Downregulation of AATK enhances susceptibility to ferroptosis by promoting endosome recycling in gefitinib-resistant lung cancer cells

Ferroptosis has been characterised by disruption of the cell membrane through iron-related lipid peroxidation. However, regulation of iron homeostasis in lung cancer cells which are resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains unclear. Transcriptome analysis identified a significant downregulation of apoptosis-associated tyrosine kinase (AATK) mRNA expression in gefitinib-resistant PC9 (PC9-GR) cells, which were found to be more susceptible to ferroptosis inducers. An in-depth analysis of publicly available datasets revealed that downregulation of AATK mRNA was associated with lymph node metastasis and poor prognosis in patients with lung adenocarcinoma. Knockdown of AATK sensitized PC9, HCC827 and H441 cells to the ferroptosis inducer RSL3, whereas ectopic expression of AATK reduced RSL3-induced cell death in PC9-GR and HCC827-GR cells. Compared to PC9 cells, PC9-GR cells exhibited higher transferrin uptake, endosome recycling rate, and increased intracellular iron levels. Blocking iron transport reduced RSL3-induced ferroptosis in PC9-GR cells. Mechanistic studies showed that AATK localized to both early and recycling endosomes. Knockdown of AATK facilitated endosome recycling and elevated intracellular ferrous iron (Fe2+) levels in PC9 cells. Conversely, ectopic expression of AATK delayed endosome recycling and reduced intracellular Fe2+ levels in PC9-GR cells. Inhibition of AATK downregulation-induced iron accumulation decreased RSL3-induced ferroptosis. Taken together, our study indicates that the downregulation of AATK contributes to endosome recycling and iron accumulation, leading to an increased susceptibility to ferroptosis in EGFR-TKI-resistant lung cancer cells. Overall design: The PC9-GR cell line was established by stepwise escalation of gefitinib from 0.5 µM to 10 µM. The PC9-GR cells were routinely maintained in the complete medium containing 10 µM gefitinib. Prior to the total RNA extraction, PC9-GR cells were cultured in a complete medium without gefitinib for a duration of 7 days. The total RNA samples were obtained from 3 independent biological repeats of PC9 and PC9-GR cell cultures.