Fuzheng Kang'ai Formula suppresses NSCLC by inducing a functional crosstalk between autophagy and ferroptosis via EZH2/DOT1L/H3K79me2 signaling.

[BACKGROUND] Fuzheng Kang'ai Formula (FZKA) is a clinically effective traditional Chinese medicine compound with demonstrated efficacy against non-small cell lung cancer (NSCLC). This study systematically investigated its anti-tumor mechanisms, focusing on the interplay between autophagy and ferroptosis.

[PURPOSE] We aimed to determine whether FZKA suppresses NSCLC through autophagy-ferroptosis crosstalk and to identify key epigenetic regulators involved in this process.

[STUDY DESIGN] The study combined in vitro assays using NSCLC cell lines with in vivo validation via subcutaneous xenograft models.

[METHODS] An Agilent 6546 LC/Q-TOF liquid chromatography-mass spectrometry system was used to detect the stability of component contents in FZKA across different batches. Autophagy was assessed through mRFP-GFP-LC3 adenovirus assay, transmission electron microscopy, and LC3B-II/I conversion. Ferroptosis was evaluated by measuring lipid peroxidation and intracellular Fe²⁺ levels. Crosstalk was examined using autophagy and ferroptosis inhibitors to monitor reciprocal effects on SLC7A11 and p-mTOR. EZH2 was investigated as an upstream regulator of mTOR and SLC7A11. Protein and mRNA expression were analyzed by Western blot and qRT-PCR. EZH2-overexpressing cells were established to evaluate functional rescue, and lysine methylation label-free quantitative proteomics was employed to identify downstream effectors. A nude mouse xenograft model was used for in vivo validation, with tumor weight and volume measured, and key proteins analyzed.

[RESULTS] The composition of FZKA remains stable across different batches. FZKA induced autophagy via mTOR suppression and Beclin1 upregulation, while promoting ferroptosis through SLC7A11 and GPX4 inhibition. Functional crosstalk was confirmed through inhibitor experiments showing mTOR and SLC7A11 as key mediators. FZKA significantly reduced EZH2 expression, and EZH2 overexpression partially reversed the effects of FZKA on mTOR and SLC7A11. Proteomic analysis revealed DOT1L/H3K79me2 as a downstream pathway of FZKA-mediated EZH2 regulation. DOT1L expression correlated with EZH2 levels, and EZH2 overexpression rescued DOT1L/H3K79me2 suppression. These results were consistently verified in vivo.

[CONCLUSION] FZKA suppresses NSCLC by regulating the EZH2/DOT1L/H3K79me2 signaling to induce crosstalk between mTOR-mediated autophagy and SLC7A11-dependent ferroptosis, leading to extensive cell death. This study reveals a novel epigenetic mechanism coordinating two cell death pathways and supports FZKA's clinical application.