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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research 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