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5-Fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents for various cancers, including cholangiocarcinoma (CCA) and colorectal cancer (CRC). However, its therapeutic efficiency has remained unsatisfactory. A better understanding of the molecular mechanisms underlying 5-FU responsiveness is therefore crucial for developing more effective treatment strategies and improving patient survival. Mixed lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, has been implicated in cancer progression and therapeutic response. However, the exact roles of MLKL in modulating chemotherapy response, particularly 5-FU, has also remained unknown. Through a comprehensive bioinformatics analysis, we identified a significant association between high MLKL expression and poor therapeutic outcomes in CCA and CRC patients treated with 5-FU. Moreover, higher MLKL expression was detected in CRC patients who were clinically unresponsive to 5-FU-based treatments compared to responders, suggesting a crucial role of MLKL in mediating 5-FU response. Of particular interest, MLKL depletion sensitized CRC cells to 5-FU and enhanced its tumor-suppressive effects in a xenograft mouse model by promoting apoptosis. We propose that MLKL suppression potentiate TNF-α/TNFR-I-mediated apoptotic signaling, potentially by prolonging TNFR-I retention within the early endosome and delaying its degradation upon 5-FU treatment. Notably, silencing of TNFR-I attenuated 5-FU-induced cell death in MLKL-knockdown cells. These findings provide novel insights into previously unrecognized roles of MLKL in modulating 5-FU responsiveness and highlight MLKL as a potential predictive and therapeutic target to improve 5-FU efficacy in precision cancer therapy.