SYT-SSX1 and SYT-SSX2 Interfere with Repression of E-Cadherin by Snail and Slug

Research Article

SYT-SSX1 and SYT-SSX2 Interfere with Repression of E-Cadherin by Snail and Slug: A Potential Mechanism for Aberrant Mesenchymal to Epithelial Transition in Human Synovial Sarcoma

Tsuyoshi Saito, Makoto Nagai, and Marc Ladanyi

Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York

Abstract open spaces [i.e., glandular epithelial differentiation (GED)] in a Synovial sarcoma is a primitive mesenchymal neoplasm background of spindle cells (Supplementary Fig. S1). Both the characterized in almost all cases by a t(X;18) fusing the SYT spindle and epithelial elements of synovial sarcoma contain transcriptional coactivator gene with either SSX1 or SSX2, the t(X;18) and are thus clonally related (2, 3). The GED in synovial with the resulting fusion gene encoding an aberrant tran- sarcoma has the hallmarks of a genuine mesenchymal to epithelial scriptional regulator.A subset of synovial sarcoma, predom- transition (MET) akin to those seen in embryonic development inantly cases with the SYT-SSX1 fusion, shows foci of (e.g., in developing kidney). Thus, the epithelial cells in synovial h morphologic epithelial differentiation in the form of nests of sarcoma express E-cadherin, keratins, a-catenin, -catenin, and glandular epithelium.The striking spontaneous mesenchymal g-catenin, whereas the spindle cells express vimentin and, focally, to epithelial differentiation in this cancer is reminiscent of a N-cadherin (4, 5). Epithelial differentiation in synovial sarcoma is developmental switch, but the only clue to its mechanistic also well documented at the ultrastructural level (6, 7). basis has been the observation that most cases of synovial An intriguing observation in synovial sarcoma is that the type of sarcoma with glandular epithelial differentiation (GED) fusion gene (SYT-SSX1 versus SYT-SSX2) is strongly correlated with contain SYT-SSX1 instead of SYT-SSX2.We report here that tumor phenotype (monophasic versus biphasic histology as defined SYT-SSX1 and SYT-SSX2 interact preferentially with Snail or by the presence of GED with lumen formation; refs. 8, 9). An Slug, respectively, and prevent these transcriptional repress- analysis of aggregate data from the three largest studies, including ors from binding to the proximal E-cadherin promoter as a total of 471 synovial sarcoma tumors (10–12), indicates that shown by coimmunoprecipitation and chromatin immuno- tumors with the SYT-SSX1 fusion are at least five times as likely to precipitation.Luciferase reporter assays reveal that SYT-SSX1 show GED compared with SYT-SSX2-bearing tumors (P < 0.0001). and SYT-SSX2 can respectively overcome the Snail- or Slug- This points to a possible role for the SYT-SSX proteins in regulating mediated repression of E-cadherin transcription.This pro- GED, but how these alternative forms of the SYT-SSX chimeric vides a mechanism by which E-cadherin expression, a transcriptional protein might exert such effects has been unknown. prerequisite of epithelial differentiation, is aberrantly dere- Understanding the mechanistic basis of the strong association pressed in synovial sarcoma and may also explain the between t(X;18) fusion type and biphasic histology should provide association of GED with the SYT-SSX1 fusion because it inter- insights into the basic histogenetic processes of architectural feres with Snail, the stronger repressor of the E-cadherin epithelial differentiation and developmental switching between promoter.Thus, our data provide a mechanistic basis for the mesenchymal and epithelial phenotypes. observed heterogeneity in the acquisition of epithelial charac- E-cadherin (CDH1) is a protein, whose extracellular domain acts teristics in synovial sarcoma and highlight the potential role as a molecular zipper mediating cell-cell adhesion whereas its of differential interactions with Snail or Slug in modulating cytoplasmic tail is linked to the actin cytoskeleton via catenins. this phenotypic transition. (Cancer Res 2006; 66(14): 6919-27) Multiple lines of evidence have defined it as a critical determinant of the epithelial phenotype, and its loss seems central to the Introduction process of epithelial to mesenchymal transition (EMT; refs. 13–15). EMT, like its reverse counterpart, MET, is not only a key process in Synovial sarcomas are primitive mesenchymal tumors that embryonic development but has also been implicated in tumor f account for 10% of all soft tissue sarcomas. Synovial sarcomas progression. Absence of E-cadherin expression is seen in synovial contain a t(X;18)(p11.2;q11.2), representing the fusion of SYT (also sarcoma lacking GED (i.e., monophasic synovial sarcoma), and this known as SS18) with either SSX1 or SSX2 or rarely with SSX4 (1, 2). is associated with either overexpression of the Snail transcriptional There are two major morphologic forms of synovial sarcoma: repressor or inactivating mutations in the E-cadherin gene (16, 17). monophasic, entirely composed of spindle cells with or without Interestingly, inactivating mutations in E-cadherin are observed solid epithelial areas, and biphasic, containing epithelial cells lining only in monophasic synovial sarcoma with SYT-SSX1 (16). This is intriguing because, as described above, it is known that synovial sarcoma with SYT-SSX1 is much more likely to display GED than Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). SYT-SSX2-positive synovial sarcoma. Together, these data sug- Current address for T. Saito: Department of Diagnostic Pathology, Tokyo Medical gested a model, in which the capacity for GED may be intrinsically University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. E-mail: saitot@ greater in synovial sarcoma cases with SYT-SSX1 than those with tokyo-med.ac.jp. Requests for reprints: Marc Ladanyi, Department of Pathology, Memorial Sloan- SYT-SSX2 but is abrogated in many SYT-SSX1 cases by mutational Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-6369; or transcriptional down-regulation of E-cadherin. In contrast to the Fax: 212-717-3515; E-mail: [email protected]. I2006 American Association for Cancer Research. findings in synovial sarcoma, E-cadherin is only rarely expressed in doi:10.1158/0008-5472.CAN-05-3697 other spindle cell sarcomas (4, 18). www.aacrjournals.org 6919 Cancer Res 2006; 66: (14). July 15, 2006

