SMARCB1/INI1 Genetic Inactivation Is Responsible for Tumorigenic Properties of Epithelioid Sarcoma Cell Line VAESBJ

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

Molecular Cancer Cancer Therapeutics Insights Therapeutics

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

Abstract Epithelioid 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 the majority 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 line VAESBJ. Restoration of SMARCB1 expression significantly affected VAESBJ cell proliferation, anchorage-independent growth, and cell migration properties, thus supporting the causative role of SMARCB1 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.

Introduction The SMARCB1 gene located at 22q11 chromosomal Epithelioid sarcoma (International Classification of Dis- region encodes for an invariant subunit of SWI/SNF eases for Oncology code number 8804/3) is a rare mes- chromatin remodeling complex and has been reported to enchymal neoplasm that displays variable epithelioid act as a tumor suppressor gene in infantile malignant morphology, presents a high rate of recurrences and rhabdoid tumor (MRT; refs. 3, 4), a highly aggressive metastases, and frequently poses diagnostic dilemmas neoplasm affecting renal or extra-renal soft tissue, and cerebral tissue in pediatric patients. Previously published (1). It usually affects young adults and arises in the distal SMARCB1 extremities (classic-type epithelioid sarcoma) or, more studies indicate that /INI1 gene is involved in the control of genomic stability and in the regulation rarely, in proximal sites of the trunk (proximal-type epi- SMARCB1 thelioid sarcoma). Proximal-type epithelioid sarcoma is of cell-cycle progression (5). stimulates the more frequently associated with epithelioid or rhabdoid p16/Rb tumor suppressor pathway by activation of morphology and higher mitotic activity (2). CDKN2A and inhibition of Cdk/cyclinD (6). As a result, in MRT cell lines, it has been shown that SMARCB1 loss is Authors' Affiliations: 1Experimental Oncology 1, 2Clinical and Experi- associated with responsiveness to Cdk/cyclin inhibitors, mental Onco-Hematology Unit, Centro di Riferimento Oncologico, Aviano; such as 4-HPR (7) and flavopiridol (8). In addition, the and 3Department of Pathology, Treviso Regional Hospital, Italy. þ in vivo spontaneous tumorigenesis in SMARCB1 / Note: Supplementary data for this article are available at Molecular Cancer knockout mice is increased by TP53-null genetic back- Therapeutics Online (http://mct.aacrjournals.org/). ground (9) and is prevented by CCND1 ablation (10), R. Maestro, and P. Modena share senior co-authorship. further supporting the interaction with crucial molecules Corrected online July 27, 2021. controlling cell-cycle progression. We first reported evidence of SMARCB1 inactivation in Corresponding Author: Piergiorgio Modena, Unit of Experimental Oncol- ogy 1, Centro di Riferimento Oncologico, via F. Gallini 2, 33081 Aviano (PN), epithelioid sarcoma (11), an event frequently associated Italy. Phone: 39-043496-6596; Fax: 39-043465-9659; E-mail: with homozygous gene deletions (12). The relevance of [email protected] SMARCB1 in epithelioid sarcoma pathogenesis was then doi: 10.1158/1535-7163.MCT-13-0005 supported by other reports of subtle intragenic mutations, 2013 American Association for Cancer Research. including small deletions and point mutations (13, 14).

