Targeting regulation of cyclin dependent kinase 9 as a novel therapeutic strategy in synovial sarcoma.

Synovial sarcomas hold a low genomic complexity, making it distinct from other types of soft-tissue sarcomas. Many studies focused on targeting the SS18-SSX fusion protein, which presents in over 90% of human synovial sarcomas. This protein acts as an oncogenic promoter in the tumorigenesis of synovial sarcomas, making it an ideal therapeutic target. However, to date there have been no effective strategies targeting SS18-SSX for the treatment of synovial sarcomas. Therefore, it is an urgent need to identify alternative therapeutic targets. More recently, CDK9, a protein involved in RNA transcription regulation, has been investigated for its role in the pathogenesis of cancer. However, the expression and function of CDK9 in synovial sarcomas remains to be elucidated. In the present study, we found that CDK9 was to be largely localized to the cell nucleus, and highly expressed in all tested human synovial sarcoma cell lines and over 90% of human sarcoma tissue microarray samples. High-CDK9 expression was associated with a poorer patient prognosis of human sarcomas. Inhibition of CDK9, with either siRNA or a CDK9 inhibitor, prevented synovial sarcoma cell growth and proliferation in a dose-dependent manner. This was also accompanied with a reduction in the phosphorylation of RNA polymerase II and an increase in the expression of anti-apoptotic proteins. Moreover, CDK9 inhibition decreased sarcoma cell spheroid formation and cell motility. Collectively, these findings highlight the importance of CDK9 in human synovial sarcoma cell growth and proliferation. Therefore, CDK9 may represent a promising target for the treatment of synovial sarcomas. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:510-521, 2019.

Preclinical Evaluation of Nintedanib, a Triple Angiokinase Inhibitor, in Soft-tissue Sarcoma: Potential Therapeutic Implication for Synovial Sarcoma.

Sarcomas are rare cancers that make up about 1% of all cancers in adults; however, they occur more commonly among children and young adolescents. Sarcomas are genetically complex and are often difficult to treat given the lack of clinical efficacy of any of the currently available therapies. Receptor tyrosine kinases (RTK) such as c-Kit, c-Met, PDGFR, IGF-1R, as well as FGFR have all been reported to be involved in driving tumor development and progression in adult and pediatric soft-tissue sarcoma. These driver kinases often act as critical determinants of tumor cell proliferation and targeting these signal transduction pathways remains an attractive therapeutic approach. Nintedanib, a potent triple angiokinase inhibitor, targets PDGFR, VEGFR, and FGFR pathways critical for tumor angiogenesis and vasculature. In this study, we evaluated the preclinical efficacy of nintedanib in soft-tissue sarcoma cell lines. Nintedanib treatment resulted in significant antiproliferative effect in vitro in cell lines with high expression of RTK drug targets. Furthermore, treatment with nintedanib showed significant downregulation of downstream phosphorylated AKT and ERK1/2. Finally, treatment with nintedanib resulted in significant tumor growth suppression in mouse xenograft model of synovial sarcoma. Notably, both the in vitro and in vivo efficacy of nintedanib was superior to that of imatinib, another multikinase inhibitor, previously tested with minimal success in clinical trials in sarcoma. Overall, the data from this study provide a strong rationale to warrant further clinical exploration of this drug in patients with synovial sarcoma. Mol Cancer Ther; 17(11); 2329-40. ©2018 AACR.

Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma.

