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SS18-SSX fusion protein-induced Wnt/β-catenin signaling is a therapeutic target in synovial sarcoma

Abstract

Synovial sarcoma is a high-grade soft tissue malignancy characterized by a specific reciprocal translocation t(X;18), which leads to the fusion of the SS18 (SYT) gene to one of three SSX genes (SSX1, SSX2 or SSX4). The resulting chimeric SS18-SSX protein is suggested to act as an oncogenic transcriptional regulator. Despite multimodal therapeutic approaches, metastatic disease is often lethal and the development of novel targeted therapeutic strategies is required. Several expression-profiling studies identified distinct gene expression signatures, implying a consistent role of Wnt/β-catenin signaling in synovial sarcoma tumorigenesis. Here we investigate the functional and therapeutic relevance of Wnt/β-catenin pathway activation in vitro and in vivo. Immunohistochemical analyses of nuclear β-catenin and Wnt downstream targets revealed activation of canonical Wnt signaling in a significant subset of 30 primary synovial sarcoma specimens. Functional aspects of Wnt signaling including dependence of Tcf/β-catenin complex activity on the SS18-SSX fusion proteins were analyzed. Efficient SS18-SSX-dependent activation of the Tcf/β-catenin transcriptional complex was confirmed by TOPflash reporter luciferase assays and immunoblotting. In five human synovial sarcoma cell lines, inhibition of the Tcf/β-catenin protein–protein interaction significantly blocked the canonical Wnt/β-catenin signaling cascade, accompanied by the effective downregulation of Wnt targets (AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin) and the specific suppression of cell viability associated with the induction of apoptosis. In SYO-1 synovial sarcoma xenografts, administration of small molecule Tcf/β-catenin complex inhibitors significantly reduced tumor growth, associated with diminished AXIN2 protein levels. In summary, SS18-SSX-induced Wnt/β-catenin signaling appears to be of crucial biological importance in synovial sarcoma tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

Introduction

Synovial sarcoma is an aggressive malignancy comprising 7–10% of all soft tissue tumors with a predominance in adolescents and young adults.1 The molecular hallmark of synovial sarcoma is a pathognomonic reciprocal translocation t(X;18)(p11;q11), leading to the fusion of SS18 (SYT) to one of the homologs SSX genes (most frequently SSX1 or SSX2, in rare cases SSX4), generating oncogenic SS18-SSX chimeric proteins.2, 3, 4 Although the pathognomonic SS18-SSX fusion proteins seem to have a crucial role in synovial sarcoma tumorigenesis and progression, the specific biological function and the mechanism of action remain to be defined. Neither SS18, the SSX proteins, nor the chimeric SS18-SSX oncoproteins have known DNA-binding motifs; however, they have been reported to contribute to the dysregulation of gene expression through association with SWI/SNF and Polycomb chromatin remodeling complexes.5, 6, 7, 8 On the basis of several microarray expression-profiling studies, one of the pathways recurrently found deregulated in synovial sarcoma is the Wnt/β-catenin signaling pathway.9, 10, 11, 12, 13

Wnt/β-catenin signaling has fundamental roles in the regulation of diverse biological processes, including embryogenesis, cell proliferation, survival and tissue regeneration. The central key mediator of the canonical Wnt pathway is β-catenin, which is, as long as the pathway is inactive, degraded through a multiprotein complex including axis inhibition protein (AXIN), adenomatous polyposis coli (APC), casein-kinase-1 (CK1) and glycogen synthase kinase-3beta (GSK-3β). Upon activation of the signaling cascade by binding of Wnt ligands to frizzled receptors (FZD), the multiprotein destruction complex is disrupted and β-catenin translocates to the nucleus, where it interacts with transcription factors of the T-cell factor (Tcf) and lymphoid enhancer factor (Lef) family inducing expression of downstream targets, including AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin.14, 15 Increasing evidence suggests that aberrant activation of Wnt/β-catenin signaling is associated with tumor development and progression in various types of cancer.16, 17, 18 In a variety of malignancies, oncogenic pathway activation is derived from genetic alterations in central signaling components, including β-catenin and APC. In synovial sarcoma, oncogenic mutations of Wnt pathway components have been reported in a minor frequency,19, 20, 21 suggesting a functional key role of the SS18-SSX chimeric oncoprotein.

The objective of the present study is to explore the functional relevance of Wnt/β-catenin signaling in synovial sarcoma tumorigenesis including its molecular dependence on the pathognomonic SS18-SSX fusion proteins and to preclinically test novel molecularly targeted approaches employing small molecule inhibitors of the Tcf/β-catenin protein complex.22, 23, 24

Results

Expression of Wnt/β-catenin signaling components in primary synovial sarcoma and tumor-derived cell lines