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SMARCB1 in VAESBJ Cell Line

Subsequent studies reported the occurrence of SMARCB1 Western blot analysis and quantitative PCR analysis. In protein and/or genetic alterations in pithelioid sarcoma in vivo experiments, 106 cells from pBabe-SMARCB1 ranging from 60% to 93% (12–16). Although these dif- and pBabe-control infected VAESBJ cells from 3 inde- ferences may be attributable to the different molecular pendent infection experiments were subcutaneously approaches undertaken to assess SMARCB1 inactiva- injected in the flank of nude mice. Tumor growth was tion, the lack of appropriate preclinical models for this monitored over time and calculated using the formula enigmatic sarcoma subtype has so far prevented the 1/2(r3). Once mice were sacrificed, tumors were explan- assessment of the causal role in epithelioid sarcoma ted, weighted, and photographed. Animal experimen- pathogenesis. tation was approved by Institutional Review Board and Here, we provide evidence that the epithelioid sar- conducted according to National laws. coma cell line VAESBJ carry a homozygous SMARCB1 deletion and that in this cell line SMARCB1 actually acts Protein expression as a tumor suppressor. Moreover, we provide evidence For Western blot analysis, protein lysates were pre- that SMARCB1 inactivation results in hyperactivation pared in radioimmunoprecipitation assay buffer (Sigma), of ERBB1/EGFR and HGFR/MET pathways, thus disclos- 40 micrograms of cell lysate were loaded on 4%–15% ing novel avenues for the treatment of patients with epi- gradient PAGE gels (Bio-Rad) and electroblotted onto thelioid sarcoma. polyvinylidene difluoride membranes (Amersham Bio- sciences). Subsequently, membranes were incubated 1 hour at room temperature in a solution of TBST [10 Materials and Methods mmol/L Tris-HCl (pH 8.0), 0.15 mol/L NaCl, and Cancer cell lines 0.05% Tween 20] supplemented with 5% nonfat dry milk. VAESBJ was purchased from Interlab Cell Line Collec- For immunodetection, the anti-BAF47/SNF5 antibody tion and the other cell lines were from American Type Cul- (BD Transduction Laboratories) was used diluted 1:250. ture Collection (ATCC). Sarcoma subtypes represented After overnight incubation at 4C with the primary anti- were osteosarcoma (CRL1543/HOS; MG63), rhabdomyo- body, membranes were washed in TBST, followed by sarcoma (CRL7862/Hs729.T, CRL7726/ T174, CRL7763/ AlexaFluor680– or IRDye800CW-conjugated goat-anti- TE381.T), epithelioid sarcoma (VAESBJ, ATCC number mouse or goat-antirabbit antibodies (from Invitrogen and CRL-2138), fibrosarcoma (HTB152/Hs913.T; MES-SA), Li-Cor, respectively). Odyssey infrared imaging system leiomyosarcoma (HTB88/SK-LMS-1), renal rhabdoid sar- (Li-Cor) was used for detection. Protein loading equiva- coma (G401), and renal leiomyoblastoma (G402). Hek293 lence was assessed using an anti-Gapdh antibody (Sigma). and IMR90 are immortalized noncancer cell lines origin- Additional antibodies used are: TP53 (DO-1 1:1,000; Santa ally derived from human embryonic kidney and fibro- Cruz), CCND1 (DCS-6 1:500; Santa Cruz), CDKN2A/p16 blasts, respectively. Cells were grown in RPMI-1640 (C-20 1:1,000; Santa Cruz), CDKN1A/p21 (H-164 1:500; supplemented with 10% heat-inactivated FBS, in a humid- Santa Cruz), PARP (1:1,000; Cell Signaling), CASP3 ified incubator at 37 Cand5%CO2. Cell lines were authen- (1:1,000; Cell Signaling), CASP7 (1:1,000; Cell Signaling), ticated regularly by microsatellite DNA fingerprinting. P(Tyr1068)-EGFR (1:1,000; Cell Signaling), EGFR (1:1,000, To establish the G402 and VAESBJ xenograft, 5 106 cells Cell Signaling), P(Ser473)-AKT (1:1,000; Cell Signaling), were injected subcutaneously in the flank of nude mice. AKT (1:1,000; Cell Signaling), P-ERK1/2 (1:1,000; Cell After 3 weeks, the tumor was removed, formalin-fixed, Signaling), ERK1/2 (1:1,000; Cell Signaling), P(Tyr1003)- and processed as reported for immunohistochemistry anal- MET (1:1,000; Cell Signaling), MET (1:1,000; Santa Cruz), ysis. For infection experiments, viral supernatants were and Vinculin (1:10,000; Santa Cruz). Immunopheno- prepared using pBabe-SMARCB1 (a kind gift by Dr. Ber- typing with CD34, cytokeratins, epithelial membrane nard E. Weissman, University of North Carolina, Chapel antigen, and CD31 antibodies was conducted for uniform Hill, NC) and pBabe-control vectors using standard calci- pathologic reexamination of xenograft tumors. Protein um–phosphate transfection method in LynxA cells (17). expression of SMARCB1 was investigated by immuno- Supernatants were used for infection with polybrene 4 histochemistry using anti-BAF47/SNF5 antibody 1:100 mg/mL by centrifugation at 1,600 rpm for 1 hour and over- (BD Transduction Laboratories). Endogenous peroxidise night incubation at 32 C. Medium was then replaced and was blocked with 0.3% hydrogen peroxide in methanol 48 hours later, puromycin selection was started for 4 days. for 30 minutes. For antigen retrieval, the slides were Bulk cell population was used in all experiments after immersed in citrate buffer solution 5 mmol/L pH 6 and 24-hour recovery from antibiotic selection. For transfection heated in autoclave at 95C for 15 minutes. Immuno- experiments, cells were transfected using the Dharmafect histochemistry analysis was done using Ultra vision Transfection Reagent (Thermo Scientific) according to the detection system (LabVision). Expression of phosphory- instructions of the manufacturer and CCND1 was silenced lated receptor kinases was detected using the RTK in VAESBJ cell line using CCND1 siRNA (s229 Ambion) Proteome Profiler Array kit (R&D Systems). The pro- and compared with off-target control siRNA (AM4611 cedures were carried out according to the manufac- Ambion). Cells were collected at different time-points turer’s protocol using 300 mg of protein lysate per after transfection and screened for CCND1 expression by array and signals generated by horseradish peroxidase-

