The Acetyltransferase ARD1 Induces Glutathione Synthesis to Facilitate Ferroptosis Evasion in Hepatocellular Carcinoma

Abstract Aberrant upregulation of the intracellular antioxidant glutathione (GSH) is implicated in promoting tumor proliferation, inducing drug resistance, and inhibiting ferroptosis across various malignancies, including hepatocellular carcinoma (HCC). Targeting the mechanism underlying GSH upregulation in HCC could represent a therapeutic strategy to improve patient outcomes. In this study, we employed a genome-wide CRISPR/Cas9 screen and targeted metabolomics to identify the acetyltransferase ARD1 as a pivotal facilitator of de novo GSH synthesis in HCC. Notably, ARD1 upregulation was positively correlated with elevated GSH levels and poor prognosis in patients with HCC. In vivo and in vitro functional assays revealed that ARD1 promoted HCC cell proliferation and inhibited ferroptosis in a GSH-dependent manner. LC/MS-MS–based stable isotope labeling revealed that ARD1 increased GSH levels by stabilizing γ-glutamylcysteine ligase catalytic (GCLC) subunit mRNA, which was mediated by the RNA-binding protein PABPC1. Mechanistically, ARD1 acetylated PABPC1 at Ki67, augmenting its cytoplasmic retention by disrupting PABPC1–importin α7 complex formation. Cytoplasmic PABPC1 then interacted with eIF4G to collaboratively stabilize GCLC mRNA, preventing its degradation, increasing GSH synthesis, and ultimately conferring ferroptosis resistance in HCC cells. Furthermore, oxidative stress induced by hydrogen peroxide suppressed ARD1 ubiquitination and degradation, thereby promoting PABPC1 cytoplasmic translocation and inducing GCLC expression. ARD1 suppression promoted sorafenib-mediated ferroptosis in xenografts derived from patients with HCC tumors with high ARD1 and GCLC expression. Overall, this research uncovers an oxidative stress–ARD1–PABPC1–GCLC axis with a crucial role in GSH metabolic reprogramming and ferroptosis regulation in HCC and reveals a strategy for ferroptosis-based targeted therapy for HCC. Significance: The acetyltransferase ARD1 promotes hepatocellular carcinoma progression, inhibits ferroptosis by upregulating GCLC to facilitate de novo synthesis of glutathione, and can be targeted to improve sorafenib efficacy.