Chemotherapy-induced CDA expression renders resistant non-small cell lung cancer cells sensitive to 5′-deoxy-5-fluorocytidine (5′-DFCR)

Abstract

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.

DNA methylome and single-cell transcriptome analyses reveal CDA as a potential druggable target for ALK inhibitor–resistant lung cancer therapy

“…Contrary to our expectation, immunohistochemistry of CDA showed no significant difference between pre-and post-ALK inhibitor therapy (Fig. 6e, left), which might be attributable to inter-or intratumoral heterogeneity of CDA expression even in the same patient 38 . Representative CDA expression in tumors from pre-or post-TKI therapy is shown in Fig.…”

Schedule-Dependent Treatment Increases Chemotherapy Efficacy in Malignant Pleural Mesothelioma

“…Visual examination revealed that a significant fraction of the cells had changed their morphology after treatment, namely, increased cell size and granularity, which is reminiscent of the induction of cellular senescence, as we have shown previously [ 3 , 4 , 9 ]. To confirm the induction of senescence, cells were reseeded at low density 6 days after therapy (day 10, see Figure 1 B).…”

“…The acquisition of a senescence phenotype is also associated with an increase in cell size and granularity (reviewed in [ 14 ]), which can be quantified by flow cytometry as an increase in the intensity of forward (FSC) and side scatter (SSC), respectively [ 9 , 15 ]. Indeed, the fraction of cells characterized by increased FSC and SSC intensity (F/S-high) was already maximally increased during the treatment phase of concomitant therapy (day 3), whereas the peak in the frequency of F/S-high cells was only reached on day 7 and day 10 after 24 h and 48 h of MTA treatment, respectively ( Figure 2 C,D).…”

“…MTA limits tumor growth by inhibiting three metabolic enzymes, e.g., the folate-dependent enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and the purine biosynthetic enzyme, glycinamide ribonucleotide formyltransferase (GARFT) [ 5 ]. Thus, treatment with MTA leads to reduced biosynthesis of purines and pyrimidines, thereby inducing an imbalance in the nucleotide pool, and subsequently to the formation of single-stranded DNA at replication forks, which, if not repaired, can lead to the formation of double-stranded DNA breaks [ 6 , 7 , 8 , 9 ]. Cisplatin is an alkylating agent that causes direct DNA damage, such as intra-strand and inter-strand DNA cross-links, which are repaired by nucleotide excision repair, which is dependent on sufficiently high nucleotide levels [ 10 ].…”

Aldolase A and Phospholipase D1 Synergistically Resist Alkylating Agents and Radiation in Lung Cancer

“…Although alkylating agents and radiation exposure have anti-cancer effects, therapeutic resistance is common (1,2). Previous studies have shown that these approaches can effectively increase cell apoptosis, DNA damage, and proliferation inhibition (3).…”