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conjugated secondary antibody were visualized and were fixed 72 hours later. No treatment, DMSO or ethanol quantified with Chemidoc imaging system (Bio-Rad). vector control served as calibrator sample. Combination index of drug combinations was calculated with Compu- Real-time PCR Syn software (Combosyn Inc.). SMARCB1 mRNA expression was analyzed by quantitative real-time PCR. Total RNA was extracted using Anchorage-independent growth TRIzol reagent (Ambion) and 1 mg RNA was retrotran- Cells (105) were resuspended in 0.35% agar complete scribed using Superscript II reverse transcriptase (Gibco) medium and seeded on 0.5% bottom agar medium in 6 cm with random primers. Ten nanograms of cDNA were petri dishes. After 2 weeks, plates were stained with used as template in 20 mL PCR reactions with 1 TaqMan iodonitrotetrazolium violet (1 mg/mL, Sigma) and clones Universal PCR master mix (Applera). Relative quantifi- were counted at the microscope. cation of gene expression was conducted in triplicate using TaqMan Assays on Demand on an ABI Prism b-Galactosidase assay 7900HT Sequence Detection System (Applera) by com- The b-galactosidase (b-Gal) staining was used as a C parative t method, using the hypoxanthine phosphor- surrogate marker of senescence. Cells were washed once ibosyltransferase (HPRT) gene (HPRT PDAR, 4326321E) in PBS (pH 7.2), fixed with 0.5% glutaraldehyde for 15 as endogenous reference control and 293 cell line as minutes, and washed in PBS (pH 7.2) supplemented with SMARCB1 calibrator. assay used was Hs00268260_m1, 1 mmol/L MgCl2. Cells were stained with senescence- encompassing exons 4–5. associated b-Gal (SA-b-Gal) stain solution [1 mg/mL 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 0.12 Mutational analysis mmol/L K3Fe(CN)6, 0.12 mmol/L K4Fe(CN)6, 1 mmol/L Mutational analysis was conducted by exon amplifica- MgCl2 in PBS (pH 6)] and incubated overnight at 37 C and tion and sequencing was conducted as previously 5% CO2. The b-Gal–positive cells in 20 microscope fields described (11). For multiplex ligation-dependent probe were counted under bright field on a Olympus IX70 amplification (MLPA), tumor DNA (100 ng) was subjected microscope and representative photographs were taken. to DNA copy number analysis using MLPA kits P258-B1, P294-A1, and P171 (MRC-Holland), following manufac- Migration assay turer instructions and together with normal DNA samples Transwell permeable supports, 6.5 mm diameter in- and cancer cell lines with known SMARCB1 gene copy serts, 8.0 mm pore size, polycarbonate membrane (Corning number alterations as controls. Fragment separation was Inc.) were used to conduct migration assay. Cells (105)in conducted on an ABI3130xl genetic analyzer (Applera). 1% FBS containing Dulbecco’s Modified Eagle Medium Raw data peak pattern evaluation was conducted using were seeded in the top chamber. The bottom chamber of GeneMapper software (Applera) and Coffalyser software the Transwell was filled with 600 mL of culture medium was used for data analysis (MRC-Holland). Homozygous containing 10% FBS. Cells were incubated at 37C for 16 deletion of VAESBJ and G402 cell lines was verified by hours. The Transwells were then removed from the 24- MLPA and delimited and cloned by sequence-tagged site well plates and stained with 0.1% crystal violet in 25% deletion mapping using PCR primer pairs available upon methanol. Nonmigrated cells were scraped off the top of request. the Transwell with a cotton swab. Migrated cells were quantified by eluting crystal violet with 1% SDS and read- Cell proliferation assay ing the absorbance at 550 nm. In parallel, equal amounts Cells were plated in multiwell plates and cell prolifer- of cells were plated in a 96-well plate, incubated at 37C ation was assessed at different time points by either Try- for 16 hours, stained with crystal violet as described, and pan blue cell counting or with sulphorhodamine B (SRB) the absorbance values obtained from Transwell elution staining (18). For SRB staining, cells (5 103) were grown were normalized over absorbance values of the 96-well in 96-well plates for 72 hours, fixed using cold 50% tri- plate to obtain the percentage of migrated cells in relation chloroacetic acid for 1 hour at 4C, then were stained to the different proliferation capacity between SMARCB1 using Sulphorhodamine B (Sigma) 0.4% in 1% acetic acid. expressing and control cells. Tris-Base 10 mmol/L was used for solubilization. The absorbance of protein biomass, which is proportional to Cell-cycle analysis the cell number, was read at 550 nm. Time zero plates were Guava cell-cycle instrument (Millipore) was used to fixed after overnight cell adhesion and served for back- carry out cell-cycle analysis. SMARCB1 reexpressing and ground subtraction and calculation of relative absorbance control cells were plated in p60 tissue culture dish. At values as described (18). Drugs used were flavopiridol different time points after plating, cells were harvested (Sigma), fenretinide (Sigma), PHA665752 HGFR/MET and fixed in ethanol 70%. Ethanol was eliminated by inhibitor (Santa Cruz), and 324674 EGFR inhibitor (Cal- centrifugation, cells were washed in PBS 1, centrifu- biochem). Drugs were resuspended in dimethyl sulfoxide gated, and stained with Guava cell-cycle reagent, incu- (DMSO) or ethanol (in case of fenretinide) and added bated for 40 minutes, shielded from light, and the data 24 hours postplating at the indicated concentrations. Cells were acquired on the Guava instrument.

