SMARCB1 INI1 Genetic Inactivation Is Responsible for ... · Monica Brenca 1, Sabrina Rossi3, Erica Lorenzetto , Elena Piccinin 1, Sara Piccinin , Francesca Maria Rossi2, Alberto Giuliano

Cancer Therapeutics Insights

SMARCB1/INI1 Genetic Inactivation Is Responsible forTumorigenic Properties of Epithelioid Sarcoma Cell LineVAESBJ

Monica Brenca1, Sabrina Rossi3, Erica Lorenzetto1, Elena Piccinin1, Sara Piccinin1, Francesca Maria Rossi2,Alberto Giuliano1, Angelo Paolo Dei Tos3, Roberta Maestro1, and Piergiorgio Modena1

AbstractEpithelioid sarcoma is a rare soft tissue neoplasm that usually arises in the distal extremities of young adults.

Epithelioid sarcoma presents a high rate of recurrences and metastases and frequently poses diagnostic

dilemmas. We previously reported loss of tumor suppressor SMARCB1 protein expression and SMARCB1

gene deletion in themajority of epithelioid sarcoma cases. Unfortunately, no appropriate preclinical models of

such genetic alteration in epithelioid sarcoma are available. In the present report, we identified lack of

SMARCB1 protein due to a homozygous deletion of exon 1 and upstream regulatory region in epithelioid

sarcoma cell lineVAESBJ. Restoration of SMARCB1 expression significantly affectedVAESBJ cell proliferation,

anchorage-independent growth, and cellmigrationproperties, thus supporting the causative role ofSMARCB1

loss in epithelioid sarcoma pathogenesis. We investigated the translational relevance of this genetic back-

ground in epithelioid sarcoma and showed that SMARCB1 ectopic expression significantly augmented

VAESBJ sensitivity to gamma irradiation and acted synergistically with flavopiridol treatment. In VAESBJ,

both activated ERBB1/EGFR and HGFR/MET impinged on AKT and ERK phosphorylation. We showed a

synergistic effect of combined inhibition of these 2 receptor tyrosine kinases using selective small-molecule

inhibitors on cell proliferation. These observations provide definitive support to the role of SMARCB1

inactivation in the pathogenesis of epithelioid sarcoma and disclose novel clues to therapeutic approaches

tailored to SMARCB1-negative epithelioid sarcoma. Mol Cancer Ther; 12(6); 1060–72. �2013 AACR.

IntroductionEpithelioid sarcoma (InternationalClassification ofDis-

eases for Oncology code number 8804/3) is a rare mes-enchymal neoplasm that displays variable epithelioidmorphology, presents a high rate of recurrences andmetastases, and frequently poses diagnostic dilemmas(1). It usually affects young adults and arises in the distalextremities (classic-type epithelioid sarcoma) or, morerarely, in proximal sites of the trunk (proximal-type epi-thelioid sarcoma). Proximal-type epithelioid sarcoma ismore frequently associated with epithelioid or rhabdoidmorphology and higher mitotic activity (2).

The SMARCB1 gene located at 22q11 chromosomalregion encodes for an invariant subunit of SWI/SNFchromatin remodeling complex and has been reported toact as a tumor suppressor gene in infantile malignantrhabdoid tumor (MRT; refs. 3, 4), a highly aggressiveneoplasm affecting renal or extra-renal soft tissue, andcerebral tissue in pediatric patients. Previously publishedstudies indicate that SMARCB1/INI1 gene is involved inthe control of genomic stability and in the regulationof cell-cycle progression (5). SMARCB1 stimulates thep16/Rb tumor suppressor pathway by activation ofCDKN2A and inhibition of Cdk/cyclinD (6). As a result,inMRT cell lines, it has been shown that SMARCB1 loss isassociated with responsiveness to Cdk/cyclin inhibitors,such as 4-HPR (7) and flavopiridol (8). In addition, thein vivo spontaneous tumorigenesis in SMARCB1þ/�

knockout mice is increased by TP53-null genetic back-ground (9) and is prevented by CCND1 ablation (10),further supporting the interaction with crucial moleculescontrolling cell-cycle progression.

We first reported evidence of SMARCB1 inactivation inepithelioid sarcoma (11), an event frequently associatedwith homozygous gene deletions (12). The relevance ofSMARCB1 in epithelioid sarcoma pathogenesis was thensupported by other reports of subtle intragenicmutations,including small deletions and point mutations (13, 14).

Authors' Affiliations: 1Experimental Oncology 1, 2Clinical and Experi-mental Onco-Hematology Unit, Centro di Riferimento Oncologico, Aviano;and 3Department of Pathology, Treviso Regional Hospital, Italy.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

R. Maestro, and P. Modena share senior co-authorship.

Corresponding Author: Piergiorgio Modena, Unit of Experimental Oncol-ogy 1, Centro di RiferimentoOncologico, via F.Gallini 2, 33081Aviano (PN),Italy. Phone: 39-043496-6596; Fax: 39-043465-9659; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0005

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 12(6) June 20131060

on October 29, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 10, 2013; DOI: 10.1158/1535-7163.MCT-13-0005

http://mct.aacrjournals.org/

Subsequent studies reported the occurrence of SMARCB1protein and/or genetic alterations in pithelioid sarcomaranging from 60% to 93% (12–16). Although these dif-ferences may be attributable to the different molecularapproaches undertaken to assess SMARCB1 inactiva-tion, the lack of appropriate preclinical models for thisenigmatic sarcoma subtype has so far prevented theassessment of the causal role in epithelioid sarcomapathogenesis.Here, we provide evidence that the epithelioid sar-

coma cell line VAESBJ carry a homozygous SMARCB1deletion and that in this cell line SMARCB1 actually actsas a tumor suppressor. Moreover, we provide evidencethat SMARCB1 inactivation results in hyperactivationof ERBB1/EGFR andHGFR/MET pathways, thus disclos-ing novel avenues for the treatment of patients with epi-thelioid sarcoma.

Materials and MethodsCancer cell linesVAESBJ was purchased from Interlab Cell Line Collec-

tion and the other cell lines were fromAmerican Type Cul-ture Collection (ATCC). Sarcoma subtypes representedwere osteosarcoma (CRL1543/HOS; MG63), rhabdomyo-sarcoma (CRL7862/Hs729.T, CRL7726/ T174, CRL7763/TE381.T), epithelioid sarcoma (VAESBJ, ATCC numberCRL-2138), fibrosarcoma (HTB152/Hs913.T; MES-SA),leiomyosarcoma (HTB88/SK-LMS-1), renal rhabdoid sar-coma (G401), and renal leiomyoblastoma (G402). Hek293and IMR90 are immortalized noncancer cell lines origin-ally derived from human embryonic kidney and fibro-blasts, respectively. Cells were grown in RPMI-1640supplemented with 10% heat-inactivated FBS, in a humid-ified incubator at 37�Cand 5%CO2. Cell lineswere authen-ticated regularly by microsatellite DNA fingerprinting.To establish the G402 and VAESBJ xenograft, 5 � 106 cellswere injected subcutaneously in the flank of nude mice.After 3 weeks, the tumor was removed, formalin-fixed,andprocessedas reported for immunohistochemistry anal-ysis. For infection experiments, viral supernatants wereprepared using pBabe-SMARCB1 (a kind gift by Dr. Ber-nard E. Weissman, University of North Carolina, ChapelHill, NC) and pBabe-control vectors using standard calci-um–phosphate transfection method in LynxA cells (17).Supernatants were used for infection with polybrene 4mg/mL by centrifugation at 1,600 rpm for 1 hour and over-night incubation at 32�C. Medium was then replaced and48 hours later, puromycin selection was started for 4 days.Bulk cell population was used in all experiments after24-hour recovery from antibiotic selection. For transfectionexperiments, cells were transfected using the DharmafectTransfection Reagent (Thermo Scientific) according to theinstructions of the manufacturer and CCND1was silencedin VAESBJ cell line using CCND1 siRNA (s229 Ambion)and compared with off-target control siRNA (AM4611Ambion). Cells were collected at different time-pointsafter transfection and screened for CCND1 expression by

Western blot analysis and quantitative PCR analysis. Inin vivo experiments, 106 cells from pBabe-SMARCB1and pBabe-control infected VAESBJ cells from 3 inde-pendent infection experiments were subcutaneouslyinjected in the flank of nude mice. Tumor growth wasmonitored over time and calculated using the formula1/2(r3). Once mice were sacrificed, tumors were explan-ted, weighted, and photographed. Animal experimen-tation was approved by Institutional Review Board andconducted according to National laws.

