The Role And Mechanism Of TBK1 And MSI2 In Radiotherapy Of Lung Cancer

BackgroundWith the increase of morbidity and mortality,malignant tumors have become the main cause of death in China.The incidence of lung cancer is as high as 17%,which is the most common malignant tumor.Non-small cell lung cancer(NSCLC),as the major lung cancer,has become the leading cause of cancer death.Lung cancer,especially NSCLC,is the biggest public health problem in our country and even in the world.In addition to traditional surgical resection,radiotherapy is the most effective treatment for lung cancer and is recommended as the main treatment for NSCLC.However,the complications of radiotherapy and the resistance to radiotherapy seriously hinder the implementation of radiotherapy and the effect of radiotherapy,and seriously affect the prognosis and survival of cancer patients.Radiationinduced pulmonary fibrosis(RIPF),as a common serious complication in the late stage of radiotherapy for lung cancer,limits the dose and frequency of radiotherapy.At present,there are no effective preventive and therapeutic measures in clinic.Similarly,during radiotherapy,lung cancer may produce radioresistance which severely weakens the killing effect of cancer.Increasing the dose and frequency of radiotherapy to improve the effect of radiotherapy is bound to increase the risk of complications of radiotherapy.The effect can only be improved by increasing the radiosensitivity of lung cancer.But there is no effective method to improve the sensitivity of lung cancer to radiotherapy at present.Therefore,it is of great significance for lung cancer radiotherapy to seek effective preventive and therapeutic measures for RIPF and improve radiosensitivity.So far,the pathogenesis of RIPF remains unclear.A large number of studies have reported that tissue and organ fibrosis is closely related to epithelial-mesenchymal transition(EMT),and EMT plays an important role in the occurrence and development of pulmonary fibrosis.Alveolar epithelial cells are transformed into activated fibroblasts through EMT and produce extracellular matrix proteins such as collagen in the interstitium of lung,which leads to the formation of pulmonary fibrosis.Recently,it has been reported that ionizing radiation could induce EMT in normal alveolar epithelial cells,which is closely related to RIPF in lung tissue induced by radiation.Targeting radiation-induced EMT may become an important breakthrough in preventing and treating RIPF which is a major complication of lung cancer radiotherapy.Previous studies have found that TBK1 promotes EMT in lung cancer cells induced by radiation by regulating ZEB1,which is a classical transcription factor of EMT.TBK1 may play an important role in the development of radiation-induced EMT in lung cancer cells.However,the role of TBK1 in radiation-induced EMT and RIPF in normal lung tissues has not been reported.The first part of our study will explore the role and mechanism of TBK1 in the development of RIPF through targeting EMT of normal alveolar epithelial cells for the first time.The aim is to provide effective prevention and treatment targets for the prevention and treatment of common and serious complications of lung cancer radiotherapy.In order to improve the radiotherapeutic effect of lung cancer,especially NSCLC,besides effective prevention and treatment of its complications,it is equally important to improve the radiosensitivity of lung cancer.Therefore,in the second part,we will focus on the radiosensitivity of lung cancer and explore the role of TBK1 in radiosensitivity of lung cancer for the fisrt time to study the role of TBK1 in radiotherapy of lung cancer more comprehensively.In addition,we have found that MSI2,a molecule that plays an important role in EMT of lung cancer,may also play an important role in radiotherapy of lung cancer.In the second part,we will explore the role and mechanism of MSI2 in radiosensitivity of NSCLC for the first time,and provide an effective target for radiosensitization of lung cancer.ObjectivesIn this study,we started with solving the two major problems that seriously affect the radiotherapy effect of lung cancer: complications of lung cancer radiotherapy and resistance to lung cancer radiotherapy.The study was carried out through in vivo,in vitro experiments and clinical sample detection to study the role and mechanism of TBK1 in radiation-induced EMT in normal alveolar epithelial cells and to explore the effect and mechanism of TBK1 and MSI2 on radiosensitivity of lung cancer.The aim of this study is to provide a new target for the effective prevention and treatment of RIPF,which is a common and serious complication of radiotherapy for lung cancer,to provide an effective new strategy for radiosensitization of lung cancer,and then to improve the effect of clinical radiotherapy of lung cancer synthetically and effectively.Methods1.Cell experimentWestern blot,immunofluorescence and morphological observation were used to detect the EMT and its degree of RLE-6TN and MLE-12 cells after irradiation.Radiosensitivity of A549,H460 and H1299 cells were studied by cell clone formation,proliferation,apoptosis and cell cycle assay.DNA damage was detected by comet assay and immunoconfocal assay.Interfering plasmids or overexpression plasmids of TBK1,MSI2 and RBM17 were constructed respectively,and their effects and mechanisms were studied by knocking down or overexpressing the corresponding molecules of cells by transfection or lentivirus infection.AKT,ERK,TBK1 and ATR specific inhibitors