Pemetrexed induces ROS generation and cellular senescence by attenuating TS-mediated thymidylate metabolism to reverse gefitinib resistance in non-small cell lung cancer

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are strongly recommended for non-small cell lung carcinoma (NSCLC) patients harboring active EGFR mutations, while drug resistance inevitably makes exploring the resistance mechanisms and seeking effective therapeutic strategies urgent endeavors. Thymidylate synthetase (TYMS or TS) is a dominant enzyme in thymidylate nucleotide metabolism. In this study, based on public database analysis and examination of gene sets from 140 NSCLC patients that received EGFR-TKI therapy, we found a significantly positive correlation between TS expression and overall survival (OS) and disease-free survival (DFS) in lung adenocarcinoma. Twenty-four tissue specimens from NSCLC patients exhibited upregulated TS mRNA expression in NSCLC patients resistant to gefitinib. The human NSCLC cell line PC9, which is sensitive to gefitinib, and relatively resistant PC9/GR cells were used to demonstrate that knockdown of TS restored the sensitivity of resistant cells to gefitinib. Furthermore, pemetrexed effectively suppressed TS-mediated thymidylate metabolism and induced ROS generation and cellular senescence, thereby hampering carcinogenesis and restoring cell sensitivity to gefitinib. The combination of pemetrexed and gefitinib damaged the proliferation, migration and invasion capabilities of gefitinib-resistant cells, exhibiting a synergistic anticancer effect. Our findings illuminate the potential mechanism of TS-triggered gefitinib resistance and indicate that inhibition of TS by pemetrexed can potentiate the effect of gefitinib in NSCLC cells resistant to gefitinib. Pemetrexed combined with gefitinib has potent anti-progression potential in gefitinib-resistant NSCLC. This suggests that NSCLC patients with both high TS expression and EGFR-driving mutations might benefit more from a combination strategy of EGFR-TKIs and pemetrexed-based chemotherapy than EGFR-TKI monotherapy, which has profound clinical implications and considerable therapeutic value.