Abstract
Background: The progression of prostate cancer is closely associated with dysregulation of tumor suppressor genes. P16 (CDKN2A), a key cell cycle regulator, is frequently downregulated in malignant phenotypes, yet the role of its post-translational modifications, particularly palmitoylation, remains unclear. This study aimed to investigate the regulatory mechanism of palmitoylation on P16 stability and explore the functional role of ZDHHC14 in this process, as well as its impact on the malignant behaviors of prostate cancer cells. Methods: CDKN2A was overexpressed in prostate cancer cells (PC-3 and DU-145) via plasmid transfection. The inhibitory effects of P16 on proliferation, migration, and invasion were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, colony formation assay, and Transwell assays. The palmitoylation modification site (Cys 72) and its regulatory role in P16 stability were analyzed through palmitoylation inhibitor [2-bromopalmitate (2-BP)] and agonist [hexadecyl acetyl phosphate (HAM)] treatments, site-directed mutagenesis (C72S), ubiquitination assays, and molecular docking. Bioinformatics screening, small interfering RNA (siRNA) knockdown, and co-immunoprecipitation (Co-IP) were employed to validate the interaction between ZDHHC14 and P16 and their functional relationship. Results: Overexpression of CDKN2A significantly inhibited the proliferation, migration, and invasion of prostate cancer cells. Palmitoylation at the Cys 72 site enhanced P16 stability by suppressing its ubiquitination-dependent degradation. ZDHHC14 was identified as the palmitoyltransferase responsible for modifying P16, and its expression positively correlated with patient survival. Knockdown of ZDHHC14 markedly reduced P16 protein levels, and their direct interaction was confirmed to mediate the palmitoylation process. Conclusions: This study reveals for the first time that ZDHHC14 enhances P16 stability and suppresses its ubiquitination-mediated degradation via palmitoylation at the Cys 72 residue, thereby exerting antitumor effects. These findings provide a theoretical foundation for developing therapeutic strategies targeting the ZDHHC14/P16 axis in prostate cancer.