Synovial sarcoma is a highly aggressive but rare form of soft tissue malignancy that primarily affects the extremities of the arms or legs, for which current chemotherapeutic agents have not been proven to be very effective. The cyclin-dependent kinase 4/6-retinoblastoma protein (CDK4/6-Rb) pathway of cell cycle control is known to be aberrant in a large proportion of cancers. Recently, CDK4 inhibitors have successfully been used pre-clinically for the treatment of many human cancers, and in 2015, following the success of clinical trials, the FDA approved the first selective CDK4/6 inhibitor, palbociclib, for the treatment of endocrine therapy resistant breast cancers. However, the expression and therapeutic potential of targeting CDK4 in synovial sarcoma remains unclear. In the present study, we report that CDK4 is highly expressed in human synovial sarcoma, and high CDK4 expressions are associated with poor prognosis in sarcomas patients and the clinical stage and the TNM grade in synovial sarcoma patients. Knockdown of CDK4 with specific small interference RNAs inhibits cell proliferation and enhances apoptotic effects in synovial sarcoma cells. CDK4 inhibitor palbociclib suppresses synovial sarcoma cell proliferation and growth in a dose and time-dependent manner. Palbociclib also inhibits the CDK4/6-Rb signaling pathway and promotes cell apoptosis without changing CDK4/6 protein levels, suggesting that palbociclib only represses the hyper-activation, not the expression of CDK4/6. Flow cytometry analysis reveals that palbociclib induces G1 cell-cycle arrest and apoptotic effects by targeting the CDK4/6-Rb pathway in synovial sarcoma cells. Furthermore, wound healing assays demonstrate that inhibition of the CDK4/6-Rb pathway by palbociclib significantly decreases synovial sarcoma cell migration in vitro. Our study highlights the importance of the CDK4/6-Rb pathway in human synovial sarcoma pathogenesis, and the role of the current selective CDK4/6 inhibitor, palbociclib, as a potential promising targeted therapeutic agent in the treatment of human synovial sarcoma.

Phosphoproteomic Profiling Reveals ALK and MET as Novel Actionable Targets across Synovial Sarcoma Subtypes.

Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry-based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2-17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res; 77(16); 4279-92. ©2017 AACR.

Essential role of mitogen-activated protein kinases in IL-17A-induced MMP-3 expression in human synovial sarcoma cells.

BACKGROUND: The tumor cells were needed to rearrange the extracellular matrix (ECM) and reorganize their cytoskeleton to facilitate the cell motility during the tumor invasion. The proinflammatory cytokine interleukin-17A (IL-17A) is reported to up-regulate tumor invasiveness via ECM degradation by matrix metalloproteinases (MMPs). However the precise effects of IL-17A-dependent invasion remain to be characterized. The aim of this study was to elucidate the mechanisms underlying IL-17A-induced MMP-3 expression in the human synovial sarcoma cells HS-SY-II. METHODS: HS-SY-II cells were incubated with IL-17A. In some experiments, the cells were pre-incubated with an anti-IL-17 receptor polyclonal antibody (IL-17R Ab) or inhibitors for signaling cascade prior to addition of IL-17A. The expression of MMP-3 was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blotting. IL-17R expression in HS-SY-II cells was assessed by immunofluorescence microscopy, while the phosphorylation of signaling molecules was measured by western blotting. RESULTS: IL-17A increased MMP-3 mRNA and protein expression. HS-SY-II cells express the IL-17R on their surface and blockage of IL-17A-IL-17R binding by IL-17R Ab suppressed IL-17A-mediated induction of MMP-3. IL-17A induced the phosphorylation of three components of the mitogen-activated protein kinase (MAPK) pathway including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Pre-treatment of the cells with inhibitors of ERK1/2, p38 MAPK, and JNK attenuated the IL-17A-induced phosphorylation of activator protein-1 (AP-1) subunits and the expression of MMP-3 mRNA. CONCLUSION: Our results indicate an essential role for MAPKs in the induction of MMP-3 in synovial sarcoma cells, through AP-1 activation.

Vorinostat synergizes with ridaforolimus and abrogates the ridaforolimus-induced activation of AKT in synovial sarcoma cells.

