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

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.