Biotech), p-(Tyr416)-SRC, p-(Tyr527)-SRC,STAT3, p-(Tyr705)-STAT3, FAK, p-(Tyr576/577)-FAK, p44/42MAPK, p-(Thr202/Tyr204)-p44/42 MAPK, AKT, p-(Ser473)-AKT, IGF-IR, p-(Tyr1131/Tyr1146)-IGF-IR (all Cell SignalingTechnologies), CSK, and PTP1B (Abcam). Secondary antibodylabeling aswell as filter development were conducted using theECL kit (Amersham) as described before (26).

Flow cytometryAbout 8 � 105 cells were grown in 75-cm2 cell culture

flasks in medium supplemented with 2% FBS. After a pre-incubation of 18 hours, they were treated with 0.1 or 0.6mmol/L dasatinib for 48 hours. For flow cytometric immu-nophenotyping, 1 � 106 cells were fixed on ice in ice-cold 2%paraformaldehyde for 10 minutes. They were then washed inPBS, collected by centrifugation, resuspended and incubatedin ice-cold PBS with 0.25% Triton X-100 for 5 minutes on ice.After another washing step, cells were resuspended in 100 mLPBS/0.5% bovine serum albumin (BSA) containing an AlexaFluor 647–labeled phospho-(Ser10)-histone H3 antibody(Cell Signaling Technology; 1:20) and a phycoerythrin-labeled cleaved PARP (Asp214) antibody (BD Biosciences;1:5) and incubated for 30 minutes at room temperature.After an additional washing step, 500 mL PBS containing 10mg/mL 40,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich)was added to stain DNA, and cells were incubated for anadditional 30 minutes at room temperature. Analysis wasconducted using a three-laser LSRII analytical flow cyt-ometer (BD Biosciences). Each experiment was carried outat least in duplicate. At least 30,000 events were recorded perexperiment. Only single cells were included in the analysis.Data were analyzed using FlowJo (Tree Star) analysis soft-ware. To document SRC specificity of the effects of dasatinib,the experiment was additionally carried out in CME-1 cells

48 hours after SRC siRNA transfection, which was done asdescribed above.

Analysis of apoptosis by DAPI stainingCells were cultured as described above and treated with

0.03 or 0.3 mmol/L dasatinib. They were harvested andwashed in PBS, fixed in 3.7% paraformaldehyde for 10minutes at room temperature, and washed again. Afterincubation with 1 mg/mL DAPI (Sigma-Aldrich) for 10 min-utes and 2 further washing steps, cells were mounted onappropriate slides using Fluoromount-G medium (SouthernBiotechnologies Associates). Nuclei were visualized andphotographed using a Leica DMLB fluorescence microscope.Apoptotic cells were morphologically defined by chromatincondensation and fragmentation. For each assay, at least 300cells were analyzed in triplicate.

Wound scratch, migration, and invasion assaysCellular motility, migration, and invasion were analyzed by

wound scratch, Boyden chamber, and invasion chamberassays. To exclude influences of growth effects on the resultsof migration and invasion assays, dasatinib was used in con-centrations of 1 or 3 nmol/L, that is concentrations, whichwereshown to exert no significant effects on cell viability in MTTassays. CME-1 and SYO-1 cells were grown to confluence in 6-well dishes under serum-reduced conditions as describedabove. Cell monolayers were wounded by scratching with asterile 100-mL pipette tip, mediumwas exchanged and the cellswere treated with dasatinib or dimethyl sulfoxide (DMSO) ascontrol. Photographs were taken at time points 0, 12, and 24hours, using an AxioCam digital camera and the AxioVisionsoftware (Zeiss). Boyden chamber motility assays were con-ducted with CME-1, SYO-1, and 1273/99 cells. A total of 2� 104to 7 � 104 tumor cells in 150 mL medium containing 2% FBSwere added into the upper Transwell chambers (6.5-mmdiameter, 8.0-mm pore size, Corning Costar Corporation) andtreated with dasatinib or DMSO as control for 24 hours. FBSconcentration in the lower chamber (500 mL) was 10%. Inva-sion chambers with Matrigel coating (6.4-mm diameter, poresize 8 mm; BD) were used to assess the effect of dasatinib oninvasion in CME-1 and SYO-1 cells. Essentially, cells wereseeded and treated as for migration assays. Dishes wereincubated in a 37�C incubator (5% CO2) for 24 hours. Afterremoval of the nonmigrating cells from the upper chamberwith a cotton swab, the membranes were fixed with 4%paraformaldehyde for 15 minutes, stained with Harris hema-toxylin for 15 to 20 minutes, washed, removed from the plasticholders, and mounted on glass slides with Aquatex (Merck).Migrated cells were counted in five �10 fields (�100 magni-fication). All assays were conducted at least in triplicate. Todocument SRC specificity of the effects of dasatinib, Boydenchamber and invasion chamber assays were additionally con-ducted in CME-1 cells 48 hours after SRC siRNA transfection,which was done as described above.

