Abstract 5836: Activation of Wnt/beta-catenin alters the Ewing sarcoma secretome - Implications for tumor:tumor microenvironment crosstalk

Abstract

Abstract Ewing sarcoma is the second most common bone cancer in children and adolescents. Patients with metastatic disease have less than a 25% chance of survival making it imperative to understand the mechanisms that drive metastasis. We previously showed that activation of Wnt/β-catenin signaling in Ewing sarcoma cells alters the tumor cell transcriptome, promoting transition to a more metastatic cellular phenotype. In addition, primary tumors with high-level Wnt activation were associated with worse clinical outcomes, supporting a role for canonical Wnt signaling in metastatic progression. Significantly, extracellular matrix (ECM) protein genes were among the most affected by Wnt/β-catenin, suggesting that activation of the pathway might lead to alterations in the local tumor microenvironment (TME). Crosstalk between the TME and tumor cells regulates fundamental aspects of the metastatic cascade including invasion, migration, and angiogenesis. It is our goal to define the impact of Wnt/β-catenin signaling on the TME and elucidate how these changes contribute to metastasis. Our specific hypothesis is that activation of Wnt/β-catenin in Ewing sarcoma cells leads to changes in the tumor secretome that result in altered TME composition and tumor:TME crosstalk. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence and absence of Wnt3a was collected for proteomic analysis. We used label-free mass spectrometry with spectral counting to quantify proteins that passed a ≤1% FDR threshold. Significance was defined as greater than 4.5 or less than 0.2 fold difference in counts between Wnt3a and control conditions. Western blot of conditioned media was used to validate identified proteins. Preliminary analysis of the TC32 cell line identified 49 proteins up regulated in Wnt3a treated media in comparison to control. Significantly, Tenascin C (TNC), was among the most significantly up regulated proteins, validating our prior data that TNC is a transcriptional target of Wnt/β-catenin and a promoter of the metastatic phenotype in vivo. Gene ontology analysis of the significantly differentially expressed proteins revealed enrichment of proteins involved in ECM-receptor signaling, ECM organization, cell adhesion, and collagen catabolism. These biologic processes all play critical roles in tumor:TME interactions and together contribute to the early steps of adhesion, migration and invasion that are responsible for initiation of metastasis. These preliminary studies confirm that Wnt/β-catenin-dependent changes in the Ewing sarcoma transcriptome are associated with changes in tumor cell secretome. We are now validating these findings in additional cell lines and updated results will be presented. These studies will elucidate how changes in tumor cells can lead to changes in the local TME and in tumor:TME crosstalk that promote metastatic progression of Ewing sarcoma. Citation Format: Allegra Hawkins, Colin Sperring, Venkatesha Basrur, Alexey Nesvizhskii, Elizabeth Lawlor. Activation of Wnt/beta-catenin alters the Ewing sarcoma secretome - Implications for tumor:tumor microenvironment crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5836. doi:10.1158/1538-7445.AM2017-5836