Based on these data, we hypothesized that GED in this mesen- deacetylase (HDAC1, HDAC2, and HDAC3), SYT-SSX1/HisMyc or SYT-SSX2/ chymal neoplasm is modulated by SYT-SSX and that SYT-SSX1 HisMyc was transfected into 293T cells and then Western blotting was done and SYT-SSX2 might differentially regulate the E-cadherin gene after immunoprecipitation with anti-myc antibody or normal mouse IgG, possibly by interactions with specific DNA-binding transcription and the membrane was probed with antibodies to HDAC1, HDAC2, and HDAC3 (Abcam, Inc., Cambridge, MA). factors. Because E-cadherin expression seems to be regulated Western blot. Western blotting was done after preparation of cell lysates primarily by transcriptional repressors (Snail, Slug, SIP1, and Twist; with radioimmunoprecipitation assay buffer. Nitrocellulose membranes refs. 13, 14, 19, 20), we examined whether its transcriptional repres- were preincubated with 5% nonfat dry milk in TBS-Tween 20before sion by Snail and Slug in synovial sarcoma is altered by SYT-SSX. incubation with specific primary antibodies for 2 hours. Specific molecules We report here that SYT-SSX1 and SYT-SSX2 interact preferentially were visualized with horseradish peroxidase–labeled anti-mouse or anti- with either Snail or Slug, respectively, and prevent these repressors goat secondary antibodies and enhanced chemiluminescence (Amersham from binding to the proximal E-cadherin promoter, thereby inter- Biosciences). fering with transcriptional repression of the E-cadherin gene to Production of polyclonal antibody against SSX COOH-terminal different degrees and potentially triggering an epithelial differen- region. The sequence of the synthetic peptide used as immunogen was the tiation program leading to a MET with GED in synovial sarcoma. following: C-LVIYEEISDPEEDD-NH2, representing a conserved sequence in the SSXRD shared by SSX1 and SSX2 and included in both SYT-SSX1 and SYT-SSX2. This antibody also recognizes native SSX1 and SSX2 proteins (Supplementary Figs. S2 and S3). Rabbit polyclonal antibody production Materials and Methods was done by AnaSpec (San Jose, CA). Construction of plasmids. The SYT-SSX expression vectors pCXN2- Establishment of SYT-SSX1 and SYT-SSX2-inducible cell lines. T-Rex SYT-SSX1 and pCXN2-SYT-SSX2 were graciously provided by Dr. Shinya 293 cells and T-Rex HeLa cells (Invitrogen) were cultured in 10-cm dishes Tanaka (Hokkaido University, Sapporo, Japan; ref. 21). The inserts were with DMEM supplemented with 10% fetal bovine serum (FBS; Tet system– digested by EcoRI and NotI and subcloned into vectors pcDNA4/myc-His approved FBS; BD Biosciences, Franklin Lakes, NJ). At 70% confluence, and pcDNA4/TO/myc-His (Invitrogen, Carlsbad, CA). We also generated cells were transfected with 2 Ag DNA of SYT-SSX1/TO or SYT-SSX2/TO, deletion mutants SYT-SSX1RD(À)/HisMyc and SYT-SSX2RD(À)/HisMyc respectively, using Fugene 6 transfection reagent. At 48 hours after trans- lacking the COOH-terminal repression domain of SSX (also known as fection, drug selection was started in fresh medium supplemented with SSXRD) and a full-length SYT expression vector, pcDNA4/SYT/HisMyc. 400 Ag/mL Zeocin (Invitrogen) for 6 weeks and drug-resistant colonies were The full-length cDNAs for SSX1 and SSX2 were amplified using as template isolated. Cells from isolated colonies were treated with 1 Ag/mL tetracycline cDNA from K562 leukemia cells, and these cDNA fragments were cloned (Invitrogen) to induce SYT-SSX expression. into pcDNA4/myc-His. The full-length Snail and Slug cDNAs were amplified Chromatin immunoprecipitation assays. Chromatin immunoprecipi- from HeLa cells. The amplified fragments were subcloned into the tation (ChIP) assays were done with a ChIP assay kit (Upstate expression vector p3xFLAG-CMV-10(Sigma, St. Louis, MO). Biotechnology, Inc., Charlottesville, VA), with minor modifications. Briefly, Luciferase reporter gene constructs containing wild-type human 293T cells were transiently transfected with either SYT-SSX1/HisMyc or E-cadherin promoter sequences (from À76 to À490) were generated by SYT-SSX2/HisMyc expression plasmid for 48 hours. To improve the amplifying the promoter region and ligating the fragment into the KpnI and efficiency of the ChIP assay, we treated the cells first with 10mmol/L BglII sites of the pGL3-enhancer vector (Promega, Madison, WI). Mutated dimethyl adipimidate, a protein cross-linking agent, and 0.25% DMSO in reporter constructs, in which three E-box sequences in the promoter were PBS for 45 minutes (24, 25). Formaldehyde was then added at a final changed from CANNTG to AANNTA (13), were generated using the Quick concentration of 1% for 10minutes at 37 jC to cross-link myc-tagged SYT- Mutagenesis kit (Stratagene, La Jolla, CA). SSX or endogenous SYT-SSX to its DNA target sites in vivo. The cells were Cell culture and transfection. Two synovial sarcoma cell lines, HS-SY-II harvested, suspended with SDS lysis buffer [1% SDS, 10mmol/L EDTA, and SYO-1, were used. HS-SY-II was originally established from a SYT-SSX1- 50mmol/L Tris-HCl (pH 8.3)], and incubated on ice for 10minutes. Lysates positive monophasic synovial sarcoma (22) and SYO-1 from a SYT-SSX2- were sonicated, and debris was removed by centrifugation for 10minutes positive biphasic synovial sarcoma (23). at 14,000 Â g. An aliquot of each chromatin solution (200 AL) was set aside HEK293 cells or HeLa cells were seeded in six-well plates at density of and designated as the input fraction. Supernatants were diluted 3-fold in 2 Â 105/mL and allowed to grow for 24 hours before transfection and done immunoprecipitation buffer and precleared with Sepharose A/G plus using Fugene 6 (Roche, Alameda, CA). For experiments assessing activation agarose beads that had been preabsorbed with salmon sperm DNA. The of E-cadherin reporter gene constructs by endogenous transcription factors, precleared chromatin solution was incubated with either anti-myc mouse 1.0 Ag of empty vector or appropriate expression plasmids, 0.35 Agof monoclonal antibody (for 293T cells; Invitrogen) or original rabbit SSX or reporter gene, and 20ng of pRL-SV40were transfected per well. To examine Snail (H-130, Santa Cruz Biotechnology) or Slug (H-140, Santa Cruz the effect of Snail and Slug in transactivation assays, 1.0 Ag of empty vector, Biotechnology) antibodies (for SYO-1 and HS-SY-II cells) or normal rabbit or 3xFLAG/Snail, or 3xFLAG/Slug was cotransfected with the above set of mouse IgG for 16 hours at 4jC. The immune complexes were then collected plasmids. with the addition of Sepharose A/G plus agarose beads followed by several In vitro coimmunoprecipitation. Total cell lysates from HEK293T cells washes with appropriate buffers according to the manufacturer’s protocol. were prepared after cotransfection of either SYT-SSX1/HisMyc, SYT-SSX2/ Each sample was eluted with freshly prepared 1% SDS and 0.1 mol/L HisMyc, SYT-SSX1RD(À)/HisMyc, SYT-SSX2RD(À)/HisMyc, SYT/HisMyc, NaHCO3, and then cross-links were reversed with the addition of 5 mol/L SSX1/HisMyc, or SSX2/HisMyc with 3xFLAG/Snail or 3xFLAG/Slug. Total NaCl. Chromatin-associated proteins were digested with proteinase K cell lysates were divided into three aliquots followed by the addition of (10mg/mL), and the immunoprecipitated DNA was recovered by phenol/ mouse antiserum against myc (Invitrogen) or normal mouse IgG (Santa chloroform extraction and ethanol precipitation and analyzed by PCR. The Cruz Biotechnology, Santa Cruz, CA), and another aliquot was saved for the primer pairs for the detection of the E-cadherin promoter were as follows: transfection control (input). After incubation with overnight rotation at E-cadherin-ChIP, 5¶-CGAACCCAGTGGAATCAGAA-3¶ (forward 1), 5¶-GCGG- 4jC, protein G-Sepharose beads (Amersham Biosciences, Buckinghamshire, GCTGGAGTCTGAACTG-3¶ (reverse 1; amplicon from À359 to À63), 5¶-TG- United Kingdom) were added to the reactions, which were then shaken on GTGGTGTGCACCTGTACT-3¶ (forward 2), and 5¶-GACCTGCACGGTTCT- a rotary shaker at 4jC for 1 hour. The beads were washed thrice with plate GATTC-3¶ (reverse 2; amplicon from À600 to À329). The primer pair for the immunoperoxidase assay buffer, boiled with loading buffer, and electro- amplification of the 3¶-untranslated region (UTR) of the E-cadherin gene phoresed in 4% to 12% MOPS (Invitrogen). After transfer to nitrocellulose was as follows: E-cadherin-ChIP-3¶-UTR, 5¶-CAAGTGCCTGCTTTTGATGA-3¶ membranes, the membranes were incubated with anti-FLAG M2 antibody (forward) and 5¶-GCTTGAACTGCCGAAAAATC-3¶ (reverse). PCR products (Sigma). Furthermore, to see the interaction between SYT-SSX and histone were resolved and visualized with ethidium bromide.