RhoA and Rac activation assayCME-1 and SYO-1 cells were cultured as described above

to yield a density of 30% to 60% after 72 hours. Cells were

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Cancer Res; 73(8) April 15, 2013 Cancer Research2520




then treated with 0.1 mmol/L of dasatinib for 20 minutes.DMSO was used as control. Lysates were prepared andimmediately snap-frozen in liquid nitrogen. Protein lysateswith equalized concentrations were analyzed. The assaywas conducted in duplicate according to the manufacturer'sinstructions (Cytoskeleton).

In vivo experiments in SYO-1 xenograftsAll mouse procedures were conducted in accordance with

the National and European Union guidelines and permissionwas obtained from the local authorities. A total of 5� 106 SYO-1 synovial sarcoma cells were injected subcutaneously into theright flank of 5-week-old BALB/c nude mice (Charles RiverLaboratories). Tumor growth was assessed daily by measuringthe tumor volume calculated as length�width� height�p/6.Treatment was started when the tumor volume reached 100mm3. Dasatinib was injected intraperitoneally (i.p.) in a dailydosage of 10mg/kg (days 1–7). As no side effects were observedduring the first week of treatment, the dosage was escalated to20mg/kg (days 8–14). After 15 days of treatment, animals weresacrificed, tumors were explanted, and the tumor tissue wasformalin-fixed and embedded in paraffin.

ResultsPhosphokinase arrays

Phosphokinase array analysis of 1273/99 and HS-SY-IIsynovial sarcoma cells detected phospho-(Tyr416)-SRC asthe most strongly phosphorylated protein kinase of 46represented targets. Independent Western blotting con-firmed this finding and detected p-(Tyr416)-SRC levels ofdifferent intensity in CME-1, SYO-1, FUJI, 1273/99, and HS-SY-II synovial sarcoma cells (Fig. 1; Supplementary TableS1). Because of their low proliferative rate, HS-SY-II synovialsarcoma cells were not suitable for further functionalassays.

Synovial sarcomas display elevated levels of Tyr416-phosphorylated SRC

In a set of 30 synovial sarcomas, immunohistochemicalstainings revealed strong expression levels of Tyr416-phos-phorylated SRC in 13% of the samples, 60% showed moderate,and 27% weak expression levels. In contrast, strong Tyr527-phosphorylated SRC was detectable in only 7% of the samples,whereas moderate or weak expression was found in 10% and30% of the tumors, respectively; in 53% of the samples, no

Figure 1. A, Tyr416-phosphorylatedSRC was found to be the moststrongly phosphorylated kinase in aphosphokinase array analysis of1273/99 synovial sarcoma cells.B, expression of p-(Tyr416)-SRCwasconfirmed by Western blot analysis.C, representative case of a biphasicsynovial sarcoma displaying highexpression of p-(Tyr416)-SRC, weakexpression of p-(Tyr527)-SRC, highCSK levels, and weak PTP1Bexpression.

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expression of Tyr527-phosphorylated SRC was detectable.Expression levels for CSK were strong in 30%, moderate in17%, and weak in 50% of the samples, no CSK expression wasfound in one synovial sarcoma. Expression levels for PTP1Bwere strong in 13%, moderate in 37%, and weak in 40% of thesamples, no expression of PTP1B was found in 10% of thesamples. No significant correlation between SRC phosphory-lation status and CSK or PTP1B protein expression levels wasdetected. Tyr416-phosphorylated SRC expression in the tumorsamples did not correlate with the patients' age, sex, tumorlocation, and tumor size or tumor grade. As in the immuno-histochemical analysis, no correlation between SRC activityand PTP1B or CSK levels was observed in the cell lines analyzedin Western blots (Fig. 1 and data not shown).