To determine the involvement of Wnt/β-catenin signaling in synovial sarcoma tumorigenesis, the expressions of nuclear β-catenin and Wnt targets were examined in a set of 30 primary synovial sarcoma specimens by immunohistochemical analysis. Significant nuclear staining of β-catenin was found in 73% (22/30) of cases (Figure 1a). Five out of 30 tumors (17%) showed no nuclear β-catenin immunoreactivity. Consistently, several Wnt targets were highly expressed in synovial sarcoma including AXIN2, DKK1, Survivin, c-MYC and CyclinD1 (Figure 1a). In all, 63% of the tumors displayed strong expression levels of AXIN2 and 37% showed weak expression. DKK1 was strongly expressed in 40% (12/30) of cases, weakly in 57% (17/30) and, in only a single case, no DKK1 expression was detectable. Strong expression of Survivin was detectable in all tumor samples. Expression levels for c-MYC were strong in 30% of the samples, weak in 27% and 43% of the tumors were negative for c-MYC. In concordance, c-MYC-negative tumor samples showed lower β-catenin nuclear staining scores compared with c-MYC-positive cases. Strong expression of CyclinD1 was detected in 32% of the tumors, 25% of the samples displayed weak expression levels and 12 tumors did not show any expression of CyclinD1. There was a significant overlap of nuclear β-catenin immunoreactivity and the strong expression of at least two Wnt targets in 23 out of 25 tumors (92%). However, distinct cases (5/30) displayed target expression without evidence of nuclear β-catenin accumulation, pointing to a more elaborate regulatory context. Expression of Wnt target genes did not correlate with the patients’ age, gender, the translocation subtype or tumor size (Figure 1 and Supplementary Table S1).

Figure 1
figure1

Activation of Wnt/β-catenin signaling in synovial sarcoma. (a) Immunohistochemical stainings showing nuclear localization of β-catenin and expression of Wnt/β-catenin targets in a representative case of synovial sarcoma (original magnification: × 20, inset × 40). (b) Detection of the cytoplasmic (C) and nuclear (N) fractions of β-catenin in five synovial sarcoma cell lines, nuclear localization being an indicator of pathway activity. GAPDH and LaminB1 were used as controls for the cytoplasmic and nuclear fractions, respectively. (c) Immunoblotting for phospho-β-catenin in synovial sarcoma cell lines. (d) Elevated expression levels of Wnt signaling targets in total protein extracts of synovial sarcoma cell lines.

In accordance with the immunohistochemical results in primary tumor tissues, elevated β-catenin protein levels were found in nuclear extracts and immunostainings of synovial sarcoma cell lines, corresponding to the transcriptionally active pool of β-catenin (Figure 1b and data not shown). MCF-7 breast carcinoma cells (known to have an activated Wnt/β-catenin signaling cascade) and MDA-MB-453 (showing only very faint nuclear levels of β-catenin) were included as controls (Supplementary Figure S1).25, 26 The fraction of β-catenin phosphorylated at Ser45, Ser33, Ser37 and Thr41 (all destabilizing phosphorylation steps exerted by CK1 and GSK-3β)27 was low compared with the fraction of β-catenin phosphorylated at Ser552 and Ser675 (inducing β-catenin accumulation in the nucleus)28 (Figure 1c). Indeed, protein levels of Wnt signaling target genes were elevated in all but one synovial sarcoma cell line (Figure 1d).

As mutations in CTNNB1 (encoding β-catenin) or APC might be responsible for Wnt/β-catenin pathway activation, we screened the CTNNB1 exon 3 sequence (encoding the regulatory degradation targeting box of β-catenin) and the entire APC coding region for mutations. All synovial sarcoma cell lines were wild type for APC, and none of them displayed mutations in the regulatory phospho-sites of the degradation targeting box of β-catenin. In SYO-1 cells, we detected a p.G34L point mutation (CTNNB1). Alterations in codon 34 have been reported to lack transforming transcriptional activation potential.29

Activation of Wnt/β-catenin signaling is induced by SS18-SSX

To evaluate whether Tcf/β-catenin transcriptional activity is molecularly dependent on the SS18-SSX fusion proteins, HEK293 cells were transfected with SS18-SSX1, SS18-SSX2, SS18, SSX1 and SSX2 expression plasmids. In luciferase reporter assays employing the TOP-/FOPflash system, expression of SS18-SSX significantly increased TOPflash reporter activity by 15- to 20-fold compared with SS18, SSX1, SSX2 or the mutant ΔN131 β-catenin30 control (Figure 2a). We next determined the functional requirement of β-catenin for SS18-SSX-induced TOPflash reporter activity. Knockdown of β-catenin by small interfering RNA (siRNA) significantly reduced the TOPflash reporter activity enhanced by SS18-SSX1 and SS18-SSX2 (Figure 2c). These results suggest that β-catenin is required for SS18-SSX-associated induction of Tcf/β-catenin-mediated transcriptional activity. In agreement with published results,31 expression of SS18-SSX or mutant ΔN131 β-catenin induced nuclear recruitment and accumulation of β-catenin (Supplementary Figure S2). In contrast, no changes in cellular localization of β-catenin were detected in cells transient transfected with SS18, SSX1 or SSX2.

