Abstract A21: The metastasis-regulator Tenascin C is activated by Wnt/beta-catenin in Ewing sarcoma
Authors: Pedersen Elisabeth A. , Menon Rajasree , Thomas Dafydd G. , Bailey Kelly M. , Noord Raelene A. Van , Chugh Rashmi , Fearon Eric R. , Lawlor Elizabeth R. 
Published: May 2016

Abstract
A pathogenic role for Wnt/beta-catenin signaling in Ewing sarcoma, an aggressive and highly metastatic tumor characterized by pathognomonic EWS/ETS fusion proteins, has yet to be elucidated. To address the potential importance of Wnt/beta-catenin in Ewing sarcoma we performed immunohistochemical staining of primary tumor specimens and discovered that nuclear beta-catenin is highly heterogeneous, both within and between tumors, and that all tumors with evidence of beta-catenin activation were associated with clinical relapse. These studies led us to hypothesize that activation of Wnt/beta-catenin in Ewing sarcoma cells promotes acquisition of an aggressive phenotype. To test this hypothesis we generated TCF/LEF-reporter cells and performed RNA-seq and functional studies of Wnt/beta-catenin activated cells. Pathway activation was achieved by provision of Wnt3a, inhibition of GSK3b, and by ectopic expression of constitutively active beta-catenin. LGR5 expression was assessed by in situ hybridization and potentiation of the Wnt signal by R-spondin determined by reporter assays and qRT-PCR. Knockdown of gene expression was achieved by lentiviral transduction of targeted shRNAs. Migration was assessed in transwell assays and phalloidin and cortactin staining was used to visualize the cytoskeleton. Metastatic potential was determined by a tail vein injection model. Our results confirm that only subpopulations of Ewing sarcoma cells activate beta-catenin in response to exogenous Wnt ligand. Responsive cells were found to be more migratory than non-responsive cells and cytoskeleton changes, including increased cell spreading and formation of migration-associated podosome structures, were observed in the activated cell populations. RNA sequencing of Wnt/beta-catenin activated cells revealed changes in expression of over a thousand genes. Interestingly, gene set enrichment analysis (GSEA) showed that the Wnt/beta-catenin modulated genes were highly inversely correlated with EWS/ETS-regulated genes. Specifically, Wnt/beta-catenin induced genes are repressed by EWS/ETS and vice versa, thus revealing transcriptional antagonism between EWS/ETS and Wnt/beta-catenin signaling. Confirmation of this antagonism was demonstrated by the finding that the cytoskeletal phenotype of Wnt/beta-catenin activated cells in part phenocopies that of EWS/ETS knockdown cells. At a molecular level TNC (tenascin C) was identified to be among the most induced genes in Wnt-activated cells. A combination of pharmacologic and genetic approaches confirmed that TNC is a beta-catenin target in Ewing sarcoma cells and that it is also transcriptionally repressed by EWS/ETS. Tenascin C is a matricellular protein that is an established regulator of metastatic phenotypes including cancer cell migration, invasion, and survival during metastatic dissemination. Studies of TNC-knockdown cells showed that loss of TNC has no significant impact on cell proliferation but results in reduced cell migration and invasion. In addition, cells with reduced expression of TNC showed a significant reduction in metastatic potential in vivo. Together these studies reveal marked cellular heterogeneity of Wnt/beta-catenin activation in Ewing sarcoma and demonstrate that the distinct gene expression signature associated with Wnt pathway activation in tumor cells is associated with transcriptional antagonism of EWS/ETS. Paradoxically, this antagonism promotes transition to a more aggressive cellular phenotype with enhanced metastatic potential that is associated with changes in the cytoskeleton and upregulation of TNC. Together these data support the innovative hypothesis that, in Ewing sarcoma, cell plasticity between non-metastatic and metastatic cellular phenotypes is dependent on the balance between EWS/ETS and Wnt/beta-catenin signaling.
Citation Format: Elisabeth A. Pedersen, Rajasree Menon, Dafydd G. Thomas, Kelly M. Bailey, Raelene A. Van Noord, Rashmi Chugh, Eric R. Fearon, Elizabeth R. Lawlor. The metastasis-regulator Tenascin C is activated by Wnt/beta-catenin in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A21.