Abstract

Background: Hepatocellular carcinoma (HCC), a highly aggressive malignancy with poor prognosis, is characterized by hyperactivation of the epidermal growth factor receptor (EGFR) signaling pathway. Glutaminase (GLS) is commonly overexpressed in numerous malignant tumors and acts as an oncogene to support cell growth and tumor progression, making it a target for cancer treatment. This study aimed to elucidate the underlying mechanisms of EGFR activation in driving glutaminolysis reprogramming and conferring ferroptosis resistance in HCC. Methods: Untargeted metabolomics, stable isotope-assisted metabolomic analysis, and RNA sequencing analysis were utilized to elucidate the mechanisms underlying glutaminolysis reprogramming upon EGFR activation. Immunoprecipitation, RNA pulldown, and dual-luciferase reporter assays were employed to examine the regulatory role of Wilms’ tumor 1-associated protein (WTAP) phosphorylation in GLS alternative splicing. Flow cytometry, cell viability assays, tumor-bearing mouse models, and HCC clinical specimens were used to validate the role of the AKT–WTAP–GLS axis in ferroptosis resistance and tumor progression. Results: Here, we demonstrated that AKT activated by EGFR signaling phosphorylated WTAP S176 and increased WTAP binding to methyltransferase-like protein 3. The enhanced interaction promoted the site-specific N6-methyladenosine (m 6 A) modification of GLS pre-mRNA, which in turn favored the alternative splicing of GLS toward glutaminase C (GAC) over kidney-type glutaminase. This switch led to increased glutamine utilization and glutathione/nicotinamide adenine dinucleotide phosphate (reduced form) biosynthesis, thereby alleviating ferroptosis and promoting tumor growth in mice. In addition, the levels of WTAP pS176 and GAC expression, which were mutually correlated, were positively associated with poor prognosis of patients with HCC. Conclusions: These findings uncover a critical mechanism by which tumor cells counter