SRC activation in synovial sarcoma is induced by SS18/SSX translocation

To functionally understand the mechanism of SRC acti-vation in synovial sarcoma, T-REx-293 cells were stablytransfected with vectors containing SS18/SSX1, SS18/SSX2,SSX1, SSX2, or SS18 cDNA to obtain an inducible cell culturemodel of the synovial sarcoma–specific chimeric transloca-tion proteins. Western blot analysis showed elevated levelsof activated p-(Tyr416)-SRC in T-REx-293 cells transfectedwith SS18/SSX1 and SS18/SSX2 (Fig. 2A); expression levels ofPTP1B and CSK were not affected (data not shown). As it hasbeen shown previously that receptor tyrosine kinase path-ways including IGF-IR signaling are of particular importancein synovial sarcomas, we analyzed promoter-specific expres-sion levels of IGF2 showing upregulation of promoter P2-and P4-dependent IGF2 transcripts in SS18/SSX1 and SS18/SSX2-expressing T-REx-293 cells (Fig. 2B). Stimulation of1273/99, FUJI and CME-1 synovial sarcoma cells with recom-binant human IGF-II protein was associated with anincrease of phosphorylation of IGF-IR at Tyr1131, AKT atSer473, and SRC at Tyr416, which revealed IGF-IR signalingas a functionally relevant mechanism leading to SRC acti-vation (Fig. 2C). A minor induction of phosphorylation ofSRC at Tyr416 was observed upon SS18 overexpression aloneas well; however, this activation was not associated withIGF2 transcriptional induction. Inversely, siRNA knockdownof the IGF-IR in CME-1 cells was associated with a signif-icant decrease of p-(Tyr416)-SRC levels (SupplementaryFig. S2).

SRC inhibition by dasatinib or RNA interference impairsgrowth of synovial sarcoma cells

siRNA-mediated knockdown of SRC resulted in a significantdecrease of growth of CME-1 and 1273/99 cells in MTT assays(t test: P < 0.001; Fig. 3A and data not shown). All analyzedsynovial sarcoma cell lines displayed dose-dependent growthinhibition upon treatment with the SRC inhibitor dasatinib(Fig. 3B). This effect was particularly distinct in nanomolarconcentrations of the inhibitor. Among the 4 synovial sarcomacell lines investigated CME-1 (GI50¼ 0.008 mmol/L), FUJI (GI50¼ 0.01mmol/L), and SYO-1 (GI50¼ 0.013mmol/L)were found tobe slightly more sensitive to dasatinib than 1273/99 cells (GI50¼ 0.077 mmol/L).

Inhibition of SRC affects phosphorylation of itsinteraction partners

To assess the effect of SRC inhibition on its interactionpartners in synovial sarcomas, cells were treated with increas-ing concentrations of dasatinib (0.01–3 mmol/L) for 60minutes(Fig. 3C). Dose-dependent dephosphorylation of p-(Tyr416)-SRC, p-(Ser473)-AKT, p-(Tyr576/577)-FAK, p-(Tyr705)-STAT3,and p-(Tyr1131)-IGF-IR was observed in all synovial sarcomacell lines with nanomolar concentrations of dasatinib. Simi-larly, siRNA knockdown of SRC led to the dephosphorylation of

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Figure 2. A, elevated levels of activated p-(Tyr416)-SRC in T-REx-293cells expressing SS18/SSX1 and SS18/SSX2. B, induction of promoterP2- and P4-dependent IGF2 transcripts inSS18/SSX1- andSS18/SSX2-expressing T-REx-293 cells. C, induction of phosphorylation of IGF-IR(Tyr1131), SRC (Tyr416), and AKT (Ser473) upon stimulation of 1273/99,FUJI, and CME-1 cells with IGF-II.

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FAK (data not shown). Interestingly, p-(Thr202/Tyr204)-p44/42 MAPK levels increased in 1273/99 and FUJI after treatmentwith higher doses of dasatinib.

Dasatinib treatment increases apoptosis and decreasesmitotic rate in synovial sarcoma cellsTo determine the effect of dasatinib on the apoptotic and

mitotic rate of synovial sarcoma cells, flow cytometricanalyses were conducted. Cleaved PARP (Asp214) was usedas a marker of apoptosis, and phospho-(Ser10)-histone H3was used as a marker of mitosis. CME-1, 1273/99, and SYO-1cell lines showed significantly increased rates of apoptosisand decreased mitotic fractions after treatment with dasa-tinib in concentrations of 0.1 and 0.6 mmol/L (Fig. 4A,Supplementary Table S2). These results were confirmed bymicroscopic analyses of DAPI-stained synovial sarcoma cellstreated with dasatinib, showing increasing rates of chroma-

tin condensation and fragmentation in a dose-dependentmanner in CME-1 and SYO-1 cells (Fig. 4B). As an indicatorof SRC specificity of the effects observed, dasatinib treat-ment of CME-1 cells after SRC knockdown did not showsignificant effects in terms of proliferation and apoptosis inflow cytometry (Supplementary Fig. S1).

