Inhibition Of PEGFR^THR654-Mediated Activiation Of DNA-PKcs Enhances Radiosensitivity In Cervical Carcinoma Cell

Objective:New evidence suggests that increased nuclear localization of the EGFR is associated with treatment resistance and poor prognosis of tumors.Radiation therapy increases EGFR expression in cancer cells, which potentially promotes radioresistance. Radiation-induced phosphorylation of EGFR on threonine 654 (pEGFRThr654) within the nuclear localization sequence (NLS) is responsible for radioresistance through the activation of DNA-PK linking to non-homologous end-joining (NHEJ) DNA repair. EGFR inhibitors can be used for the treatment of cervical cancer has been the focus of the debate.We examined if cetuximab/ Gefitinib down-regulates radiation-induced increased expression of pEGFRThr654 and pDNA-PKcsThr2609,an indicator of DNA-PKcs activity in human Cervical cancer cells.Methods:CaSki、C-33A、A-431 cells were exposed to a dose of 4Gy with 6 MV X-ray, the expression of EGFR were detected by Western blot.And then CaSki cells in the present or absent of Cetuximab/ Gefitinib, and the proteins were extracted using cytoplasmic and nuclear protein extraction kit after 40 min of irradiation. The expression of EGFR、pEGFRThr654、DNA-PKcs and pDNA-PKcsThr2609 in the cytoplasm and nuclus were detected by Western blot.Cloning assay was used to study the radiosensitivity of CaSki cells after Cetuximab/ Gefitinib treatment,and the radiobiology parameters were calculated from the curve fitted by linear-quadratic model [SF=e∧(-αD-βD2)] and the single-hit multitarget model [SF=1-(1-e-D/D0)N].SPSS 16.0 software was used for statistical analysis.Results:1、The expression of nuclei EGFR were increased in CaSki 、 C-33A and A-431 cells after irradiation especially in CaSki cell.2、 The expression of EGFR and pEGFRThr654, DNA-PKcs and pDNA-PKcsThr2609 in the nuclei were significantly increased in irradiated CaSki cells compared to nonirradiated CaSki cells (P<0.05).3、Gefitinib individually increased the expression of nuclear EGFR (P=0.011) and pDNA-PKcsThr2609 (P=0.022) but not pEGFRThr654 (P=0.971) and DNA-PK (P=0.881) in nonirradiated CaSki cells. However, the expression of pDNA-PKcsThr2609 in the nuclei of CaSki cells were significantly reduced by Gefitinib combined with irradiation, compared to irradiation alone (P=0.001),the values of Do. Dq、SF2、α/β and SER were 2.396 Gy、0.860Gy、50.94%、11.3456Gy and 1.41 respectively.4、Cetuximab individually increased the expression of nuclear EGFR (P=0.033) and pDNA-PKcsThr2609 (P=0.012) but not pEGFRThr654 (P=0.887) and DNA-PK (P=0.275) in nonirradiated CaSki cells. However, the expression of EGFR and pEGFRThr654 、DNA-PK and pDNA-PKcsThr2609 in the nuclei of CaSki cells were significantly reduced by Cetuximab combined with irradiation (P<0.05),the values of D0、Dq、SF2、α/β and SER were 1.572Gy、0.385Gy、31.70%、 49.3735Gy and 2.34 respectively.And cloning assay analysis showed that Cetuximab can make CaSki cells radioresistant which better than Gefitinib.Conclusion:Our findings show that pEGFRThr654 is the key point of radiation-induced EGFR nuclear transfer. Gefitinib and cetuximab as a ligand inhibitor of EGFR may cause feedback upregulation of EGFR expression, but not facilitate pEGFRThr654 expression in CaSki cells. Cetuximab inhibits radiation-induced translocation of pEGFRThr654 through down-regulating activation of DNA-PKcs,which can increase the radiation sensitivity in CaSki cells. But Gefitinib could not knocked down the pEGFRThr654 in the nuclei.Therefore, radiation therapy with concurrent cetuximab may benefit patients with cervical squamous carcinoma.