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Abstract 957: Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma


 Introduction: Metastatic Ewing sarcoma (ES) has a dismal prognosis, and clarifying the molecular and cellular mechanism of metastasis is the most pressing need to improving prognosis. Recently, the heterogenous expression of the driving fusion gene EWS-FLI1, as well as canonical Wnt/β-catenin signaling activity has been increasingly recognized as a potential mediator of metastatic progression. However, targeting the pathway has been difficult due to its complexity and cross-activity, and not much is known of the mechanism of how ES cells maintain Wnt signaling heterogeneity. Cadherin-11 (CDH11) is a member of the cadherin super-family, which modulates calcium ion-dependent cell-cell adhesion and signal transduction, and is highly expressed in ES. CDH11 has been found to regulate the expression level of β-catenin, and play a key role in enhancing the stem cell, migration and invasion in other carcinomas, however the relationship between CDH11 and β-catenin in ES is not clear.
 Methods: Wnt/β-catenin dependent transcriptional activity of ES cells in vitro and in vivo were assessed by flow cytometry, immune fluorescence, as well as single cell RNA-seq. β-catenin and CDH11 knock-down cell lines were generated, and affected on invasion/migration, and metastatic progression was assessed in vitro and in vivo. Furthermore, the interaction of CDH11 with β-catenin, and the contribution of the protein-protein interaction to the heterogenous activation of Wnt/β-catenin was assessed by immunofluorescence, flowcytometry, Western blot, and co-immunoprecipitation.
 Results: At baseline, only a small fraction of ES cells have activated Wnt/β-catenin dependent transcriptional activity both in vitro and in vivo, which can respond rapidly with Wnt ligand stimulation. β-catenin knockdown reduced clonogenicity in vitro, and metastatic tumor burden in the orthotopic implantation amputation mouse model of ES. We further identified that most of the tested ES cells had abundant β-catenin bound to CDH11 in the cell membrane, which would rapidly be released through the cytoplasm and into the nucleus within 2 hours of adding exogenous Wnt ligand. CDH11 knockdown reduced β-catenin expression, rapid activation of signaling by Wnt ligands, and metastasis progression.
 Conclusion: Our data suggests that the cell surface CDH11- β-catenin complex contributes to the heterogenous and dynamic activation of the Wnt/β-catenin pathway in ES that drives ES metastasis. Targeting the CDH11- β-catenin complex can present a novel strategy to interfere with Wnt driven metastasis in ES.
 Citation Format: Ryota Shirai, Tyler Biebighauser, Sarah Nelson-Taylor, Avery Bodlak, Timothy Porfilio, Deandra Walker, Naoki Oike, Masanori Hayashi. Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 957.