HDAC3-mediated deacetylation of p21 stabilizes protein levels and promotes 5-FU resistance in colorectal cancer cells

5-Fluorouracil (5-FU) remains a cornerstone in colorectal cancer (CRC) chemotherapy; however, its clinical efficacy is often compromised by the development of resistance. Histone deacetylase 3 (HDAC3) has been implicated in chemoresistance across various cancers, yet its precise role in mediating 5-FU resistance in CRC remains poorly understood. In this study, we established a 5-FU-resistant CRC cell line (HCT116/5-FU) by gradually exposing parental HCT116 cells to increasing 5-FU concentrations. Screening of HDAC family members revealed significant upregulation of HDAC3 at both mRNA and protein levels in resistant cells. Mechanistically, we show that HDAC3 promotes 5-FU resistance by destabilizing the cyclin-dependent kinase inhibitor p21 through deacetylation, enhancing its ubiquitination and subsequent degradation. This HDAC3-mediated reduction in p21 levels disrupts cell cycle control, contributing to chemoresistance. Importantly, treatment with the HDAC3-specific inhibitor RGFP966 restored p21 stability, reduced colony formation, and sensitized HCT116/5-FU cells to 5-FU. Xenograft experiments further validated the synergistic efficacy of RGFP966 and 5-FU in vivo. These findings identify HDAC3 as a critical regulator of 5-FU resistance through modulation of p21 stability and suggest that combining HDAC3 inhibitors with conventional chemotherapy represents a promising strategy to overcome chemoresistance in CRC patients.