PAD4-mediated citrullination of IGF2BP2 stabilizes MCM mRNAs to drive intrahepatic cholangiocarcinoma progression

INTRODUCTION: Peptidyl arginine deiminase 4 (PAD4) catalyzes the deamination of arginine residues to citrulline, a post-translational modification known as citrullination, which regulates protein structure, function, and localization. Despite growing evidence connects PAD4 to cancer progression, its role in intrahepatic cholangiocarcinoma (ICC) remains largely unexplored. OBJECTIVES: This study aimed to elucidate the role and underlying mechanisms of PAD4 in the regulation of intrahepatic cholangiocarcinoma proliferation, as well as to explore its clinical relevance and therapeutic potential. METHODS: Hydrodynamic tail vein injection (HTVI) and subcutaneous xenograft models were used to evaluate the role of PAD4 in vivo. In vitro functional assays were performed to assess the effects of PAD4 on ICC cell proliferation. Putative targets were identified by RNA-seq and Co-IP/MS analyses. Molecular interactions were examined using GST pull-down, surface plasmon resonance (SPR), and RNA immunoprecipitation-PCR. The clinical relevance of PAD4 was evaluated in ICC patient samples using immunohistochemical analysis. RESULTS: A-modified MCM2-7 transcripts, thereby stabilizing and elevating MCM mRNAs expression. Clinically, high PAD4 expression correlated with poor prognosis in ICC patients, while PAD4 and IGF2BP2 co-expression predicted worse outcomes. In vivo, combined inhibition of PAD4 and IGF2BP2 synergistically suppressed ICC growth. CONCLUSION: Our findings identify a tumor-intrinsic PAD4-IGF2BP2 axis that drives ICC progression through citrullination-dependent stabilization of MCM transcripts. Dual targeting of PAD4 and IGF2BP2 represents a promising therapeutic strategy for ICC.