Department of General Thoracic Surgery, Inselspital, Bern University Hospital, Murtenstrasse 50, 3008, Bern, Switzerland.
Department of BioMedical Research, University of Bern, Bern, Switzerland.
J Exp Clin Cancer Res. 2021 Apr 19;40(1):138. doi: 10.1186/s13046-021-01938-2.
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.
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.
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.
Chemotherapy increases CDA and TYMP expression thereby rendering resistant lung cancer cells susceptible to subsequent 5'-DFCR treatment.
培美曲塞（MTA）联合顺铂联合治疗被认为是晚期非小细胞肺癌（NSCLC）患者的标准治疗方法。然而，在晚期 NSCLC 中，5 年生存率低于 10%，主要是由于对治疗的耐药性。我们之前已经表明，在 NSCLC 细胞系 A549 中，在用 MTA 和顺铂联合治疗后，间充质样、化疗耐药的副克隆细胞的比例增加。胞苷脱氨酶（CDA）和胸苷磷酸化酶（TYMP）是嘧啶补救途径的关键酶。5'-去氧-5-氟胞苷（5'-DFCR）是胞苷类似物（卡培他滨的代谢物），它被 CDA 转化，然后被 TYMP 转化为 5-氟尿嘧啶，这是一种常用于治疗实体瘤的化疗药物。本研究的目的是鉴定并利用化疗诱导的代谢适应性来靶向耐药癌细胞。
使用细胞活力和集落形成测定法来定量 MTA 和顺铂联合治疗以及与时间表相关的 5'-DFCR 给药对 A549 副克隆细胞和 NSCLC 细胞系生长和存活的疗效。通过 Western blot 监测 CDA 和 TYMP 蛋白表达。最后，通过流式细胞术分析治疗后随时间推移 EMT 表型、DNA 损伤反应激活和细胞周期分布。通过免疫组织化学法检测接受新辅助化疗前后患者肿瘤组织中的 CDA 表达。
我们进行了线粒体代谢抑制剂的小规模筛选，结果表明 5'-DFCR 选择性靶向具有高 CDA 和 TYMP 表达的化疗耐药 A549 副克隆细胞。在细胞系 A549 中，CDA 和 TYMP 的表达随着时间的推移呈时间依赖性增加，在依赖 KRAS 的 NSCLC 细胞系 H358 和 H411 中也观察到了这种情况。在 MTA 和顺铂联合治疗后的第二天添加 5'-DFCR 是根除化疗耐药 NSCLC 细胞最有效的治疗方法。此外，从治疗诱导的 DNA 损伤中恢复延迟，并伴有衰老诱导和获得混合 EMT 表型。在患者肿瘤的亚组中，新辅助化疗后 CDA 表达也增加。
化疗增加了 CDA 和 TYMP 的表达，从而使耐药肺癌细胞对随后的 5'-DFCR 治疗敏感。