Protein expressionFor Western blot analysis, protein lysates were pre-

pared in radioimmunoprecipitation assay buffer (Sigma),40 micrograms of cell lysate were loaded on 4%–15%gradient PAGE gels (Bio-Rad) and electroblotted ontopolyvinylidene difluoride membranes (Amersham Bio-sciences). Subsequently, membranes were incubated 1hour at room temperature in a solution of TBST [10mmol/L Tris-HCl (pH 8.0), 0.15 mol/L NaCl, and0.05% Tween 20] supplemented with 5% nonfat dry milk.For immunodetection, the anti-BAF47/SNF5 antibody(BD Transduction Laboratories) was used diluted 1:250.After overnight incubation at 4�C with the primary anti-body, membranes were washed in TBST, followed byAlexaFluor680– or IRDye800CW-conjugated goat-anti-mouse or goat-antirabbit antibodies (from Invitrogen andLi-Cor, respectively). Odyssey infrared imaging system(Li-Cor) was used for detection. Protein loading equiva-lencewasassessedusingananti-Gapdhantibody (Sigma).Additional antibodies used are: TP53 (DO-1 1:1,000; SantaCruz), CCND1 (DCS-6 1:500; Santa Cruz), CDKN2A/p16(C-20 1:1,000; Santa Cruz), CDKN1A/p21 (H-164 1:500;Santa Cruz), PARP (1:1,000; Cell Signaling), CASP3(1:1,000; Cell Signaling), CASP7 (1:1,000; Cell Signaling),P(Tyr1068)-EGFR (1:1,000; Cell Signaling), EGFR (1:1,000,Cell Signaling), P(Ser473)-AKT (1:1,000; Cell Signaling),AKT (1:1,000; Cell Signaling), P-ERK1/2 (1:1,000; CellSignaling), ERK1/2 (1:1,000; Cell Signaling), P(Tyr1003)-MET (1:1,000; Cell Signaling), MET (1:1,000; Santa Cruz),and Vinculin (1:10,000; Santa Cruz). Immunopheno-typing with CD34, cytokeratins, epithelial membraneantigen, andCD31 antibodieswas conducted for uniformpathologic reexamination of xenograft tumors. Proteinexpression of SMARCB1 was investigated by immuno-histochemistry using anti-BAF47/SNF5 antibody 1:100(BD Transduction Laboratories). Endogenous peroxidisewas blocked with 0.3% hydrogen peroxide in methanolfor 30 minutes. For antigen retrieval, the slides wereimmersed in citrate buffer solution 5 mmol/L pH 6 andheated in autoclave at 95�C for 15 minutes. Immuno-histochemistry analysis was done using Ultra visiondetection system (LabVision). Expression of phosphory-lated receptor kinases was detected using the RTKProteome Profiler Array kit (R&D Systems). The pro-cedures were carried out according to the manufac-turer’s protocol using 300 mg of protein lysate perarray and signals generated by horseradish peroxidase-

SMARCB1 in VAESBJ Cell Line

www.aacrjournals.org Mol Cancer Ther; 12(6) June 2013 1061




conjugated secondary antibody were visualized andquantified with Chemidoc imaging system (Bio-Rad).

Real-time PCRSMARCB1 mRNA expression was analyzed by quan-

titative real-time PCR. Total RNA was extracted usingTRIzol reagent (Ambion) and 1 mg RNA was retrotran-scribed using Superscript II reverse transcriptase (Gibco)with random primers. Ten nanograms of cDNA wereused as template in 20 mL PCR reactionswith 1� TaqManUniversal PCR master mix (Applera). Relative quantifi-cation of gene expression was conducted in triplicateusing TaqMan Assays on Demand on an ABI Prism7900HT Sequence Detection System (Applera) by com-parative Ct method, using the hypoxanthine phosphor-ibosyltransferase (HPRT) gene (HPRT PDAR, 4326321E)as endogenous reference control and 293 cell line ascalibrator. SMARCB1 assay used was Hs00268260_m1,encompassing exons 4–5.

Mutational analysisMutational analysis was conducted by exon amplifica-

tion and sequencing was conducted as previouslydescribed (11). For multiplex ligation-dependent probeamplification (MLPA), tumorDNA(100ng)was subjectedto DNA copy number analysis using MLPA kits P258-B1,P294-A1, and P171 (MRC-Holland), following manufac-turer instructions and togetherwithnormalDNAsamplesand cancer cell lines with known SMARCB1 gene copynumber alterations as controls. Fragment separation wasconducted on an ABI3130xl genetic analyzer (Applera).Raw data peak pattern evaluation was conducted usingGeneMapper software (Applera) and Coffalyser softwarewas used for data analysis (MRC-Holland). Homozygousdeletion of VAESBJ and G402 cell lines was verified byMLPA and delimited and cloned by sequence-tagged sitedeletion mapping using PCR primer pairs available uponrequest.

Cell proliferation assayCells were plated in multiwell plates and cell prolifer-

ation was assessed at different time points by either Try-pan blue cell counting or with sulphorhodamine B (SRB)staining (18). For SRB staining, cells (5 � 103) were grownin 96-well plates for 72 hours, fixed using cold 50% tri-chloroacetic acid for 1 hour at 4�C, then were stainedusing Sulphorhodamine B (Sigma) 0.4% in 1% acetic acid.Tris-Base 10 mmol/L was used for solubilization. Theabsorbance of protein biomass, which is proportional tothe cell number,was read at 550 nm.Time zero plateswerefixed after overnight cell adhesion and served for back-ground subtraction and calculation of relative absorbancevalues as described (18). Drugs used were flavopiridol(Sigma), fenretinide (Sigma), PHA665752 HGFR/METinhibitor (Santa Cruz), and 324674 EGFR inhibitor (Cal-biochem). Drugs were resuspended in dimethyl sulfoxide(DMSO) or ethanol (in case of fenretinide) and added24 hours postplating at the indicated concentrations. Cells

were fixed 72 hours later. No treatment, DMSO or ethanolvector control served as calibrator sample. Combinationindex of drug combinations was calculated with Compu-Syn software (Combosyn Inc.).

