The Effect And Mechanism Of Afatinib On The Radiosensitivity Of Nasopharyngeal Carcinoma Cells

Objective: Nasopharyngeal carcinoma(NPC)is one of the most common head and neck malignant carcinoma in China,with regional clustering and racial preference,as well as a higher incidence in China than that in Europe and America.Radiotherapy is one of the crucial treatments for NPC at present.It is considered that the poor radiosensitivity of NPC cells is a major cause leading to the poor treatment effect and the tendency to recurrence or metastasis.Therefore,finding effective methods to improve the radiosensitivity of NPC cells is the key to improving the prognosis of patients.Ionizing radiation(IR)can rapidly activate a large number of epidermal growth factor receptor(EGFR),and blocking EGFR could improve the radiosensitivity of NPC.However,the current EGFR-targeted drugs used for NPC patients are not efficacy to improve the outcomes,it is an urge to search for new strategies.Human epidermal growth factor receptor 2(HER2),which interacts and shares similar downstream signaling pathways with EGFR,can be activated by IR as well,is a potential factor for NPC patients with less sensitivity to EGFR-targeted drugs.Therefore,to seek for novel drugs to enhance the radiosensitivity of NPC cells,we studied the effects of afatinib(EGFR,HER2 dual tyrosine kinase inhibitor)on the radiosensitivity of NPC cells and its molecular mechanism.Methods: Firstly,the MTT assay was used to detect the inhibition effect of afatinib and erlotinib on the proliferation of different NPC cell lines.Flow cytometry and immunofluorescence experiments were performed to test apoptosis and DNA damage.Meanwhile,the expression levels of key proteins in apoptosis were determined via Western blot.Colony survival assay was used to analyze the effects of afatinib and erlotinib on colony survival of NPC cells upon ionizing radiation.Then,TMT labeled phosphorylated proteomics technique was applied to study the protein phosphorylation of NPC cells towards afatinib treatment.Furthermore,the molecular mechanism of afatinib enhancing the radiosensitivity of NPC was explored through structural domain analysis,GO function analysis,KEGG signal pathway and protein-protein interaction network.Finally,we applied Western blot assay to determine the protein phosphorylation levels of EGFR,HER2 and downstream viral proteins Erk1/2 and Akt,as well as the phosphorylation change of CD44 and STAT3 screened by TMT labeled phosphorylation proteomics.And wound healing experiment,transwell migration experiment and Western blot were performed to study the epithelial-mesenchymal transition(EMT)associated markers,including epithelial phenotype and mesenchymal phenotypes related proteins.Results: Briefly,this study found that: 1.Afatinib could significantly inhibit cell proliferation,and DNA damage repair ability of NPC cells after IR,while increase cell apoptosis after IR,thus improving the radiosensitivity of NPC cells.2.A total of 170 differentially expressed phosphorylated proteins were identified by TMT labeled phosphorylated proteomics.And combined with bioinformatics analysis,we revealed that differential phosphorylated proteins are mainly involved in EGFR,HER2 and downstream signal transduction,as well as the EMT process and CD44/STAT3 signaling pathway.3.Furthermore,we verified that afatinib could impaire the activation of EGFR,HER2 and the related downstream signaling pathways,as well as the EMT process and CD44/STAT3 signaling pathway.Conclusions: In this study,we mainly demonstrated that the effect and mechanisms of afatinib enhanced the radiosensitivity of NPC cells.Our findings revealed the significance of inhibiting HER2 and EGFR in improving the radiosensitivity of NPC cells.In addition,our results further clarified the underly mechanism that afatinib dampened the EGFR/HER2 signaling pathway,EMT process regulated and CD44/STAT3 signaling pathway to strengthen the radiosensitivity of NPC cells.In conclusion,our study provided a novel strategy and theoretical basis for further accelerating the radiosensitivity of NPC,which would be a benefit to improve the outcomes of NPC patients.