Abstract
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 up-regulated 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.
Highlights
From the ‡Departments of Pediatrics, §Pathology, and ¶Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan
Activation of canonical Wnt signaling in Ewing sarcoma cells leads to a significant increase in the transcription of genes that encode extracellular matrix (ECM) proteins as well as other proteins that play key roles in tumor: tumor microenvironment (TME) interaction and crosstalk
Consistent with our mass spec results, Collagen I was readily detected in the media of Wnt-activated CHLA10 cells but was not increased beyond the high levels that were already secreted under basal conditions (Fig. 5E). Together these studies confirm that activation of Wnt/betacatenin signaling in Ewing sarcoma cells leads to increased expression and secretion of ECM proteins that have the potential to impact on tumor progression by altering tumor: TME crosstalk
Summary
From the ‡Departments of Pediatrics, §Pathology, and ¶Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan. The Ewing sarcoma secretome reveals that Wnt/betacatenin activated tumor cells upregulate secretion of ECM proteins. We have investigated whether activation of canonical Wnt signaling in Ewing sarcoma cells impacts on their secretion of ECM proteins.
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