Anchorage-independent growthCells (105) were resuspended in 0.35% agar complete

medium and seeded on 0.5%bottom agarmedium in 6 cmpetri dishes. After 2 weeks, plates were stained withiodonitrotetrazolium violet (1 mg/mL, Sigma) and cloneswere counted at the microscope.

b-Galactosidase assayThe b-galactosidase (b-Gal) staining was used as a

surrogate marker of senescence. Cells were washed oncein PBS (pH 7.2), fixed with 0.5% glutaraldehyde for 15minutes, and washed in PBS (pH 7.2) supplemented with1 mmol/L MgCl2. Cells were stained with senescence-associated b-Gal (SA-b-Gal) stain solution [1 mg/mL5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 0.12mmol/L K3Fe(CN)6, 0.12 mmol/L K4Fe(CN)6, 1 mmol/LMgCl2 in PBS (pH6)] and incubated overnight at 37�Cand5% CO2. The b-Gal–positive cells in 20 microscope fieldswere counted under bright field on a Olympus IX70microscope and representative photographs were taken.

Migration assayTranswell permeable supports, 6.5 mm diameter in-

serts, 8.0mmpore size, polycarbonatemembrane (CorningInc.) were used to conduct migration assay. Cells (105) in1% FBS containing Dulbecco’s Modified Eagle Mediumwere seeded in the top chamber. The bottom chamber ofthe Transwell was filled with 600 mL of culture mediumcontaining 10% FBS. Cells were incubated at 37�C for 16hours. The Transwells were then removed from the 24-well plates and stained with 0.1% crystal violet in 25%methanol. Nonmigrated cells were scraped off the top ofthe Transwell with a cotton swab. Migrated cells werequantified by eluting crystal violet with 1% SDS and read-ing the absorbance at 550 nm. In parallel, equal amountsof cells were plated in a 96-well plate, incubated at 37�Cfor 16 hours, stained with crystal violet as described, andthe absorbance values obtained from Transwell elutionwere normalized over absorbance values of the 96-wellplate to obtain the percentage of migrated cells in relationto the different proliferation capacity between SMARCB1expressing and control cells.

Cell-cycle analysisGuava cell-cycle instrument (Millipore) was used to

carry out cell-cycle analysis. SMARCB1 reexpressing andcontrol cells were plated in p60 tissue culture dish. Atdifferent time points after plating, cells were harvestedand fixed in ethanol 70%. Ethanol was eliminated bycentrifugation, cells were washed in PBS 1�, centrifu-gated, and stained with Guava cell-cycle reagent, incu-bated for 40 minutes, shielded from light, and the datawere acquired on the Guava instrument.

Brenca et al.

Mol Cancer Ther; 12(6) June 2013 Molecular Cancer Therapeutics1062




Bromodeoxyuridine assayBrdUrd Flow Kit (BD Pharmingen) was used to quan-

tify cells that were actively synthesizing DNA. Cells werepulsed with 2 hours of bromodeoxyuridine (BrdUrd)incorporation washed and released for additional 2 hoursin complete medium. Cells were permeabilized and pro-cessed according to the manufacturer’s instructions.Stained cells were analyzed with flow cytometer (Beck-man Coulter).

Caspase-3/7 assayCaspase-3/7 activity was assessed using Caspase-Glo

3/7 assay (Promega). Cells were irradiated in suspensionusing a Gammacell 1000 Elite biologic irradiator (NordionInc.), plated in 96 white-walled plates and analyzed after48 hours. Caspase 3/7 GLO Reagent was added to eachwell and gently mixed for 1 minute. The luminescence ofeach sample was measured with a plate-reading lumin-ometer 30 minutes after staining. As a control, all samplesanalyzed were plated also in a 96-multiwell and stainedwith SRB, as indicated above. To take into account thedifferent proliferation between SMARCB1-expressing andcontrol cells, the measured Caspase-Glo 3/7 assay lumi-nescence was normalized over SRB absorbance values.

Clonogenic assayCells (103) were plated in 6 cm culture dishes and

incubated for 10 days in a humidified incubator at 37�Cand5%CO2.Plateswere stainedwith 0.1%crystal violet in25% methanol, cells were photographed and then elutedwith 1% SDS. An equal amount of initial cells was imme-diately fixed and corresponding absorbance readingswere used to normalize for variations in cell seedingamong experimental conditions.

Cell death evaluationCells (2� 105) were irradiated in suspension and plated

in 6-well plates. After 48 hours of treatment, cells wereharvested,washed inPBS1� and stainedwithAnnexinV-FITC (Clontech; 20mg/mL) and 7 amino Actinomycin D(BD Pharmingen). Following incubation at room temper-ature for 15 minutes in the dark, cells were analyzed withFACScan flow cytometer.

Statistical analysisComparisons between 2 classes have been carried out

by 2-sample unpaired Student t test. Comparisonsbetween 3 or more classes have been carried out byrepeated-measures one-way ANOVA. Two classes’ com-parisons with multiple measurement points have beencarried out by 2-way ANOVA. The GI50 values werecalculated by nonlinear regression curve fit of dose versusresponse based on Hill equation. All statistical analyseswere carried out using Prism 5 software (GraphPad Inc.).At least 3 independent biologic replicate experimentswere carried out and average � SD of these replicateswere considered. P values less than 0.05 were consideredstatistically significant.

ResultsSMARCB1 protein expression in sarcoma cell lines

Western blot analysis of a panel of commercially avail-able sarcoma cell lines revealed that, in addition to pre-viously reported rhabdoid sarcoma cell lines, the VAESBJand G402 cell lines displayed a dramatic reduction ofSMARCB1 protein expression (Fig. 1A and Supplemen-tary Fig. S1A). To ascertain the histology of these cellslines, subcutaneous xenograft tumors were established innude mice. Morphology (Fig. 1B) and immunophenotype(CD34 and EMA positive, SMARCB1 negative, Fig. 1C–E)of VAESBJ xenografts were coherent with an origin of thiscell line from an epithelioid sarcoma, as originallydescribed (19). On the contrary, morphology, immuno-phenotype analysis of the G402 xenografts (CD34 andSMARCB1 negative, SMA, and EMA positive, Supple-mentary Fig. S1B–S1E) as well as available clinical data (akidney cancer in an infant 6-month-old patient), suggestthat G402 originated from amisclassified renal malignantrhabdoid tumor, rather than from a leiomyoblastoma aslisted in ATCC.

SMARCB1 is homozygously deleted in VAESBJ andG402 sarcoma cell lines

Exon amplification and MLPA analysis indicatedthat VAESBJ and G402 lost the chromosome region en-compassing SMARCB1 exon 1 and exon 3, respectively(Fig. 2A and Supplementary Fig. S1F and S1G). In parti-cular, PCR-based chromosomal walking allowed theidentification of an 8.4 kb homozygous deletion encom-passing the genomic region from MMP11 exon 3 toSMARCB1 exon 1 in VAESBJ (Supplementary Fig. S2Aand S2B). Primer pairs flanking the region of deletionallowed cloning and sequencing of the breakpoint inVAESBJ (Fig. 2B). Interestingly, high-resolution single-nucleotide polymorphism array-based copy numberanalysis of this cell line carried out by the Sanger Cen-tre (Hinxton, Cambridge; http://www.sanger.ac.uk/cgi-bin/genetics/CGP/cghviewer/CghHome.cgi) fail-ed to identify the presence of such a homozygousdeletion (Supplementary Fig. S2C and S2D), thus sup-porting the subtle nature of such genetic alteration.Moreover, real-time PCR analysis using primers encom-passing exons 4-5 (Fig. 2C) evidenced that the mRNAexpression of SMARCB1 in VAESBJ was significantlyimpaired compared with control 293T cells, consistentwith lack of exon 1/upstream regulatory regions andindicating that no aberrant MMP11-SMARCB1 fusiontranscript was significantly expressed as a result ofthis deletion. Real-time PCR analysis revealed a highlyreduced expression also in G402, consistent with non-sense-mediated mRNA decay, as predicted by theformation of a premature stop codon due to exon 3 dele-tion. Additional MLPA analysis of VAESBJ cells reveal-ed concurrent homozygous deletion of CDKN2A andCDKN2B loci encoding p16, p14, and p15 proteins(Fig. 2D).






