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.
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.
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).