Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

<b>Background/Objectives</b>: Ewing sarcoma is an aggressive pediatric sarcoma of bone and soft tissues, with metastatic disease being the most significant prognostic factor of poor outcome. We have previously reported that WNT974, a selective Porcn inhibitor, delays the onset of metastases in three different xenograft models of Ewing sarcoma with no effect on primary tumor growth, suggesting a specific role of the drug in metastasis. The goal of this work was to define the role of Wnt signaling in this process. <b>Methods</b>: We evaluated transcriptional changes in Wnt ligands upon Porcn inhibition using real-time PCR. Boyden chamber assays were used to quantify migration upon Wnt inhibition and addition of recombinant Wnt ligands. Changes in FAK, Src, ALCAM, and MCAM after treatment with WNT974 were evaluated using Western blots, immunofluorescence, and phalloidin staining. Wnt5a knock-out Ewing sarcoma cell lines were generated using Crispr-Cas9 editing to evaluate changes in migration and cytoskeletal arrangements. <b>Results</b>: We show that a non-canonical pathway responsive to Wnt5a drives Ewing sarcoma migration. Ewing sarcoma cells modulate their endogenous transcription of Wnt5a upon differing concentrations of exogenous Wnt5a exposure, suggesting an important feedback-dependent response. We demonstrate changes to FAK phosphorylation, cross-linking of filamentous actin by vinculin to ALCAM, and alterations in post-translational modifications of ALCAM, which all affect Ewing sarcoma cell migration. Crispr-Cas9 editing of Wnt5a results in an inability of the cells to migrate with a global lack of filamentous actin in the cell cytoskeleton. <b>Conclusions</b>: These findings suggest that a Wnt5a-dependent signaling pathway drives the cytoskeletal changes and cell adhesion molecule changes necessary for early steps of migration in the metastatic cascade.