Figure 2
figure2

Synovial sarcoma-associated SS18-SSX fusion proteins stimulate Tcf/β-catenin-mediated transcriptional activity. (a) HEK293 cells were co-transfected with indicated SS18-SSX, SS18 or SSX expression vector, and TOP-/FOPflash luciferase reporter plasmids to study Tcf/β-catenin-mediated transcriptional activity. Luciferase reporter activities were measured 24 h after transfection and normalized to Renilla luciferase activities. Co-expression of SS18-SSX1 and SS18-SSX2 significantly increased TOPflash reporter activity compared with SS18, SSX1 or SSX2, confirming stimulated Tcf/β-catenin activity. Experiments were performed in triplicates; results are expressed as mean±s.d. (b) Elevated Wnt target protein levels of AXIN2, CDC25A, DKK1 and CyclinD1 in HT1080 cells expressing the SS18-SSX fusion proteins. (c) Significant reduction of TOPflash reporter activity upon siRNA-mediated knockdown of β-catenin (inset) in HEK293 cells co-transfected with SS18-SSX1 and SS18-SSX2 expression vectors and TOPflash reporter plasmids, indicating requirement of β-catenin for SS18-SSX stimulated reporter activation. Luciferase reporter activities were measured 72 h after siRNA transfection and were normalized to Renilla luciferase activities.

Comparable to what has been observed in NIH3T3 cells,31 the induction of Wnt target expression in HEK293 cells upon SS18-SSX expression was rather weak though TOP-Flash assays indicated a strong activation of β-catenin/TCF transcriptional activity. We therefore chose HT1080 fibrosarcoma cells as an additional in vitro model to further investigate the expression of Wnt/β-catenin signaling downstream targets, as these cells have been reported to express nuclear β-catenin at low levels, indicating basal pathway activity.17 Immunoblot analyses showed elevated levels of AXIN2, CDC25A, DKK1 and CyclinD1 upon expression of the SS18-SSX fusion proteins (Figure 2b). Destabilizing phosphorylation of β-catenin at residues Ser45, Ser33, Ser37 and Thr41 remained constant. Likewise, no relevant changes in the fraction of β-catenin phosphorylated at Ser552 and Ser675 were found (data not shown).

PKF115–584, CGP049090 and PKF118–310 suppress cell viability of synovial sarcoma cell lines in vitro

To investigate the biological effects of treatment with small molecule inhibitors of the Tcf/β-catenin complex (PKF115–584, CGP049090 and PKF118–310), synovial sarcoma and control cell lines (MCF-7 and MDA-MB-453) were exposed to increasing concentrations (0.15–1.25 μM) of these compounds for 72 h. All three substances were effective in reducing synovial sarcoma and MCF-7 cell viability with IC50 values ranging from 0.17– to 1.82 μM (Figure 3a and Table 1). SYO-1, CME-1 and HS-SY-II cells were more sensitive to treatment compared with FUJI and 1273/99 cells, but no correlation between the SS18-SSX translocation subtype and the exerted effect was observed. IC50 values of synovial sarcoma cell lines were comparable to those of MCF-7 control cells with known activation of Wnt/β-catenin signaling and similar to previously reported IC50 values in colon and prostate cancer cell lines.22 In contrast, MDA-MB-453 control cells with almost undetectable nuclear levels of β-catenin showed only minor responses. These results argue in favor of Wnt/β-catenin specificity of the substances’ mode of action. Combination of Tcf/β-catenin complex inhibition with conventional chemotherapeutic agents (Vincristine, Doxorubicin and Actinomycin D; 0.1–1000 ng/ml) resulted in additive effects on SYO-1 cell viability (Figure 3b).

Figure 3
figure3

In vitro cytotoxic effects of PKF115–584, CGP049090 and PKF118–310 on synovial sarcoma cell lines. (a) Cell viability of synovial sarcoma cell lines was inhibited by treatment with increasing concentrations (0.15–1.25 μM) of PKF115–584, CGP049090 and PKF118–310. MCF-7 and MDA-MB-453 breast cancer cells were included as positive and negative controls, respectively. At least three independent experiments were performed (each in quintuplicate), results are expressed as mean±s.e. (b) Combined treatment of SYO-1 cells with conventional chemotherapeutic agents (Vincristine, Doxorubicin and Actinomycin D; 0.1–1000 ng/ml) and small molecular inhibitors of the Tcf/β-catenin protein–protein interaction (0.1-0.2 μM; concentration resulting in 20–30% growth inhibition) resulted in additive effects.

Table 1 IC50 values for PKF115–584, CGP049090 and PKF118–310 in synovial sarcoma and breast carcinoma cell lines

PKF115–584, CGP049090 and PKF118–310 inhibit Tcf/β-catenin interaction and mediated transcriptional activity

Effects of treatment with PKF115–584, CGP049090 and PKF118–310 on Tcf/β-catenin-mediated transcriptional activity in synovial sarcoma cell lines were assessed employing the TOP-/FOPflash luciferase reporter assay. Significant dose-dependent (0.5–2 μM) inhibition of reporter activity was observed in SYO-1, HS-SY-II (Figure 4a) and CME-1 (Supplementary Figure S3a) cells. Furthermore, increased reporter activity after co-expression of mutant ΔN131 β-catenin was diminished in treated SYO-1 cells (Figure 4b), indicating specific suppression of Wnt/β-catenin signaling in the setting of mesenchymal tumor cells. Immunoblotting of AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin revealed a dose- and time-dependent downregulation in SYO-1, HS-SY-II and CME-1 cells (Figures 4c–d, Supplementary Figure S3b and data not shown), with no alterations in the nuclear fractions of β-catenin observed (data not shown).

