Activation of Wnt/β-Catenin in Ewing Sarcoma Cells Antagonizes EWS/ETS Function and Promotes Phenotypic Transition to More Metastatic Cell States

Abstract

Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/beta-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated beta-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/beta-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/beta-catenin activated tumor cells. Consistent with this, Wnt/beta-catenin activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/beta-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype and up regulation of EWS/ETS-repressed genes. Notably, activation of Wnt/beta-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS-repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/beta-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in de-repression of metastasis-associated gene programs.

The Ewing Sarcoma Secretome and Its Response to Activation of Wnt/beta-catenin Signaling

“…To achieve this we interrogated three independent, publicly available databases (13,24,25) and assessed correlations between expression of LEF1 and TNC and COL1A1 in primary patient tumors. We have previously shown that LEF1 is a robust biomarker of Wnt/beta-catenin activation in Ewing sarcoma cells and tumors (14). As shown, expression of TNC was significantly positively correlated with LEF1 in all datasets and COL1A1 correlated with LEF1 in two of the three patient cohorts ( Figure 5C).…”

“…Both of these ECM proteins are well-established mediators of tumor engraftment and metastasis (46,48). We previously showed that stimulation of Ewing sarcoma cells with Wnt3a for 48 hours leads to a two-fold increase in expression of TNC mRNA (14). Analysis of cells following the five-day stimulation protocol used for secretome analysis confirmed robust upregulation of TNC mRNA to greater than five-fold ( Figure 5A).…”

“…We previously showed that activation of Wnt/beta-catenin in Ewing sarcoma cells induces transition to a more migratory cellular phenotype and enhances metastatic engraftment (14). In the current study, we have investigated whether activation of canonical Wnt signaling in Ewing sarcoma cells impacts on their secretion of ECM proteins.…”

“…To begin to understand the mechanisms through which upregulation of the Wnt/beta-catenin signaling axis contribute to metastatic phenotypes and poor outcomes in Ewing sarcoma, we performed pathways analysis on our previously reported RNA-sequencing study of Wnt/betacatenin activated cells (14). In this previous study, 1157 transcripts were identified as being significantly induced by Wnt activation and 1221 were significantly induced in Wnt activation with potentiation by Rspondin2.…”

“…We previously observed that the response of Ewing sarcoma cells to exogenous Wnt3a ligand is highly heterogeneous (14). Thus, we used flow cytometry analysis of TCF/LEF-GFP reporter cells, as previously described (14) Figure 3D).…”