Abstract 3270: Distinct and heterogeneous response of Ewing sarcoma cells to canonical Wnt signaling

Abstract

Abstract Canonical Wnt/β-catenin signaling underlies the pathogenesis of many colorectal carcinomas and other cancer types. However, the role of the canonical Wnt signaling pathway in Ewing sarcoma (ES) pathogenesis is unclear. Current data suggest that Wnts may function as morphogens rather than mitogens in ES. Thus, we hypothesized that the response of ES cells to Wnt ligands is distinct from other cells and dependent on cellular context. To investigate the response of ES cells to Wnt signaling, we transduced ES cell lines with 7xTCF-GFP (7TGP) or 7xTCF-luciferase (7TFP) reporter vectors, and stimulated cells with control or WNT3a conditioned media (CM) with or without recombinant R-spondin 2 (RSPO2), a molecule that strongly potentiates canonical Wnt signaling. We used in situ hybridization to determine the expression of LGR5, the receptor for RSPO2. To isolate highly Wnt-responsive cells, we sorted the top 10% of GFP-positive 7TGP-transduced cells that had been stimulated by Wnt3a CM +/- RSPO2, and performed RNA-sequencing (RNA-seq). Using edgeR, we generated lists of differentially expressed genes either up- or down-regulated by Wnt signaling (p<0.05). We next interrogated pathways enriched in these lists using DAVID and gene set enrichment analysis (GSEA). From these studies, we observed that the activation of Wnt signaling varied among cell lines and among distinct cells within a given cell line. We hypothesized that this inter- and intra-cell line heterogeneity in the response to Wnt pathway activation was due in part to variations in expression of LGR5, and in situ hybridization confirmed that there was marked variability of LGR5 expression among individual cells in both cell lines and primary tumors. RNA-seq analyses revealed that the most significantly induced or repressed genes in response to Wnt pathway activation included many genes not previously shown to be regulated by Wnt signaling, in addition to a limited number of previously described Wnt targets. Further, DAVID and GSEA revealed that genes in developmental pathways were enriched in the induced gene set, while genes associated with cell cycle regulation were enriched in the repressed genes. Notably, GSEA revealed that numerous genes induced by Wnt activation are repressed in response to the ES-specific oncogene EWS/FLI1, and many genes repressed by Wnt are induced by EWS/FLI1. This led us to hypothesize that Wnt signaling may transcriptionally antagonize EWS/FLI1 signaling, and phalloidin staining confirmed that Wnt stimulation induces changes in the cytoskeleton that are reminiscent of an EWS/FLI1 knockdown phenotype. Together, these data show that canonical Wnt signaling in LGR5-positive Ewing sarcoma cells results in distinct transcriptional programs that may depend in part on the relative basal levels of LGR5 expression. Further, the findings suggest a novel mechanism by which the functional activity of EWS/FLI1 may be altered by Wnt signals in the tumor microenvironment. Citation Format: Elisabeth Anne Pedersen, Rajasree Menon, Dafydd Thomas, Eric Fearon, Elizabeth Lawlor. Distinct and heterogeneous response of Ewing sarcoma cells to canonical Wnt signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3270. doi:10.1158/1538-7445.AM2015-3270