Truncated SSX Protein Suppresses Synovial Sarcoma Cell Proliferation by Inhibiting the Localization of SS18-SSX Fusion Protein

Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18) (p11.2;q11.2) is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18) and the truncated SSX moiety (tSSX) of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.     

Subnuclear distribution of SSX regulates its function

SSX, a family of genes clustered on the X chromosome, has been identified as a cancer–testis antigen and also forms a part of the SYT–SSX fusion gene found in synovial sarcoma, implying that it has an important role in tumorigenesis. However, knowledge of the molecular regulation of SSX is still limited. In this study, we demonstrate that SSX or its SYT fusion protein is distributed as nuclear speckles, in which it is co-localized with B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1), which is a core factor of polycomb repressor complex 1. The C-terminal residues of SSX are indispensable for the nuclear speckle distribution, while the N-terminal domain is necessary for the recruitment of Bmi1, indicating that intact SSX must be needed for interaction with Bmi1 both spatially and functionally. In addition, the N-terminus of SSX also proved to contain an intrinsic nucleolar localization signal, which mediates the nucleolar translocation of SSX in particular kinds of cell stress such as the oxidation of hydrogen peroxide or heat shock. This stress-induced translocation is reversible and accompanied by HSP 70 or p14ARF traffic, suggesting that SSX is a stress response gene. It is of note that nucleolar translocation of SSX can result in disassociation of SSX from Bmi1, with consequent down-regulation of Bmi1 activity. These novel findings regarding distinct domains of SSX and its interaction with Bmi1 may shed light on the mechanism by which synovial sarcoma develops and on the up-regulation of SSX in cancer cells.     

Identification and characterization of mouse SSX genes: a multigene family on the X chromosome with restricted cancer/testis expression

Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.     

The synovial sarcoma SYT-SSX2 oncogene remodels the cytoskeleton through activation of the ephrin pathway

Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway.     

The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone

In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow and which assembles a gate to maintain the specific ciliary proteome. Here we characterize the function of a novel centriolar satellite protein, synovial sarcoma X breakpoint–interacting protein 2 (SSX2IP), in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using small interfering RNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body. Cep290 takes a central role in gating proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, somatostatin receptor 3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome, and Rab8.     

新型肝癌相关性抗原HCA519及其变异体HCA90的基因克隆和表达

通过利用肝癌病人体内血清中所含的对肿瘤抗原产生的特异性抗体筛选肝癌组织cDNA表达文库的方法 (SEREX) ,筛选得到了可以诱导肝癌病人抗体免疫应答的两个新抗原HCA5 19基因(GenBankAF14 6 731)及其变异体HCA90基因 (GenBankAF 2 872 6 5 ) .它们定位于染色体 2 0q11 2 ,HCA5 19含 18个外显子 ,HCA90含 19个外显子 .其中HCA90所特有的外显子序列长 10 8bp ,属Alu重复序列片段 ,插入于HCA5 19外显子 10和 11之间 ,原为HCA5 19内含子序列 .该插入片段位于HCA5 19开放阅读框架之内 ,不改变HCA5 19的读码框 ,使HCA5 19编码的 74 7个氨基酸增长至HCA90的 783个氨基酸 .通过Northern杂交和RT PCR分析发现 ,HCA5 19和HCA90基因分别在 9 9例和 6 9例肝癌组织中高表达 ,RT PCR显示 ,它们在正常肝组织和其它正常组织中有极低水平转录本表达 .而这种低表达转录本在Northern杂交中不能被检测到 .HCA5 19蛋白被首次发现在肿瘤病人中能够诱导机体的抗体免疫应答 ,为一个新的肿瘤相关性抗原分子 .其变异体HCA90抗原基因为首次发现的新基因 .其功能可能与细胞的恶性增殖相关 ,并可进一步研究其作为临床肿瘤治疗和诊断的靶分子的可行性     

SSX2IP: an emerging role in cancer

We describe the emerging role of Synovial Sarcoma X breakpoint 2 Interacting Protein (SSX2IP) in cancer and its still largely unknown function in human cells. In rodents, SSX2IP has been shown to play a role in adherens junctions and cell adhesion, while in chickens SSX2IP was identified by virtue of its regulation by the light cycle and circadian rhythms. In humans, SSX2IP was identified through its interaction with the cancer-testis gene SSX2. However SSX2IP is expressed in a range of normal and fetal tissues unlike SSX2. SSX2IP containing constructs indicated that SSX2IP could be expressed in the nucleus and cytoplasm of transfected human cells, however, SSX2IP expression has been subsequently shown to peak on the surface of myeloid leukaemia cells during mitosis. Here we discuss the current knowledge of SSX2IP function in several species and the growing evidence that SSX2IP may be a suitable target for leukaemia immunotherapy.     

Establishment and proteomic characterization of a novel synovial sarcoma cell line,NCC-SS2-C1

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18–SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18–SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18–SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.