cGAS Downregulation Contributes to EGFR-TKI Resistance in NSCLC through the p-Nrf2-SIRT3-ROS/Ferroptosis Axis.

Abstract

Background: Although epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibition produces substantial initial benefit in EGFR-mutant non-small cell lung cancer, durable disease control is frequently compromised by the emergence of drug-resistant tumor cells. We therefore examined whether loss of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) supports the resistant phenotype by altering redox control and the cellular threshold for ferroptotic injury. Methods: The Gene Expression Omnibus (GEO) datasets GSE172002 and GSE236654 were analyzed to identify resistance-associated pathways. cGAS was depleted in parental cells and restored in resistant derivatives, followed by phenotypic, redox, mitochondrial, and signaling assessments in cultured cells and xenografts; pathway relationships were further examined by rescue experiments and structural modeling. Results: Bioinformatics analysis indicated significant alteration of DNA repair-related pathways in epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI)-resistant models. Resistant PC-9/GR and H1975/OR cells displayed increased half-maximal inhibitory concentration (IC₅₀) values and attenuated inhibition of phosphorylated EGFR (p-EGFR), Phosphorylated Protein Kinase B1 (p-AKT1), and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) after matched EGFR-TKI treatment. In parental cells, EGFR-TKI exposure was associated with increased DNA damage, cytosolic double-stranded DNA (dsDNA) accumulation, cGAS induction, ferroptosis-related staining patterns, reduced glutathione (GSH), and increased malondialdehyde (MDA), whereas these changes were less evident in resistant cells and were partly attenuated by Ferrostatin-1. Functionally, cGAS knockdown was associated with enhanced proliferative, migratory/invasive, and xenograft growth phenotypes, together with a redox pattern consistent with reduced ferroptosis susceptibility. Conversely, cGAS overexpression in resistant cells produced opposite effects. Altering cGAS abundance redistributed total and Ser40-phosphorylated nuclear factor erythroid 2-related factor 2 (Nrf2) between cellular compartments and concurrently changed sirtuin 3 (SIRT3) abundance and deacetylase activity. Nrf2 or SIRT3 manipulation partially reversed cGAS-associated phenotypes. Conclusions: These findings identify low cGAS abundance as a feature of the resistant state and support its involvement in an Nrf2-SIRT3-dependent antioxidant program that raises the threshold for ferroptotic damage.