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To investigate changes in histone modifications at the E-cadherin tation experiments revealed that SYT-SSX1 interacts strongly with promoter after induction of the SYT-SSX, ChIP assays were done in HeLa Snail whereas SYT-SSX2 does not (Fig. 1A). Because transcriptional T-Rex SYT-SSX-inducible cell lines with or without tetracycline treatment repression by Snail may be modulated in a competitive fashion by using anti-acetyl histone H3 and anti-acetyl histone H4 antibodies (Upstate Slug through lower affinity binding of the same target sequences Biotechnology) for immunoprecipitation and the same primer pair for PCR (26), we also examined the interaction of SYT-SSX1 and SYT-SSX2 detection. The bound fraction DNA was extracted from the fraction bound to protein A-agarose/salmon sperm DNA, and the unbound fraction DNA with Slug. In contrast to Snail, Slug appeared to be bound more was extracted from cell lysate (supernatant) in the same tube. efficiently by SYT-SSX2 than SYT-SSX1 (Fig. 1A). Because SYT-SSX1 To see whether Snail or Slug still remains bound to the proximal and SYT-SSX2 differ only within their SSX-derived portions, this E-cadherin promoter in the presence of SYT-SSX1 or SYT-SSX2, respectively, raised the question if native SSX can also interact with Snail/Slug. mock, SYT-SSX1, or SYT-SSX2 expression vector was cotransfected with However, coimmunoprecipitation experiments revealed that native Snail or Slug expression vector into 293T cells, and ChIP assay was done SSX proteins do not interact with either Snail or Slug (data not by immunoprecipitation with anti-FLAG antibody (for Snail or Slug). shown). Furthermore, full-length SYT also did not interact with RNA interference. To investigate the effect of endogenous SYT-SSX on either Snail or Slug (data not shown). These results suggested that E-cadherin expression, we used RNA interference by a small interfering RNA this interaction between SYT-SSX and Snail/Slug is a gain of (siRNA) duplex against SYT-SSX. In brief, 24 hours before transfection, 80% function of the fusion proteins relative to their native counterparts. confluent cells were trypsinized and diluted with fresh medium without antibiotics at 3 Â 105/mL and transferred to six-well plates (2 ml/well). SYT-SSX proteins interfere with transcriptional repression Transfection of siRNAs was carried out using Oligofectamine reagent of E-cadherin by Snail/Slug. We first confirmed the reported (Invitrogen) and 0.84 Ag of siRNA duplex. The sense and antisense siRNA transcriptional repression of E-cadherin by Snail and Slug in HeLa sequences were 5¶-CCAGAUCAUGCCCAAGAAGdTdT-3¶ and 5¶-CUU- cells (Supplementary Fig. S2A and B; refs. 13, 14, 26). The levels of CUUGGGCAUGAUCUGGdTdT, respectively (Dharmacon, Chicago, IL). repression of the E-cadherin promoter in this system differ Quantitative real-time reverse transcription-PCR. To evaluate the somewhat from Snail/Slug-dependent repression of this promoter change in the expression levels of SYT-SSX and E-cadherin transcripts after in Madin-Darby canine kidney (MDCK) cells (Supplementary SYT-SSX knockdown or SYT-SSX induction, real-time quantitative reverse Fig. S2C; ref. 26) but are consistent with findings in breast transcription-PCR (RT-PCR) was done on an iCycler (Bio-Rad, Hercules, CA) carcinoma cells (27). We then examined whether SYT-SSX can using Taqman assays. The levels of SYT-SSX and E-cadherin transcripts transactivate the E-cadherin promoter. Transfection of SYT-SSX were normalized to the expression level of the transcript of the TATA box- f binding protein gene (16). The sequences used were as follows: SYT-SSX, expression vectors into HeLa cells produced 20-fold (SYT-SSX1) 5¶-CAGCAGAGGCCTTATGGATATGA-3¶ (forward primer), 5¶-TTTGTG- to 30-fold (SYT-SSX2) transactivation of the E-cadherin reporter GGCCAGATGCTTC-3¶ (reverse primer), and 5¶-ATCATGCCCAAGAAGC- (Fig. 1B). However, the same experiment done in 293 cells resulted CAGCAGAGG-3¶ (probe) and E-cadherin, 5¶-CTTCTCTCACGCTGTG- in only 2-fold (SYT-SSX1) to 3-fold (SYT-SSX2) transactivation TCATC-3¶ (forward primer), 5¶-CTCCTGTGTTCCTGTTAATGGT-3¶ (reverse (Fig. 1C). These contrasting results in different cell lines may reflect primer), and 5¶-TACAATGCCGCCATCGCTTACAC-3¶ (probe). Plasmids for differences in the available interacting proteins in these two cell standard curves were generated by cloning cDNA fragments of SYT-SSX and lines. Specifically, endogenous Snail and Slug are inactivated by E-cadherin into the pCRII TOPO vector (Invitrogen). phosphorylation in E-cadherin-positive cell lines, such as 293 cells, but are strongly expressed and active in HeLa cells (28). Thus, Results transactivation of the E-cadherin reporter by SYT-SSX proteins may SYT-SSX1 and SYT-SSX2 interact with either Snail or Slug as be, at least partly, dependent on the presence of Snail/Slug, raising a gain of function. SYT-SSX expression vectors were cotransfected the possibility that SYT-SSX proteins exert their effect on this with Snail expression plasmid into 293T cells. Coimmunoprecipi- reporter in part by overcoming transcriptional repression by