BACKGROUND: Curative treatments for patients with metastatic synovial sarcoma (SS) do not exist, and such patients have a poor prognosis. We explored combinations of molecularly-targeted and cytotoxic agents to identify synergistic treatment combinations in SS cells. METHODS: Two SS cell lines (HS-SY-II and SYO-I) were treated with single agents or combinations of molecularly targeted therapies (HDAC inhibitor, vorinostat; mTOR inhibitor, ridaforolimus) and cytotoxic agents. After 72 hours, cell viability was measured using the MTS cell proliferation assay. Combination Indices (CI) were calculated to determine whether each combination was synergistic, additive, or antagonistic. Western Blot analysis assessed alterations in total and phospho-AKT protein levels in response to drug treatment. RESULTS: We determined the single-agent IC50 for ridaforolimus, vorinostat, doxorubicin, and melphalan in HS-SY-II and SYO-I. Synergism was apparent in cells co-treated with ridaforolimus and vorinostat: CI was 0.28 and 0.63 in HS-SY-II and SYO-I, respectively. Ridaforolimus/doxorubicin and ridaforolimus/melphalan exhibited synergism in both cell lines. An additive effect was observed with combination of vorinostat/doxorubicin in both cell lines. Vorinostat/melphalan was synergistic in HS-SY-II and additive in SYO-I. Western blot analysis demonstrated that ridaforolimus increased pAKT-ser473 levels; this effect was abrogated by vorinostat co-treatment. CONCLUSIONS: The combination of ridaforolimus and vorinostat demonstrates in vitro synergism in SS. Addition of vorinostat abrogated ridaforolimus-induced AKT activation. Since AKT activation is a possible mechanism of resistance to mTOR inhibitors, adding vorinostat (or another HDAC inhibitor) may be a route to circumvent AKT-mediated resistance to mTOR inhibitors.

Antiangiogenic approaches for the treatment of advanced synovial sarcomas.

PURPOSE OF REVIEW: Synovial sarcomas are regarded as chemosensitive tumors compared to other types of soft tissue sarcomas, however, prognosis for advanced refractory disease remains poor. In light of the vascular nature of sarcomas, current molecularly targeted therapies aim at an antiangiogenic approach to management of this disease. RECENT FINDINGS: Recent studies with oral vascular endothelial growth factor receptor (VEGFR) inhibitors such as sunitinib, sorafenib and cediranib have shown disease stabilization in patients with advanced synovial sarcoma. Forty-nine percent of patients with synovial sarcoma on the phase II trial of pazopanib had no evidence of disease progression at 12 weeks. SUMMARY: The overall impact of chemotherapy on survival has been minimal in advanced soft tissue sarcomas. Anti-VEGF therapies have resulted in improved outcomes for patients with various solid tumors, and have shown preliminary evidence of activity in synovial sarcomas. Combinations of anti-VEGF therapies with agents targeting other pathways dysregulated in sarcomas have the potential to improve the outcome of this difficult-to-treat disease.

A novel multi-kinase inhibitor pazopanib suppresses growth of synovial sarcoma cells through inhibition of the PI3K-AKT pathway.

Synovial sarcoma is an aggressive soft tissue sarcoma with only a modest response to conventional cytotoxic agents. In the present study, we evaluated the potential antitumor effects of a novel anti-angiogenesis agent, pazopanib, against synovial sarcoma cells. We found that pazopanib directly inhibited the growth of synovial sarcoma cells by inducing G1 arrest. Multiplex analyses revealed that the PI3K-AKT pathway was highly suppressed in pazopanib-sensitive synovial sarcoma cells. Furthermore, administration of pazopanib highly suppressed the tumor growth in a xenograft model. Taken together, these results suggest pazopanib as a possible agent against synovial sarcoma and may warrant further clinical studies.

Simultaneous inhibition of Src and Aurora kinases by SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion and angiogenesis in vivo.

Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo.

Adaptor protein Crk induces Src-dependent activation of p38 MAPK in regulation of synovial sarcoma cell proliferation.

