Background: Pemetrexed (MTA) plus cisplatin combination therapy is considered the standard of care for patients with advanced non-small-cell lung cancer (NSCLC). However, in advanced NSCLC, the 5-year survival rate is below 10%, mainly due to resistance to therapy. We have previously shown that the fraction of mesenchymal-like, chemotherapy-resistant paraclone cells increased after MTA and cisplatin combination therapy in the NSCLC cell line A549. Cytidine deaminase (CDA) and thymidine phosphorylase (TYMP) are key enzymes of the pyrimidine salvage pathway. 5'-deoxy-5-fluorocytidine (5'-DFCR) is a cytidine analogue (metabolite of capecitabine), which is converted by CDA and subsequently by TYMP into 5-fluorouracil, a chemotherapeutic agent frequently used to treat solid tumors. The aim of this study was to identify and exploit chemotherapy-induced metabolic adaptations to target resistant cancer cells.
Methods: Cell viability and colony formation assays were used to quantify the efficacy of MTA and cisplatin treatment in combination with schedule-dependent addition of 5'-DFCR on growth and survival of A549 paraclone cells and NSCLC cell lines. CDA and TYMP protein expression were monitored by Western blot. Finally, flow cytometry was used to analyze the EMT phenotype, DNA damage response activation and cell cycle distribution over time after treatment. CDA expression was measured by immunohistochemistry in tumor tissues of patients before and after neoadjuvant chemotherapy.
Results: We performed a small-scale screen of mitochondrial metabolism inhibitors, which revealed that 5'-DFCR selectively targets chemotherapy-resistant A549 paraclone cells characterized by high CDA and TYMP expression. In the cell line A549, CDA and TYMP expression was further increased by chemotherapy in a time-dependent manner, which was also observed in the KRAS-addicted NSCLC cell lines H358 and H411. The addition of 5'-DFCR on the second day after MTA and cisplatin combination therapy was the most efficient treatment to eradicate chemotherapy-resistant NSCLC cells. Moreover, recovery from treatment-induced DNA damage was delayed and accompanied by senescence induction and acquisition of a hybrid-EMT phenotype. In a subset of patient tumors, CDA expression was also increased after treatment with neoadjuvant chemotherapy.
Conclusions: Chemotherapy increases CDA and TYMP expression thereby rendering resistant lung cancer cells susceptible to subsequent 5'-DFCR treatment.
Keywords: 5′-DFCR; Chemotherapy resistant; Cisplatin; Cytidine deaminase (CDA); DNA damage; Non-small cell lung cancer; Pemetrexed; Thymidine phosphorylase (TYMP).