IQGAP3 Interacts With Rad17 To Recruit The Mre11/Rad50/Nbs1 Complex And Contributes To Radioresistance In Lung Cancer

Purpose:Lung cancer is one of the leading causes of cancer-related mortality worldwide.Currently,surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy are the principal treatment approaches for lung cancer.Among these approaches,radiotherapy has been considered the preferential treatment strategy for lung cancer patients with unresectable locally advanced NSCLC(Non-small cell lung cancer).However,local relapse and distant metastasis occur frequently in lung cancer patients due to radioresistance.Therefore,molecular events associated with radioresistance need to be elucidated to improve the prognosis of lung cancer patients.The IQ motif containing GTPase-activating protein(IQGAP)family of proteins is an evolutionarily conserved family IQGAP3 has been implicated in diverse cellular processes including neuronal morphogenesis,cell proliferation and motility as well as epithelial-mesenchymal transition.However,its role in lung cancer radioresistance remain to be clarified.Methods:(1)The databases were used to examine IQGAP3 mRNA levels in lung cancer tissues.(2)Western Blot was used to detect IQGAP3 protein levels in human lung cancer cell lines.(3)The protein expression of IQGAP3 were detected by immunohistochemistry(IHC)staining in lung adenocarcinoma tissues and adjacent tissues.The correlation between IQGAP3 protein level and overall survival was analyzed by Kaplan-Meier method.(4)The effects of IQGAP3 on cell proliferation in lung cancer were determined by cell colony formation assay,Ed U and CSFE assays.(5)Nude mice xenograft tumor model was used to examine the effect of IQGAP3 on tumorigenesis in lung cancer at the animal level.(6)Neutral comet assay,Rad51 foci formation and cell survival assays after irradiation(IR)were utilized to investigate the effects of IQGAP3 on the lung cancer radiosensitivity.(7)The effect of IQGAP3 on radiosensitivity in lung cancer was investigated by nude mouse xenograft models after IR.(8)Co-immunoprecipitation assay was used to detect the interaction between IQGAP3 and Rad17.(9)Western Blot and Immunofluorescence assay were utilized to detect Rad17 protein level and Rad17 foci formation in IQGAP3-depleted lung cancer cells after IR.(10)The effects of IQGAP3 on the Nbs1,Mre11 and Rad50 foci formation in lung cancer were detected by IF assay after IR.(11)We detected phospho-ATM,phospho-Chk2,phospho-ATR and phospho-Chk1 protein level and foci formation in IQGAP3 deficiency lung cancer cells by Western Blot and IF assay after IR.(12)Rescue experiments were used to explore whether IQGAP3 performs its functions in a Rad17-dependent manner.(13)Using immunohistochemistry staining,the expression of Rad17 were detected in lung adenocarcinoma tissues.Kaplan-Meier method was used to analyze the correlation between Rad17 protein level and overall survival.Theχ~2 test was used to analyze the correlation between the expression of IQGAP3 and Rad17 in lung cancer tissues.Results:(1)The databases analysis showed that IQGAP3 mRNA levels in lung cancer tissues were higher than that in normal lung tissues.(2)IQGAP3 protein levels in lung cancer cells were higher than that in human bronchial epithelial cells.(3)IHC experiments demonstrated that IQGAP3 was overproduced and correlates with poor clinical outcomes in lung cancer patients.(4)Loss of IQGAP3 suppressed lung cancer cell growth and proliferation in vitro.(5)The tumor sizes and weights in mice were decreased when IQGAP3 was targeted by sgRNAs in vivo.(6)Loss of IQGAP3 promotes DNA damage and inhibited DNA repair in lung cancer cells.(7)IQGAP3 depletion enhanced lung cancer radiosensitivity in vivo.(8)IQGAP3 associated with Rad17 in lung cancer cells.(9)IQGAP3 silencing downregulated Rad17 protein level and foci formation in lung cancer cells.(10)IQGAP3 is involved in the recruitment of the Mre11/Rad50/Nbs1 complex to DSBs in a Rad17-dependent manner.(11)IQGAP3 silencing downregulated the protein level and foci formation of phospho-ATM,phospho-Chk2,phospho-ATR and phospho-Chk1protein levels.and the foci formation following irradiation(12)Rescue experiments demonstrated that the effects of IQGAP3 knockdown depend mainly on Rad17.(13)Rad17 was highly expressed and associated with adverse prognosis in lung cancer patients.The expression of IQGAP3 in lung cancer tissues was positive correlated with the expression of Rad17.Conclusion:IQGAP3 is overexpressed and correlates with poor clinical outcomes in lung cancer.IQGAP3 drives oncogenesis and radioresistance in lung cancer in vitro and in vivo.Mechanistically,IQGAP3 interacts with Rad17 and controls its expression to activate the ATM/Chk2 and ATR/Chk1 signaling pathways by recruiting the Mre11/Rad50/Nbs1complex in response to DNA damage.Clinically,IQGAP3 overexpression positively correlates with Rad17 upregulation in human lung cancer tissues.Therefore,our findings suggest that IQGAP3 may serve as a promising therapeutic target for lung cancer radiotherapy.