The adaptor protein Crk mediates intracellular signaling related to cell motility and proliferation and is implicated in human tumorigenesis. The role of Crk in the growth of human sarcoma has remained unclear, however. The present study shows that Crk-induced activation of Src and subsequent signaling by p38 mitogen-activated protein kinase (MAPK) contribute to the enhanced proliferation of human synovial sarcoma cells. Depletion of Crk by RNA interference markedly inhibited proliferation of the synovial sarcoma cell lines HS-SYII, SYO-1, and Fuji as well as prevented anchorage-independent growth. Conversely, reconstitution with CrkII by authentic small interfering RNA-resistant Crk gene restored proliferation in Crk-silenced SYO-1 cells. Crk-depleted synovial sarcoma cells manifested enhanced transcriptional activity and expression of the p16(INK4A) gene, resulting in their accumulation in G(1) phase of the cell cycle. In response to hepatocyte growth factor stimulation, Crk prominently induced the tyrosine phosphorylation of Grb2-associated binder 1 through activation of Src and focal adhesion kinase, and the Src family kinase inhibitor PP2 almost completely inhibited the proliferation of SYO-1 cells. Crk also induced the phosphorylation of p38 MAPK, and SB203580, a p38 MAPK-specific inhibitor, increased expression of p16(INK4A) gene in SYO-1 cells. Furthermore, SB203580 or depletion of p38 MAPK by small interfering RNA suppressed both the phosphorylation of Akt triggered by hepatocyte growth factor and the proliferation of SYO-1 cells. These results suggest that Crk promotes proliferation of human synovial sarcoma cells through activation of Src and its downstream signaling by a novel p38 MAPK-Akt pathway, with these signaling molecules providing potent new targets for molecular therapeutics.

Sorafenib induces growth inhibition and apoptosis in human synovial sarcoma cells via inhibiting the RAF/MEK/ERK signaling pathway.

Synovial sarcoma is a soft tissue sarcoma with poor prognosis and lack of response to conventional cytotoxic chemotherapy. The regulatory mechanisms for the rapid proliferation of synovial sarcoma cells and the particular aggressiveness of this sarcoma remain poorly understood. Mitogen-activated protein kinase (MAPK) cascades have been shown to play important roles in synovial sarcoma survival. Sorafenib (Nexavar, BAY 43-9006), a potent recombinant activated factor (RAF) inhibitor, inhibits the MAPK signaling pathway both in vitro and in vivo. In this study, we examined the inhibitory proliferation effects of sorafenib in synovial sarcoma growth and evaluated whether sorafenib modulates MAPK and tumor apoptosis cascades in human synovial sarcoma cell lines SW982 and HS-SY-II. Our results indicated that sorafenib effectively inhibited cellular proliferation and induces apoptosis of these two cells. Sorafenib inhibited the phosphorylation of MEK and ERK, downregulated cyclin D1 and Rb levels, caused G(1) arrest and S phase decrease, and induced apoptosis as confirmed by flow cytometry and the TUNEL assay. Furthermore, Bcl-xl and Mcl-1 levels significantly decreased, whereas expression levels of the proteins bcl-2 and bax were unchanged in response to sorafenib treatment in SW982 and HS-SY-II cells. In conclusion, our findings demonstrate that sorafenib is effective for growth inhibition of synovial sarcoma cell lines in vitro and suggest that sorafenib may be a new therapeutic option for patients with synovial sarcoma.

Matrix metalloproteinase expression in high grade soft tissue sarcomas.

