Exosomes are small RNA- and protein-containing extracellular vesicles (EVs) that are thought to mediate hetero- and homotypic intercellular communication between normal and malignant cells.Tumour-derived exosomes are believed to promote re-programming of the tumour-associated stroma to favour tumour growth and metastasis. Currently, exosomes have been intensively studied in carcinomas. However, little is known about their existence and possible role in sarcomas. Here, we report on the identification of vesicles with exosomal features derived from Ewing's sarcoma(ES), the second most common soft-tissue or bone cancer in children and adolescents. ES cell line-derived EV shave been isolated by ultracentrifugation and analysed by flow-cytometric assessment of the exosome-associated proteins CD63 and CD81 as well as by electron microscopy. They proved to contain ES-specific transcripts including EWS-FLI1, which were suitable for the sensitive detection of ES cell line-derived exosomes by qRT-PCRin a pre-clinical model for patient plasma. Microarray analysis of ES cell line-derived exosomes revealed that they share a common transcriptional signature potentially involved in G-protein-coupled signalling, neurotransmitter signalling and stemness. In summary, our results imply that ES-derived exosomes could eventually serve as biomarkers for minimal residual disease diagnostics in peripheral blood and prompt further investigation of their potential biological role in modification of the ES-associated microenvironment
Abstract Introduction: We reported blocking of BET bromodomain binding proteins (BRDs) by use of an inhibitor (JQ1) and the associated strong downregulation of the predominant EWS-ETS protein EWS-FLI1 in Ewing sarcoma (ES). Here we analyzed in depth the mechanistic effects of this treatment by EWS-FLI1 interaction studies and the evaluation of combined targeted treatment options. Experimental procedures: Function of BRDs was analyzed by application of specific inhibitors (JQ1, I-BET151), RNA interference (RNAi) with the generation of stable and inducible knockdowns or knockouts by the generation of BRD4 CRISPR/Cas9 cell lines. To analyze the resulting changes Co-IP, ChIP-qPCR, RT-PCR, Western blotting, cell cycle analysis, proliferation and invasion assays, whole transcriptome analysis via microarrays, as well as xenograft mouse models were utilized. Results: By use of JQ1 or iBET we strikingly observed a strong downregulation of the predominant EWS-ETS protein EWS-FLI1, and subsequent microarray analysis revealed JQ1 treatment to block the typical ES associated expression program. The effect on this expression program was partially mimicked by RNAi for BRD3 or BRD4 but not by BRD2. However, knockout studies of BRD4 by CRISPR/Cas9 as well as knockdowns of individual BRD2, 3, or 4 did not recapitulate JQ1-mediated proliferation restrictions and blockade and tumor development in xenograft mice as observed for JQ1. However, co-immunoprecipitation experiments revealed a DNA-independent interaction of BRD4 with EWS/FLI1 and additional interaction with CDK9. Treatment of ES cells with a specific CDK9 inhibitor demonstrated a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. Furthermore, CDK9 inhibition induced apoptosis in ES as depicted by downregulation of XIAP and CFLAR and consequently cleavage of Caspase 8, PARP and increased CASP3 activity, similar to JQ1. Combined treatment of ES with BRD and CDK9 inhibitors in a preclinical model was more effective than individual drug application alone. Conclusion: Translocation-driven tumors such as ES are very susceptible to combined treatment with epigenetic inhibitors. Here we demonstrate that treatment with inhibitors targeting the p-TEFb complex could interrupt communication between EWS-FLI1, BRD4, and CDK9, further impeding EWS-ETS transcriptional activity and its associated pathognomonic expression program. Citation Format: Tim Hensel, Chiara Giorgi, Fiona Becker-Dettling, Julia Calzada-Wack, Oxana Schmidt, Shudong Wang, Beat W. Schaefer, Stefan Burdach, Guenther H.S. Richter. Combined targeting of the Ewing sarcoma transcriptional program by blocking epigenetic readers and transcription initiation via EWS-FLI1 [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B02.
Ewing's sarcoma (ES) is the second most common bone-associated malignancy in children and is driven by the fusion oncogene EWS/FLI1 and characterised by rapid growth and early metastasis. Here, we explored the role of the Zyxin-related protein thyroid receptor interacting protein 6 (TRIP6) in ES. The Zyxin family comprises seven homologous proteins involved in migration and proliferation of many cell types of which Zyxin has been described as a tumour suppressor in ES.By interrogation of published microarray data (n = 1254), we observed that of all Zyxin proteins, only TRIP6 is highly overexpressed in primary ES compared with normal tissues. Re-analysis of published EWS/FLI1 gain- and loss-of-function microarray experiments as well as chromatin-immunoprecipitation assays revealed that TRIP6 overexpression is not mediated by EWS/FLI1. Microarray and subsequent gene-set enrichment analyses of ES cells with and without RNA interference-mediated TRIP6 knockdown demonstrated that TRIP6 expression confers a pro-proliferative and pro-invasive transcriptional signature to ES cells. While short-term proliferation was not considerably affected by TRIP6 knockdown, silencing of the protein significantly reduced migration, invasion, long-term proliferation and clonogenicity of ES cells in vitro as well as tumourigenicity in vivo.Taken together, our data indicate that TRIP6 acts, in contrast to Zyxin, as an oncogene that partially accounts for the autonomous migratory, invasive and proliferative properties of ES cells independent of EWS/FLI1.
Transfection by DNA isolated from bacteriophage T3 was studied using Escherichia coli 921/0 as host. The following conditions were found optimal: Competent E. coli 921/0 were obtained by harvesting the bacteria at the onset of late exponential growth (5 X 10(8) cells/ml) and treating the latter with 0.05 M CaCl2. Hereafter, the microbes were suspended in 50 mM Tris-HCl buffer (pH 7.2) and the concentration adjusted to 7 X 10(9) cells/ml. T3 DNA was added and the suspension kept at 0 degrees C for 15 min. Determination of the number of infectious centers was then carried out in the usual way. The efficiency of transfection under these conditions amounted to 10(4) p. f. u./microgram DNA. Preincubation of competent bacteria with T4 DNA at 0 degrees C before the addition of T3 DNA reduced the number of infectious centers. However, if T3- and T4 DNA were added simultaneously no decrease of the transfection efficiency occurred. Calf thymus DNA was without influence on transfection.
Abstract The molecular events during the anti‐tumor response induced by interleukin (IL)‐4 were investigated by quantitative polymerase chain reaction. The growth of Chinese hamster ovary cells transfected to produce IL‐4 (CHO.T1) was strongly suppressed when cells were injected intraperitoneally into nude mice and this suppression was accompanied by the rapid accumulation of activated macro‐phages. Peritoneal cells from such mice were analyzed for mRNA induced by IL‐4. Correlating with a high local IL‐4 concentration, several transcripts were found to be up‐regulated during the early phase of the anti‐tumor response [IL‐4 receptor, IL‐5, tumor necrosis factor (TNF) and interferon (IFN)‐γ]. The functional relevance of the elevated mRNA levels was analyzed by injection of CHO.T1 cells together with anti‐cytokine monoclonal antibodies (mAb). In contrast to anti‐IL‐5 and anti‐TNF mAb, an anti‐IFN‐γ mAb interfered with the anti‐tumor response demonstrating the involvement of IFN‐γ during the IL‐4‐induced tumor suppression. Tumor growth in anti‐IFN‐γ mAb‐treated animals was significantly delayed in comparison to anti‐IL‐4 mAb‐treated mice, suggesting that IFN‐γ‐independent effector cells may also be involved.