Figure 1. A, alternative interactions of SYT-SSX1 and SYT-SSX2 with either Snail or Slug. 293T Cells were cotransfected with pcDNA4-HMB-SYT-SSX (À1orÀ2) and p3xFLAG-CMV-10-Snail or Slug. Total cell lysate was extracted 48 hours after transfection. After immunoprecipitation (IP) and Western blotting, membranes were probed with anti-FLAG antibody (M2) to detect Snail or Slug. B, transactivation of the E-cadherin promoter by SYT-SSX with or without cotransfection of Snail or Slug expression vector in HeLa cells. Both forms of SYT-SSX can overcome transcriptional repression by Snail or Slug, but their efficiencies differ in parallel with the coimmunoprecipitation findings. Note that each of the relative luciferase values of the three experiments (mock, Snail, or Slug cotransfection) was normalized separately to that of the corresponding mock cotransfection (light blue). Thus, the data cannot be directly compared between the three experiments. C, transactivation of the E-cadherin promoter by SYT-SSX without cotransfection of Snail or Slug expression vector in 293 cells. D, transactivation of the E-cadherin promoter with three mutated E-boxes by SYT-SSX with or without cotransfection of Snail or Slug expression vector in HeLa cells. The derepressive effect of SYT-SSX on Snail/Slug-mediated repression of the E-cadherin promoter reporter is abolished. However, SYT-SSX1 and SYT-SSX2 nonetheless exert a modest activating effect on this mutant reporter.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research endogenous Snail/Slug. To test this, Snail/Slug expression vectors endogenous Snail/Slug), we asked if SYT-SSX is present at the were cotransfected with either SYT-SSX1 or SYT-SSX2 and endogenous E-cadherin promoter in the absence of Snail/Slug. E-cadherin reporter vector in HeLa cells. Cotransfection of Snail HEK293T cells were transiently transfected with myc-tagged SYT- or Slug expression vectors into HeLa cells along with SYT-SSX SSX expression plasmids. ChIP was done with anti-myc antibody. vectors showed that the latter can overcome the transcriptional This revealed that both forms of SYT-SSX are present in vivo at repression of an E-cadherin reporter construct by Snail or Slug the E-cadherin promoter in the absence of Snail/Slug, suggesting (Fig. 1B). Interestingly, the derepressive effect was stronger for that this minor component of SYT-SSX-associated transactivation SYT-SSX1 on Snail cotransfection and for SYT-SSX2 on Slug is through a mechanism other than Snail/Slug interaction (Fig. 3B). cotransfection, in line with the preferential interactions of these Next, we confirmed that endogenous Snail or Slug is present in vivo respective proteins shown by the above coimmunoprecipitation at the E-cadherin promoter in synovial sarcoma cell lines (Fig. 3C, studies. Moreover, when the assays were repeated with an top) and that endogenous SYT-SSX is also present in vivo at the E-cadherin reporter vector in which the three E-box sequences E-cadherin promoter in synovial sarcoma cell lines using a custom known to be bound by Snail and Slug (13) were mutated, the polyclonal SSX antibody (Fig. 3C, bottom). The suitability of this derepressive effect of SYT-SSX was abolished (Fig. 1D). SYT-SSX antibody for immunoprecipitation of SYT-SSX was confirmed by also exerted a modest transactivating effect on the mutated transient transfection of SYT-SSX expression plasmid into E-cadherin promoter probably through a mechanism other than HEK293T cells (Supplementary Figs. S4 and S5). Although these Snail/Slug interaction (Fig. 1D). In contrast, this derepressive effect experiments confirmed the presence of Snail/Slug and SYT-SSX at could not be observed for full-length native SYT, SSX1, or SSX2 the E-cadherin promoter in synovial sarcoma cell lines, it remained (Supplementary Fig. S3A and B). Taken together, these data suggest unclear whether Snail or Slug remains localized to the proximal that SYT-SSX1 and SYT-SSX2 interact preferentially with Snail or E-cadherin promoter when bound by SYT-SSX proteins. To address Slug, respectively, and interfere with the normal functions of these this, we did several ChIP experiments. Snail or Slug expression transcriptional repressors at least in these model settings. vector was transfected into 293T cells with either mock or SYT- SSX repression domain is necessary for interaction of SSX1 or SYT-SSX2 expression vector. These experiments revealed SYT-SSX with Snail/Slug. SYT-SSX1 and SYT-SSX2 contain both that Snail binding to the E-cadherin promoter appears lower in the transcriptional activation and repressor domains (Fig. 2A). Our presence of SYT-SSX1 than in the presence of SYT-SSX2 (Fig. 3D, observation of contrasting interactions of SYT-SSX1 and SYT-SSX2 top) and that, conversely, Slug binding to the E-cadherin promoter with Snail or Slug prompted us to ask if the SSX repression domain, is lower in the presence of SYT-SSX2 than in the presence of SYT- the most divergent portion of SYT-SSX1 and SYT-SSX2, might be SSX1 (Fig. 3D, bottom). Taken together with the data from responsible for this derepressive effect. Coimmunoprecipitation coimmunoprecipitation and luciferase reporter assays, these results following the cotransfection of either SYT-SSX1RD(À)orSYT- support a model in which SYT-SSX1 and SYT-SSX2 interact with SSX2RD(À) with Snail/Slug revealed that these COOH-terminal either Snail or Slug and prevent Snail or Slug from binding to the deletion mutants (see diagram in Fig. 2A) could not interact with proximal E-cadherin promoter, resulting in a derepressive effect on Snail/Slug (data not shown). Luciferase reporter assays showed the E-cadherin promoter. The relatively weak signals in the SYT- that these COOH-terminal deletion mutants could transactivate SSX lanes in Fig. 3C may also be consistent with this interpretation, E-cadherin promoter only very weakly (<3-fold) in HeLa cells (with if indeed most of the functional interaction of SYT-SSX with this high endogenous Snail/Slug; Fig. 2B), reminiscent of the results in promoter occurs through the physical interaction with Snail or 293 cells (with low endogenous Snail/Slug) transfected with full- Slug that reduces their binding to the E-boxes and thereby also length SYT-SSX (Fig. 1C). Furthermore, the aforementioned removes SYT-SSX proteins from the vicinity of this promoter. derepressive effect of full-length SYT-SSX in the setting of Snail/ Induction of endogenous E-cadherin expression by SYT-SSX. Slug cotransfection was disrupted in these COOH-terminal deletion To gauge the effect of exogenous SYT-SSX on endogenous mutants (Fig. 2B). These results suggest that the SSX repression E-cadherin expression, total RNA was extracted from 293 T-Rex domain of SYT-SSX is necessary for the interaction between SYT- cells and HeLa T-Rex cells stably transfected with an inducible SSX and Snail/Slug and the resulting derepressive effect on Snail/ SYT-SSX expression plasmid. E-cadherin transcript levels were Slug target promoters. measured with or without induction of SYT-SSX by tetracycline SYT-SSX is also present at the E-cadherin promoter (Fig. 4A) at five time points (24, 48, 72, 96, and 120hours after independent of Snail or Slug. Next, to understand how SYT- induction). Quantitative RT-PCR revealed only a minimal increase SSX proteins exert their transactivating effects on the mutated (f2-fold) in E-cadherin expression on induction of SYT-SSX E-cadherin promoter lacking canonical E-boxes in HeLa cells and in 293 T-Rex cells (Fig. 4B). In contrast, in HeLa T-Rex cells, on the intact E-cadherin promoter in 293 cells (with low E-cadherin transcript levels were significantly up-regulated in a