SMARCB1 loss sustains VAESBJ tumorigenicproperties

To establish the relevance of SMARCB1 inactivation inVAESBJ cell line, we reintroduced SMARCB1 via retrovi-ral infection (Fig. 3A). After virus infection, cells wereselected and the bulk cell populationwas analyzed for cellproliferation, cell-cycle distribution, senescence, anchor-age-independent growth, and cell migration. For compar-ison, selected experiments were conducted in parallel inG401malignant rhabdoid tumor cell line (SupplementaryFig. S3), which was previously reported to be affected bySMARCB1 restoration (7, 8, 20).

Ectopic SMARCB1 expression significantly reducedcell proliferation without affecting the extent of sponta-neous cell death, as assessed by Trypan blue staining(P ¼ 0.019, 0.014, and 0.004 at 24, 48, and 72 hours,respectively, 2-way ANOVA P < 0.001 for globaltrend, Fig. 3B). The observed reduction in cell prolifer-

ation correlated with a reduction of cells in S and G2–Mphases and an increase in the G0–G1 phase, as assessedby flow cytometry (Fig. 3C). Concordantly, BrdUrdincorporation and clonogenic potential were reduced inSMARCB1-engineered cells (P ¼ 0.025, Fig. 3D andP ¼ 0.001, Fig. 3E respectively). Also anchorage-inde-pendent growth was reduced in SMARCB1 reexpressingVAESBJ cells (P¼ 0.019, Fig. 3F), in part likely as a resultof impaired proliferation. Different from the rhabdoidcell line G401, the ectopic expression of SMARCB1 inVAESBJ failed to result in the induction of prematuresenescence, as determined by SA-b-Gal staining (Sup-plementary Fig. S3). Moreover, similar to what wasdescribed for rhabdoid cell lines (21), SMARCB1 reex-pression significantly reduced VAESBJ cell migration, asassayed by Transwell chamber assay (P < 0.001, Fig. 3Gand Supplementary Fig. S3). Concordant with theobserved reduction of anchorage-independent growth,

CR

L7726

G401

293

VA

ES

BJ

IMR

90

HO

S

Hs729.T

T174

TE

381.T

MG

63

Hs913T

SK

-LM

S-1

ME

S-S

A

Mark

er

α-SMARCB1

α-GAPDH

A

B C

ED

kDa

47

37

Figure 1. SMARCB1 alteration inVAESBJ sarcoma cell line. A,Western blot analysis in a panel ofsarcoma cell lines showing lack ofSMARCB1 protein expression inVAESBJ. B–E, hematoxylin–eosin(B) and immunophenotype (C–E) ofVAESBJ mouse xenograft tumors,showing C, SMARCB1-negative;D, CD34-positive; and E, EMA-positive tumor cells.

Brenca et al.





also in vivo tumorigenesis was significantly reduced andtumors grown were significantly smaller in SMARCB1-reexpressing VAESBJ cells (P < 0.0001 and P ¼ 0.036respectively, Fig. 3H).

SMARCB1 restoration sensitizes VAESBJ cell line toirradiation

The SWI/SNF chromatin-remodeling complex andSMARCB1 itself have been reported to influence the

VAESBJ gene dosage analysis

B 8435 bp deletion

VAESBJ

G402

D

A

Co

py

nu

mb

er

SMARCB1 intron 1MMP11 intron 2

C

VA

ES

BJ

G4

02

ME

S-S

A

T1

74

29

3

MG

63

HO

S

12

73

99

LO

G10 S

MA

RC

B1

mR

NA

ex

pre

ss

ion

1.5

1

0.5

0

–0.5

–1

–1.5

–2

Figure 2. A, SMARCB1 gene dosage analysis by MLPA identified intragenic deletions in VAESBJ and G402 cell lines. B, electropherogram obtainedfrom direct sequencing of the PCR product encompassing the genomic boundaries of SMARCB1 homozygous deletion in VAESBJ cell line. C, quantitativemRNAexpression analysis ofSMARCB1, showing highly reduced expression in VAESBJandG402 cell lines comparedwithSMARCB1wild-type cell lines. D,copy-number analysis of VAESBJ pseudo-triploid cell line by MLPA identifies SMARCB1 and CDKN2A/B homozygous deletion as the sole geneticabnormalities.






response to DNA damage (22). We here show thatSMARCB1 reexpression increased sensitivity to gammairradiation in VAESBJ cell line. Reduced cell viability (P¼0.039, 0.045, 0.02 at 200, 400, and 800 rad, respectively,2-way ANOVA P ¼ 0.085, Fig. 4A) and increased celldeath (P ¼ 0.001, 0.013, 0.002, 0.02 at 100, 200, 400, and800 rad, respectively, 2-way ANOVA P ¼ 0.008, Fig. 4B)were associated with increased of caspase-3/7 activity(P ¼ 0.014 at 1200 rad, Fig. 4C) and caspase-mediatedprotein cleavage (Fig. 4D). The enhanced SMARCB1-mediated sensitization to irradiation was also confirmedby Annexin V/7-AAD staining (P ¼ 0.022 and P ¼ 0.002,

untreated vs. 800 rad and 1,200 rad–treated cells, re-spectively, Fig. 4E).

VAESBJ cell line is susceptible to flavopiridolinhibition and to concurrent inhibition of activatedERBB1/EGFR and HGFR/MET oncogenic pathways

WeexploitedVAESBJ epithelioid sarcoma cellmodel toinvestigate the response to pharmacologic treatmentsthat proved promising in preclinical models of MRT.Fenretinide (7) and flavopiridol (8) are reported to act aseffective CDKs/cyclins inhibitors in the cellular context ofrhabdoid tumor cell lines. We compared the cytotoxicity

GFP

SMARCB1

γ-Tubulin

26

47

50

kDa

A

0

1

2

3

4

5

72 h48 h24 h0

Ra

tio

of

liv

e c

ell

s

Time postplating

*

*

*

pBabeControl

pBabeSMARCB1

B

C

–10

–5

0

96 h72 h48 h24 h

% D

iffe

ren

ce

in

ce

ll-c

yc

le p

ha

se

(pB

ab

eS

MA

RC

B1

–p

Ba

be

Co

ntr

ol)

Time postplating

% G0–G

1

% S

%G2–M

0

0.5

1

1.5

2

2.5

3

No

rmalized

ab

so

rba

nc

e v

alu

e

*

pBabeControl pBabeSMARCB1

E F

0

0.5

1N

orm

alized

ab

so

rban

ce v

alu

e

pBabeControl pBabeSMARCB1

G

0

10

20

30

40

*

pBabeSMARCB1pBabeControl

No

. o

f co

lon

ies/f

ield

*

pBabeSMARCB1 pBabeControl

D+15

+10

+5

50

40

30

20

10

0

% B

rdU

rd-p

osit

ive c

ells

BrdUrd +

*

pBabeSMARCB1pBabeControl

*

Tu

mo

r w

eig

ht

(mg

) 200

150

100

50

0Tu

mo

r vo

lum

e (

mm

3)