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Bromodeoxyuridine assay Results BrdUrd Flow Kit (BD Pharmingen) was used to quan- SMARCB1 protein expression in sarcoma cell lines tify cells that were actively synthesizing DNA. Cells were Western blot analysis of a panel of commercially avail- pulsed with 2 hours of bromodeoxyuridine (BrdUrd) able sarcoma cell lines revealed that, in addition to pre- incorporation washed and released for additional 2 hours viously reported rhabdoid sarcoma cell lines, the VAESBJ in complete medium. Cells were permeabilized and pro- and G402 cell lines displayed a dramatic reduction of cessed according to the manufacturer’s instructions. SMARCB1 protein expression (Fig. 1A and Supplemen- Stained cells were analyzed with flow cytometer (Beck- tary Fig. S1A). To ascertain the histology of these cells man Coulter). lines, subcutaneous xenograft tumors were established in nude mice. Morphology (Fig. 1B) and immunophenotype Caspase-3/7 assay (CD34 and EMA positive, SMARCB1 negative, Fig. 1C–E) Caspase-3/7 activity was assessed using Caspase-Glo of VAESBJ xenografts were coherent with an origin of this 3/7 assay (Promega). Cells were irradiated in suspension cell line from an epithelioid sarcoma, as originally using a Gammacell 1000 Elite biologic irradiator (Nordion described (19). On the contrary, morphology, immuno- Inc.), plated in 96 white-walled plates and analyzed after phenotype analysis of the G402 xenografts (CD34 and 48 hours. Caspase 3/7 GLO Reagent was added to each SMARCB1 negative, SMA, and EMA positive, Supple- well and gently mixed for 1 minute. The luminescence of mentary Fig. S1B–S1E) as well as available clinical data (a each sample was measured with a plate-reading lumin- kidney cancer in an infant 6-month-old patient), suggest ometer 30 minutes after staining. As a control, all samples that G402 originated from a misclassified renal malignant analyzed were plated also in a 96-multiwell and stained rhabdoid tumor, rather than from a leiomyoblastoma as with SRB, as indicated above. To take into account the listed in ATCC. different proliferation between SMARCB1-expressing and control cells, the measured Caspase-Glo 3/7 assay luminescence was normalized over SRB absorbance values. SMARCB1 is homozygously deleted in VAESBJ and G402 sarcoma cell lines Clonogenic assay Exon amplification and MLPA analysis indicated Cells (103) were plated in 6 cm culture dishes and that VAESBJ and G402 lost the chromosome region en- incubated for 10 days in a humidified incubator at 37C compassing SMARCB1 exon 1 and exon 3, respectively and 5% CO2. Plates were stained with 0.1% crystal violet in (Fig. 2A and Supplementary Fig. S1F and S1G). In parti- 25% methanol, cells were photographed and then eluted cular, PCR-based chromosomal walking allowed the with 1% SDS. An equal amount of initial cells was imme- identification of an 8.4 kb homozygous deletion encom- diately fixed and corresponding absorbance readings passing the genomic region from MMP11 exon 3 to were used to normalize for variations in cell seeding SMARCB1 exon 1 in VAESBJ (Supplementary Fig. S2A among experimental conditions. and S2B). Primer pairs flanking the region of deletion allowed cloning and sequencing of the breakpoint in Cell death evaluation VAESBJ (Fig. 2B). Interestingly, high-resolution single- Cells (2 105) were irradiated in suspension and plated nucleotide polymorphism array-based copy number in 6-well plates. After 48 hours of treatment, cells were analysis of this cell line carried out by the Sanger Cen- harvested, washed in PBS 1 and stained with Annexin V- tre (Hinxton, Cambridge; http://www.sanger.ac.uk/ FITC (Clontech; 20mg/mL) and 7 amino Actinomycin D cgi-bin/genetics/CGP/cghviewer/CghHome.cgi) fail- (BD Pharmingen). Following incubation at room temper- ed to identify the presence of such a homozygous ature for 15 minutes in the dark, cells were analyzed with deletion (Supplementary Fig. S2C and S2D), thus sup- FACScan flow cytometer. porting the subtle nature of such genetic alteration. Moreover, real-time PCR analysis using primers encom- Statistical analysis passing exons 4-5 (Fig. 2C) evidenced that the mRNA Comparisons between 2 classes have been carried out expression of SMARCB1 in VAESBJ was significantly by 2-sample unpaired Student t test. Comparisons impaired compared with control 293T cells, consistent between 3 or more classes have been carried out by with lack of exon 1/upstream regulatory regions and repeated-measures one-way ANOVA. Two classes’ com- indicating that no aberrant MMP11-SMARCB1 fusion parisons with multiple measurement points have been transcript was significantly expressed as a result of carried out by 2-way ANOVA. The GI50 values were this deletion. Real-time PCR analysis revealed a highly calculated by nonlinear regression curve fit of dose versus reduced expression also in G402, consistent with non- response based on Hill equation. All statistical analyses sense-mediated mRNA decay, as predicted by the were carried out using Prism 5 software (GraphPad Inc.). formation of a premature stop codon due to exon 3 dele- At least 3 independent biologic replicate experiments tion. Additional MLPA analysis of VAESBJ cells reveal- were carried out and average SD of these replicates ed concurrent homozygous deletion of CDKN2A and were considered. P values less than 0.05 were considered CDKN2B loci encoding p16, p14, and p15 proteins statistically significant. (Fig. 2D).

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kDa CRL7726 G401 293 VAESBJ IMR90 HOS Hs729.T T174 TE381.T MG63 Hs913T SK-LMS-1 MES-SA Marker

α-SMARCB1 47

α-GAPDH 37

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

D E

SMARCB1 loss sustains VAESBJ tumorigenic ation correlated with a reduction of cells in S and G2–M properties phases and an increase in the G0–G1 phase, as assessed To establish the relevance of SMARCB1 inactivation in by flow cytometry (Fig. 3C). Concordantly, BrdUrd VAESBJ cell line, we reintroduced SMARCB1 via retrovi- incorporation and clonogenic potential were reduced in ral infection (Fig. 3A). After virus infection, cells were SMARCB1-engineered cells (P ¼ 0.025, Fig. 3D and selected and the bulk cell population was analyzed for cell P ¼ 0.001, Fig. 3E respectively). Also anchorage-inde- proliferation, cell-cycle distribution, senescence, anchor- pendent growth was reduced in SMARCB1 reexpressing age-independent growth, and cell migration. For compar- VAESBJ cells (P ¼ 0.019, Fig. 3F), in part likely as a result ison, selected experiments were conducted in parallel in of impaired proliferation. Different from the rhabdoid G401 malignant rhabdoid tumor cell line (Supplementary cell line G401, the ectopic expression of SMARCB1 in Fig. S3), which was previously reported to be affected by VAESBJ failed to result in the induction of premature SMARCB1 restoration (7, 8, 20). senescence, as determined by SA-b-Gal staining (Sup- Ectopic SMARCB1 expression significantly reduced plementary Fig. S3). Moreover, similar to what was cell proliferation without affecting the extent of sponta- described for rhabdoid cell lines (21), SMARCB1 reex- neous cell death, as assessed by Trypan blue staining pression significantly reduced VAESBJ cell migration, as (P ¼ 0.019, 0.014, and 0.004 at 24, 48, and 72 hours, assayed by Transwell chamber assay (P < 0.001, Fig. 3G respectively, 2-way ANOVA P < 0.001 for global and Supplementary Fig. S3). Concordant with the trend, Fig. 3B). The observed reduction in cell prolifer- observed reduction of anchorage-independent growth,