Figure 2. A, schematic diagram of domain structure of the SYT, SSX, and SYT-SSX proteins. Amino acid residues representing the boundaries of selected domains. The scale is approximate. SNH, SYT NH2-terminal domain; QPGY, SYT glutamine-, proline-, glycine-, and tyrosine- rich domain; KRAB, Kru¨ppel-associated box; DD, SSX divergent domain; SSXRD, SSX repressor domain. Bottom, COOH-terminal deletion mutant. B, the SSX repression domain of the SYT-SSX is necessary for the derepressive effect. COOH-terminal deletion mutants of SYT-SSX can transactivate the E-cadherin promoter only very weakly (<3-fold) in HeLa cells. Moreover, the aforementioned derepressive effect on Snail/Slug was disrupted in these COOH-terminal deletion mutants.

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Figure 3. ChIP assays at the E-cadherin promoter. A, map of the E-cadherin promoter showing the location of the three E-boxes and the sites of amplification for ChIP analyses. B, ChIP in 293T cells transfected with SYT-SSX1 shows that exogenously expressed SYT-SSX is present at the E-cadherin promoter. C, top, endogenous Snail or Slug is present at the proximal E-cadherin promoter in synovial sarcoma cell lines; bottom, ChIP in SYO-1 and HS-SY-II cell lines shows that endogenous SYT-SSX protein is also present at the E-cadherin promoter in synovial sarcoma cells. Primer pairs 1 and 2 amplify the E-cadherin promoter region from À359 to À63 and from À600 to À329, respectively. D, top, snail binds to the E-cadherin promoter to a lesser extent in the presence of SYT-SSX1; bottom, Slug binds to the E-cadherin promoter to a lesser extent in the presence of SYT-SSX2. time-dependent fashion after SYT-SSX induction for both SYT- investigated how SYT-SSX proteins exert their derepressive effect SSX1 (Fig. 4C) and SYT-SSX2 (Fig. 4D). These results parallel those on E-cadherin transcription. Because Snail requires HDAC activity of the luciferase reporter assays described above and suggest to repress the E-cadherin promoter, as manifested by deacetylation that the effects of SYT-SSX proteins on the E-cadherin promoter of histones H3 and H4 (25), we studied the effect of SYT-SSX1- are enhanced in cells that contain high levels of Snail/Slug, such mediated suppression of Snail function on histone modifications at as HeLa. the E-cadherin promoter. We did ChIP assays with antibodies Knockdown of SYT-SSX decreases E-cadherin expression in against acetyl histones H3 and H4 in SYT-SSX1-inducible HeLa synovial sarcoma cell lines. Knockdown of SYT-SSX was achieved T-Rex cell lines. The precipitated DNA was subjected to PCR with by transient transfection of a siRNA targeting the SYT-SSX fusion primers for the E-cadherin proximal promoter region (E-cadherin- transcript junction (the sequence at the junction is identical in ChIP forward primer 1 pair). This showed that SYT-SSX1 induction SYT-SSX1 and SYT-SSX2). This siRNA caused up to 75% knockdown is associated with a significant increase in the levels of acetylated of the endogenous SYT-SSX transcript at 48 hours after transfection histones H3 and H4 at the E-cadherin promoter (Supplementary in both synovial sarcoma cell lines compared with a control Fig. S6A). SYT-SSX2 induction in HeLa T-Rex cells did not change nonspecific siRNA. Quantitative RT-PCR showed that E-cadherin the acetylation status of histones H3 and H4 at the E-cadherin mRNA levels in both synovial sarcoma cell lines were significantly promoter (data not shown), consistent with the notion that its decreased at 8 hours after transfection of the SYT-SSX siRNA preferential interaction with the weaker transcriptional repressor duplex (Fig. 5). The E-cadherin quantitative RT-PCR was done at an Slug would result in little or no alteration of Snail-mediated early time point to minimize secondary effects mediated by other repression. Although we have shown that some SYT-SSX is present possible interactions of SYT-SSX proteins. at the E-cadherin promoter independent of the interaction with Transactivation of the E-cadherin gene by SYT-SSX1 is either Snail or Slug, it remained unclear whether this increased associated with histone hyperacetylation caused by dissocia- acetylation of histones H3 and H4 is caused by the direct tion of Snail from the E-cadherin promoter. Next, we interaction of SYT-SSX1 protein and HDACs to cause acetylation www.aacrjournals.org 6923 Cancer Res 2006; 66: (14). July 15, 2006

Figure 4. Effect of SYT-SSX induction on endogenous E-cadherin expression in heterologous cell lines. A, robust induction of SYT-SSX in HeLa T-Rex cells. B, E-cadherin transcript levels increased slightly after the induction of SYT-SSX in 293 T-Rex cells. C and D, E-cadherin transcript increased markedly after SYT-SSX1 (C) and SYT-SSX2 (D) induction in HeLa T-Rex cells. Data at each time point were normalized to the data from corresponding noninduced cells. These results are consistent with those of luciferase reporter assays and suggest that E-cadherin promoter activity is strongly enhanced by SYT-SSX only in cell lines that express Snail/Slug (such as HeLa).