Days postinjection

pBabeControl

pBabeSMARCB1

700

600

500

400

300

200

100

03 7 10 14 17 21 24

H

pBab

eCont

rol

pBab

e SM

ARCB

1

Par

enta

l Figure 3. Effects of SMARCB1restoration in VAESBJ sarcomacell line. A, Western blot analysisfollowing restoration of SMARCB1expression or control GFP gene byectopic retroviral infection. B, cellproliferation assay. The ratio of livecells by Trypan blue exclusion issignificantly decreased inSMARCB1 expressing cellscompared with control (2-wayANOVA P < 0.001). C, cell-cycleanalysis. The difference ofpercentage of cells in specific cell-cycle phases between SMARCB1reexpressing and control cells isreported, indicating thatSMARCB1 increases thepercentage of cells in G0–G1phaseof cell cycle while decreasing thepercentage of cells in S and G2–M.D, BrdUrd incorporation assay,showing significantly reducedDNA synthesis in SMARCB1reexpressing VAESBJ cells (P ¼0.025). E, clonogenic assay.Crystal violet relative absorbancevalues is significantly decreasedafter SMARCB1 reexpressionversus control (P < 0.001). F,reduced anchorage-independentgrowth is evident in SMARCB1expressing cells compared withcontrol (P ¼ 0.019). G, migrationcapability is significantly reducedin SMARCB1 reexpressing cellscompared with control (P < 0.001).H, left, reduced VAESBJ in vivotumorigenesis in nude mice uponSMARCB1 restoration (2-wayANOVA P < 0.0001). Right,corresponding reduction in weightof explanted tumors (P¼0.036). Allgraphs display average � SD of 3independent experiments.�, P < 0.05 by 2-tailed unpairedStudent t test.

Brenca et al.





profile of these 2drugs inVAESBJ and controlG401 cancercell lines (Fig. 5A). Although the sensitivity to Flavopir-idol was similar for the 2 cell lines (G401 50% growthinhibition,GI50¼160nmol/L;VAESBJGI50¼ 118nmol/L),VAESBJ displayed a lower sensitivity to fenretinidecompared with G401 (GI50 ¼ 45 mmol/L and GI50 ¼ 1.2mmol/L, respectively). This suggests that SMARCB1 inac-tivation plays a minor role in fenretinide sensitivity.Furthermore, flavopiridol efficiently downmodulatedCCND1 and upregulated both p53 and p21 (Fig. 5B,left), consistent with a significant reduction of cell pro-liferation and increased cell death (one-way ANOVAP < 0.001 and P ¼ 0.008, respectively; Fig. 5B, right).Indeed, the siRNA-mediated silencing of CCND1 (Fig.5C and D) potently inhibited cell proliferation (Fig. 5E,P ¼ 0.006), as did flavopiridol. Finally, flavopiridoleffect was significantly potentiated upon SMARCB1restoration (P < 0.001 and P ¼ 0.012 at 100 nmol/L and300 nmol/L, respectively; Fig. 5F), suggesting the pres-ence of a synergistic effect.To gain insights on the oncogenic pathways underlying

VAESBJ tumorigenic phenotype that can be exploited fortargeted therapeutic interventions, we conducted phos-

pho–protein array analyses. These assays identified sig-nificant expression of activated/phosphorylated ERBB1/EGFR and HGFR/MET (Fig. 6A), that in turn impinge onAKT and ERK phosphorylation (Fig. 6B). Pharmacologicinhibition of HGFR/MET with PHA665752 resulted inimpaired cell motility and correlated with reduced FAKkinase phosphorylation/activation (Fig. 6E), whereas thecombined inhibition of both ERBB1/EGFR and HGFR/MET using selective small-molecule inhibitors Calbio-chem 324674 and PHA665752, respectively, displayed asynergistic effect on proliferation reduction comparedwith single-agent treatment (Fig. 6C and D). Notably,Western blot analysis and immunohistochemistry analy-sis of epithelioid sarcoma tumor samples showed thatmultiple cases presented ERBB1/EGFR and/or HGFR/MET overexpression and activation (Fig. 6F).

DiscussionWehere show that SMARCB1 loss plays a crucial role in

the tumorigenic phenotype of VAESBJ cell line, thusproviding definitive evidence of the causative role ofSMARCB1 loss in epithelioid sarcoma pathogenesis.SMARCB1 is a core subunit of the mammalian SWI/SNF

Figure 4. Gamma irradiationtreatment in VAESBJ cell line afterSMARCB1 restoration. Cellproliferation reduction (2-wayANOVA P ¼ 0.085, A) and increasedcell death (2-way ANOVA P¼ 0.008,B) in SMARCB1 reexpressing cellsas assessed by SRB assay andTrypan blue count, respectively,following 48 hours g-irradiationtreatment. SMARCB1 restorationincreased caspase 3/7 and PARPactivity in VAESBJ cell line treatedwith high dose of g radiation, asassessed by caspase assay (C) andWestern blot analysis (D). E,increased apoptosis is evident byAnnexinV/7AAD staining andsubsequent fluorescence-activatedcell sorting (FACS) analysis. Graphsdisplay average �SD of onerepresentative out of 3 independentexperiments. �, P < 0.05 by 2-tailedunpaired Student t test.

23.3%

29.7%

pBabeSMARCB1+800 RAD

6.2%

5.4%

pBabe Control+800 RAD

0

20

40

60

80

100

120

Rela

tive a

bso

rban

ce (

SR

B a

ssay)

0

4

8

12

16

20

800RAD400RAD200RAD100RADNT

% o

f d

ea

d c

ell

s

A

B

0

1

2

3

4

5

1,200 RAD800 RADNT

Re

lati

ve

lu

min

es

ce

nc

e

Caspase-3/7 assay

C

pBabe

control

pBabe

SMARCB1

PRO-CASP3

Cleaved CASP3

PARPCleaved PARP

GAPDH

PRO-CASP7

Cleaved CASP7

0 0 rad

D

E

*

***

****

*

*

NT 100RAD 200RAD 400RAD 800RAD

pBabeControl

pBabeSMARCB1

pBabeControl

pBabeSMARCB1

pBabeControl

pBabeSMARCB1

0

10

20

30

40

50

% In

cre

ase o

f p

osit

ive c

ells

pB

ab

eS

MA

RC

B1 o

ver

co

ntr

ol

AnnexinV FACS analysis

NT

800RAD

1,200RAD

7-A

AD

Annexin V Annexin V

800

1,2

00

800

1,2

00

ApoptoticApoptotic

Early apoptotic

0 102 103 104 105

010

210

310

410

5

0 102 103 104 105

Early apoptoticAliveAlive






ATP-dependent chromatin remodeling multisubunitcomplexes. By epigenetically affecting histone-DNAcontacts and nucleosome remodeling, the SWI/SNFcomplexes regulate a broad range of cellular pathways(23, 24). Loss of SMARCB1 has been shown to occur at

high frequency in MRT and epithelioid sarcoma.Accordingly, genetic inactivation of SMARCB1 tumorsuppressor gene has been documented in several MRT-derived cell lines. Instead, occasional reports ofSMARCB1 mutations in cell lines of other tumor types,

0

20

40

60

80

100

120

Re

lati

ve

ab

so

rba

nc

e(S

RB

as

sa

y)

NT Flavo

30 nmol/L

Flav

100 nmol/L

Flav

300 nmol/L

pBabeControl

pBabeSMARCB1

No

. o

fli

ve

ce

lls

(x1

03)

% d

ea

d c

ellsV

iab

le c

ells (

××10

5)