Combination of SRC inhibition and conventionalchemotherapy results in additive effects on cell growth

To determine the effects of combinations of conventionalchemotherapy (vincristine, doxorubicin, actinomycin D) anddasatinib on the growth of synovial sarcoma cells, we inves-tigated 1273/99 cells, which were the least responsive tomonotreatments with dasatinib. Cells were exposed to increas-ing concentrations of conventional cytotoxic drugs and to aconcentration of dasatinib that led to 20% to 30% growthinhibition after 72 hours. The combinations did not fulfill the

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Figure 3. A, significant growth inhibition (MTT proliferation assay) upon siRNA-mediated knockdown of SRC (inset) in CME-1 synovial sarcoma cells.��, P < 0.001, Student t test. B, dose-dependent growth inhibition (MTT proliferation assay) in 4 synovial sarcoma cell lines treated with the SRC inhibitordasatinib. C, dose-dependent dephosphorylation of p-(Tyr416)-SRC, p-(Tyr1131)-IGF-IR, p-(Tyr576/577)-FAK, p-(Ser473)-AKT, and p-(Tyr705)-STAT3 insynovial sarcoma cells upon treatment with increasing concentrations of dasatinib (0.01–3 mmol/L).






criteria of synergy as defined above; the effects observedresulted from an additive, obviously independent action ofthe SRC inhibitor and conventional chemotherapeutic agents(Fig. 4C).

Dasatinib inhibits motility and invasive potential ofsynovial sarcoma cells associated with an increasedactivity of RhoA and diminished Rac activity

As SRC has been shown to modulate motility and inva-siveness of tumor cells, we investigated the effect of SRCinhibition by dasatinib on cell migration and invasion. CME-1, SYO-1, and 1273/99 synovial sarcoma cells (treated withdasatinib in doses not affecting cell viability) showed a dose-dependent decrease of migratory and invasive potential inBoyden chamber and invasion chamber assays. Accordingly,wound healing was impaired in scratch assays in CME-1 andSYO-1 treated with dasatinib. In ELISA-based RhoA and Racactivation assays, treatment with dasatinib resulted in sig-

nificantly increased levels of activated RhoA and decreasedlevels of activated Rac in SYO-1 and CME-1 cells (Fig. 5A–D,data not shown). As an indicator of SRC specificity of theeffects observed, dasatinib treatment of CME-1 cells afterSRC knockdown did not show significant effects in terms ofmigration and invasion in Boyden chamber and invasionchamber assays (Supplementary Fig. S1).

Dasatinib displays antitumor activity in synovialsarcoma xenografts in vivo

The antitumor activity of dasatinib was tested in vivo in axenograft model of SYO-1 synovial sarcoma cells. The inhibitorsignificantly reduced tumor growth rate (Fig. 6A). No signif-icant changes in the weight of the tumor-bearing mice wereobserved (data not shown). Consistent with the in vitro results,treatment was associated with diminished levels of Tyr416-phosphorylated SRC, a significant reduction of the mitoticfraction (t test: P < 0.001) and a significant increase of the

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Figure 4. A, significantly increased rate of apoptosis [cleaved PARP (Asp214)] and decreased mitotic fraction [phospho-(Ser10)-histone H3] in CME-1and SYO-1 synovial sarcoma cells upon treatment with 0.6 mmol/L dasatinib as determined by flow cytometry. B, microscopic analyses of DAPI-stainedcells concerning chromatin condensation and fragmentation. ��, P < 0.01; ��, P < 0.001, Student t test. C, coincubation of 1273/99 cells with conventionalcytotoxic drugs and 0.0075 mmol/L dasatinib resulted in additive effects.

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apoptotic fraction (t test: P < 0.001) compared with controltumors (Fig. 6B and C).

DiscussionConsiderable progress has been made in the understanding

of soft-tissue tumors in the recent years. However, apart fromfew examples such as c-KIT or platelet-derived growth factor(PDGF) receptor inhibition in GIST and dermatofibrosarcomaprotuberans (5, 6), the translation of molecular results intoclinical care in terms of molecularly based therapies is still rarein this group of neoplasias. Despite elaborate treatment pro-

tocols involving radical surgery and standardized chemo- andradiotherapy, prognosis is poor in advanced cases of synovialsarcoma. Therefore, the identification of molecular targets,which are at the same time biologically essential and accessibleto specific therapeutic drugs, represents an important issue forthe development of innovative therapeutic approaches.

