# Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma

## TL;DR

This study shows that Wnt5a, a protein in a non-canonical signaling pathway, promotes migration in Ewing sarcoma by altering cell structure and adhesion, offering new targets for preventing metastasis.

## Contribution

The study identifies Wnt5a as a key driver of Ewing sarcoma migration through non-canonical Wnt signaling, revealing novel mechanisms for targeting metastasis.

## Key findings

- Wnt5a drives Ewing sarcoma migration via non-canonical signaling, altering cytoskeleton and cell adhesion.
- Wnt5a knockout cells show reduced migration and disrupted filamentous actin structures.
- Modulation of Wnt5a affects phosphorylation of FAK and post-translational modifications of ALCAM.

## Abstract

Ewing sarcoma is the second most common bone tumor in children, adolescents and young adults. Over the last three decades, the five-year survival rate for metastatic Ewing sarcoma has not improved, highlighting the need to better understand the mechanisms driving metastasis. Wnt signaling has been implicated in driving migration, invasion, and metastasis in various cancers. In this study, we show that a non-canonical Wnt signaling pathway drives Ewing sarcoma migration, with Wnt5a being a key regulator of this mechanism. We demonstrate changes in the cytoskeleton and in cell adhesion molecule expression with the activation of this pathway, supporting the role of non-canonical Wnt signaling in altering cell intrinsic properties to drive migration. Our work highlights the need to target Wnt5a-responsive non-canonical pathways, along with conventional beta-catenin-dependent canonical pathways, when developing clinical trials to improve patient outcomes by preventing metastatic relapse.

Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.