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A VAESBJ

G402

B 8435 bp deletion C 1.5

MMP11 intron 2 SMARCB1 intron 1 1 VAESBJ G402 MES-SA T174 293 MG63 HOS 127399 0.5 0 SMARCB1

10 –0.5

LOG –1 mRNA expression –1.5 –2 D VAESBJ gene dosage analysis Copy number

Figure 2. A, SMARCB1 gene dosage analysis by MLPA identiﬁed intragenic deletions in VAESBJ and G402 cell lines. B, electropherogram obtained from direct sequencing of the PCR product encompassing the genomic boundaries of SMARCB1 homozygous deletion in VAESBJ cell line. C, quantitative mRNA expression analysis of SMARCB1, showing highly reduced expression in VAESBJ and G402 cell lines compared with SMARCB1 wild-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 genetic abnormalities.

also in vivo tumorigenesis was significantly reduced and SMARCB1 restoration sensitizes VAESBJ cell line to tumors grown were significantly smaller in SMARCB1- irradiation reexpressing VAESBJ cells (P < 0.0001 and P ¼ 0.036 The SWI/SNF chromatin-remodeling complex and respectively, Fig. 3H). SMARCB1 itself have been reported to influence the

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A B 5 Figure 3. Effects of SMARCB1 4 * kDa restoration in VAESBJ sarcoma pBabe pBabe Parental Control SMARCB1 3 * cell line. A, Western blot analysis following restoration of SMARCB1 47 SMARCB1 * pBabeControl 2 expression or control GFP gene by pBabeSMARCB1 26 GFP 1 ectopic retroviral infection. B, cell Ratio of live cells proliferation assay. The ratio of live 50 γ-Tubulin 0 cells by Trypan blue exclusion is 0 24 h 48 h 72 h Time postplating signiﬁcantly decreased in C D SMARCB1 expressing cells +15 50 compared with control (2-way ANOVA P < 0.001). C, cell-cycle +10 40 analysis. The difference of * percentage of cells in speciﬁc cell- 30 +5 cycle phases between SMARCB1 % G –G 0 1 20 reexpressing and control cells is 0 % S reported, indicating that 10 %G2–M SMARCB1 increases the

% BrdUrd-positive cells – –5 0 percentage of cells in G0 G1 phase BrdUrd + of cell cycle while decreasing the

% Difference in cell-cycle phase – (pBabeSMARCB1 –pBabeControl) –10 pBabeControl pBabeSMARCB1 percentage of cells in S and G2 M. 24 h 48 h 72 h 96 h Time postplating D, BrdUrd incorporation assay, showing significantly reduced EF3 G 40 1 DNA synthesis in SMARCB1 2.5 * reexpressing VAESBJ cells (P ¼ 2 * 30 * 0.025). E, clonogenic assay. Crystal violet relative absorbance 1.5 20 0.5 values is significantly decreased 1 Normalized Normalized 10 after SMARCB1 reexpression 0.5 absorbance value absorbance value versus control (P < 0.001). F, No. of colonies/field 0 0 0 reduced anchorage-independent pBabeControl pBabeSMARCB1 pBabeControl pBabeSMARCB1 pBabeControl pBabeSMARCB1 growth is evident in SMARCB1 expressing cells compared with control (P ¼ 0.019). G, migration capability is significantly reduced in SMARCB1 reexpressing cells H 700 compared with control (P < 0.001). H, left, reduced VAESBJ in vivo 600

) pBabeControl

3 tumorigenesis in nude mice upon 200 * 500 pBabeSMARCB1 SMARCB1 restoration (2-way 150 ANOVA P < 0.0001). Right, 400 corresponding reduction in weight 100 300 of explanted tumors (P ¼ 0.036). All graphs display average SD of 3 50 200 independent experiments. Tumor volume (mm Tumor weight (mg) P 100 0 , < 0.05 by 2-tailed unpaired Student t test. 0 pBabeControl pBabeSMARCB1 3 7 10 14 17 21 24 Days postinjection

response to DNA damage (22). We here show that untreated vs. 800 rad and 1,200 rad–treated cells, re- SMARCB1 reexpression increased sensitivity to gamma spectively, Fig. 4E). irradiation in VAESBJ cell line. Reduced cell viability (P ¼ 0.039, 0.045, 0.02 at 200, 400, and 800 rad, respectively, VAESBJ cell line is susceptible to flavopiridol 2-way ANOVA P ¼ 0.085, Fig. 4A) and increased cell inhibition and to concurrent inhibition of activated death (P ¼ 0.001, 0.013, 0.002, 0.02 at 100, 200, 400, and ERBB1/EGFR and HGFR/MET oncogenic pathways 800 rad, respectively, 2-way ANOVA P ¼ 0.008, Fig. 4B) We exploited VAESBJ epithelioid sarcoma cell model to were associated with increased of caspase-3/7 activity investigate the response to pharmacologic treatments (P ¼ 0.014 at 1200 rad, Fig. 4C) and caspase-mediated that proved promising in preclinical models of MRT. protein cleavage (Fig. 4D). The enhanced SMARCB1- Fenretinide (7) and flavopiridol (8) are reported to act as mediated sensitization to irradiation was also confirmed effective CDKs/cyclins inhibitors in the cellular context of by Annexin V/7-AAD staining (P ¼ 0.022 and P ¼ 0.002, rhabdoid tumor cell lines. We compared the cytotoxicity