of histones as opposed to histone acetylation resulting from expression levels of its transcriptional repressor Snail (16). Our activation of transcription by the SYT-SSX1-induced dissociation present data link these two sets of observations and suggest that of Snail from E-cadherin promoter. To address this issue, we did a the interaction of SYT-SSX with members of the Snail family of coimmunoprecipitation experiment to examine the interaction transcription factors may determine the propensity for GED in between SYT-SSX and HDAC1, HDAC2, and HDAC3. This revealed synovial sarcoma. no such interaction (Supplementary Fig. S6B). Taken together with Snail family transcription factors have been implicated in the ChIP data, these results indicate that the increased histone embryonic development, mesoderm differentiation, neural crest acetylation observed after SYT-SSX1 induction results from the formation, neural development, apoptosis, and neoplastic EMT transcriptional activation (derepression) of the E-cadherin gene (30). In epithelial cells, the induction of EMT by Snail or Slug caused by the dissociation of Snail from the E-cadherin promoter is mediated by the direct transcriptional repression of E-cadherin, and, therefore, that histone acetylation is an indirect effect of reversing its intercellular adhesion functions (13, 14, 26). In embry- SYT-SSX. onic development, the transcriptional repression of E-cadherin is reversible (31–33). Indeed, EMT as a whole can be a reversible process in different tissues during embryonic development Discussion (31, 32). Therefore, we hypothesized that, in some synovial sarcoma The strong correlation of SYT-SSX fusion type and GED in tumor cells, the transcriptional repression of E-cadherin is synovial sarcoma has long suggested a role for this fusion abrogated due to interactions of SYT-SSX1 and SYT-SSX2 with oncoprotein in the control of epithelial differentiation in this Snail and other E-cadherin transcriptional repressors, leading to sarcoma, but the underlying mechanisms have remained obscure. their functional inactivation and resulting in the acquisition of This is due in part to the fact that the regulatory targets of the epithelial characteristics. SYT-SSX proteins have been difficult to define because these Our data provide multiple lines of evidence supporting an fusion proteins lack a DNA-binding domain and few interacting interaction of SYT-SSX with these transcriptional repressors of transcription factors have been identified (29). In parallel with E-cadherin. Moreover, these interactions have a reciprocal aspect: these observations, E-cadherin down-regulation has been reported SYT-SSX1 interacts with Snail and SYT-SSX2 with Slug. Coimmu- to be associated with the loss or absence of GED in synovial noprecipitation experiments to detect potential interactions sarcoma and this loss of E-cadherin expression is associated either between either full-length SYT or full-length SSX and Snail/Slug with inactivating mutations of E-cadherin or with higher were negative. Thus, these interactions seem specific to

Figure 5. Knockdown of endogenous SYT-SSX in synovial sarcoma cell lines results in decreased endogenous E-cadherin expression. Real-time quantitative RT-PCR revealed that E-cadherin transcript levels decreased significantly in the SYT-SSX siRNA-transfected synovial sarcoma cell lines HS-SY-II (A) and SYO-1 (B).

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SYT-SSX fusion gene products, indicating that this is a gain of and Slug. The finding that E-cadherin transcription was increased function of SYT-SSX relative to the corresponding native proteins. in the presence of Snail/Slug in SYT-SSX-inducible cell lines sup- Further experiments to evaluate the transactivation of the ports a positive role of SYT-SSX fusion proteins in the up- E-cadherin promoter by SYT-SSX proteins in the presence of the regulation of the E-cadherin gene in E-cadherin-negative cell lines. high levels of Snail/Slug in HeLa cells showed that these alternative Furthermore, this differential interaction of SYT-SSX1 and SYT- interactions could overcome repression of this promoter by Snail SSX2 with either Snail or Slug may provide a mechanistic basis for the finding that SYT-SSX2 is at least 5-fold less likely than SYT- SSX1 to be associated with GED in synovial sarcoma tumors (see Introduction) because it has been suggested that Slug functions as a weaker repressor than Snail at the mouse E-cadherin promoter (26), an observation that we have further confirmed using the human E-cadherin promoter reporter vector in MDCK cells (Supplementary Fig. S2C). In addition, it has been also reported that, in MDCK cells, Snail is a stronger repressor than Slug at the promoter of human Claudin-1, which has two E-box sequences and encodes a tight junction protein (34). Thus, Snail-mediated repression of the E-cadherin promoter may be modulated in a competitive fashion by Slug through lower affinity binding of the same E-box sequences. Taken together, these findings suggest a model in which the alternative interactions of SYT-SSX1 and SYT- SSX2 with either Snail or Slug, respectively, alter the availability of one or the other of these repressors to bind the three E-boxes in the human E-cadherin promoter. In synovial sarcoma cells with SYT-SSX1, the fusion protein interacts with Snail and prevents it from binding to the E-cadherin promoter, preventing it from repressing E-cadherin transcription but not impairing the repres- sive function of Slug. Because Slug was shown to have a lower affinity to E-box sequences than Snail (26), this may explain the greater prevalence of E-cadherin expression and GED in synovial sarcoma with the SYT-SSX1 fusion. Conversely, in synovial sarcoma cells with SYT-SSX2, the fusion protein interacts with Slug and prevents it from binding to the E-cadherin promoter but does not alter the function of Snail. This results in stronger transcriptional repression of E-cadherin and hence a lower likelihood of GED in this subset of synovial sarcoma (Fig. 6A). This model is also consistent with the recent data showing an inverse correlation between E-cadherin and Snail expression levels in synovial sarcoma (16). Figure 6B integrates the present functional data with previous data on E-cadherin mutations and Snail expression levels in synovial sarcoma into a model of the determinants of epithelial differentiation in this mesenchymal cancer. Although SYT-SSX1 caused a stronger induction of E-cadherin at early time points after SYT-SSX induction, we noted that, at later time points, SYT-SSX2 caused a stronger up-regulation of E-cadherin than SYT-SSX1 did. This may seem unexpected given Figure 6. A, proposed model for the role of alternative interactions of that Slug (bound by SYT-SSX2) is a weaker repressor of SYT-SSX1 and SYT-SSX2 with either Snail or Slug in derepression of the E-cadherin transcription in synovial sarcoma. a, in the absence of SYT-SSX, E-cadherin than Snail (bound by SYT-SSX1) at least in MDCK E-cadherin expression is transcriptionally silenced in cells of mesenchymal origin cells (26). However, this is otherwise consistent with our finding by the binding of Snail or Slug to three E-boxes in the E-cadherin promoter; b, in synovial sarcoma cells with SYT-SSX1, SYT-SSX1 protein interacts with that, in HeLa cells, Slug represses the E-cadherin promoter Snail and keeps it from binding to the E-cadherin promoter, preventing it from equally or more strongly than Snail (Supplementary Fig. S4A and repressing E-cadherin transcription without impairing Slug, the weaker repressor B) as also reported in a subset of breast cancer cell lines (27). at this promoter; c, conversely, in synovial sarcoma cells with SYT-SSX2, SYT-SSX2 protein binds Slug, preventing it from repressing E-cadherin Alternatively, this result in HeLa cells may in part reflect transcription without impairing Snail, the stronger repressor at this promoter, and functional differences between SYT-SSX1 and SYT-SSX2 unrelated hence, this subset of synovial sarcoma shows a lower likelihood of E-cadherin to their interactions with Snail or Slug because we have observed expression and subsequent GED. B, proposed model of determinants of epithelial differentiation pathways in synovial sarcoma. The differences in the a stronger intrinsic transactivation by SYT-SSX2 than SYT-SSX1 degree of transcriptional derepression of E-cadherin by SYT-SSX1 and when either is fused to the GAL4 DNA-binding domain as assayed SYT-SSX2 result in different propensities for epithelial differentiation. Asubset of 1 SYT-SSX1-bearing synovial sarcoma cases escapes epithelial differentiation in a GAL4 reporter system. by down-regulating E-cadherin either by transcriptional repression by high levels of Snail or through E-cadherin-inactivating mutations (16). Arrows, thickness is roughly proportional to the percentage of cases in each pathway. Overall, f25% of all synovial sarcomas are biphasic (almost all SYT-SSX1 positive) and 75% are monophasic (half SYT-SSX1 positive and half SYT-SSX2 positive). 1 T. Saito and M. Ladanyi, unpublished data. www.aacrjournals.org 6925 Cancer Res 2006; 66: (14). July 15, 2006

