Abstract
<title>Abstract</title> <p>Radiotherapy is a mainstay treatment for esophageal squamous cell carcinoma (ESCC), but radioresistance remains a major clinical challenge. Ferroptosis has been implicated in cancer therapy responses, yet how ESCC cells evade ferroptosis to acquire radioresistance is unclear. Here, ESCC cell lines (ECA109 and TE-1) and a subcutaneous xenograft mouse model were employed. GPX4 was modulated by shRNA or RSL3, and methyltransferaselike 3(METTL3) was knocked down by shRNA or inhibited by DAA or STM2457. Glutathione peroxidase 4 (GPX4) was overexpressed using a plasmid. Radiosensitivity was assessed by CCK-8, colony formation, and tumor growth assays, while ferroptosis was evaluated by ROS and Fe²⁺ measurements. m6A modification of GPX4 mRNA was analyzed by MeRIP-qPCR, protein interactions by Co-IP and IF, O-GalNAcylation sites by site-directed mutagenesis and VVL pull-down, and protein stability by CHX chase assays. Irradiation (4 Gy) upregulated GPX4, and GPX4 knockdown or RSL3 significantly enhanced radiosensitivity. Mechanistically, irradiation induced METTL3, which installed m6A modifications on GPX4 mRNA, increasing its stability and expression. METTL3 knockdown reduced GPX4, promoted ferroptosis, and sensitized cells to radiation, effects rescued by GPX4 overexpression. The O-GalNAc transferase Glycosyltransferase 2(GALNT2) interacted with METTL3 and mediated its O-GalNAcylation at S64. GALNT2 overexpression upregulated METTL3 and GPX4, suppressed ferroptosis, and promoted radioresistance, which was reversed by METTL3 knockdown or STM2457. Our study identifies a GALNT2-METTL3-GPX4 axis that promotes radioresistance in ESCC by suppressing ferroptosis. Targeting this axis via METTL3 or GALNT2 inhibition may represent a promising strategy to overcome radioresistance in ESCC.</p>