On the basis of a phosphokinase screen, we identified theSRC tyrosine kinase as one of themost strongly phosphorylatedkinases in synovial sarcoma cells. Its particular relevance wasconfirmed immunohistochemically in biopsies of 30 synovialsarcomas, in which Tyr416-phosphorylated, that is activated,

Figure 5. Inhibition of cellularmotility and invasiveness of synovialsarcoma cells treated with dasatinib.A, Boyden chamber membranesof CME-1 cells treated with0.003 mmol/L dasatinib for 24 hours.A and B, comparable reduction ofmotility and invasiveness in invasionchamber assays. C, representativewound scratches in SYO-1 synovialsarcoma cells treated with 0.003mmol/L dasatinib (originalmagnification, �20). D, significantlyincreased levels of activated RhoAand decreased levels of activatedRac in SYO-1 cells treated withdasatinib. �, P < 0.05; ��, P < 0.001,Student t test.

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SRC was found to be expressed in the majority of the cases. Aconsistent pattern of dysregulation of the SRC-regulatingproteins CSK and PTP1B, analogous to what has been shownin some epithelial tumors could be excluded in synovialsarcomas (15–17, 30). Interestingly, SRC was found to beactivated through the SS18/SSX translocation proteins. Thisactivation was associated with an IGF-IR–dependent mecha-nism based on transcriptional induction of IGF2, which linksSRC activation to the characteristic molecular aberration ofsynovial sarcomas. As shown, expression of the transcriptionalcofactor SS18 alone is capable to (indirectly) induce SRCphosphorylation at lower levels as well; however, this appearsto be independent from IGF2 induction. This finding under-lines the oncogenic character of the SS18/SSX fusion proteinsand distinguishes components of the IGF/SRC context fromother therapeutic targets as molecularly based and tumor-specific. However, the finding of consistent expression offurther growth factor receptors, such as PDGFR and EGFR,in synovial sarcomas makes it probable that other than IGF-IR–dependent pathways may mediate SRC activation in syno-vial sarcomas as well (31). Considering the IGF-IR and the SRCkinases as potential therapeutic targets, its central positionwithin different oncogenic signaling pathways makes SRC an

attractive candidate for specifically directed approaches. Asshown here, synovial sarcomas display a fundamental depen-dence on SRC signals with regard to cellular proliferation andsurvival. This was observed in vitro in siRNA-mediatedapproaches and after pharmacologic intervention with theSRC inhibitor dasatinib as well as in vivo in murine synovialsarcoma xenografts. A role for dasatinib is clinically well-established in the treatment of chronic myelogenous leukemiaand Philadelphia chromosome–positive acute lymphoblasticleukemia (ALL), in which the substance inhibits Abl kinases(32, 33). Effectivity of dasatinib has previously been shown forcells derived from solid tumors aswell, includingmesenchymalneoplasias, such as GIST and chondrosarcoma (34, 35). Inchondrosarcoma, growth effects observed upon treatmentwith dasatinib were not consistently associated with dimin-ished p-(Tyr416)-SRC levels, which makes SRC-independentmodes of action probable (35). In contrast, in synovial sarco-mas, dephosphorylation of SRC and its targets was a consistentfeature detectable upon treatment with dasatinib. The lowdrug dosages resulting in dephosphorylation of the SRC targetsargue in favor of SRC-dependent effects and against effectsexerted through direct interaction of dasatinib with IGF-IR,FAK, and AKT (36). As an indirect proof of specific SRC-related

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Figure 6. A, significantly reduced tumor growth in vivo in dasatinib-treated SYO-1 xenografts associated with diminished levels (B, C) of Tyr416-phosphorylated SRC, a reduction of the p-(Ser10)-histone H3–positive mitotic cell fraction, and an induction of the cleaved caspase-3 (Asp175)–positiveapoptotic cell fraction. ��, P < 0.001, Student t test.