Figure 4
figure4

PKF115–584, CGP049090 and PKF118–310 inhibit Tcf/β-catenin interaction and transcriptional activity in synovial sarcoma cell lines. (a) SYO-1 and HS-SY-II cells were transfected with the TOPflash luciferase reporter plasmid and treated with increasing concentrations (0.5–2 μM; 24 h) of PKF115–584, CGP049090 and PKF118–310. In response to treatment with all three compounds, significant dose-dependent inhibition of luciferase activity was observed in both cell lines. Experiments were performed in triplicates, results are expressed as mean±s.d. (b) In SYO-1 cells, TOPflash reporter activity was significantly enhanced after co-expression of mutant ΔN131 β-catenin and reversed upon treatment with PKF115–584, CGP049090 and PKF118–310 (1.5 μM). (c) Compounds inhibit Tcf/β-catenin-regulated expression of AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin in SYO-1 and (d) HS-SY-II cells. Synovial sarcoma cells were treated with increasing concentrations of the substances (0.5–2 μM) for 15 h. Changes in target expression were determined by immunoblotting.

PKF115–584, CGP049090 and PKF118–310 inhibit cell proliferation by inducing apoptosis and decreasing mitotic activity in synovial sarcoma cell lines

Performing flow cytometric analysis, poly-adenosine diphosphate (ADP)-ribose polymerase (PARP; Asp214) cleavage was used as a marker for apoptosis and phospho-histone H3 (Ser10) was employed as a marker for mitosis. After treatment with PKF115-584, CGP049090 and PKF118-310 (0.1 μM), SYO-1 and CME-1 cells showed significantly increased rates of apoptosis, accompanied by decreased mitotic fractions (Figure 5a, Table 2 and Supplementary Figure S3c). Consistently, immunoblot analyses of treated synovial sarcoma cells demonstrated an induction of caspase-3 cleavage in a dose-dependent manner (Figure 5b and Supplementary Figure S3d). Similar results were observed in HS-SY-II cells (data not shown).

Figure 5
figure5

PKF115–584, CGP049090 and PKF118–310 induce apoptosis and impair proliferation of SYO-1 synovial sarcoma cells. (a) Representative results of flow cytometric analysis of cleaved PARP (Asp214) and phospho-histone H3 (Ser10) in treated SYO-1 cells. DMSO was employed as control. Significantly increased rates of apoptosis (cleaved PARP) and decreased mitotic fractions (phospho-histone H3) were detected upon treatment with indicated compounds (0.1 μM; 24 h). (b) Increasing concentrations of PKF115-584, CGP049090 and PKF118–310 resulted in induced caspase-3 cleavage in SYO-1 cells.

Table 2 Results of flow cytometric analysis in synovial sarcoma cell lines

Knockdown of CTNNB1 and SS18-SSX2 affect cell viability and Wnt target expression in synovial sarcoma cell lines

To document the functional role of β-catenin by an independent non-pharmacological approach, synovial sarcoma cell lines (SYO-1, HS-SY-II and CME-1) were transfected with siRNAs directed against human CTNNB1 (encoding β-catenin). In MTT assays, all analyzed synovial sarcoma cell lines displayed significantly reduced cell viabilities (***P<0.001; **P<0.01) in comparison with non-targeting control siRNA (Figure 6a and Supplementary Figure S4a). Consistently, siRNA-mediated knockdown of β-catenin leads to reduced Wnt target gene expression, combined with the induction of caspase-3 cleavage (Figure 6b and Supplementary Figure S4b). To inversely prove the contribution of SS18-SSX2 to Tcf/β-catenin-mediated transcriptional activity, synovial sarcoma cells (SYO-1, FUJI, 1273/99 and CME-1) were transfected with a set of published and validated siRNA duplex oligos targeting the SSX2 portion of the chimeric oncoprotein.31, 32 Upon decreased SS18-SSX2 expression, all analyzed cell lines displayed reduced Wnt target expression (Figure 6c), implying that the SS18-SSX2 fusion protein is involved in the regulation of Tcf/β-catenin-mediated transcriptional activity. Consistent with the elevated expression levels of Wnt/β-catenin signaling targets upon SS18-SSX transfection (Figure 2b), expression of AXIN2, DKK1 and CyclinD1 was inversely suppressed compared with non-targeting negative control siRNA (Figure 6c).

Figure 6
figure6

Effects of siRNA-mediated knockdown of β-catenin and SS18-SSX2 on cell viability and Tcf/β-catenin transcriptional activity. (a) Significant reduction of cell viability (MTT assay) upon siRNA-mediated knockdown of β-catenin (encoded by CTNNB1) in SYO-1 and HS-SY-II synovial sarcoma cells (***P<0.001; **P<0.01). Experiments were performed in quintuplicates and results of a representative experiment are expressed as mean±s.d. (b) Immunoblotting analyses demonstrate a significant induction of caspase-3 cleavage (Asp175) and a decrease in Wnt/β-catenin signaling target expression following CTNNB1 or (c) SS18-SSX2-specific siRNA transfection in four different synovial sarcoma cell lines. Efficient siRNA-mediated SS18-SSX2 knockdown was confirmed with an antibody targeting the N terminus of the SS18 protein.

