Shen Lin, Li Zhanzhan, Shen Liangfang
Department of Oncology, Xiangya Hospital, Central South University, Changsha 410008, People's Republic of China.
Cancer Manag Res. 2020 Dec 9;12:12667-12678. doi: 10.2147/CMAR.S260028. eCollection 2020.
Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment. Protein tyrosine phosphorylation has emerged as a key device in the control of resistance to therapy in cancer cells.
Using tandem mass tag (TMT) labeling and phospho-antibody affinity enrichment followed by high-resolution LC-MS/MS analysis, quantitative tyrosine phosphorylome analysis was performed in CNE2 (parental) and its radioresistant subline CNE2-IR.
Altogether, 233 tyrosine phosphorylation sites in 179 protein groups were identified, among which 179 sites in 140 proteins were quantified. Among the quantified proteins, 38 tyrosine phosphorylation proteins are up-regulated and 18 tyrosine phosphorylation proteins are down-regulated in CNE2-IR vs CNE2. Increased tyrosine phosphorylation in multiple receptor/protein tyrosine kinases (EPHA2, EGFR, IGF1R, ABL1 and LYN) was identified in CNE2-IR vs CNE2 cells. Intensive bioinformatic analyses revealed robust activation of multiple biological processes/pathways including E-cadherin stabilization, cell-cell adhesion, and cell junction organization in radioresistant CNE2-IR cells. Specifically, we observed that the CNE2 cells incubated with EphrinA1-Fc exhibited higher EPHA2 Y772 phosphorylation and lower E-cadherin expression, as compared with PBS control. Furthermore, an ATP-competitive EPHA2 RTK inhibitor (ALW-II-41-27, ALW) reduced EPHA2 Y772 phosphorylation and increased the expression of E-cadherin in CNE2-IR cells. Colony formation analysis showed that EFNA1 (EFNA1 is the ligand of EPHA2) treatment in CNE2 significantly promoted colony formation after 6Gy irradiation; while incubation with EPHA2 inhibitor ALW-II-41-27 in CNE2-IR cells impaired colony formation after irradiation, as compared with solvent control (DMSO).
In conclusion, phosphoproteomic approach allowed us to link tyrosine kinases signaling with radioresistance in NPC. Further studies are necessary to delineate the molecular function of EPHA2/E-cadherin signaling in radioresistant NPC and to explore rational combination therapy and its underlying mechanism.
放射抗性是鼻咽癌（NPC）治疗中的一个主要挑战。蛋白质酪氨酸磷酸化已成为癌细胞治疗抗性控制中的关键机制。
采用串联质谱标签（TMT）标记和磷酸化抗体亲和富集，随后进行高分辨率液相色谱-质谱/质谱分析，在CNE2（亲本）及其放射抗性亚系CNE2-IR中进行定量酪氨酸磷酸化蛋白质组分析。
共鉴定出179个蛋白质组中的233个酪氨酸磷酸化位点，其中140个蛋白质中的179个位点被定量。在定量的蛋白质中，与CNE2相比，CNE2-IR中有38个酪氨酸磷酸化蛋白上调，18个酪氨酸磷酸化蛋白下调。与CNE2细胞相比，在CNE2-IR中鉴定出多种受体/蛋白酪氨酸激酶（EPHA2、EGFR、IGF1R、ABL1和LYN）中酪氨酸磷酸化增加。深入的生物信息学分析揭示了放射抗性CNE2-IR细胞中多种生物学过程/途径的强烈激活，包括E-钙黏蛋白稳定、细胞间黏附和细胞连接组织。具体而言，我们观察到与PBS对照相比，用EphrinA1-Fc孵育的CNE2细胞表现出更高的EPHA2 Y772磷酸化和更低的E-钙黏蛋白表达。此外，一种ATP竞争性EPHA2 RTK抑制剂（ALW-II-41-27，ALW）降低了CNE2-IR细胞中EPHA2 Y772磷酸化并增加了E-钙黏蛋白的表达。集落形成分析表明，在CNE2中用EFNA1（EFNA1是EPHA2的配体）处理在6Gy照射后显著促进了集落形成；而与溶剂对照（DMSO）相比，在CNE2-IR细胞中用EPHA2抑制剂ALW-II-41-27孵育会损害照射后的集落形成。
总之，磷酸化蛋白质组学方法使我们能够将酪氨酸激酶信号与NPC中的放射抗性联系起来。有必要进一步研究以阐明EPHA2/E-钙黏蛋白信号在放射抗性NPC中的分子功能，并探索合理的联合治疗及其潜在机制。