25

20

15

10

5

0NT Flavo

30 nmol/L

Flavo

100 nmol/L

Flavo 300

nmol/L

25

20

15

10

5

0

0

0.5

1

1.5

Re

lati

ve

qu

an

tity Real-time PCR

+

+

– + –+ – + –

– +– +– +––

siRNA OT

siRNA CCND1

96 h48 h24 h 72 h

CCND1

γ-Tubulin

TP53

CCND1

CDKN1A/p21

GAPDH

Flavopiridol nmol/L

0 30 100 300

0

10

20

30

40

50

60

70

80

90

100

110

120

1,0001001010

10

20

30

40

50

60

70

80

90

100

110

120

1001010.1

VAESBJ

G401

Flavopiridol (nmol/L) Fenretinide (μmol/L)

Re

lati

ve

ab

so

rba

nc

e (

SR

B a

ss

ay

)

Re

lati

ve

ab

so

rba

nc

e (

SR

B a

ss

ay

)A

B

D

C

F

siRNA OT siRNA CCND1

Time posttrasfection

siRNA OT

siRNA CCND1*

*

*

E 400

350

300

250

200

150

100

50

0

*

*

24 h 48 h 72 h 96 h

Figure 5. A, cell cytotoxicity assay of flavopiridol (left) and fenretinide (right) treatment in VAES BJ and G401 cell lines. The average �SD of 3 independentexperiments is plotted. B, Western blot analysis of VAESBJ cells treated with increasing doses of flavopiridol for 48 hours, showing upregulation of TP53and CDKN1A/p21 and downregulation of CCND1 proteins. Concurrent reduction of cell proliferation and increased cell death is evident by Trypan blueexclusion cell counting (one-way ANOVA P < 0.001 and P ¼ 0.008, respectively). C, efficient CCND1 mRNA downmodulation by siRNA transfection asassayed by quantitative RT-PCR 24 hours posttrasfection. D, efficient CCND1 protein silencing by siRNA transfection as assayed by Western blotanalysis at different time-points posttrasfection. E, the siRNA CCND1-induced cell proliferation reduction is evident by Trypan blue exclusion assay (2-wayANOVA P ¼ 0.006). F, increasing doses of flavopiridol differentially reduced cell proliferation in control and SMARCB1 reexpressing VAESBJ cells. B–F,graphs display average �SD of one representative out of 3 independent experiments. �, P < 0.05 by 2-tailed unpaired Student t test.

Brenca et al.





Ap-ERBB1/EGFR

p-HGFR/MET

p-ERK1/2

ERK1/2

Vinculin

Serum

EGF

PHA665752(HGFR/Met inh.)

324674(EGFR inh.)

B

p-EGFR

EGFR

p-HGFR

HGFR

p-AKT

AKT

− +− − − + − + +

− +− − − − + ++

+ ++ − +− − − −

− − − −− − − −+

GI50 (µmol/L)Drug rm

EGFR inh. Calbiochem324674 0.98400.24649 0.30373

HGFR/Met inh. PHA665752 0.99710.17865 8.93801

EGFR+Met inh. Combined 0.97460.24279 0.04450

D2

1.5

1

0.5

00.2 0.3 0.4 0.5 0.6 0.7 0.8

Co

mb

ina

tio

n in

de

x (

CI)

Fraction affected (Fa)

E

0

0.5

1

–PHA +PHA

*

No

rma

lize

d

ab

so

rban

ce

0

10

20

30

40

50

60

70

80

90

100

0 0.04 0.15 0.3 52.51.250.6 HGFR inh. (μμmol/L)

0 0.08 0.3 52.51.250.6 EGFR inh. (μmol/L)10

Rela

tive a

bso

rban

ce (

SR

B a

ssay)

HGFRi

EGFRi

Combined

HGFRi / EGFRi

C

p FAK (Tyr 397)

p FAK (Tyr 576-577)

FAK

γ-Tubulin

p FAK (Tyr 925)

p-HGFR

HGFR

+ PHA–

F

€

p EGFR

EGFR

ES

1

ES

2

ES

3

ES

4

ES

5

ES

6

ES

7

ES

8

γ-Tubulin

p HGFR

HGFR

EGFR HGFR

Figure 6. A, phospho–protein array analysis of VAESBJ cell line, showing significant expression of activated/phosphorylated ERBB1/EGFR and HGFR/METbut not other receptor kinases investigated. B, Western blot analysis of protein expression in VAESBJ cell line, showing that both activated EGFR andHGFR/MET impinge on AKT and ERK phosphorylation. C, sulphorodamine-B cell cytotoxicity assay showing dose–response plots of EGFR inhibitor324674, HGFR/MET inhibitor PHA665752, and combined treatment with both inhibitors. D, combination index (CI) plot showing synergism (CI<1) of EGFRinhibitor 324674 and HGFR/MET inhibitor PHA665752 at all doses of combined treatments tested. E, reduction of migration capability upon treatment withHGFR/MET inhibitor PHA665752 (�, P ¼ 0.012 by 2-tailed unpaired Student t test). F, evidence of EGFR and HGFR/MET expression and activation inepithelioid sarcoma tumor samples by Western blot and immunohistochemistry.






such as rhabdomyosarcoma (e.g., A204 line; ref. 25) andWilms’ tumor (e.g., G401 line; ref. 26), have been sub-sequently invalidated by reevaluation of such cell linesas misclassified malignant rhabdoid tumors (27, 28). Toour opinion, this is also the case of G402 cell line,originally indicated as a leiomyoblastoma, that we showto carry an exon 3 SMARCB1 deletion. Morphologic andimmunophenotypic characterization of this cell line,together with the available clinical data regarding aninfant renal tumor (ATCC, www.atcc.org), allowed usto establish that G402 is also a bona fide MRT cell line.On the contrary, morphology and immunophenotypeof the VAESBJ xenograft (including CD34-positivestaining) as well as available clinical data on the tumorof origin, reporting a paraspinal sarcoma in a patientaged 42 years (19), support the diagnosis of epithelioidsarcoma.

We provide evidence that VAESBJ carry a homozygousdeletion of SMARCB1 involving exon 1. This result indi-cates that VAESBJ represents a unique model to investi-gate the role of SMARCB1 inactivation in the context ofepithelioid sarcoma. Because in MRTs cells, SMARCB1tumor suppressor activity has been shown to impinge oncell proliferation (29, 30), apoptosis (31), senescence(20, 27), and cell motility (21), we investigated thesephenotypes in SMARCB1-deleted VAESBJ epithelioidsarcoma cell line. Here, we provide evidence thatSMARCB1 restoration in this cell line affects cell prolif-eration, enhances the sensitivity to genotoxic stress, andreduces cell migration. Notably, different from what wasreported forMRT cell models, restoration of SMARCB1 inVAESBJ expression failed to result in premature senes-cence. In MRTs cells, such a response relied on p16activation (20). The fact that VAESBJ cells carry homozy-gous deletion of CDKN2A/p16 locus may account for theattenuation of the senescent phenotype in this cell line.

We exploitedVAESBJ epithelioid sarcoma cellmodel togain insight into activated oncogenic pathways andresponse to targeted as well as conventional genotoxictreatments. We here provide evidence that VAESBJ cellsdisplay activation of both the receptor tyrosine kinaseERBB1/EGFR and HGFR/MET. Recently, a role for EGFRactivation has been proposed in epithelioid sarcoma (32).Although VAESBJ does not carry EGFR mutation, it dis-played sustained, EGFR-independent, phospho-AKTactivation, and showed limited response to EGFR inhibi-tion (32). We found that the HGFR/MET pathway coop-erates with EGFR in sustaining AKT and ERK phosphor-ylation. Concordantly, combined inhibition of ERBB1/EGFR and HGFR/MET pathways resulted in enhancedand synergic growth suppression. Thus, our observationsprovide an explanation for the incomplete response ofVAESBJ cell line to EGFR inhibition (32).