Because the SSX repression domain SSXRD (35), which is endogenous Snail or Slug, such as HEK293T cells. Together, these included in the SYT-SSX fusion proteins, is the portion that is most results suggest that SYT-SSX proteins can also transactivate the divergent between SYT-SSX1 and SYT-SSX2, we examined the role E-cadherin gene through a mechanism independent of interac- of this domain in these interactions, which differ so sharply tions with Snail or Slug, but the in vivo relevance of this between the two forms of the fusion protein. Indeed, SYT-SSX phenomenon is unclear because the endogenous E-cadherin constructs in which the SSXRD was deleted could not interact with transcript is only weakly increased on induction of SYT-SSX in Snail/Slug and could not derepress the effect of excess amount of 293 T-Rex cell lines. Snail/Slug on the E-cadherin promoter in HeLa cells, indicating In summary, differential interactions of SYT-SSX1 and SYT-SSX2 that the SSXRD is necessary for these interactions. with either Snail or Slug may provide a mechanistic basis for the It has been shown that Snail mediates E-cadherin repression by observed heterogeneity in the acquisition of epithelial character- the recruitment of the mSin3A/HDAC1/HDAC2 complex (25). istics in this unique mesenchymal cancer (Fig. 6B). However, it is mSin3A, a component of the HDAC complex, can interact with likely that this model is an oversimplification for at least two native SYT and SYT-SSX (36). SYT-SSX may also interact with the reasons. Firstly, the relative concentrations of Snail or Slug and chromatin remodeling factor hBRM/hSNF2a (37). Histone deace- SYT-SSX, as well as that of other E-cadherin repressors (E12/E47, tylation may also play role in transcriptional repression by Slug ZEB1, SIP1, and Twist; refs. 19, 39–41) and potential coregulators, (38). Along these lines, we found that the acetylation of histones may also be important in regulating the role of E-cadherin in H3 and H4 at the E-cadherin promoter increased on induction of epithelial differentiation in synovial sarcoma cells. Secondly, Snail SYT-SSX1 in HeLa cells. Furthermore, we showed that this may repress many other genes involved in epithelial differentiation, increased histone acetylation observed after SYT-SSX1 induction including among others the ELF3 transcription factor gene (42), was dependent on the dissociation of Snail from the E-cadherin previously shown by us to be overexpressed in biphasic synovial promoter and, therefore, that histone acetylation is an indirect sarcoma (16, 43). SYT-SSX fusion proteins may interfere with the effect of SYT-SSX. The minimal alterations in acetylated histones repression of these other significant Snail targets as well. at the E-cadherin promoter following induction of SYT-SSX2 in Nonetheless, our findings strengthen the concept that the protein HeLa cells suggested that its preferential interaction with Slug interactions with Snail or Slug are important in the modulation of results in little or no alteration of histone modifications, possibly MET and EMT in mesenchymal differentiation and neoplasia. indicating that these are mainly triggered by Snail at this promoter. However, it should be also noted that SYT-SSX proteins had some activity on the E-cadherin reporter plasmid that seemed Acknowledgments independent of Snail/Slug based on luciferase reporter assays in Received 10/13/2005; revised 5/2/2006; accepted 5/16/2006. HeLa cells using an E-cadherin reporter plasmid, in which all Grant support: The Uehara Memorial Foundation Postdoctoral Fellowship, Japan three E-box sequences, and based on ChIP assays that showed (T. Saito) and NIH grant PO1 CA47179 (M. Ladanyi). The costs of publication of this article were defrayed in part by the payment of page specific binding of SYT-SSX to the E-cadherin promoter without charges. This article must therefore be hereby marked advertisement in accordance cotransfection of Snail or Slug even in cells expressing little or no with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. SYT-SSX1 and SYT-SSX2 Interfere with Repression of E-Cadherin by Snail and Slug: A Potential Mechanism for Aberrant Mesenchymal to Epithelial Transition in Human Synovial Sarcoma

Tsuyoshi Saito, Makoto Nagai and Marc Ladanyi

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