In vivo efficacy of CGP049090 in a murine xenograft model of synovial sarcoma

To investigate the in vivo efficacy of Tcf/β-catenin complex inhibition on tumor growth and progression in a xenograft mouse model of human synovial sarcoma, we selected CGP049090 as previously published small molecule inhibitor with no major toxic side effects observed.22, 33, 34 SYO-1 cells were injected subcutaneously into the lower flank of nude mice to initiate tumor formation. When the tumor volume reached around 100 mm3, tumor-bearing mice were treated daily with 25 mg/kg CGP049090 (n=6) or DMSO vehicle (n=6) for 14 days. CGP049090 administration resulted in a significant reduction in tumor volume (Figure 7a and Table 3; *P<0.05) compared with the DMSO vehicle control group and did not induce any significant effects on body weights (Figure 7b and Table 3). Immunohistochemical analysis of tumor sections revealed that Tcf/β-catenin inhibition was associated with decreased AXIN2 protein levels (Figure 7c). The percentage of phospho-histone H3 (Ser10)- positive mitotic cells was significantly decreased in CGP049090-treated tumors compared with vehicle-treated controls (Figures 7c and d; ***P<0.001); as cleaved caspase-3 (Asp175) levels were significantly increased upon CGP049090 treatment (Figures 7c and e; **P<0.01).

Figure 7
figure7

CGP049090 inhibits synovial sarcoma xenograft growth in vivo. Mice bearing SYO-1 tumors were randomized (n=6 per group) and treated daily with CGP049090 (25 mg/kg) or DMSO vehicle control for 14 days. (a) Mean tumor volume±s.e. at given time points and (b) mean of body weight during killing for SYO-1 xenografts are shown. Mice treated with CGP049090 exhibited significantly reduced tumor volumes (*P<0.05). (c) Xenograft tumor tissues from vehicle-treated (left) and CGP049090-treated (right) mice were subjected to immunohistochemical analyses of AXIN2, phospho-histone H3 (Ser10) and cleaved caspase-3 (Asp175) (original magnification, × 40). (d) Phospho-histone H3 (Ser10)-positive mitotic cells and (e) cleaved caspase-3 (Asp175)-positive apoptotic cells were counted from randomly selected areas and are indicated as mean±s.e. Tumor growth reduction was associated with decreased levels of phospho-histone H3 (Ser10) (***P<0.001) and an induction of cleaved caspase-3 (Asp175)-positive apoptotic cell fraction (**P<0.01).

Table 3 Effects of CGP049090 treatment on tumor volume and body weight of SYO-1 xenografts

Discussion

Synovial sarcoma is a highly malignant mesenchymal tumor with a dismal prognosis in patients with advanced metastasized disease. Current therapeutic concepts are mainly based on radical surgery and conventional radiotherapeutic protocols. Synovial sarcoma is characterized by specific oncogenic SS18-SSX fusion proteins, which are of crucial importance in tumor development, as has convincingly been demonstrated in a conditional synovial sarcoma mouse model.4, 35, 36 The SS18-SSX fusion proteins act as transcriptional regulators through complex interaction with various partners within chromatin remodeling complexes, thereby dysregulating gene expression.5, 6, 7, 8 As it is particularly challenging to specifically target these chimeric fusion proteins within regulatory active complexes, it might be more suitable to search for specific therapeutic targets among oncogenic pathways, which are activated by the SS18-SSX translocation proteins.

One of the deregulated pathways that has recurrently been identified in cDNA expression microarray analyses of synovial sarcoma is the Wnt/β-catenin signaling pathway.9, 10, 11, 12, 13 However, its oncogenic function and potential role as a specific therapeutic target has only partially been analyzed. In a variety of malignancies, activation of Wnt signaling is due to genetic alterations in central pathway components, including CTNNB1 and APC. In synovial sarcoma, such mutations have been reported in a minor frequency, suggesting a regulatory function of the SS18-SSX chimeric oncoproteins.19, 20, 37 We therefore set out to analyze the biological role of canonical Wnt/β-catenin signaling in synovial sarcoma, to decipher its functional dependence on the pathognomonic t(X;18) translocation and to determine whether molecularly targeted interventions with Wnt signals might represent a novel therapeutic option for the treatment of synovial sarcoma.

In accordance with previous studies, we found nuclear accumulation of β-catenin, representing a strong indication of activated Wnt/β-catenin signaling to be a frequent feature in synovial sarcoma.37, 38, 39 In tumor-derived synovial sarcoma cell lines, we could additionally show that β-catenin was predominantly present in the non-phosphorylated (Ser33/-37/Thr41 and Ser45), and transcriptionally active form. Consistently, expression of Wnt/β-catenin signaling targets AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin was found strongly elevated in four out of five synovial sarcoma cell lines. The expression of these targets was increased in primary tumors with nuclear β-catenin reactivity compared with β-catenin-negative tumors. Accordingly, in 1273/99 synovial sarcoma cells, low nuclear β-catenin levels were associated with minor expression levels of Wnt targets, serving as further evidence for the Wnt-dependent regulation of these targets in synovial sarcoma.

To comprehensively understand the oncogenic mechanisms leading to aberrant activation of Wnt/β-catenin signaling, we explored the molecular dependence of Wnt signals on the pathognomonic SS18-SSX fusion proteins. In response to SS18-SSX expression, significantly induced TOPflash reporter luciferase activity was seen in HEK293 cells, demonstrating a pronounced induction of Tcf/β-catenin-mediated transcriptional activity.

