Abstract 3085: Tenascin C is a canonical Wnt target gene in Ewing sarcoma and its expression is potentiated by R-spondin

Ewing sarcoma (ES) is a highly aggressive bone and soft tissue tumor, and for patients with metastatic disease, survival is dismally low. We have recently shown that primary ES tumors and cell lines express the stem cell marker LGR5, and that this expression correlates with aggressive disease. Further, we have shown that Wnt/β-catenin signaling can be strongly induced in ES cells and that signaling is potentiated by the LGR5 ligand, R-spondin 2 (RSPO2). Interestingly, however, we found no impact of Wnt signaling on cell proliferation leading us to hypothesize that the highly context-dependent nature of Wnt target genes induces a variant response in ES. To determine the consequences of Wnt signaling in ES we first aimed to define canonical Wnt target genes, in particular target genes whose expression was potentiated by RSPO2. LGR5-expressing ES cells were exposed to Wnt3a conditioned media (CM) and/or RSPO2 and then FACS-sorted on the basis of Wnt activity using a 7xTCF-GFP fluorescent reporter. RNA-sequencing was performed on sorted populations using the Illumina HiSeq platform. Using real-space FPKM values from triplicate sorts, we identified genes that were reproducibly induced by Wnt3a CM and further increased by the addition of RSPO2. Significantly, the metastasis-associated extracellular matrix protein tenascin C (TNC) was identified to be among the most robustly induced gene targets. In addition, comparison of our list of up-regulated genes with datasets in the Molecular Signatures Database (MolSigDB) revealed that the genes that were induced by Wnt3a/RSPO2 in ES are associated with metastasis pathways in other cancers. Quantitative real-time PCR and immunocytochemistry studies of multiple ES cell lines validated that tenascin C is indeed induced by Wnt3a CM and potentiated by RSPO2. In addition, potentiation was directly related to LGR5 expression. Conversely, with the exception of AXIN2, expression of other well-established canonical Wnt targets in epithelial cancers was unaffected. To test if Wnt signaling and/or tenascin C contributes to ES cell migration and invasion, cells were stimulated with Wnt3a CM +/- RSPO2 and transwell migration assays performed using Boyden chambers and the Roche xCelligence system. Addition of Wnt3a CM increased cellular migration and this effect was potentiated in LGR5-expressing cells by the addition of RSPO2. Conversely, knockdown of TNC inhibited ES cell migration and invasion. Thus, we have shown that canonical Wnt signaling induces metastasis-associated transcriptional programs in ES, in particular the matrix-associated gene TNC, and that expression of TNC is potentiated by the LGR5-ligand RSPO2. Furthermore, we have identified that autocrine production of tenascin C promotes ES cellular migration and invasion, thus implicating the RSPO2-Wnt-TNC axis as a candidate effector of tumor metastasis. Citation Format: Elisabeth Anne Pedersen, Christopher A. Scannell, Rajasree Menon, Elizabeth R. Lawlor. Tenascin C is a canonical Wnt target gene in Ewing sarcoma and its expression is potentiated by R-spondin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3085. doi:10.1158/1538-7445.AM2014-3085