SRC inhibition represents a potential therapeutic strategy in liposarcoma.

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.

TGF-ß1-dependent induction and nuclear translocation of FHL2 promotes keratin expression in pilomatricoma.

Pilomatricoma is a tumour derived from hair matrix cells, which shows progressive keratin expression. Tumorigenesis is frequently associated with activating mutations in ß-catenin gene inducing nuclear expression of ß-catenin protein. The present study analysed the role of transforming growth factor-ß1 (TGF-ß1) 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-ß1 and nuclear FHL2 expression in the outer root sheath layer separated from nuclear ß-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-ß1 and nuclear FHL2 in tumour cells, which mostly lacked nuclear ß-catenin expression. Tumoural proliferation (ki67) was associated with nuclear ß-catenin staining but not with expression of nuclear FHL2. In early pilomatricomas, TGF-ß1 expression was observed in few peripheral tumour cells showing absent or faint nuclear FHL2 co-staining. TGF-ß1 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-ß1 expression; in case of enhanced TGF-ß1 expression associated with keratin 10 staining. TGF-ß1-treatment of cultured human keratinocytes induced nuclear and cytoplasmatic FHL2 staining as well as keratin 14 staining. Accordingly, siRNA-mediated FHL2 knockdown of TGF-ß1-stimulated keratinocytes reduced keratin 14 staining. In conclusion, tumoural TGF-ß1 secretion seems to induce nuclear translocation of co-factor FHL2 mediating progressive keratin expression in pilomatricoma.

SRC signaling is crucial in the growth of synovial sarcoma cells.

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.

p75(NTR) induces apoptosis in medulloblastoma cells.

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.

Phosphatidylinositol-3'-kinase/AKT signaling is essential in synovial sarcoma.

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-3ß and p-(Ser2448)-mTOR and the cell cycle regulators Cyclin D1 and p27(KIP1) 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-3ß 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-3ß and p-mTOR, indicating activation of the PI3K/AKT signaling cascade in synovial sarcomas; Cyclin D1 and p27(KIP1) 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-3ß and mTOR. Mechanistically, PI3K pathway inhibition lead to enhanced apoptosis and decreased cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) 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.