ESRP2-dependent CD44 isoform switching is functionally linked to epithelial-mesenchymal plasticity in bladder cancer.

Epithelial-mesenchymal transition (EMT) drives phenotypic plasticity in bladder cancer and contributes to tumor progression, therapeutic resistance, and recurrence. While epithelial splicing regulatory proteins (ESRPs) are established modulators of EMT-associated splicing programs, the downstream mechanisms through which they influence tumor cell state remain incompletely understood. Here, we identify CD44 isoform switching as a functionally relevant component of ESRP2-dependent control of epithelial plasticity. Using epithelial (RT4) and mesenchymal (UM-UC-3) bladder cancer models, we show that distinct cellular states are characterized by different CD44 isoform landscapes, with epithelial cells enriched in CD44 variant isoforms and mesenchymal cells dominated by the standard isoform CD44s. Functional perturbation studies revealed that ESRP2 exerts a stronger effect than ESRP1 on CD44 isoform composition, promoting epithelial-associated CD44 variant isoforms while suppressing CD44s. Induction of EMT in RT4 cells was accompanied by downregulation of ESRP1/2 and a shift toward CD44s enrichment. Conversely, depletion of CD44s attenuated EMT-associated transcriptional and morphological changes and partially reversed the effects of ESRP2 loss on EMT marker expression. In mesenchymal UM-UC-3 cells, CD44s depletion reduced migratory capacity and impaired spheroid growth, further supporting a functional contribution of CD44 isoform balance to mesenchymal behavior. Collectively, our findings indicate that ESRP2-dependent regulation of CD44 isoform composition is closely linked to epithelial-mesenchymal plasticity in bladder cancer. These findings provide insight into splicing-associated regulation of tumor cell state and underscore the importance of isoform-resolved analyses for understanding cancer progression.