Similar toMRTs cell lines (7, 10), flavopiridol treatmentin VAESBJ induced both CCND1 protein downmodula-tion and cell-cycle arrest. Unexpectedly, we observed asynergistic effect on cell-cycle block with combined Fla-vopiridol treatment and SMARCB1 ectopic reexpression,

indicating that flavopiridol affects cell proliferation andviability through pathways at least in part independent ofSMARCB1 tumor suppressor.

The cancer genome of pediatric MRTs is extraordi-narily stable and the only recurrent alteration reportedso far is SMARCB1 inactivation (33), showing that inspecific cellular contexts unique genetic alterations aresufficient to sustain tumorigenesis. In contrast, epithe-lioid sarcoma (11, 34) and VAESBJ epithelioid sarcomamodel system (ref. 35; see also http://www.sanger.ac.uk/cgi-bin/genetics/CGP/cghviewer/CghHome.cgi),despite the shared feature of recurrent SMARCB1 inac-tivation, show a much more unstable genome, thussuggesting that in the adult setting, the cancer-initiatingcell of epithelioid sarcoma requires the overriding ofmultiple pathways, including the one involved in thecontrol of genome integrity.

The p16/CCND1/CDK4/RB/E2F pathway inacti-vation has been initially reported as the prominentdownstream effect of SMARCB1 inactivation in MRTs(20, 30, 36). However, more recently, engineered mousemodels showed that SMARCB1 haploinsufficiency coop-erates with both RB and TP53 haploinsufficiency toincrease penetrance and reduce latency of tumor onset(9, 22, 37) and in vitro studies showed that SMARCB1inactivation globally affects the epigenetic status of thecancer genome (38, 39). We observed that VAESBJ cellsretain a wild-type TP53 but carry a homozygous deletionof CDKN2A locus, thus displaying impaired p16/RB andp14/TP53 responses. Overall, these data suggest thatSMARCB1 collaborates with the inactivation of both TP53and RB signals to promote tumorigenesis by, at least inpart, independent pathways.

A previous report described the involvement ofSMARCB1 in the modulation of DNA repair after UVirradiation (40). We here show that indeed SMARCB1inactivation is responsible for resistance to gammairradiation and its restoration significantly augmentsthe apoptotic response to irradiation-induced DNAdamage.

In conclusion, we provide the first characterization ofa cell model of epithelioid sarcoma carrying geneticinactivation of SMARCB1 by homozygous gene dele-tion. Our observations provide definitive support to therole of SMARCB1 inactivation in the pathogenesis ofepithelioid sarcoma and disclose novel clues to thera-peutic approaches tailored to SMARCB1-negative epi-thelioid sarcoma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Brenca, R. Maestro, P. ModenaDevelopment of methodology: M. Brenca, E. Lorenzetto, A.P. Dei Tos,P. ModenaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Rossi, E. Piccinin, S. Piccinin, F.M. Rossi,A. Giuliano, A.P. Dei Tos, P. Modena

Brenca et al.





Analysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):M. Brenca, S. Rossi, E. Piccinin, A. Giuliano,A.P. Dei Tos, P. ModenaWriting, review, and/or revision of the manuscript: M. Brenca, S. Rossi,A.P. Dei Tos, R. Maestro, P. ModenaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Piccinin, P. ModenaStudy supervision: R. Maestro, P. Modena

AcknowledgmentsThe authors thank Dr. Bernard E. Weissman, University of North

Carolina, NC, for providing SMARCB1 vectors and Flavia Pivetta fortechnical assistance.

Grant SupportThis work was supported by the Italian Ministry of Research and

Health to R. Maestro (MIUR-Fondo per gli Investimenti della Ricerca diBase; Ricerca Finalizzata, 5 � 1000 funding), the Italian Association forCancer Research to P. Modena (Associazione Italiana per la Ricerca sulCancro).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 22, 2013; revised March 20, 2013; accepted March 29,2013; published OnlineFirst April 10, 2013.

References1. Fletcher CCD, Unni KK, Mertens F, editors. Pathology and genetics of

tumors of soft tissue and bone. Lyon, France: IARC Press; 2002.2. Dei Tos AP, Wagner AJ, Modena P, Comandone A, Leyvraz S.

Epithelioid soft tissue tumors. Semin Oncol 2009;36:347–57.3. Versteege I, Sevenet N, Lange J, Rousseau-Merck MF, Ambros P,

Handgretinger R, et al. Truncating mutations of hSNF5/INI1 in aggres-sive paediatric cancer. Nature 1998;394:203–6.

4. Biegel JA, Zhou JY, Rorke LB,StenstromC,Wainwright LM, FogelgrenB. Germ-line and acquired mutations of INI1 in atypical teratoid andrhabdoid tumors. Cancer Res 1999;59:74–9.

5. Vries RG, Bezrookove V, Zuijderduijn LM, Kia SK, Houweling A,Oruetxebarria I, et al. Cancer-associated mutations in chromatinremodeler hSNF5 promote chromosomal instability by compromisingthe mitotic checkpoint. Genes Dev 2005;19:665–70.

6. Imbalzano AN, Jones SN. Snf5 tumor suppressor couples chromatinremodeling, checkpoint control, and chromosomal stability. CancerCell 2005;7:294–5.

7. Alarcon-Vargas D, Zhang Z, Agarwal B, Challagulla K,Mani S, KalpanaGV. Targeting cyclin D1, a downstream effector of INI1/hSNF5, inrhabdoid tumors. Oncogene 2006;25:722–34.

8. Smith ME, Cimica V, Chinni S, Challagulla K, Mani S, Kalpana GV.Rhabdoid tumor growth is inhibited by flavopiridol. Clin Cancer Res2008;14:523–32.

9. Isakoff MS, Sansam CG, Tamayo P, Subramanian A, Evans JA,Fillmore CM, et al. Inactivation of the Snf5 tumor suppressorstimulates cell cycle progression and cooperates with p53 loss inoncogenic transformation. Proc Natl Acad Sci U S A 2005;102:17745–50.

10. Tsikitis M, Zhang Z, Edelman W, Zagzag D, Kalpana GV. Geneticablation of cyclin D1 abrogates genesis of rhabdoid tumors resultingfrom Ini1 loss. Proc Natl Acad Sci U S A 2005;102:12129–34.

11. Modena P, Lualdi E, Facchinetti F, Galli L, Teixeira MR, Pilotti S, et al.SMARCB1/INI1 tumor suppressor gene is frequently inactivated inepithelioid sarcomas. Cancer Res 2005;65:4012–9.

12. Gasparini P, Facchinetti F, Boeri M, Lorenzetto E, Livio A, Gronchi A,et al. Prognostic determinants in epithelioid sarcoma. Eur J Cancer2011;47:287–95.

13. Flucke U, Slootweg PJ, Mentzel T, Pauwels P, Hulsebos TJ. Re:Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: auseful tool in distinguishing epithelioid sarcoma from malignant rhab-doid tumor: direct evidence of mutational inactivation of SMARCB1/INI1 in epithelioid sarcoma. Hum Pathol 2009;40:1361–2; author reply1362–4.

