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Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. Patients remain at high risk for early and late metastasis. A high-throughput drug screen consisting of over 900 tool compounds and epigenetic modifiers, representing over 100 drug classes, was undertaken in a panel of synovial sarcoma cell lines to uncover novel sensitizing agents and targetable pathways. Top scoring drug categories were found to be HDAC inhibitors and proteasomal targeting agents. We find that the HDAC inhibitor quisinostat disrupts the SS18-SSX driving protein complex, thereby reestablishing expression of EGR1 and CDKN2A tumor suppressors. In combination with proteasome inhibition, HDAC inhibitors synergize to decrease cell viability and elicit apoptosis. Quisinostat inhibits aggresome formation in response to proteasome inhibition, and combination treatment leads to elevated endoplasmic reticulum stress, activation of pro-apoptotic effector proteins BIM and BIK, phosphorylation of BCL-2, increased levels of reactive oxygen species, and suppression of tumor growth in a murine model of synovial sarcoma. This study identifies and provides mechanistic support for a particular susceptibility of synovial sarcoma to the combination of quisinostat and proteasome inhibition.

Background Heterogeneity and low incidence comprise the biggest challenge in sarcoma diagnosis and treatment. Chemotherapy, although efficient for some sarcoma subtypes, generally results in poor clinical responses and is mostly recommended for advanced disease. Specific genomic aberrations have been identified in some sarcoma subtypes but few of them can be targeted with approved drugs. Methods We cultured and characterised patient-derived sarcoma cells and evaluated their sensitivity to 525 anti-cancer agents including both approved and non-approved drugs. In total, 14 sarcomas and 5 healthy mesenchymal primary cell cultures were studied. The sarcoma biopsies and derived cells were characterised by gene panel sequencing, cancer driver gene expression and by detecting specific fusion oncoproteins in situ in sarcomas with translocations. Results Soft tissue sarcoma cultures were established from patient biopsies with a success rate of 58%. The genomic profile and drug sensitivity testing on these samples helped to identify targeted inhibitors active on sarcomas. The cSrc inhibitor Dasatinib was identified as an active drug in sarcomas carrying chromosomal translocations. The drug sensitivity of the patient sarcoma cells ex vivo correlated with the response to the former treatment of the patient. Conclusions Our results show that patient-derived sarcoma cells cultured in vitro are relevant and practical models for genotypic and phenotypic screens aiming to identify efficient drugs to treat sarcoma patients with poor treatment options.

Synovial sarcoma (SS) is driven by a unique t(18;X) chromosomal translocation resulting in expression of the SS18-SSX fusion oncoprotein, a transcriptional regulator with both activating and repressing functions. However, the manner in which SS18-SSX contributes to the development of SS is not entirely known. Here, we show that SS18-SSX drives the expression of Preferentially Expressed Antigen in Melanoma (PRAME), which is highly expressed in SS but whose function remains poorly understood. The fusion protein directly binds and activates the PRAME promoter and we found that expression of SS18-SSX and PRAME are positively correlated. We provide evidence that PRAME modulates retinoic acid (RA) signaling, forming a ternary complex with the RA receptor α (RARα) and the Enhancer of Zeste Homolog 2 (EZH2). Knockdown of PRAME suppressed the response to all- trans retinoic acid (ATRA) supporting PRAME’s role in modulating RA-signaling. Notably, we demonstrate that combined pharmacological inhibition of EZH2 and treatment with ATRA reconstituted RA signaling followed by reduced proliferation and induction of cellular senescence. In conclusion, our data provides new insights on the role of the SS18-SSX fusion protein in regulation of PRAME expression and RA signaling, highlighting the therapeutic potential of disrupting the RARα-PRAME-EZH2 complex in SS. Schematic presentation of the proposed model . A The RARα-PRAME-EZH2 ternary complex in SS . The fusion SS18-SSX oncoprotein binds to the PRAME promoter and activates its expression. PRAME in turn interacts with RARα-RXR heterodimers as well as with EZH2, and the complex binds to retinoic acid response elements (RAREs) in the DNA. This results in transcriptional repression of retinoic acid (RA) responsive genes and thus inhibition of RA-signaling, allowing tumor cell proliferation. B Therapeutic strategy . Treatment with an EZH2 inhibitor, such as GSK343, or activation of RAR receptors via all- trans retinoic acid (ATRA), disrupts the RARα-PRAME-EZH2 ternary complex and restores RA-signaling. Exposure to GSK343 or ATRA results in inhibition of cell proliferation and induction of cellular senescence, where GSK343 shows a dominant effect. The Figure was created with Biorender.com.

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

SSX is a transcription factor with elusive oncogenic functions expressed in a variety of human tumors of epithelial and mesenchymal origin. It has raised substantial interest as a target for cancer therapy since it elicits humoral responses and displays restricted expression to cancer, spermatogonia and mesenchymal stem cells. Here, we investigated the oncogenic properties of SSX by employing a RNA interference to knock-down the endogenous expression of SSX in melanoma and osteosarcoma cell lines. Depletion of SSX expression resulted in reduced proliferation with cells accumulating in the G1 phase of the cell cycle. We found that the growth promoting and survival properties of SSX are mediated in part though modulation of MAPK/Erk and Wnt signaling pathways, since SSX silencing inhibited Erk-mediated signaling and transcription of cMYC and Akt-1. We also found that SSX forms a transient complex with β-catenin at the G1-S phase boundary resulting in the altered expression of β-catenin target genes such as E-cadherin, snail-2 and vimentin, involved in epithelial-mesenchymal transitions. Importantly the silencing of SSX expression in in vivo significantly impaired the growth of melanoma tumor xenografts. Tumor biopsies from SSX silenced tumors displayed reduced cyclin A staining, indicative of low proliferation and predominantly cycloplasmic β-catenin compared to SSX expressing tumors. The present study demonstrates a previously unknown function of SSX, that as an oncogene and as a tumor target for the development of novel anti-cancer drugs.

