EGFR Nuclear Translocation-mediated Radiation Resistance And Clincal Research In Cervical Cancer

Part 1EFFECT OF EGFR NUCLEAR TRANSLOCATONON PROGNOSIS Of CERVERCAL CANCERWITH CHEMORADIOTHERAPYObjective: Epidermal growth factor receptor(EGFR)had the role mediating cell surface signaling cascade and translocating to the nucleus to regulate many cellular processes including transcription regulation and Nonhomologous end joining DNA damage repair.A large number of study reported that the expression of EGFR was correlated with development progression and prognosis of lung cancer? hepatocellular carcinoma? stomach cancer ? head and neck cancer and so on.But results about the relationship between prognosis or chemoradiation sensitivity and expression of EGFR in cervical cancer remained controversial or uncertain.New evidence suggested that expression of nuclear EGFR was found in many tumors that leaded to the poor prognosis and treatment resistance.Studies have reported that radiationactivated PKN1 one member of Protein Kinase C(PKC)family and induced the formation of EGFR dimers.EGFR phosphorylation of at threonine 654 site(EGFR,pThr654)that was activated by PKN1 might be one of the key points of the nuclear translocation of EGFR.This paper studied expression of membrane EGFR(m EGFR),phosphorylated PKN1(PKN1 pThr774),EGFR pThr6p54 in the Local middle-late cervical cancer to investigate the prognostic role of nuclear translocation of EGFR for patients with middle-advanced cervical cancer treated by chemoradiotherapy.Methods: This study collected pre-treatment tissue samples of 129 cervical cancer patients with IIA-IIIB(FIGO)stage,treated with concurrent chemoradiotherapy between January 2007 and December 2012.Proteins expression of EGFR(EGFR),EGFR pThr654 and PKN1 pThr774 were examined by immunohistochemistry.Expression level of these proteins?the relations of their expressions with clinicopathologic factors and prognosis of cervical cancer patients were analyzed.Meanwhile we tried to find the relationship among expression levels of EGFR,EGFR pThr654,PKN1 pThr774 protein.Results:Microscopic observation showed that m EGFR was expressed on the cell membrane,PKN1 pThr774 was expressed in both cytoplasm and nucleus,but EGFR pThr654 was expressed only in the nucleus.According to the cut-off values of EGFR,EGFR pThr654,PKN1 pThr774 expression levels calculated by X-tile software,in the 129 cervical cancer patients,84 cases(65.1%,84/129)had low expression of EGFR and 45 cases(34.9%,45/129)had high EGFR expression;high EGFR pThr654 and PKN1 pThr774 staining were present in 32cases(24.8%,32/129)and 37cases(28.7%,37/129),respectively;low EGFR pThr654 and PKN1 pThr774 staining were present in97 cases(75.2%,97/129)and 92 cases(71.3%,92/129),respectively.Expressionof EGFR pThr654 was positively correlated to Expression of EGFR(r=0.256,P=0.015)and PKN1 pThr774(r=0.217,P=0.04),statistically.Multivariate analysis showed that high EGFR(HR=28.469,95%CI=4.626-86.348;P=0.019)and EGFR pThr654(HR=45.137,95%CI=6.312-110.850;P=0.000)expression?HGB less than or equal to 100 g/L were independent predictors for poor overall survival(OS);high EGFR(HR=32.278,95%CI=6.615-89.134P=0.001)and EGFR pThr654(HR=40.009,95%CI=4.425-100.962,P=0.000)expression?Lymph node metastasis status were independent predictors for poor progress-free survival(PFS);high EGFR pThr654(HR=30.028,95%CI= 6.103-70.095 P=0.011)expression was the only independent predictors for poor local relapse-free survival(LRFS).conclusion : Nuclear EGFR is found in tissues of cervical cancer patients;The high expression of m EGFR and nuclear EGFR pThr654 is associated with a poorer survival after radical radiotherapy for cervical cancer,suggesting that this may be one of the factors contributing to the resistance to chemoradiotherapy;MEGFR,EGFR pThr654 and PKN1 pThr774 can be used as predictors to the sensitivity and prognosis of cervical cancer treated by chemoradiotherapy and as therapeutic targets.Part 2Effect Of RADIATION ON EGFR NUCLEARTRANSLOCATION AND EXPRESSION OF PROTEINSIN CERVICAL CANCER CELL LINESObjective: The epidermal growth factor receptor(EGFR)can be directly transferred to the nucleus,activate DNA-dependent kinase(DNA-PK),enhancenon-homologous end-joining DNA-double strand break repair to DNA damage,reduce radiation sensitivity,that is a new signal pathway mediated by EGFR.Our previous research has shown that cervical cancer tissues had the expression of nuclear EGFR,m EGFR and EGFR pThr654 PKN1 pThr774 may be proteins involved in the EGFR nuclear