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.
背景/目的：尤文肉瘤是一种侵袭性儿童骨与软组织肉瘤，转移性病变是预后不良的最重要因素。我们先前报道过，选择性Porcn抑制剂WNT974在三种不同的尤文肉瘤异种移植模型中能延缓转移发生，但对原发肿瘤生长无影响，提示该药物在转移过程中具有特异性作用。本研究旨在明确Wnt信号通路在此过程中的作用机制。 方法：采用实时荧光定量PCR检测Porcn抑制后Wnt配体的转录变化。通过Boyden小室实验评估Wnt抑制及重组Wnt配体添加对细胞迁移的影响。运用蛋白质印迹、免疫荧光和鬼笔环肽染色技术分析WNT974处理后FAK、Src、ALCAM和MCAM的变化。利用Crispr-Cas9基因编辑技术构建Wnt5a敲除的尤文肉瘤细胞系，以评估其对细胞迁移和细胞骨架排列的影响。 结果：研究发现，响应Wnt5a的非经典信号通路驱动尤文肉瘤细胞迁移。尤文肉瘤细胞在不同浓度外源性Wnt5a刺激下会调节内源性Wnt5a转录，表明存在重要的反馈依赖性调节机制。研究证实FAK磷酸化改变、纽蛋白介导的丝状肌动蛋白与ALCAM交联，以及ALCAM翻译后修饰变化均影响尤文肉瘤细胞迁移。Wnt5a的Crispr-Cas9编辑导致细胞迁移能力丧失，并引起细胞骨架中丝状肌动蛋白全面缺失。 结论：这些发现表明，Wnt5a依赖性信号通路驱动了转移级联反应早期迁移步骤所必需的细胞骨架改变和细胞黏附分子变化。
Wnt5a Regulates Focal Adhesion Formation to Promote Migration in Ewing Sarcoma
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