As synovial sarcomas are mesenchymal tumors, we chose HT1080 fibrosarcoma cells for further analyses. Upon expression of SS18-SSX, HT1080 cells displayed elevated protein levels of AXIN2, CDC25A, DKK1 and CyclinD1. Inversely, in synovial sarcoma cell lines, downregulation of SS18-SSX2 negatively regulated Wnt signaling activity as indicated by altered target gene expression. Coherently, siRNA-mediated knockdown of β-catenin affected its transcriptional function and the expression of target genes in synovial sarcoma cell lines. Collectively, these observations highlight that the SS18-SSX fusion proteins cooperate to synovial sarcoma tumorigenesis by activation of the Tcf/β-catenin transcription complex. The mechanism of activation and regulation of the Wnt signaling pathway is subject to on-going investigation, with the SS18-SSX oncoproteins being involved in Polycomb and SWI/SNF chromatin remodeling complexes and multiprotein complexes associated with ATF2 and TLE1.40 Recently, it was shown that the oncogenic SS18-SSX fusion protein incorporates into the SWI/SNF (BAF) chromatin-remodeling complex displacing wild-type SS18, thereby leading to an induction of Sox2 expression and subsequent cellular proliferation.41 Interestingly, Sox2 has been identified to act as a transcriptional partner of β-catenin, synergizing in the transcriptional induction of the Wnt target CyclinD1 in breast cancer cells.42 Sox2 is uniformly expressed and activated in synovial sarcoma.43

Previous experiments in NIH3T3 cells demonstrated that exogenous expression of SS18-SSX2 stimulates the nuclear recruitment of β-catenin, forming a transcriptionally active complex (containing SS18-SSX2 and β-catenin) to potentiate Tcf/β-catenin-mediated reporter activity in a p300-dependent manner. However, expression of the Wnt signaling targets CyclinD1 and c-MYC was not found to be enhanced in these studies, which leads the authors to delineate a non-canonical Wnt context in synovial sarcoma.31

Small molecule inhibitors of the Tcf/β-catenin protein complex22, 23, 24 have been found to possess antitumor activities against the central key mediator of canonical Wnt/β-catenin signaling in solid tumors as hepatocellular carcinoma34 and hematopoietic malignancies as chronic lymphocytic leukemia.33 Being the first to apply these substances in a malignant mesenchymal tumor, we here show that these compounds effectively counteract Wnt/β-catenin signal transduction in five human synovial sarcoma cell lines in vitro, including both major SS18-SSX translocation subtypes. Cellular proliferation and viability were significantly reduced in a dose- and time-dependent manner, associated with decreased TOPflash luciferase reporter activities and Wnt/β-catenin target expression. Combined treatments with conventional chemotherapeutic agents (Vincristine, Doxorubicin and Actinomycin D) resulted in additive effects. Treatment with PKF115–584, CGP049090 and PKF118–310 reduced the mitotic fraction of synovial sarcoma cells and induced apoptosis. Consistent with these in vitro results, administration of CGP049090 significantly inhibited tumor growth in SYO-1 synovial sarcoma xenografts in vivo, accompanied with an induction of apoptosis and downregulation of AXIN2 expression. In summary, our data indicate that inhibition of Wnt/β-catenin signaling by treatment with small molecule inhibitors of the Tcf/β-catenin protein complex possesses antitumor potentials in synovial sarcoma xenografts.

In conclusion, the results of the current study imply that the expression of regulatory oncogenes promoting cell cycle progression and cellular proliferation is transcriptionally controlled by the Wnt/β-catenin signaling pathway in a SS18-SSX-dependent manner. Disruption of the Tcf/β-catenin protein complex via small molecule inhibitors may provide an effective therapeutic approach for the treatment of synovial sarcoma. The present preclinical testing of novel molecularly targeted strategies employing Tcf/β-catenin complex inhibitors shows potent effects in vitro and in vivo, qualifying the Wnt/β-catenin signaling pathway as a specific therapeutic target in synovial sarcoma.

Materials and methods

Patients, tumor samples and cell lines

The study included primary tumor tissues from 30 synovial sarcoma patients (11 women, 19 men; median age at diagnosis was 43 years, range 7–91 years). Clinicopathologic characteristics are summarized in Supplementary Table S1. Histologically, 22 tumors belong to the monophasic subtype, and 8 tumors were classified as biphasic. Median tumor size was 6 cm (range 0.4–19.5 cm). In all cases, fluorescence in situ hybridization (FISH) or PCR analysis confirmed the diagnosis of synovial sarcoma, revealing the pathognomonic t(X;18) translocation as described previously.44 Approval of the study by the Ethical Committee of the University of Bonn Medical Center was obtained. The human monophasic synovial sarcoma cell lines HS-SY-II (expressing SS18-SSX1), FUJI, 1273/99 and CME-1 and the biphasic SYO-1 cells (all expressing SS18-SSX2) were cultured as described previously.45, 46, 47 Presence of the pathognomonic SS18-SSX translocation was confirmed by reverse transcriptional PCR (RT–PCR) using specific primers for the translocation subtypes. The human fibrosarcoma cell line HT1080, human embryonic kidney cells HEK293 and the breast carcinoma cell lines MCF-7 and MDA-MB-453 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All monolayer cell cultures were grown under standard incubation condition (37 °C, humidified atmosphere, 5% CO2) and maintained in DMEM (HS-SY-II, SYO-1, HT1080, HEK293, MCF-7 and MDA-MB-453), RPMI 1640 (FUJI and CME-1) or F-12 (1273/99) media, supplemented with 10–15% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA). To study the effects of PKF115–584, CGP049090 and PKF118–310 treatment on Tcf/β-catenin-mediated Wnt signaling, synovial sarcoma cells were grown in six-well plates (medium supplemented with 2% FBS) before treatment with increasing concentrations of the compounds (0–2 μM). Cell lysis, protein extraction and immunoblotting were performed 15 h after treatment as described previously.48

