Hippo Pathway Modulation Alters the Proteome and Migratory Phenotype of Prostate Cancer Cells

Abstract

ABSTRACT Background The Hippo pathway effector YAP1 has been implicated in prostate cancer progression, migration, and castration resistance, yet how pharmacological YAP1 inhibition reshapes the prostate cancer proteome remains uncharacterized. Methods We used label‐free quantitative proteomics together with functional assays to characterize the proteomic and phenotypic effects of CA3, a selective YAP1 inhibitor, in androgen‐independent PC3 and androgen‐sensitive LNCaP prostate cancer cells, and validated key findings with wound‐healing, western blot, and immunodetection assays for focal adhesion kinase (FAK), vimentin, occludin, and junctional adhesion molecule A (JAM‐A). Results CA3 reduced cell viability and YAP1 protein levels in both cell lines in a dose‐ and time‐dependent manner ( p < 0.05). Nonlinear regression analysis yielded 24 h IC50 values of 1.59 µM (95% CI, 1.38–1.83) for PC3 and 0.63 µM (95% CI, 0.53–0.77) for LNCaP, with corresponding 48 h values of 0.59 µM (95% CI, 0.50–0.69) and 0.49 µM (95% CI, 0.39–0.62). CA3 showed markedly lower potency in the non‐tumorigenic PNT1A prostate epithelial cell line (IC50 18.87 µM at 24 h; 4.47 µM at 48 h), corresponding to an approximately 7‐ to 30‐fold selectivity window favoring the cancer cell lines. Label‐free quantitative proteomics identified 365 and 153 differentially expressed proteins (DEPs) in PC3 and LNCaP cells, respectively. Functional enrichment analysis revealed cell line‐dependent alterations in pathways related to RNA metabolism and translation, alongside migration‐ and adhesion‐associated processes, including focal adhesion, cytoskeletal organization, and guidance signaling. Comparison of these proteins with the Gene Ontology cell migration gene set (GO:0016477) identified EGFR, SRC, TGFB1, IL6, and SCARB1 as hub proteins in PC3 cells, and RAC1, RHOC, NF1, FSCN1, and ASPM in LNCaP cells. Functional validation confirmed that CA3 reduced wound closure in both cell lines, decreased FAK protein levels, selectively reduced vimentin in PC3 cells, increased occludin in both cell lines, and increased JAM‐A specifically in PC3 cells (all p < 0.05). Conclusions To our knowledge, this is the first study to characterize the CA3‐induced proteome in prostate cancer cells and link these changes to migration‐associated protein networks, in cell lines with differing androgen dependence.