Project description:Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta- catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells upregulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression.

Project description:Ewing sarcoma is a highly aggressive solid malignancy affecting children and young adults. Ewing sarcoma is driven primarily by EWSR1::FLI1, a fusion oncoprotein that has been notoriously difficult to target with traditional pharmacologic agents. There are numerous examples of promising preclinical combinations of small molecules that are never tested in pediatric clinical trials because agents fail to reach the market due to limited efficacy for common adult cancers. Moreover, the effectiveness of single-agent therapies for cancer treatment is often limited. To address these limitations, we selected 28 compounds that were FDA approved at the time of the study or in late stages of clinical development and known to regulate important pathways in Ewing sarcoma. We performed a drug screen in Ewing sarcoma cell lines with 180 combinations of tyrosine kinase inhibitors, cell cycle inhibitors, and conventional chemotherapy. The results of the screen revealed that a PI3K inhibitor, copanlisib, combined with a CDK4/6 inhibitor, ribociclib, exhibited strong synergistic anti-Ewing sarcoma activity. Using proteomic methods such as a reverse-phase protein array and western immunoblotting, we demonstrated that this combination induced a downregulation of the PI3K/AKT pathway as well as proteins involved in cell cycle regulation. We further confirmed these in vitro data using bulk RNA-sequencing. To evaluate the phenotypic effect of the PI3K/CDK4/6 inhibition in Ewing sarcoma lines, we performed apoptosis and cell cycle analyses using flow cytometry and demonstrated that ribociclib primarily induced a G0/G1 arrest with minimal effect on Ewing cell viability but significantly enhanced the apoptotic effect of copanlisib treatment. In two xenograft models of Ewing sarcoma, we demonstrated that the combination significantly prolonged survival compared to treatment with either vehicle or single-agent therapy alone. Our findings identify a new candidate therapy combination for Ewing sarcoma and provide a resource of additional potential synergistic combinations for future validation.

Project description:Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent mTOR inhibition might suggest a number of therapeutic combinations that could improve its clinical activity. TC32 and TC71 ES clones with acquired resistance to ridaforolimus (MK8669, mTOR inhibitor) were generated by maintaining the corresponding parental cell lines with increasing concentrations of the agents (up to 50 Î¼M using ridaforolimus) for 7 months. All parental and acquired drug resistant cell lines were tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.) according to the manufacturerâ??s protocol and validated using short-tandem repeat fingerprinting with an AmpFLSTR Identifier kit as previously described. Herein, we determine subtle differences in acquired mechanism of resistance by promising small molecule inhibitor of mTOR, were evaluated using in vitro assays to decipher the mechanism(s) by which IGF-1R inhibition induces drug resistance in Ewing sarcoma cells. The preparation of extracted proteins from sensitive and acquired resistant Ewing sarcoma cells to ridaforolimus for reverse-phase protein lysate array (RPPA) analysis were prepared using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 115 validated primary antibodies, and detected using a DakoCytomation-catalyzed system with secondary antibodies. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Log2 values were media-centered by protein to account for variability in signal intensity by time and were calculated using the formula log2 signal â?? log2 median. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between sensitive and resistant cell lines to drug treatments. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs). Biostatistical analyses comparing two groups were performed using an unpaired t-test with Gaussian distribution followed by the Welch correction. To distinguish between treatment groups, we used one-way ANOVA with the Geisser-Greenhouse correction. Differences with p values <0.05 were considered significant. Within clustered image maps (CIM), unsupervised double hierarchical clustering used the Pearson correlation distance and Wardâ??s linkage method as the clustering algorithm to link entities (proteins) and samples.

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Homo sapiens

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