Compounds

Small molecule inhibitors of the Tcf/β-catenin protein complex (PKF115–584, CGP049090 and PKF118–310) were provided by Novartis Pharma AG (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA). The final DMSO concentration did not exceed 0.1% (v/v) for all in vitro and in vivo applications. Vincristine, Doxorubicin and Actinomycin D were purchased from Sigma-Aldrich.

SS18-SSX fusion protein overexpression in HT1080 cells

The generation of expression plasmids for SS18-SSX1, SS18-SSX2, SS18, SSX1 and SSX2 was described previously.49 HT1080 cells were grown in six-well plates and transfected with 2.5 μg of plasmid DNA using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Expression was confirmed 24 h after transfection by immunoblotting and RT–PCR. As a control, HT1080 cells were transfected with the pT-REx/GW-30/lacZ plasmid (Life Technologies) expressing β-galactosidase.

Luciferase reporter assay

To assess Tcf/β-catenin-mediated transcriptional activity, TOP-/FOPflash luciferase reporter gene assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.50 HEK293 cells were transiently transfected with Firefly TOPflash or the control FOPflash plasmid DNA (Merck Millipore, Darmstadt, Germany) containing wild-type or mutant Tcf DNA-binding sites, respectively. For extrinsic activation of Wnt/β-catenin signaling, cells were co-transfected with the mutant ΔN131 β-catenin plasmid. The amount of plasmid DNA in each transfection was kept constant by addition of the empty pcDNA3.1 plasmid. After incubation for 24 h, cells were lysed and luciferase activity was measured in triplicates as described previously.51 Firefly luciferase activity was normalized to the co-transfected Renilla pRL-TK control plasmid (Promega) to account for potential differences in transfection efficiency. SYO-1, CME-1 and HS-SY-II cells were transfected with TOP-/FOPflash plasmid DNA to determine the ability of PKF115–584, CGP049090 and PKF118–310 to disrupt Tcf/β-catenin complex formation and associated transcriptional activity. After 6 h, medium containing transfection reagent was replaced with new culture medium supplemented with indicated concentrations (0.5–2 μM; 2% FBS) of each compound for additional 18 h. Luciferase reporter activity was assayed as described above.

Cell viability assay (MTT)

Cell viability was measured using the MTT cell proliferation kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. SYO-1 (5 × 103), FUJI (7.5 × 103), 1273/99 (7.5 × 103), CME-1 (5 × 103), HS-SY-II (7.5 × 103), MCF-7 (7.5 × 103), and MDA-MB-453 (7.5 × 103) cells were seeded in 96-well plates (100 μl of medium supplemented with 2% FBS) and exposed to increasing concentrations of PKF115–584, CGP049090 and PKF118–310 (0.15–1.25 μM) for 72 h. An appropriate DMSO control was included. For combined treatment with chemotherapeutic agents, SYO-1 cells were incubated with increasing concentrations of Vincristine (0.1–100 ng/ml), Doxorubicin (0.1–1000 ng/ml) and Actinomycin D (0.1–10 ng/ml), alone or in combination with PKF115-584, CGP049090 or PKF118–310 (0.1–0.2 μM; concentration resulting in 20–30% growth inhibition). Synergy was evaluated using the fractional product method.52 Differences of >10% between the observed and predicted effect were considered to signify synergistic activity between the chemotherapeutic agent and the small molecule inhibitors of the Tcf/β-catenin protein complex, a difference <10% was defined as additive.

RNA interference

Small interference RNA (siRNA) specific for human CTNNB1 (Stealth Select RNAi siRNA set of 3: HSS102461, HSS102462, HSS102460) and non-targeting negative control siRNA (BLOCK-iT Alexa Fluor Red Fluorescent Control) were purchased from Life Technologies. To target the SSX2 portion of SS18-SSX2, a set of published and validated duplex oligos was employed (sense: 5′-IndexTermAAC CAA CUA CCU CUG AGA AGA-3′; antisense: 5′-IndexTermUCU UCU CAG AGG UAG UUG GUU-3′ and sense: 5′-IndexTermCAA GAA GCC AGC AGA GGA ATT-3′; antisense: 5′-IndexTermUUC CUC UGC UGG CUU CUU GTT-3′).31, 32 SYO-1, CME-1 and HS-SY-II cells were cultured in 25 cm2 cell culture flasks (medium supplemented with 10% FBS) and transfected with indicated siRNA (75 pmol; cell density of 50%) using Lipofectamine RNAiMAX (Life Technologies). After incubation for 24 h, siRNA-transfected cells were trypsinized and re-seeded for MTT assays as described above. Knockdown efficiency was documented by immunoblotting.

