Abstract 3098: Canonical Wnt signaling is activated during the initiation of the migratory phenotype of Ewing's sarcoma family of tumors (ESFT)
Authors: Shaw Lucy A. , Gaffney Andrew P. , Payne Helen L. , Burchill Susan A. 
Published: May 2015

Abstract
Activation of canonical and noncanonical Wnt signaling pathways in the initiation of the Ewing's sarcoma family of tumors (ESFT) migratory phenotype has been investigated using an in vitro model of ESFT.
Human embryonic stem (hES) cells transfected with a doxycycline-inducible expression vector containing EWS-FLI1 (hES-EWS-FLI1) have been investigated. Expression of EWS-FLI1 was confirmed by RT-qPCR and western blot, and induction of cell surface CD99 and EWS-FLI1 target gene expression analyzed by flow cytometry and RT-qPCR, respectively. Migration was assessed following seeding of cell spheroids onto gelatin-coated plates. The pixel count of the migrated area was calculated at 72h and expressed relative to the pixel count at 0h to give relative migration (RM). Changes in the actin cytoskeleton were examined by phalloidin staining and immunofluorescence microscopy. Viable cell number was measured using the trypan blue exclusion assay. Expression of components (n=91) of the Wnt signaling pathway were analyzed using TaqMan® WNT Pathway arrays (Applied Biosystems). Activity of canonical Wnt signaling was quantified using a lentiviral TCF-luciferase reporter assay and noncanonical Wnt signaling using a Rac1 pull-down assay.
Expression of EWS-FLI1 RNA and protein was induced in hES-EWS-FLI1+ cells after 72h exposure to doxycycline (5µg/ml). Induction of EWS-FLI1 was tolerated by hES cells and had no effect on viable cell number. However, these cells grew as a substrate adherent monolayer, in contrast to the hES-EWS-FLI1- cells that retained the colony-phenotype reminiscent of the parent hES cells. hES-EWS-FLI1+ cells had fewer cell-cell adhesions and produced filopodia that were absent in hES-EWS-FLI1- cells. Interestingly, migration of hES-EWS-FLI1+ cells was significantly increased compared to that of hES-EWS-FLI1- cells (RM= 2 compared to 30, p&lt;0.01). Expression of CD99 (p&lt;0.01) and other EWS-FLI1 target genes including CAV1 (p&lt;0.05), IGF1 (p&lt;0.01) and NR0B1 (p&lt;0.0001) were significantly increased in hES-EWS-FLI1+ cells. Components (n=8) of the Wnt signaling pathway were also differentially expressed in hES-EWS-FLI1+ cells. Importantly canonical Wnt signaling was increased two-fold in hES-EWS-FLI1+ cells, within 72 hours of EWS-FLI1 expression. There was a ten-fold increase in the activation of canonical Wnt signaling following treatment of hES-EWS-FLI1+ cells with recombinant Wnt3A (200ng/ml, 24 hours) compared to that in hES-EWS-FLI1- cells (1X compared to 11X, p&lt;0.05). In contrast, the activity of the noncanonical Wnt signaling pathway was not increased in hES-EWS-FLI1+ cells and these cells did not respond to the noncanonical ligand Wnt5A.
In conclusion, activation of the canonical Wnt signaling pathway may be an important step in the initiation of ESFT. Targeting this pathway could be a useful approach to prevent relapse and metastasis.
Citation Format: Lucy A. Shaw, Andrew P. Gaffney, Helen L. Payne, Susan A. Burchill. Canonical Wnt signaling is activated during the initiation of the migratory phenotype of Ewing's sarcoma family of tumors (ESFT). [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 3098. doi:10.1158/1538-7445.AM2014-3098