Abstract PS2-12-04: Promoter Hypermethylation-Induced Silencing of FXYD1 Drives Breast Cancer Metastasis via DDX5-Mediated Wnt/β-Catenin Pathway Activation

Abstract Background: Epigenetic dysregulation, particularly promoter hypermethylation, plays a critical role in the silencing of tumor suppressor genes during cancer progression. Although FXYD1 has been associated with ion transport regulation, its epigenetic regulation and functional significance in breast cancer metastasis remain to be elucidated. Methods: FXYD1 expression was evaluated using public datasets (TCGA, GEO), breast cancer cell lines, and patient-derived tissues. Functional analyses, including proliferation assays, Transwell invasion assays, and murine xenograft metastasis models, were conducted following FXYD1 overexpression or knockdown. Mechanistic studies involved RNA sequencing, co-immunoprecipitation, DNA methylation analysis, ubiquitination assays, and genetic rescue experiments. Results: FXYD1 expression was significantly downregulated in breast cancer tissues compared with adjacent normal tissues (p<0.001, Student's t-test), and low expression was associated with advanced TNM stage and decreased overall survival (HR=1.83, 95% CI 1.2-2.8). Methylation analysis demonstrated that promoter hypermethylation was a critical mechanism underlying FXYD1 silencing. Functional restoration of FXYD1 suppressed tumor proliferation (50% reduction in colony formation, p<0.01), migration (70% decrease in wound healing, p<0.001), and lung metastasis (4-fold reduction in nodules, p<0.005). Mechanistically, FXYD1 facilitated Lysine 63 (K63)-linked polyubiquitination of DDX5 at residue K470 by recruiting the E3 ligase MAEA, leading to proteasomal degradation. This resulted in inhibition of Wnt/β-catenin signaling, as evidenced by reduced nuclear β-catenin (60% decrease) and downstream target expression (c-Myc, Cyclin D1). Rescue experiments confirmed that DDX5 re-expression (1.5-fold increase) or β-catenin stabilization (via CHIR99021) reversed FXYD1-mediated tumor suppression. Conclusions: Our findings establish FXYD1 as a previously unrecognized tumor suppressor that inhibits breast cancer metastasis via the DDX5-β-catenin axis. This study not only clarifies a novel ubiquitination regulatory mechanism but also highlights FXYD1 as a potential therapeutic target for metastasis intervention. Citation Format: P. Wen, G. Wang, F. Qu, Q. Shao, L. Wang, N. Zhang, X. Zeng. Promoter Hypermethylation-Induced Silencing of FXYD1 Drives Breast Cancer Metastasis via DDX5-Mediated Wnt/β-Catenin Pathway Activation [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-04.