Soft tissue sarcomas comprise a heterogeneous group of mesenchymal tumors with varying biological behavior ranging from indolent tumors with no or minimal metastatic risk to aggressive and frequently metastasizing tumors. Among the more common aggressive adult soft tissue sarcomas are malignant fibrous histiocytoma, synovial sarcoma and liposarcoma. Matrix metalloproteinases are enzymes which perform a homeostatic role in mesenchymal tissue and function in both tumorigenesis and metastasis. The objectives of this study are to determine the presence and relative quantity of matrix metalloproteinases (MMPs) -1, -2, -8, -9, and -13; extracellular matrix metalloproteinase inducer (EMMPRIN); and tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 in high grade soft tissue sarcoma tumor specimens using real-time PCR. The second objective is to determine if a relationship exists between quantity of EMMPRIN, MMPs, and TIMPs expressed in tumor tissue and disease-free survival. One hundred and forty patients diagnosed with high grade soft tissue sarcomas between 1995-2003 were identified. Tissue blocks and histologic slides were acquired for 41 specimens. Tumor specimens included 29 malignant fibrous histiocytomas, 3 liposarcomas and 11 synovial sarcomas. RNA was extracted and RT-PCR was performed in triplicate. No significant differences were found between the three types of high grade soft tissue sarcomas studied and the expression of MMPs. Interestingly, no relationship was found between high or low levels of MMPs when compared with disease-free survival. Our data support other research which finds variable correlation between MMP expression in soft tissue sarcomas and disease-free survival. We assert that the difference in correlation between MMP expression in carcinomas and sarcomas and disease-free survival is based on the vast phenotypic and genotypic difference between the cells of origin.

Doxorubicin induces cell senescence preferentially over apoptosis in the FU-SY-1 synovial sarcoma cell line.

Surgical resection coupled with adjuvant radiotherapy and/or doxorubicin based chemotherapy are the mainstays of synovial sarcoma (SS) treatment. Although effective as a SS adjuvant, the proposed mechanism of action of doxorubicin remains controversial. Current opinion supports DNA damage-induced apoptosis. This in vitro study used cDNA gene expression profiling to investigate whether apoptosis, alone or in combination with cell senescence, is induced by doxorubicin in SS cells. Cell cultures of the FU-SY-1 SS, the pleomorphic SW982 sarcoma, and a primary dermal fibroblast (NHDF), were exposed to 500 nM doxorubicin, and then processed for cDNA microarray analysis. The one class response option of SAM (Significance Analysis of Microarrays) was used to test for significant overexpression of 15 apoptosis-related genes and nine senescence-related genes. Drug-induced cell senescence was quantified by measuring beta-galactosidase activity. None of 15 apoptosis-related genes and only two of nine senescence-related genes were identified by SAM as significantly overexpressed in doxorubicin-treated cultures. Drug-induced senescence as reflected by beta-galactosidase activity was significantly increased (p < 0.05) only in FU-SY-1 SS cultures. Apoptosis does not appear to be a major determinant of doxorubicin-induced mortality in FU-SY-1 SS or NHDF cultures, but may impact SW982 cells via the overexpression of BAX relative to Bcl-2. Doxorubicin-induced cell senescence was prominent in FU-SY-1 SS cultures, but negligible in SW982 and NHDF cultures. Likely, both apoptosis and cell senescence contribute to doxorubicin-induced cell death in this synovial sarcoma cell line.

[Correlation between matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 expression, metastatic potential and tumor angiogenesis in synovial sarcoma and its prognostic significance].

OBJECTIVE: To study the expression of matrix metalloproteinases (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP)-2 in tumor cells of synovial sarcoma and its clinical significance. METHODS: Expression of MMP-2 and TIMP-2 in tumor cells of 72 cases of synovial sarcoma was studied by immunohistochemistry. The profile was correlated with clinicopathologic parameters, microvessel density (MVD) (analyzed by CD31 immunostaining) and survival rate. RESULTS: (1) There was a statistically significant negative correlation between expression of MMP-2 and TIMP-2 (r = -0.290 and P = 0.013). (2) The proportion of high MMP-2 expression to low TIMP-2 expression in patients with tumor metastasis was significantly higher than that in patients without metastasis (P = 0.010 and 0.002 respectively). (3) MVD of patients with high MMP-2 expression was higher than that in the low MMP-2 expression group (P = 0.005). MVD of patients with high TIMP-2 expression was lower than that in the low TIMP-2 expression group (P = 0.048). (4) Low TIMP-2 expression significantly correlated with poor prognosis of patients with synovial sarcoma, by univariate and multivariate survival analysis (P = 0.002 and 0.016 respectively). CONCLUSIONS: Expression of MMP-2 and TIMP-2 correlates with metastatic potential and tumor angiogenesis in synovial sarcoma. Low TIMP-2 expression often indicates poor prognosis and unfavorable clinical outcomes.