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A C 120 Caspase-3/7 assay 5 100 * *** pBabeControl 4 pBabeControl 80 3 60 Figure 4. Gamma irradiation pBabeSMARCB1 2 pBabeSMARCB1 40 treatment in VAESBJ cell line after 1 SMARCB1 restoration. Cell 20 proliferation reduction (2-way Relative luminescence 0 0 ANOVA P ¼ 0.085, A) and increased Relative absorbance (SRB assay) NT 100RAD 200RAD 400RAD 800RAD NT 800 RAD 1,200 RAD cell death (2-way ANOVA P ¼ 0.008, B) in SMARCB1 reexpressing cells B D pBabe pBabe control SMARCB1 as assessed by SRB assay and 20 Trypan blue count, respectively, * *** 0 800 1,200 0 800 1,200 rad following 48 hours g-irradiation 16 PARP pBabeControl treatment. SMARCB1 restoration Cleaved PARP 12 increased caspase 3/7 and PARP PRO-CASP3 activity in VAESBJ cell line treated 8 pBabeSMARCB1 Cleaved CASP3 with high dose of g radiation, as

% of dead cells dead of % PRO-CASP7 assessed by caspase assay (C) and 4 Western blot analysis (D). E, Cleaved CASP7 increased apoptosis is evident by 0 GAPDH AnnexinV/7AAD staining and NT 100RAD 200RAD 400RAD 800RAD subsequent ﬂuorescence-activated cell sorting (FACS) analysis. Graphs E pBabe Control+800 RAD pBabeSMARCB1+800 RAD

5 AnnexinV FACS analysis display average SD of one Apoptotic

10 Apoptotic representative out of 3 independent 50 NT experiments. , P < 0.05 by 2-tailed 4 unpaired Student t test. 10 40 *

3 * 7-AAD 6.2% 23.3% 800

10 30 RAD

2 20 5.4% 29.7% 1,200 010 Early apoptotic Early apoptotic 10 Alive Alive RAD

0102 103 104 105 0102 103 104 105 0 % Increase of positive cells Annexin V Annexin V pBabeSMARCB1 over control

profile of these 2 drugs in VAESBJ and control G401 cancer pho–protein array analyses. These assays identified sig- cell lines (Fig. 5A). Although the sensitivity to Flavopir- nificant expression of activated/phosphorylated ERBB1/ idol was similar for the 2 cell lines (G401 50% growth EGFR and HGFR/MET (Fig. 6A), that in turn impinge on ¼ ¼ inhibition, GI50 160 nmol/L; VAESBJ GI50 118 nmol/L), AKT and ERK phosphorylation (Fig. 6B). Pharmacologic VAESBJ displayed a lower sensitivity to fenretinide inhibition of HGFR/MET with PHA665752 resulted in ¼ ¼ compared with G401 (GI50 45 mmol/L and GI50 1.2 impaired cell motility and correlated with reduced FAK mmol/L, respectively). This suggests that SMARCB1 inac- kinase phosphorylation/activation (Fig. 6E), whereas the tivation plays a minor role in fenretinide sensitivity. combined inhibition of both ERBB1/EGFR and HGFR/ Furthermore, flavopiridol efficiently downmodulated MET using selective small-molecule inhibitors Calbio- CCND1 and upregulated both p53 and p21 (Fig. 5B, chem 324674 and PHA665752, respectively, displayed a left), consistent with a significant reduction of cell pro- synergistic effect on proliferation reduction compared liferation and increased cell death (one-way ANOVA with single-agent treatment (Fig. 6C and D). Notably, P < 0.001 and P ¼ 0.008, respectively; Fig. 5B, right). Western blot analysis and immunohistochemistry analy- Indeed, the siRNA-mediated silencing of CCND1 (Fig. sis of epithelioid sarcoma tumor samples showed that 5C and D) potently inhibited cell proliferation (Fig. 5E, multiple cases presented ERBB1/EGFR and/or HGFR/ P ¼ 0.006), as did flavopiridol. Finally, flavopiridol MET overexpression and activation (Fig. 6F). effect was significantly potentiated upon SMARCB1 restoration (P < 0.001 and P ¼ 0.012 at 100 nmol/L and Discussion 300 nmol/L, respectively; Fig. 5F), suggesting the pres- We here show that SMARCB1 loss plays a crucial role in ence of a synergistic effect. the tumorigenic phenotype of VAESBJ cell line, thus To gain insights on the oncogenic pathways underlying providing definitive evidence of the causative role of VAESBJ tumorigenic phenotype that can be exploited for SMARCB1 loss in epithelioid sarcoma pathogenesis. targeted therapeutic interventions, we conducted phos- SMARCB1 is a core subunit of the mammalian SWI/SNF

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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 independent experiments is plotted. B, Western blot analysis of VAESBJ cells treated with increasing doses of flavopiridol for 48 hours, showing upregulation of TP53 and CDKN1A/p21 and downregulation of CCND1 proteins. Concurrent reduction of cell proliferation and increased cell death is evident by Trypan blue exclusion cell counting (one-way ANOVA P < 0.001 and P ¼ 0.008, respectively). C, efficient CCND1 mRNA downmodulation by siRNA transfection as assayed by quantitative RT-PCR 24 hours posttrasfection. D, efficient CCND1 protein silencing by siRNA transfection as assayed by Western blot analysis at different time-points posttrasfection. E, the siRNA CCND1-induced cell proliferation reduction is evident by Trypan blue exclusion assay (2-way ANOVA 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.

