β‐Arrestin1 Promotes Prostate Cancer Progression via Modulating ESRP1‐Mediated Alternative Splicing of CD44

ABSTRACT Metastatic prostate cancer (PCa) is a major cause of poor prognosis and mortality, but its underlying mechanisms remain incompletely understood. This study investigated the role of β‐arrestin1 (ARRB1) and its regulation of epithelial splicing regulatory protein 1 (ESRP1)‐mediated CD44 alternative splicing in PCa progression. Immunohistochemistry, RT‐qPCR, western blotting, and analyses of three GEO cohorts showed higher ARRB1 expression in metastatic than localized PCa. Gain‐ and loss‐of‐function experiments combined with MTS, colony‐formation, wound‐healing, and Transwell assays demonstrated that ARRB1 enhanced PCa cell proliferation, migration, and invasion and induced epithelial–mesenchymal transition. Subcutaneous tumor and tail‐vein metastasis models, evaluated by tumor measurements and IVIS imaging, provided in vivo evidence of increased tumor growth and pulmonary colonization. RNA sequencing, pathway enrichment, expression analyses, and CRISPR‐Cas9‐mediated CD44 knockout supported a role for CD44 in ARRB1‐driven malignant phenotypes. ESRP1 rescue experiments showed that ESRP1 counteracted ARRB1‐induced phenotypes and altered CD44 isoform expression. Isoform‐specific transfection and functional assays identified CD44v2 and CD44v6 as potential key splice variants through which ESRP1 exerts its tumor‐suppressive effects. These findings indicate that ARRB1 promotes PCa progression by suppressing ESRP1 expression, thereby promoting CD44s expression while reducing CD44 variant isoforms. ARRB1 may therefore represent a potential therapeutic target warranting further investigation.