Novel p38 MAP kinase inhibitor R-130823 suppresses IL-6, IL-8 and MMP-13 production in spheroid culture of human synovial sarcoma cell line SW 982.

Synovial hyperplasia is a hallmark of rheumatoid arthritis (RA) and is regarded as a major destructive element of articular bone and cartilage. This pathological process is accompanied by the production of proinflammatory cytokines, prostaglandin E(2) (PGE(2)), and matrix metalloproteinases (MMPs) in synoviocytes. We studied the spontaneous production of these substances in RA synoviocytes in spheroid culture. Synovial sarcoma cell line SW 982 formed a single spheroid in non-adherent culture plates. It produced interleukin (IL)-1beta, IL-6, IL-8, PGE(2), MMP-2 and MMP-13. Neither the addition of integrin antagonizing oligopeptide (GRGDSP) nor that of vitronectin receptor inhibitor SB-265123 to the culture inhibited any production. Phosphorylation of p38 mitogen-activated protein (MAP) kinase was observed during the culture. A novel p38 MAP kinase inhibitor, R-130823, inhibited the release of IL-6, IL-8 and MMP-13 in a concentration-dependent manner, but not that of IL-1beta or MMP-2. Real-time RT-PCR analysis demonstrated that IL-6, IL-8 and MMP-13 were inhibited at the transcriptional level. R-130823 did not affect the production of PGE(2) in spheroid culture, while the addition of R-130823 suppressed IL-1beta-induced PGE(2) synthesis in monolayer culture of SW 982 cells. The results suggest that spheroid culture induced proinflammatory factors and MMPs in signaling pathways both dependent and independent of p38 MAP kinase.

Matrix metalloproteinase-2 expression correlates with morphological and immunohistochemical epithelial characteristics in synovial sarcoma.

AIMS: Synovial sarcoma is a unique mesenchymal tumour characterized by the presence of epithelial differentiation, although the mechanism involved in the epithelial morphology is still unclear. The aim of this study was to evaluate the function of matrix metalloproteinase-2 (MMP-2) in synovial sarcoma, in order to assess whether MMP-2 expression plays an important role in epithelial differentiation, or whether it contributes to a poor clinical outcome. METHODS AND RESULTS: Immunohistochemical stainings for MMP-2, cytokeratins (CKs) 7, 8, 18 and 19, and E-cadherin were performed for 58 (44 monophasic and 14 biphasic) cases of synovial sarcoma, and we compared the expression of these proteins with the histological and clinical findings. MMP-2 and E-cadherin expression was observed in 43 cases (74.1%) and in 18 cases (31.0%), respectively. Expression of these proteins was preferentially observed in the glandular components of biphasic tumours or the epithelioid areas of monophasic tumours. Statistically significant correlations were recognized between MMP-2 expression and E-cadherin expression of biphasic subtype. Moreover, there were statistically significant correlations between monophasic tumours with epithelioid areas and MMP-2 expression or E-cadherin expression. MMP-2 expression was correlated with epithelial differentiation as assessed by CK immunoreactivity. The expression of MMP-2 did not affect the overall survival rate in synovial sarcoma. CONCLUSIONS: MMP-2 expression seemed to have an important role to play in the epithelial differentiation of tumour cells in synovial sarcoma, through remodelling of the extracellular matrix and by changing the cytoskeletal interaction between the extracellular matrix and tumour cells.

[Detection and activity analysis of gelatinases in bone and soft tissue tumors].

