Quantitative Tyrosine Phosphoproteomic Analysis of Resistance to Radiotherapy in Nasopharyngeal Carcinoma Cells

Background: 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. Methods: 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. Results: 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). Conclusion: 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.