Cholangiocytes' Primary Cilia Regulate DNA Damage Response and Repair

Abstract

Primary cilia have been considered as 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 in CCA. 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, and KIF3A, followed by exposure to genotoxic agents such as cisplatin, methyl methanesulfonate (MMS), and irradiation. Cell survival, cell cycle progression, and apoptosis rates were evaluated, while DNA damage was assessed using comet assays and {gamma}H2AX quantification. An in vivo liver-specific knockout model of IFT88 was generated using the Cre/Lox recombination system. Results showed that RAD51 localized at the cilia base, while ATR, PARP1, CHK1 and CHK2 were found within the cilia. Deciliated cells displayed dysregulation in critical DDR pathways. These cells also showed reduced survival and increased S-phase arrest after genotoxic challenges. Enhanced DNA damage was observed via increased {gamma}H2AX signals and comet assay results. An increase in {gamma}H2AX expression was observed in our in vivo model, indicating elevated DNA damage. Additionally, key DDR proteins, such as ATM, p53, and p21, were downregulated in deciliated cells after irradiation. This study underscores the crucial role of primary cilia in regulating DNA repair, suggesting that targeting cilia-related mechanisms could present a novel therapeutic approach for CCA. Key PointsThe results presented delineate the following critical observations: O_LIDNA Damage Response (DDR) and DNA repair proteins localized to primary cilia structures C_LIO_LIDeciliated cells exhibited dysregulated DDR pathways, reduced survival, and S-phase arrest under genotoxic stress C_LIO_LIElevated DNA damage was evident in deciliated cells, with increased {gamma}H2AX signals and comet assay results C_LIO_LIIn vivo IFT88 knockout mice also showed increased {gamma}H2AX expression, indicating heightened DNA damage C_LIO_LIKey DDR proteins (ATM, p53, p21) were downregulated in deciliated cells after irradiation. C_LI