14. Kohashi K, Izumi T, Oda Y, Yamamoto H, Tamiya S, Taguchi T, et al.Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: auseful tool in distinguishing epithelioid sarcoma from malignant rhab-doid tumor. Hum Pathol 2009;40:349–55.

15. Hornick JL, Dal Cin P, Fletcher CD. Loss of INI1 expression is char-acteristic of both conventional and proximal-type epithelioid sarcoma.Am J Surg Pathol 2009;33:542–50.

16. Chbani L, Guillou L, Terrier P, Decouvelaere AV, Gr�egoire F, Terrier-Lacombe MJ, et al. Epithelioid sarcoma: a clinicopathologic andimmunohistochemical analysis of 106 cases from the french sarcomagroup. Am J Clin Pathol 2009;131:222–7.

17. Hannon GJ, Sun P, Carnero A, Xie LY, Maestro R, Conklin DS, et al.MaRX: an approach to genetics in mammalian cells. Science1999;283:1129–30.

18. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cyto-toxicity screening. Nat Protoc 2006;1:1112–6.

19. Helson C, Melamed M, Braverman S, Traganos F, Preti RA, Helson L.VAESBJ- an epithelioid sarcoma cell line. Int J Oncol 1995;7:51–6.

20. Oruetxebarria I, Venturini F, Kekarainen T, Houweling A, ZuijderduijnLM, Mohd-Sarip A, et al. P16INK4a is required for hSNF5 chromatinremodeler-induced cellular senescence in malignant rhabdoid tumorcells. J Biol Chem 2004;279:3807–16.

21. Caramel J, Quignon F, Delattre O. RhoA-dependent regulation of cellmigration by the tumor suppressor hSNF5/INI1. Cancer Res 2008;68:6154–61.

22. Klochendler-Yeivin A, Picarsky E, Yaniv M. Increased DNA damagesensitivity and apoptosis in cells lacking the Snf5/Ini1 subunit of theSWI/SNF chromatin remodeling complex. Mol Cell Biol 2006;26:2661–74.

23. Wilson BG, Roberts CW. SWI/SNF nucleosome remodellers andcancer. Nat Rev Cancer 2011;11:481–92.

24. Sansam CG, Roberts CW. Epigenetics and cancer: altered chromatinremodeling via Snf5 loss leads to aberrant cell cycle regulation. CellCycle 2006;5:621–4.

25. DeCristofaro MF, Betz BL, Wang W, Weissman BE. Alteration ofhSNF5/INI1/BAF47 detected in rhabdoid cell lines and primaryrhabdomyosarcomas but not Wilms' tumors. Oncogene 1999;18:7559–65.

26. Dowdy SF, Fasching CL, Araujo D, Lai KM, Livanos E, Weissman BE,et al. Suppression of tumorigenicity in Wilms tumor by the p15.5-p14region of chromosome 11. Science 1991;254:293–5.

27. Chai J, Charboneau AL, Betz BL, Weissman BE. Loss of the hSNF5gene concomitantly inactivates p21CIP/WAF1 and p16INK4a activityassociated with replicative senescence in A204 rhabdoid tumor cells.Cancer Res 2005;65:10192–8.

28. Garvin AJ, ReGG, Tarnowski BI, Hazen-Martin DJ, SensDA. TheG401cell line, utilized for studies of chromosomal changes in Wilms' tumor,is derived from a rhabdoid tumor of the kidney. Am J Pathol 1993;142:375–80.

29. Zhang ZK, Davies KP, Allen J, Zhu L, Pestell RG, Zagzag D, et al. Cellcycle arrest and repression of cyclin D1 transcription by INI1/hSNF5.Mol Cell Biol 2002;22:5975–88.

30. Betz BL, Strobeck MW, Reisman DN, Knudsen ES, Weissman BE. Re-expression of hSNF5/INI1/BAF47 in pediatric tumor cells leads to G1arrest associated with induction of p16ink4a and activation of RB.Oncogene 2002;21:5193–203.

31. Ae K, Kobayashi N, Sakuma R, Ogata T, Kuroda H, Kawaguchi N, et al.Chromatin remodeling factor encoded by ini1 induces G1 arrest andapoptosis in ini1-deficient cells. Oncogene 2002;21:3112–20.

32. Xie X, Ghadimi MP, Young ED, Belousov R, Zhu QS, Liu J, et al.Combining EGFR and mTOR blockade for the treatment of epithelioidsarcoma. Clin Cancer Res 2011;17:5901–12.

33. Lee RS, Stewart C, Carter SL, Ambrogio L, Cibulskis K, Sougnez C,et al. A remarkably simple genome underlies highly malignant pediatricrhabdoid cancers. J Clin Invest 2012;122:2983–8.






34. Lualdi E, Modena P, Debiec-Rychter M, Pedeutour F, Teixeira MR,Facchinetti F, et al. Molecular cytogenetic characterization of proximal-typeepithelioidsarcoma.GenesChromosomesCancer 2004;41:283–90.

35. Sakharpe A, Lahat G, Gulamhusein T, Liu P, Bolshakov S, Nguyen T,et al. Epithelioid sarcoma and unclassified sarcoma with epithelioidfeatures: clinicopathological variables, molecular markers, and a newexperimental model. Oncologist 2011;16:512–22.

36. Versteege I, Medjkane S, Rouillard D, Delattre O. A key role of thehSNF5/INI1 tumour suppressor in the control of the G1-S transition ofthe cell cycle. Oncogene 2002;21:6403–12.

37. Chai J, Lu X, Godfrey V, Fletcher C, Roberts CW, Van Dyke T, et al.Tumor-specific cooperation of retinoblastoma protein family and Snf5inactivation. Cancer Res 2007;67:3002–9.

38. Wilson BG, Wang X, Shen X, McKenna ES, Lemieux ME, Cho YJ,et al. Epigenetic antagonism between polycomb and SWI/SNFcomplexes during oncogenic transformation. Cancer Cell 2010;18:316–28.

39. Wang X, Sansam CG, Thom CS, Metzger D, Evans JA, Nguyen PT,et al. Oncogenesis caused by loss of the SNF5 tumor suppressor isdependent on activity of BRG1, the ATPase of the SWI/SNF chromatinremodeling complex. Cancer Res 2009;69:8094–101.

40. Ray A, Mir SN, Wani G, Zhao Q, Battu A, Zhu Q, et al. Human SNF5/INI1, a component of the human SWI/SNF chromatin remodelingcomplex, promotes nucleotide excision repair by influencing ATMrecruitment and downstream H2AX phosphorylation. Mol Cell Biol2009;29:6206–19.

Brenca et al.





2013;12:1060-1072. Published OnlineFirst April 10, 2013.Mol Cancer Ther Monica Brenca, Sabrina Rossi, Erica Lorenzetto, et al. Properties of Epithelioid Sarcoma Cell Line VAESBJ

Genetic Inactivation Is Responsible for TumorigenicINI1/SMARCB1

Updated version

10.1158/1535-7163.MCT-13-0005doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2013/04/09/1535-7163.MCT-13-0005.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/12/6/1060.full#ref-list-1

This article cites 39 articles, 18 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/12/6/1060To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-13-0005

http://mct.aacrjournals.org/content/suppl/2013/04/09/1535-7163.MCT-13-0005.DC1

http://mct.aacrjournals.org/content/12/6/1060.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/12/6/1060


SMARCB1 INI1 Genetic Inactivation Is Responsible for ... · Monica Brenca 1, Sabrina Rossi3, Erica Lorenzetto , Elena Piccinin 1, Sara Piccinin , Francesca Maria Rossi2, Alberto Giuliano

Documents