PDF file - 57K, Densitometric analysis of the top five phosphorylated kinases as found in phospho kinase arrays in 1273/99 and HS-SY-II synovial sarcoma cells. Flow cytometric analysis of three synovial sarcoma cell lines treated with different doses of dasatinib for 48 hours.

PDF file - 57K, Significant decrease of p-(Tyr416)-SRC levels upon siRNA-mediated knockdown of IGF-1R in CME-1 synovial sarcoma cells.

Liposarcomas (LS) are the most common malignant mesenchymal tumors, with an overall long‐term mortality rate of 60%. LS comprise three major subtypes, i.e., well‐differentiated/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/round cell liposarcoma (MLS) and pleomorphic liposarcoma (PLS). Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional significance of SRC in primary human LS and in LS‐derived cell lines. Immunohistochemical and Western blot analyses reveal relevant levels of activated p‐(Tyr416)‐SRC in LS of the different subtypes with particular activation in MLS and PLS. Dysregulation of the SRC modifiers CSK and PTP1B was excluded as major reason for the activation of the kinase. Consistent siRNA‐mediated knockdown of SRC or inhibition by the SRC inhibitor Dasatinib led to decreased proliferation of LS cell lines of the different subtypes, with MLS cells reacting particularly sensitive in MTT assays. Flow cytometric analyses revealed that this effect was due to a significant decrease in mitotic activity and an induction of apoptosis. SRC inhibition by Dasatinib resulted in dephosphorylation of SRC itself, its interacting partners FAK and IGF‐IR as well as its downstream target AKT. Consistent with a particular role of SRC in cell motility, Dasatinib reduced the migratory and invasive potential of MLS cells in Boyden chamber and Matrigel chamber assays. In summary, we provide evidence that SRC activation plays an important role in LS biology and therefore represents a potential therapeutic target, particularly in MLS and PLS.

Pilomatricoma is a tumour derived from hair matrix cells, which shows progressive keratin expression. Tumorigenesis is frequently associated with activating mutations in β-catenin gene inducing nuclear expression of β-catenin protein. The present study analysed the role of transforming growth factor-β1 (TGF-β1) and four-and-a-half LIM domain protein 2 (FHL2) in pilomatricoma in synopsis with their expression patterns in human anagen hair. Human anagen hair showed TGF-β1 and nuclear FHL2 expression in the outer root sheath layer separated from nuclear β-catenin staining, which was observed in cells of matrix and inner root sheath layers. Correspondingly, 41 out of 50 pilomatricomas showed co-labelling of TGF-β1 and nuclear FHL2 in tumour cells, which mostly lacked nuclear β-catenin expression. Tumoural proliferation (ki67) was associated with nuclear β-catenin staining but not with expression of nuclear FHL2. In early pilomatricomas, TGF-β1 expression was observed in few peripheral tumour cells showing absent or faint nuclear FHL2 co-staining. TGF-β1 expression extended in growing tumours going along with strong nuclear FHL2 co-labelling as well as progressive keratin 14 and keratin 1 expression. In vitro, cultured human keratinocytes showed weak to marked autocrine TGF-β1 expression; in case of enhanced TGF-β1 expression associated with keratin 10 staining. TGF-β1-treatment of cultured human keratinocytes induced nuclear and cytoplasmatic FHL2 staining as well as keratin 14 staining. Accordingly, siRNA-mediated FHL2 knockdown of TGF-β1-stimulated keratinocytes reduced keratin 14 staining. In conclusion, tumoural TGF-β1 secretion seems to induce nuclear translocation of co-factor FHL2 mediating progressive keratin expression in pilomatricoma.

Abstract Synovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a chimeric transcriptional modifier. Several receptor tyrosine kinases have been found activated in synovial sarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of the results of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of the SRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK and PTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence of SRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcoma xenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC was detected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for the activation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated with increased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway. Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation, associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure of cells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasion were dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA. Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. In summary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovial sarcoma. Cancer Res; 73(8); 2518–28. ©2013 AACR.