translocation pathway.PDK1 is one of the important proteins in PI3K/AKT pathway which can regulate the activity of DNA-PK.By detecting changes of expression of nuclear EGFR?EGFR pThr654?PKN1 pThr774?PDK1?PDK1 p Ser241,DNA-PK ?DNAPK pThr2609 before and after radiotherapy.This study observed Effect of irradiationon EGFR nuclear translocation meanwhile discussed the mechanism of nuclear transport and its possible role in radiation tolerance of cervical cancer cells.Methods: C33 A ?Ca Ski?Hela ? A431 cells were exposed to a dose of4 Gy with6 MV X-ray,the proteins were extracted using cytoplasmic and nuclear protein extraction kit after0,10,20,40 min of irradiation.The expression of EGFR,PDK1?PKN1?DNA-PK and the corresponding phosphorylated protein were detected by Western blot.Results: The expression of nuclear EGFR were increased in all four groups.Like A431 cells,The expression of EGFR pThr654,DNA-PK and DNA-PK pThr2609 in the nuclei were significantly increased in Ca Ski and Hela cells after irradiation(P<0.05).EGFR transport was associated with increased DNA-PK phosphorylation at residue T2609(P<0.05).The expression of PKN1 p T774 in the nuclei was significantly increased in Ca Ski cells after irradiation(P<0.05)and was associated with increased EGFR pThr654.IN nuclei of Hela cells The expression of PDK1 p Ser241 was correlated with increased DNA-PK pThr2609 significantly after irradiation(P<0.05)Conclusion: Nuclear translocations of EGFR are found in cervical cancer cells after irradiation.Activation of DNA-PK by nuclear EGFR is one of possible factors increasing radiation tolerance of cervical cancer cells.EGFR pThr654 may be the key point of irradiation-induced EGFR nuclear transfer regardless of adenocarcinoma of cervix or squamous carcinoma.The expression of p-PKN1-T774 in the nuclei is associated with EGFRThr654 in Ca Ski cells;nuclear PDK1 and PDK1 p Ser241 may be responsible for radiation tolerance of cervical adenocarcinoma.Our findings imply that many kinds of protein present nuclear transfer after radiation,which may be one mechanism of protection to treatment.Part 3EFFECT OF EGFR NUCEARL TRANSLOCATIONON IRRADIATION SENSITIVITY OF CEVERCALCANCER CELLSObjective: Our previous study has show Radiation-induced nuclear transportation of EGFR has been known to mediated DNA-PK-dependent role for radioresistance of cervical cancer,EGFR pThr654 may be the key point of EGFR nuclear transportation.we examined whether expression of EGFR pThr654 is involved in activation of EGFR nuclear transportation,and whether cetuximab(C225)or gefitinib down-regulates radiation-induced expression of nuclear EGFR and DNA-PK pThr2609 and sensitize human cervical cancer Ca-Ski and Hela cells to irradiation.Methords: cervical cancer Ca-Ski and Hela cells were pretreated with EGF R nuclear translocation inhibitory peptide(p T654)?control peptide?cetuximab or gefitinib for 16 h,then the cells were exposed to 4Gy 6MV-X Ray irradiation.Western blot was used to determine the expression of EGFR,EGFR pThr654,DNA-PK and DNA-PK pThr2609 at different time points.DNA damage was quantified with ?H2AX foci analysis in irradiation group(IR)?irradiation combined C225(IR+C225)group,irradiation combined gefitinib(IR+Gegefitinib)group.Response of Ca-Ski or Hela cells to irrradiation wasdetermined using colony formation assay.Dose survival curves were calculated by Graph Pad Prism5.0software.SPSS 17.0 software was used for statistical analysis.Results: The inhibitory peptides of EGFR nuclear translocation(p T654)?cetuximab and gefitinib significantly decreased the expression of EGFR?EGFR pThr654 and DNA-PK pThr2609 in Ca-Ski or Hela cells after the radiation(P<0.05).Control peptide didn't change the increase of these protein expression statistically.Interestingly,cetuximab and gefitinib also induced EGFR nuclear transfer.IR+C225 and IR+Gegefitinib enhanced number of ?H2AX foci(P<0.05).IR+C225 increased SER(1.67 vs.1)comparing with IR in Ca Ski cells.No difference of SER was found among IR?IR+C225 and IR+Gegefitinib in Hela cells(1 vs.1.015 vs.1.009).Conclusion: EGFR phosphorylation at Threonine 654 has been shown to stimulate nuclear EGFR translocation;Inhibition of nuclear translocation increases radiation sensitivity;radiation therapy with concurrent cetuximab increases Ca Ski cells but Hela cells radiation sensitivity,because of differences of signal pathway.Basic nuclear EGFR high expression may be the cause of acquired treatment tolerance;nuclear EGFR translocation during treatment may be the cause of nonacquired treatment tolerance.