This study was aimed to investigate the expression of gelatinases (MMP-2 and MMP-9) in mesenchymal tumors and the roles they act during invasion and metastasis. 45 cases of bone and soft tissue tumors with various pathologic categories were subjected to immunohistochemistry and gelatin zymography analysis. The results showed that the positive immuno-staining ratio and the activity of both MMP-2 and MMP-9 were higher in malignant tumors than in benign tumors and normal tissue. In malignant tumors, the positive stained cells usually gathered in the marginal area of tumor foci or the invading area, or formed distinctive positive-stained cell clusters, indicating their relationship with the aggressive biologic behavior of this kind of malignant tumor.

Urokinase-plasminogen-activator levels and prognosis in 69 soft-tissue sarcomas.

The local and systemic invasiveness of soft-tissue sarcomas may depend upon an interaction between the primary tumour and the extracellular matrix in which the proteolytic enzyme, urokinase plasminogen activator (uPA), may have an important role. We analyzed the expression of uPA in soft-tissue sarcoma using a luminescent immunoassay technique, and examined the relationships between different uPA levels and tumour characteristics and behaviour. We evaluated 69 adult patients with surgically treated soft-tissue sarcomas (MFH 43, leiomyosarcoma 8, liposarcoma 5, synovial sarcoma 4, others 9) of the extremities and trunk wall. Sixteen developed local recurrences, 26 developed metastases, and 5 had both. The median follow-up for survivors was 55 (30-80) months. The median uPA level was 1.4 (0.04-10.6) ng/mg protein. Increasing uPA levels correlated with increasing grade, malignant fibrous histiocytomas, leiomyosarcomas, DNA non-diploidy, tumour necrosis, local recurrence, and metastasis. Storiform-pleomorphic MFH had higher uPA levels than the myxoid variant. A cut-off value of 0.25 ng/mg protein was identified, above which local recurrence and metastasis occurred more frequently. High uPA levels appear to reflect the malignant phenotype in soft-tissue sarcoma, thus supporting the role of uPA as a prognostic indicator.

Differential response of retinoic acid pretreated human synovial sarcoma cell line to anticancer drugs.

The response of tumorigenic human synovial sarcoma (HSS) cell line to 10(-5)M optimum concentration of retinoic acid (RA) included changes in morphology, growth rate, suppression of anchorage-independent growth, induction of high alkaline phosphatase activity and excessive secretion of a 68 kDA glycoprotein of unknown function. HSS cells pretreated with 10(-5)M RA exhibited differential response to 3 potent anticancer drugs, namely cisplatin, vincristine and adriamycin. Sensitivity of the cells to cisplatin was found to be considerably enhanced after exposure to retinoic acid.

Size dependent enzyme activities of multinucleated (osteoclastic) giant cells in bone tumors.

In osteoclastic giant cells of six different tumors of bones and joints (fibrous dysplasia, proliferating giant cell tumor, malignant giant cell tumor, osteosarcoma after chemotherapy, malignant synovioma and Ewing's sarcoma) activities of tartrate-resistant acid phosphatase, NADH-tetrazolium-oxidoreductase and, in three of them, of non-specific esterase are determined by enzyme histochemical methods. Quantitative microphotometry makes it possible to determine relative enzyme activities in the cut sections of giant cells of different sizes. Giant cells of the various tumors reveal similar trends: With an increase in cell size, mean extinctions of NADH-tetrazolium-oxidoreductase and non-specific esterase decrease. Mean extinctions of tartrate-resistant acid phosphatase increase in cells of medium size, whereas the large cells reveal in part low activities. An additional ultrastructural examination of the giant cells in the proliferating giant cell tumor as well as in the osteosarcoma shows morphological signs of degeneration in the large cells. Electron probe microanalysis of the proliferating giant cell tumor exhibits evidence of phagocytosis of Ca and/or Fe containing particles. The similar size dependent reaction pattern of enzymes in osteoclastic giant cells of different tumors favors the concept of a common histogenesis, i.e. a host reaction.