Immunoblot analysis

Following primary antibodies were used according to the manufacturer’s instructions: cleaved caspase-3 (Asp175), CyclinD1, Survivin, β-catenin, and phospho(Ser33/37/Thr41)-β-catenin, phospho(Thr41/Ser45)-β-catenin, phospho(Ser552)-β-catenin, phospho(Ser675)-β-catenin (all obtained from Cell Signaling Technology, Danvers, MA, USA); AXIN2, CDC25A, c-MYC, DKK1 and GAPDH (all obtained from Abcam, Cambridge, UK); SS18 and LaminB1 (Santa Cruz Biotechnology, Dallas, TX, USA); β-actin (Sigma-Aldrich). The SS18-SSX fusion protein was detected with an antibody targeting the N terminus of SS18. Secondary antibody labeling (Bio-Rad Laboratories, Hercules, CA, USA) as well as immunoblot development was performed using the enhanced chemiluminescence detection kit (Amersham Biosciences, Little Chalfont, UK) as described before. Subcellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions.

Flow cytometry

The effect of PKF115-584, CGP049090 and PKF118-310 on synovial sarcoma apoptosis and cell proliferation was assessed by flow cytometric analysis. Briefly, SYO-1 and CME-1 cells were grown in 75 cm2 cell culture flasks (medium supplemented with 2% FBS) and treated with 0.1 μM of the compounds for 24 h. Cells were fixed in 2% paraformaldehyde for 10 min on ice, washed in PBS, collected by centrifugation and incubated in ice-cold PBS with 0.25% Triton X-100 for 5 min on ice. After an additional washing step, cells were resuspended in 100 μl PBS/0.5% BSA containing following antibodies: cleaved Poly-(ADP-ribose)-polymerase (PARP) (Asp214) (BD Biosciences, San Jose, CA, USA; phycoerythrin-labeled) and phospho-histone H3 (Ser10) (Cell Signaling Technology; Alexa Fluor 647-labeled) followed by incubation for 30 min at room temperature. Fluorescence intensity was measured using a LSRII analytical flow cytometer (BD Biosciences), and cytometric data were analyzed using the FlowJo (Tree Star, Ashland, OR, USA) software. Each experiment was carried out at least in duplicates.

In vivo efficacy of CGP049090 in synovial sarcoma xenograft studies

SYO-1 cells (5 × 106) were injected subcutaneously into the lower flank region of male BALB/c-nude mice (Charles River Laboratories, Wilmington, MA, USA; 5 weeks old). Tumor growth was monitored daily, and tumor volume was calculated according to the formula: TV=length (mm) × width (mm) × height (mm) × π/6. Treatment was initiated once the tumor volume reached 100 mm3. To evaluate the in vivo antitumor efficacy of CGP049090, tumor-bearing mice were randomly assigned into two treatment groups and received 25 mg/kg CGP049090 (n=6) or DMSO (vehicle control; n=6) every other day via intraperitoneal (i.p.) administration. After 15 days of treatment, mice were killed, with tumor volume, body weights and general physical status recorded. Tumor tissues were explanted, followed by immunohistochemical and histopathological examination as described above. All studies were performed in accordance with the standards of the National and European Union guidelines, and permission was obtained from the local authorities.

Statistical analysis

The immunohistochemical results were statistically analyzed using Fisher’s exact test. Results of MTT assays and flow cytometric analyses are represented as mean±s.d. or s.e. from n independent experiments. Two-group comparisons were analyzed by unpaired Student’s t-test. Statistical differences were considered significant at P<0.05. Statistical probability is indicated as follows: *P<0.05; **P<0.01 and ***P<0.001. The compound concentration, which is required for 50% growth inhibition (IC50 value), was calculated by non-linear regression analysis using the GraphPad Prism (GraphPad Software, La Jolla, CA, USA).

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Acknowledgements

PKF115-584, CGP049090 and PKF118–310 were generously provided by A Wood (Novartis Pharma AG, Basel, Switzerland). This study was supported by Wilhelm Sander-Stiftung, Dr Eberhard und Hilde Rüdiger Stiftung, Deutsche Krebshilfe (KoSar sarcoma competence network), BONFOR (Medical Faculty, University Hospital Bonn, Bonn) and Fortune program (Medical Faculty, University of Cologne, Cologne).

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Correspondence to W Hartmann.

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E Wardelmann has received honoraria from speakers’ bureau of Novartis Oncology, MSD and Eisai, and is a scientific consultant/advisory board member of Novartis Oncology and MSD. The remaining authors declare no conflict of interest.

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Supplementary Information accompanies this paper on the Oncogene website

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Trautmann, M., Sievers, E., Aretz, S. et al. SS18-SSX fusion protein-induced Wnt/β-catenin signaling is a therapeutic target in synovial sarcoma. Oncogene 33, 5006–5016 (2014). https://doi.org/10.1038/onc.2013.443

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Keywords

  • Synovial sarcoma
  • SYT-SSX
  • β-catenin
  • Wnt signaling pathway
  • PKF115-584
  • CGP049090
  • PKF118-310

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