ATP-dependent chromatin remodeling multisubunit high frequency in MRT and epithelioid sarcoma. complexes. By epigenetically affecting histone-DNA Accordingly, genetic inactivation of SMARCB1 tumor contacts and nucleosome remodeling, the SWI/SNF suppressor gene has been documented in several MRT- complexes regulate a broad range of cellular pathways derived cell lines. Instead, occasional reports of (23, 24). Loss of SMARCB1 has been shown to occur at SMARCB1 mutations in cell lines of other tumor types,

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SMARCB1 in VAESBJ Cell Line

A C 0 0.04 0.15 0.3 0.6 1.25 2.5 5 HGFR inh. (μmol/L) p-ERBB1/EGFR 100

80 HGFRi

60 EGFRi 50

40 Combined HGFRi / EGFRi p-HGFR/MET 30

20 B Serum + − − − − − −−− 10 Relative absorbance (SRB assay) EGF − − − − + ++− + 0 PHA665752 (HGFR/Met inh.) − − + − − − + ++ 0 0.08 0.3 0.6 1.25 2.5 5 10 EGFR inh. (μmol/L) 324674 + (EGFR inh.) − −−+ − − + + D p-EGFR 2 EGFR Drug GI50 (μmol/L) m r EGFR inh. Calbiochem324674 0.30373 0.24649 0.9840 p-HGFR 1.5 HGFR/Met inh. PHA665752 8.93801 0.17865 0.9971 HGFR EGFR+Met inh. Combined 0.04450 0.24279 0.9746 p-AKT 1 AKT 0.5 p-ERK1/2 Combination index (CI)

ERK1/2 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Vinculin Fraction affected (Fa) E F 1

* ES1 ES2 ES3 ES4 ES5 ES6 ES7 ES8

0.5 p EGFR Normalized absorbance 0 EGFR –PHA +PHA

p HGFR

HGFR € – + PHA p-HGFR γ-Tubulin

HGFR

p FAK (Tyr 397)

p FAK (Tyr 576-577)

p FAK (Tyr 925)

FAK

γ-Tubulin EGFR HGFR

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

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such as rhabdomyosarcoma (e.g., A204 line; ref. 25) and indicating that flavopiridol affects cell proliferation and Wilms’ tumor (e.g., G401 line; ref. 26), have been sub- viability through pathways at least in part independent of sequently invalidated by reevaluation of such cell lines SMARCB1 tumor suppressor. as misclassified malignant rhabdoid tumors (27, 28). To The cancer genome of pediatric MRTs is extraordi- our opinion, this is also the case of G402 cell line, narily stable and the only recurrent alteration reported originally indicated as a leiomyoblastoma, that we show so far is SMARCB1 inactivation (33), showing that in to carry an exon 3 SMARCB1 deletion. Morphologic and specific cellular contexts unique genetic alterations are immunophenotypic characterization of this cell line, sufficient to sustain tumorigenesis. In contrast, epithe- together with the available clinical data regarding an lioid sarcoma (11, 34) and VAESBJ epithelioid sarcoma infant renal tumor (ATCC, www.atcc.org), allowed us model system (ref. 35; see also http://www.sanger.ac. to establish that G402 is also a bona fide MRT cell line. uk/cgi-bin/genetics/CGP/cghviewer/CghHome.cgi), On the contrary, morphology and immunophenotype despite the shared feature of recurrent SMARCB1 inac- of the VAESBJ xenograft (including CD34-positive tivation, show a much more unstable genome, thus staining) as well as available clinical data on the tumor suggesting that in the adult setting, the cancer-initiating of origin, reporting a paraspinal sarcoma in a patient cell of epithelioid sarcoma requires the overriding of aged 42 years (19), support the diagnosis of epithelioid multiple pathways, including the one involved in the sarcoma. control of genome integrity. We provide evidence that VAESBJ carry a homozygous The p16/CCND1/CDK4/RB/E2F pathway inacti- deletion of SMARCB1 involving exon 1. This result indi- vation has been initially reported as the prominent cates that VAESBJ represents a unique model to investi- downstream effect of SMARCB1 inactivation in MRTs gate the role of SMARCB1 inactivation in the context of (20, 30, 36). However, more recently, engineered mouse epithelioid sarcoma. Because in MRTs cells, SMARCB1 models showed that SMARCB1 haploinsufficiency coop- tumor suppressor activity has been shown to impinge on erates with both RB and TP53 haploinsufficiency to cell proliferation (29, 30), apoptosis (31), senescence increase penetrance and reduce latency of tumor onset (20, 27), and cell motility (21), we investigated these (9, 22, 37) and in vitro studies showed that SMARCB1 phenotypes in SMARCB1-deleted VAESBJ epithelioid inactivation globally affects the epigenetic status of the sarcoma cell line. Here, we provide evidence that cancer genome (38, 39). We observed that VAESBJ cells SMARCB1 restoration in this cell line affects cell prolif- retain a wild-type TP53 but carry a homozygous deletion eration, enhances the sensitivity to genotoxic stress, and of CDKN2A locus, thus displaying impaired p16/RB and reduces cell migration. Notably, different from what was p14/TP53 responses. Overall, these data suggest that reported for MRT cell models, restoration of SMARCB1 in SMARCB1 collaborates with the inactivation of both TP53 VAESBJ expression failed to result in premature senes- and RB signals to promote tumorigenesis by, at least in cence. In MRTs cells, such a response relied on p16 part, independent pathways. activation (20). The fact that VAESBJ cells carry homozy- A previous report described the involvement of gous deletion of CDKN2A/p16 locus may account for the SMARCB1 in the modulation of DNA repair after UV attenuation of the senescent phenotype in this cell line. irradiation (40). We here show that indeed SMARCB1 We exploited VAESBJ epithelioid sarcoma cell model to inactivation is responsible for resistance to gamma gain insight into activated oncogenic pathways and irradiation and its restoration significantly augments response to targeted as well as conventional genotoxic the apoptotic response to irradiation-induced DNA treatments. We here provide evidence that VAESBJ cells damage. display activation of both the receptor tyrosine kinase In conclusion, we provide the first characterization of ERBB1/EGFR and HGFR/MET. Recently, a role for EGFR a cell model of epithelioid sarcoma carrying genetic activation has been proposed in epithelioid sarcoma (32). inactivation of SMARCB1 by homozygous gene dele- Although VAESBJ does not carry EGFR mutation, it dis- tion. Our observations provide definitive support to the played sustained, EGFR-independent, phospho-AKT role of SMARCB1 inactivation in the pathogenesis of activation, and showed limited response to EGFR inhibi- epithelioid sarcoma and disclose novel clues to thera- tion (32). We found that the HGFR/MET pathway coop- peutic approaches tailored to SMARCB1-negative epi- erates with EGFR in sustaining AKT and ERK phosphor- thelioid sarcoma. ylation. Concordantly, combined inhibition of ERBB1/ HGFR/ EGFR and MET pathways resulted in enhanced Disclosure of Potential Conflicts of Interest and synergic growth suppression. Thus, our observations No potential conflicts of interest were disclosed. provide an explanation for the incomplete response of VAESBJ cell line to EGFR inhibition (32). Authors' Contributions Similar to MRTs cell lines (7, 10), flavopiridol treatment Conception and design: M. Brenca, R. Maestro, P. Modena in VAESBJ induced both CCND1 protein downmodula- Development of methodology: M. Brenca, E. Lorenzetto, A.P. Dei Tos, tion and cell-cycle arrest. Unexpectedly, we observed a P. Modena Acquisition of data (provided animals, acquired and managed patients, synergistic effect on cell-cycle block with combined Fla- provided facilities, etc.): S. Rossi, E. Piccinin, S. Piccinin, F.M. Rossi, vopiridol treatment and SMARCB1 ectopic reexpression, A. Giuliano, A.P. Dei Tos, P. Modena

