Cholangiocytes' primary cilia regulate DNA damage response and repair.

Abstract

Primary cilia have been considered tumor-suppressing organelles in cholangiocarcinoma (CCA), though the mechanisms behind their protective role are not fully understood. This study investigates how the loss of primary cilia affects DNA damage response (DDR) and DNA repair processes. Human cholangiocyte cell lines were used to examine the colocalization of DNA repair proteins at the cilia and assess the impact of experimental deciliation on DNA repair pathways. Deciliation was induced using shRNA knockdown or CRISPR knockout of IFT20, IFT88, or KIF3A, followed by exposure to the genotoxic agents cisplatin, methyl methanesulfonate (MMS), or irradiation. Cell survival, cell cycle progression, and apoptosis rates were evaluated, and DNA damage was assessed using comet assays and phosphorylated H2AX (γH2AX) quantification. An in vivo liver-specific IFT88 knockout model, generated using Albumin-Cre/Lox recombination, was used to study the loss of primary cilia in the liver. Results showed that RAD51 localized predominantly at the base of the cilium, whereas Ataxia Telangiectasia and Rad3-related protein (ATR), PARP1, CHK1, and CHK2 were also detected within the ciliary shaft. Deciliated cells displayed dysregulation in critical DNA repair pathways. These cells also showed reduced survival and increased S-phase arrest after genotoxic challenges as compared with ciliated cells. Enhanced DNA damage was observed via increased γH2AX signals and comet assay results. An increase in γH2AX expression was also observed in our in vivo model, indicating elevated DNA damage. In addition, key DDR proteins such as Ataxia Telangiectasia Mutated protein (ATM), p53, and p21, were downregulated in deciliated cells after irradiation. This study underscores the crucial role of primary cilia in regulating DNA repair and suggests that targeting cilia-related mechanisms could present a novel therapeutic approach for CCA.NEW & NOTEWORTHY Our study reveals a novel link between primary cilia and DNA repair in cholangiocytes. We show that DNA damage response (DDR) and repair proteins localize to cilia, and deciliation impairs survival and induces S-phase arrest under genotoxic stress. Deciliated cells exhibit increased DNA damage after cisplatin, irradiation, or methyl methanesulfonate (MMS) challenge. Following irradiation, Ataxia Telangiectasia Mutated protein (ATM), p53, and p21 are downregulated in deciliated cells. Similarly, IFT88 knockout mice show heightened DNA damage, highlighting the role of primary cilia in genome stability.