Abstract Liposarcomas (LS) represent the most common malignant soft tissue tumors in adults. Therapeutic outcome of LS is mainly determined by the efficiency of surgery as a high tendency for local relapse is seen. Chemo- and radiotherapy represent further therapeutic options, however, specifically targeted therapies are currently not available. The oncoprotein SRC is a tyrosine kinase, which has been shown to be activated in a variety of human cancers. SRC activation plays an important role in cancer cell proliferation, survival, angiogenesis and motility. In this work we analyzed LS biopsies comprising the main different subgroups, i.e. myxoid (MLS), well/dedifferentiated (WDLS/DDLS) and pleomorphic (PLS) LS and corresponding cell lines regarding SRC activation and studied the biological effects of SRC inhibition. Based on data derived from phospho-kinase screens in LS cells, immunhistochemical stainings of (Tyr416)-phosphorylated (p)-SRC were performed in primary LS biopsies. LS cell lines treated with the SRC inhibitor dasatinib were analyzed for phosphorylation of SRC and its downstream signaling molecules, cell proliferation, migration and apoptosis. Furthermore, synergistic effects of simultaneous treatments with dasatinib and conventional cytotoxic drugs were investigated. Particularly WDLS/DDLS and MLS tumor cells showed a significant expression of p-SRC. In LS cell lines, inhibition of SRC with dasatinib resulted in a substantially impaired cellular growth accompanied by a decreased phosphorylation of SRC and its downstream targets. Flow cytometric analysis showed that effects of SRC inhibition were due to an increase in apoptosis (cleaved PARP(Asp 214)) and a decrease of cellular proliferation (phospho-(Ser10) Histone H3). Scratch and Boyden chamber assays indicated a diminished migratory potential of the tumor cells due to SRC inactivation. Simultaneous treatment of liposarcoma cell lines with dasatinib and chemotherapeutic drugs resulted in additive effects. Our data suggest that SRC kinase activation represents an important biological feature of LS which might be successfully addressed in targeted therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2855. doi:1538-7445.AM2012-2855

Abstract Synovial sarcoma is a rare malignant soft tissue tumor affecting mainly adolescents and young adults. The hallmark of synovial sarcoma is the presence of a reciprocal balanced t(X;18) translocation, leading to the fusion of the SS18 gene to either the SSX1, SSX2 or rarely the SSX4 gene, resulting in a chimeric transcriptional modifier. Therapeutic outcome of synovial sarcomas is primarily determined by the efficiency of surgery as a high tendency for local relapse is documented. Standardized chemo- and radiotherapy are further therapeutic options, however, specific targeted therapies are currently not available. Recently, several expression profiling studies in mesenchymal malignancies revealed gene expression signatures indicating WNT signaling activation in synovial sarcomas. This study was performed to examine the functional relevance of WNT signaling in synovial sarcomas and to evaluate if interference with the WNT signaling pathway might represent an option in the development of novel and highly selective drugs in the treatment of synovial sarcoma. To assess the prevalence of WNT signaling activation in a set of 30 synovial sarcoma tumor samples, nuclear staining of beta-catenin was analyzed immunohistochemically. Nuclear beta-catenin signals were observed in a significant subset of these tumors, indicating activation of the WNT signaling pathway. In order to evaluate whether WNT activation is molecularly dependent on the SS18/SSX fusion proteins, tetracycline-inducible systems overexpressing the SS18/SSX fusion proteins were established in T-Rex293 cells. In luciferase reporter assays employing the TOP-/FOPflash system, expression of SS18/SSX proteins effectively activated TCF/beta-catenin mediated transcriptional activity, which was associated with nuclear recruitment of beta-catenin. Five human synovial sarcoma cell lines were subsequently treated with small molecular inhibitors of WNT signalling. In MTT assays, a significant dose-dependent inhibition of cellular growth was observed, which was accompanied by decreased expression of the WNT downstream targets c-Myc and Cyclin D1. In flow cytometric analyses, the growth effects exerted by the inhibitors were shown to be due to a reduction of cellular proliferation combined with an increase of apoptosis. In summary, our data emphasize the pivotal role of WNT signaling in synovial sarcoma and indicate its functional dependence on the characteristic SS18/SSX translocations. Furthermore, our study demonstrates that targeting the WNT signaling pathway provides a specific, molecularly founded therapeutic strategy in the treatment of synovial sarcoma. Additional functional studies in vitro and in vivo are required to further understand the role of WNT signaling and its therapeutic applicability in synovial sarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3016. doi:1538-7445.AM2012-3016