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Analysis and interpretation of data (e.g., statistical analysis, biostatis- Grant Support tics, computational analysis): M. Brenca, S. Rossi, E. Piccinin, A. Giuliano, This work was supported by the Italian Ministry of Research and A.P. Dei Tos, P. Modena Health to R. Maestro (MIUR-Fondo per gli Investimenti della Ricerca di Writing, review, and/or revision of the manuscript: M. Brenca, S. Rossi, Base; Ricerca Finalizzata, 5 1000 funding), the Italian Association for A.P. Dei Tos, R. Maestro, P. Modena Cancer Research to P. Modena (Associazione Italiana per la Ricerca sul Administrative, technical, or material support (i.e., reporting or orga- Cancro). nizing data, constructing databases): S. Piccinin, P. Modena The costs of publication of this article were defrayed in part by the Study supervision: R. Maestro, P. Modena payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acknowledgments this fact. The authors thank Dr. Bernard E. Weissman, University of North Carolina, NC, for providing SMARCB1 vectors and Flavia Pivetta for Received January 22, 2013; revised March 20, 2013; accepted March 29, technical assistance. 2013; published OnlineFirst April 10, 2013.

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Correction: SMARCB1/INI1 Genetic Inactivation Is Responsible for Tumorigenic Properties of Epithelioid Sarcoma Cell Line VAESBJ Monica Brenca, Sabrina Rossi, Erica Lorenzetto, Elena Piccinin, Sara Piccinin, Francesca Maria Rossi, Alberto Giuliano, Angelo Paolo Dei Tos, Roberta Maestro, and Piergiorgio Modena

In the original version of this article (1), Fig. 5D contained two erroneously swapped blots and one accidentally duplicated blot. The error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.

Reference 1. Brenca M, Rossi S, Lorenzetto E, Piccinin E, Piccinin S, Rossi FM, et al. SMARCB1/INI1 Genetic inactivation is responsible for tumorigenic properties of epithelioid sarcoma cell line VAESBJ. Mol Cancer Ther 2013; 12:1060–72.

Published online August 2, 2021. Mol Cancer Ther 2021;20:1495 doi: 10.1158/1535-7163.MCT-21-0451 Ó2021 American Association for Cancer Research

AACRJournals.org | 1495 Published OnlineFirst April 10, 2013; DOI: 10.1158/1535-7163.MCT-13-0005

SMARCB1/INI1 Genetic Inactivation Is Responsible for Tumorigenic Properties of Epithelioid Sarcoma Cell Line VAESBJ

Monica Brenca, Sabrina Rossi, Erica Lorenzetto, et al.

Mol Cancer Ther 2013;12:1060-1072. Published OnlineFirst April 10, 2013.

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