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ARD1 facilitates ferroptosis evasion via the induction of glutathione synthesis through catalysis of PABPC1 acetylation in HCC [CRISPR screen]

ABSTRACT: Aberrant upregulation of intracellular antioxidant glutathione (GSH) is implicated in promoting tumor proliferation, inducing drug resistance, and inhibiting ferroptosis across various malignancies, including hepatocellular carcinoma (HCC). However, the mechanism underlying the GSH metabolism reprogramming in HCC remains poorly understood. In this study, we employed a genome-wide CRISPR‒Cas9 screen and RNA-seq to identify ARD1 as a pivotal facilitator of de novo GSH synthesis in HCC. Notably, ARD1 upregulation is positively correlated with elevated GSH levels and a poor prognosis in HCC patients. In vivo and in vitro functional assays revealed that ARD1 promotes HCC cell proliferation and inhibits ferroptosis in a GSH-dependent manner. LC‒MS/MS-based stable isotope labeling revealed that ARD1 increases GSH levels by stabilizing the γ-glutamylcysteine ligase catalytic subunit (GCLC) transcript. Overall, this research underscores the crucial role of ARD1 in GSH metabolic reprogramming and ferroptosis regulation in HCC and reveals a novel strategy for ferroptosis-based targeted therapy for HCC.

ORGANISM(S): synthetic construct

PROVIDER: GSE280151 | GEO | 2025/08/02

REPOSITORIES: GEO

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Project description:Glutathione (GSH) is a critical endogenous antioxidant that protects against intracellular oxidative stress. As such, pathological alterations in GSH levels are linked to a myriad of diseases including cancer, neurodegeneration and cataract. The rate limiting step in GSH biosynthesis is catalyzed by the glutamate cysteine ligase catalytic subunit (GCLC). The high expression of GCLC in the lens supports the synthesis of millimolar concentrations of GSH in this tissue. Herein, we describe the morphological consequences of deleting (knocking out) Gclc from surface ectoderm-derived ocular tissues (using the Le-Cre transgene; Gclc KO) which includes an overt microphthalmia phenotype and severely disrupted formation of multiple ocular structures (i.e., cornea, iris, lens, retina). Controlling for the Le-Cre transgene revealed that the deletion of Gclc significantly exacerbated the microphthalmia phenotype in Le-Cre hemizygous mice and resulted in dysregulated gene expression that was unique to only the lenses of KO mice. We further characterized the impaired lens development by conducting an RNA-seq experiment on KO and Gclc control (CON) mouse lens at the day of birth. RNA-sequencing revealed significant differences between Gclc knockout (KO) and Gclc control (CON) lenses, including down-regulation of crystallins and lens fiber cell identity genes, and up-regulation of lens epithelial cell identity genes. In addition, genes related to the immune system (e.g., immune system process, inflammatory response, neutrophil chemotaxis) were upregulated, and genes related to eye/lens development were downregulated. TRANSFAC analysis of differentially expressed genes (DEGs) in the lens of Gclc KO mice implicated PAX6 as a key upstream regulator of Gclc KO sensitive genes. This was further supported by a strong positive correlation between the transcriptomes of the lenses of Gclc KO and Pax6 KO mice. Strikingly, the dysregulation of PAX6-regulated genes in Gclc KO mice was observed despite no change in the ocular localization of PAX6 or decrease in the expression of PAX6 in the lens. In vitro experiments demonstrated that suppression of intracellular GSH concentrations resulted in impairment of PAX6 transactivation activity. Taken together, the present results elucidate a novel mechanism wherein intracellular GSH concentrations may modulate PAX6 activity.

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ARD1 facilitates ferroptosis evasion via the induction of glutathione synthesis through catalysis of PABPC1 acetylation in HCC [CRISPR screen]

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