The classic medulloblastoma (CMB) and the desmoplastic medulloblastoma (DMB) subtypes represent the major medulloblastoma variants. In contrast to CMB, DMB display high levels of the low-affinity nerve growth factor receptor p75(NTR) . Given the reports of a better clinical course of DMB, we hypothesized that p75(NTR) might act as a tumor suppressor in medulloblastomas. In a large set of medulloblastomas, p75(NTR) was screened for mutations, and its mRNA expression and the DNA methylation status of its 5'-region were assessed. p75(NTR) immunostainings were performed in wild-type murine cerebella and medulloblastomas arising in patched heterozygous mice, and murine cerebellar granule cell precursors (GCP) were analyzed in vitro. Medulloblastoma cells engineered to express p75(NTR) were characterized flow cytometrically and morphologically. One CMB displayed a mutation of the p75(NTR) coding sequence. p75(NTR) mRNA levels clearly delineated DMB and CMB; however, CpG island hypermethylation was excluded as the cause of low p75(NTR) expression in CMB. Sonic Hedgehog-treated GCP showed elevated p75(NTR) expression, and strong expression of p75(NTR) was detected in the external granule cell layer of wild-type mice and in murine ptc(±) medulloblastomas. CMB cells overexpressing p75(NTR) displayed a significant increase in apoptosis. In summary, our data link activated Hedgehog signaling in DMB with p75(NTR) expression and characterize p75(NTR) as a biologically relevant inductor of apoptosis in MB.

Synovial sarcomas account for 5–10% of all malignant soft tissue tumors. They have been shown to express different membranous growth factor receptors, many of them signaling via intracellular kinase cascades. In our study, the functional role of PI3K/AKT signals in synovial sarcoma is analyzed with regard to tumor biology and therapeutic applicability. Immunohistochemical stainings of (Ser473)‐phosphorylated (p)‐AKT, its targets p‐(Ser9)‐GSK‐3β and p‐(Ser2448)‐mTOR and the cell cycle regulators Cyclin D1 and p27KIP1 were performed in 36 synovial sarcomas. The PIK3CA gene was screened for mutations. In vitro, four synovial sarcoma cell lines were treated with the PI3K inhibitor LY294002. Phosphorylation of AKT, GSK‐3β and mTOR was assessed, and cellular proliferation and apoptosis were analyzed to functionally characterize the effects of PI3K inhibition. Finally, coincubations of LY294002 with cytotoxic drugs were performed. Most tumors showed significant expression levels of p‐AKT, p‐GSK‐3β and p‐mTOR, indicating activation of the PI3K/AKT signaling cascade in synovial sarcomas; Cyclin D1 and p27KIP1 were differentially expressed. Mutations in the PIK3CA gene could be excluded. In vitro, PI3K inhibition diminished synovial sarcoma cell growth accompanied by reduced phosphorylation of AKT, GSK‐3β and mTOR. Mechanistically, PI3K pathway inhibition lead to enhanced apoptosis and decreased cellular proliferation linked to reduced Cyclin D1 and increased p27KIP1 levels. Simultaneous treatment of synovial sarcoma cell lines with LY294002 and cytotoxic drugs resulted in additive effects. In summary, PI3K signaling plays an essential role in growth control of synovial sarcomas and might be successfully targeted in multimodal therapeutic strategies.