Nanoparticle (NP)-based contrast agents enabling different imaging modalities are sought for non-invasive bio-diagnostics. A hybrid material, combining optical and X-ray fluorescence is presented as a bioimaging contrast agent. Core NPs based on metallic rhodium (Rh) have been demonstrated to be potential X-ray Fluorescence Computed Tomography (XFCT) contrast agents. Microwave-assisted hydrothermal method is used for NP synthesis, yielding large-scale NPs within a significantly short reaction time. Rh NP synthesis is performed by using a custom designed sugar ligand (LODAN), constituting a strong reducing agent in aqueous solution, which yields NPs with primary amines as surface functional groups. The amino groups on Rh NPs are used to directly conjugate excitation-independent nitrogen-doped carbon quantum dots (CQDs), which are synthesized through citrate pyrolysis in ammonia solution. CQDs provided the Rh NPs with optical fluorescence properties and improved their biocompatibility, as demonstrated in vitro by Real-Time Cell Analysis (RTCA) on a macrophage cell line (RAW 264.7). The multimodal characteristics of the hybrid NPs are confirmed with confocal microscopy, and X-ray Fluorescence (XRF) phantom experiments.

Diffraction-limited resolution and low penetration depth are fundamental constraints in optical microscopy and in vivo imaging. Recently, liquid-jet X-ray technology has enabled the generation of X-rays with high-power intensities in laboratory settings. By allowing the observation of cellular processes in their natural state, liquid-jet soft X-ray microscopy (SXM) can provide morphological information on living cells without staining. Furthermore, X-ray fluorescence imaging (XFI) permits the tracking of contrast agents in vivo with high elemental specificity, going beyond attenuation contrast. In this study, we established a methodology to investigate nanoparticle (NP) interactions in vitro and in vivo, solely based on X-ray imaging. We employed soft (0.5 keV) and hard (24 keV) X-rays for cellular studies and preclinical evaluations, respectively. Our results demonstrated the possibility of localizing NPs in the intracellular environment via SXM and evaluating their biodistribution with in vivo multiplexed XFI. We envisage that laboratory liquid-jet X-ray technology will significantly contribute to advancing our understanding of biological systems in the field of nanomedical research.

Medulloblastoma is one of the most common malignant brain tumor types in children, with an overall survival of 70%. Mortality is associated with metastatic relapsed tumors. Rho-associated kinases (ROCKs), important for epithelial-mesenchymal transition (EMT) and proper nervous system development, have previously been identified as a promising drug target to inhibit cancer growth and metastatic spread. Here, we show that ROCKs are expressed in medulloblastoma, with higher ROCK2 mRNA expression in metastatic compared to non-metastatic tumors. By evaluating three ROCK inhibitors in a panel of medulloblastoma cell lines we demonstrated that medulloblastoma cells were sensitive for pharmacological ROCK inhibition. The specific ROCK inhibitor RKI-1447 inhibited the tumorigenicity in medulloblastoma cells as well as impeded cell migration and invasion. Differential gene expression analysis suggested that ROCK inhibition was associated with the downregulation of signaling pathways important in proliferation and metastasis e.g., TNFα via NFκβ, TGFβ, and EMT. Expression of key proteins in these pathways such as RHOA, RHOB, JUN, and vimentin was downregulated in ROCK inhibited cells. Finally, we showed that ROCK inhibition by RKI-1447 suppressed medulloblastoma growth and proliferation in vivo. Collectively, our results suggest that ROCK inhibition presents a potential new therapeutic option in medulloblastoma, especially for children with metastatic disease.

Morphologically controllable synthesis of Rh nanoparticles (NPs) was achieved by the use of additives during polyol synthesis. The effect of salts and surfactant additives including PVP, sodium acetate, sodium citrate, CTAB, CTAC, and potassium bromide on Rh NPs morphology was investigated. When PVP was used as the only additive, trigonal NPs were obtained. Additives containing Br− ions (CTAB and KBr) resulted in NPs with a cubic morphology, while those with carboxyl groups (sodium citrate and acetate) formed spheroid NPs. The use of Cl− ions (CTAC) resulted in a mixture of polygon morphologies. Cytotoxicity of these NPs was evaluated on macrophages and ovarian cancer cell lines. Membrane integrity and cellular activity are both influenced to a similar extent, for both the cell lines, with respect to the morphology of Rh NPs. The cells exposed to trigonal Rh NPs showed the highest viability, among the NP series. Particles with a mixed polygon morphology had the highest cytotoxic impact, followed by cubic and spherical NPs. The Rh NPs were further demonstrated as contrast agents for X-ray fluorescence computed tomography (XFCT) in a small-animal imaging setting. This work provides a detailed route for the synthesis, morphology control, and characterization of Rh NPs as viable contrast agents for XFCT bio-imaging.