Dihydroberberine regulates the ferroptosis-autophagy positive feedback loop in colorectal cancer by targeting PANX2.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide and remains a major clinical challenge, underscoring the urgent need for novel therapeutic targets and treatment strategies. Ferroptosis, a form of cell death triggered by iron-dependent lipid peroxidation, is emerging as a promising new anti-cancer therapeutic strategy. PURPOSE: This study aims to identify a key target regulating the ferroptosis process in CRC, screen for small molecule modulators against this target, and elucidate their potential anti-tumor mechanisms. METHODS: We developed a Drug Discovery Strategy for Targeted Ferroptosis Therapy Based on Bioinformatics-Machine Learning Integration for the Treatment of CRC (DDTF-BMLI-CRC), aiming to identify key ferroptosis regulators. The functional role of this factor in CRC and ferroptosis was validated through knockdown and overexpression techniques, establishing it as a potential therapeutic target. Subsequently, candidate compounds were screened from natural product and FDA databases using a dual-scoring model combining machine learning and deep learning. The direct binding of candidate compounds to target proteins was validated through molecular docking, molecular dynamics simulations, DARTS, CETSA, and SPR techniques. Finally, a series of in vitro and in vivo experiments were conducted to systematically evaluate their anti-tumor effects and potential mechanisms. RESULTS: PANX2 was identified as a key ferroptosis-suppressing gene in CRC. We discovered the natural small molecule dihydroberberine (dhBBR) to be a potent and direct inhibitor of the PANX2 protein. In vitro, dhBBR significantly inhibited the proliferation, migration, and invasion of CRC cells while inducing ferroptosis. In vivo, dhBBR effectively suppressed xenograft tumor growth. Mechanistic studies revealed that dhBBR-induced ferroptotic stress activates autophagy, which in turn promotes GPX4 degradation, thereby amplifying the ferroptotic effect and establishing a ferroptosis-autophagy positive feedback loop. Crucially, PANX2 knockdown largely abolished the additional anti-tumor effect of dhBBR, and dhBBR did not further suppress tumor growth beyond PANX2 knockdown alone. CONCLUSION: This study demonstrates that PANX2 knockdown suppresses CRC progression by inducing ferroptosis. Furthermore, we identified dhBBR for the first time as a PANX2-targeting small-molecule inhibitor. Our research reveals a novel therapeutic strategy targeting the PANX2-mediated ferroptosis-autophagy axis and provides a highly promising candidate compound for the treatment of CRC.