Michels et al.





action of dasatinib in synovial sarcoma cells, CME-1 did notdisplay any significant effects upon dasatinib treatment aftersiRNA-mediated SRC knockdown. Interestingly, SRC inhibitionwas associated with a loss of phosphorylation of the IGF-IR atTyr1131, which indicates activation of IGF-IR tyrosine kinaseactivity usually detectable upon ligand binding. This finding isparticularly relevant for the option of IGF-IR directed thera-peutic approaches, which have been proposed for synovialsarcomas recently (20, 37). Beyond that, because of the centralposition of SRC within intracellular signaling networks and itsobvious capacity of cross-activating pathways as documentedhere, it is conceivable that targeting SRCas a central componentintegrating different signaling activities might be advantageouscompared with individual receptor-directed approaches. Asshown here, combined treatment of synovial sarcoma cells withchemotherapeutic drugs and dasatinib results in additive butnot in synergistic effects. Therefore, SRC inhibitors might beuseful in innovative therapeutic approaches, in which targetingof an activated pathway with specific inhibitory substancesallows the reduction of the individual compounds' dosages,thereby minimizing toxicity. In our in vivo experiments, dasa-tinib was found to be highly effective with regard to tumorgrowth and was well tolerated by the animals.As known for a variety of epithelial tumors (36, 38), on the

basis of our data, the SRC signaling network appears to be ofcrucial relevance for cellularmigration and invasion in synovialsarcomas. In all assays applied here, doses of dasatinib, whichdid not affect cellular proliferation, resulted in significantlyimpairedmigratory and invasive capacities. These effects wereassociated with an SRC-dependent shift in activation levels ofRac and RhoA, small GTPases essentially involved in theregulation of cell mobility processes. This finding provides afunctional background of the effects observed here and sub-stantiates specificity, as increased levels of activated RhoA areassociated with stress fiber formation, whereas diminishedlevels of activated Rac go along with the impairment of a"motile" phenotype (18). This finding is of particular impor-tance with regard to therapeutic concepts, as prognosticallyunfavorable cases of synovial sarcomas frequently developmetastases. Using a dual-inhibition approach of SRC andAurora kinases by SU6656, Arai and colleagues recentlyobserved high antitumor effectiveness in synovial sarcomaxenografts involving antiangiogenic mechanisms. This finding

provides further evidence of the crucial role of SRCwith regardto complex aspects of tumor biology and underlines its role in aoncogenic signaling network (39).

In summary, our data in detail substantiate previous find-ings on the relevance of SRC in synovial sarcomas (40). For thefirst time, it is systematically shown that the SRC signalingnetwork is commonly activated in synovial sarcomas and thattargeting SRC results in substantial effects on tumor cellgrowth and motility. These findings argue in favor of SRC asa potential therapeutic target in synovial sarcomas.

Disclosure of Potential Conflicts of InterestE.Wardelmannhas honoraria from speakers' bureau fromNovartis Oncology,

MSD, and Eisai and is a consultant/advisory board member for NovartisOncology and MSD. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: S. Michels, M. Trautmann, R. B€uttner, W. HartmannDevelopment of methodology: S. Michels, M. Trautmann, E. Sievers, D.Kindler, E. Endl, A. Kawai, W. HartmannAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Michels, E. Sievers, M. Renner, N. Friedrichs, P.Wurst, R. Penzel, O. Larsson, S. Tanaka, G. Mechtersheimer, E. Wardelmann, R.B€uttner, W. HartmannAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Michels, M. Trautmann, D. Kindler, S. Huss, P.Wurst, L. Heukamp, R. Penzel, R. B€uttner, W. HartmannWriting, review, and/or revision of the manuscript: S. Michels, M. Traut-mann, E. Sievers, D. Kindler, M. Renner, N. Friedrichs, O. Larsson, P. Schirmacher,E. Wardelmann, R. B€uttner, W. HartmannAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Trautmann, J. Kirfel, S. Steiner, A.Kawai, H. Sonobe, P. SchirmacherStudy supervision: W. Hartmann

AcknowledgmentsThe authors thank Alexandra Florin for excellent technical support.

Grant SupportThis study was supported by the Deutsche Krebshilfe (KoSar-Sarcoma Net),

The Wilhelm Sander-Stiftung, the BONFOR program of the Medical Faculty,University of Bonn, the Fortune program of the Medical Faculty, University ofCologne, and grant HBFG-109-517 to the Flow Cytometry Core Facility at theInstitute of Molecular Medicine, University of Bonn.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 3, 2012; revised December 28, 2012; accepted January 16, 2013;published OnlineFirst April 10, 2013.

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2013;73:2518-2528. Published OnlineFirst April 15, 2013.Cancer Res Sebastian Michels, Marcel Trautmann, Elisabeth Sievers, et al. SRC Signaling Is Crucial in the Growth of Synovial Sarcoma Cells

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