PF-04691502,SCH772984,Amlexanox and VE-821 were used to investigate the intermolecular regulation.The interaction between proteins was investigated by immunoprecipitation.2.Animal experimentAll mice were randomly divided into different groups and administered by gavage.Radiation-induced pulmonary fibrosis model of C57BL/6 mice was established with a dose of 20 Gy irradiation on lung and the mice were kept for 3 to 12 months after irradiation.C57BL/6 mice were subjected to whole body irradiation(7.5 Gy or 8.5 Gy)and kept for 1 month to observe the survival of mice after irradiation.The tumors of nude mice were locally irradiated with a dose of 15 Gy 1 month after tumor loading in nude mice and then measured and analyzed 15 days later.Western blot and immunofluorescence were used to detect EMT and its degree in lung tissue of mice.The development of radiation-induced pulmonary fibrosis in mice was analyzed by HE staining,MASSON staining and hydroxyproline detection.3.Clinical sample assayWe collected 80 lung cancer tissue samples and para-cancer lung tissue samples from Shanghai Lung Hospital.Tissue microarray was made for immunohistochemical detection.Then we used scanner to scan and observe,and made quantitative analysis by immunohistochemical score.4.Statistical analysisStatistical analyses were performed using GraphPad Prism.All quantitative data were presented as mean ± SEM from at least three independent experiments.Student two-tailed unpaired t tests were used to compare differences between two groups.One-way ANOVA followed by Dunnett multiple comparison tests were used to compare more than two groups.Kaplan-Meier analysis was performed for survival analysis,and significance between survival curves was determined by a log rank test.Statistical significance was set at P <0.05.Results1.Role of TBK1 in radiation-induced EMT and RIPFWe first confirmed in vitro that irradiation could induce EMT in alveolar epithelium after irradiation.The expression of epithelial marker decreased,the expression of mesenchymal markers increased,and the cell morphology changed from cuboidal appearance to extending pseudopods,elongating and swelling.In particular,70% of the cells showed this change 48 hours after 8Gy irradiation.Radiation could increase the expression of TBK1 in alveolar epithelial cells,while knocking down TBK1 could reverse the radiationinduced EMT.Amlexanox,an inhibitor of TBK1 had radioprotective effect on alveolar epithelial cells.In vivo experiments showed that Amlexanox reversed EMT in lung tissue of mice,significantly reduced RIPF in mice,and improved the survival rate of mice after whole body irradiation.2.The mechanism of TBK1 in radiation-induced EMTBoth AKT and ERK were activated after irradiation in cell experiments.Inhibiting the activation of AKT and ERK could partially reverse the radiation-induced EMT.Knocking down TBK1 could restrain the activation of AKT and ERK respectively,but inhibiting the activation of AKT or ERK did not affect the increase expression of TBK1 after irradiation.In addition,inhibiting AKT could weaken the activation of ERK after irradiation,while inhibiting ERK did not affect the activation of AKT after irradiation.These data suggest that TBK1 is the upstream of AKT and ERK,and AKT may be the upstream of ERK.TBK1 may play a role in radiation-induced EMT through the AKT-ERK pathway.3.Effect of TBK1 on radiosensitivity of NSCLCInhibiting TBK1 by knocking down TBK1 or using TBK1 inhibitors could reduce the proliferation of few NSCLC cell lines,but there was no significant change in the cloning ability,proliferation level and apoptotic level of most NSCLC cell lines after irradiation.Inhibiting TBK1 did not increase the radiosensitivity of NSCLC,but also did not increase its radioresistance.4.The role of MSI2 in the genesis and development of NSCLCThe colony forming ability and proliferation level of lung cancer cells decreased after knocking down MSI2 in vitro,but the colony forming ability and proliferation level increased after the RESCUE of MSI2.In vivo,the size of tumors from sh-MSI2+Mock group was significantly smaller than that of the control group,which indicated that MSI2 promoted the genesis and development of NSCLC.5.Effect of MSI2 on radiosensitivity of NSCLCKnocking down MSI2 could decrease the colony forming ability,proliferation level and increase apoptotic level of cells after irradiation in vitro experiments,while the RESCUE of MSI2 could reverse these effects.The size of tumors was significantly reduced with knocking down MSI2 after irradiation in vivo tumor-bearing experiments.Knocking down MSI2 increased the radiosensitivity of lung cancer cells.MSI2 could promote the radioresistance of lung cancer cells.6.The mechanism of MSI2 on radiosensitivity of NSCLCWe screened that RBM17 interacted with MSI2 after irradiation by immunoprecipitation-mass spectrometry.Knocking down RBM17 also had radiosensitization effect in vivo and in vitro experiments.MSI2 and RBM17 cooperated with each other to promote radiosensitization of NSCLC.Vitro experiments showed that MSI2 and RBM17 began to enter into the nucleus 0.5h after irradiation,then entered more obviously 8h after irradiation,and went out of nucleus 8h after irradiation at last.We found MSI2,RBM17 and ATR interacted after irradiation by immunoprecipitation.Inhibiting RBM17 harmed the entrance of MSI2 to nucleus and the interaction with ATR,while inhibiting MSI2 did not affect the entrance of RBM17 to nucleus and the interaction with ATR.Using ATR inhibitor did not affect the entrance of MSI2 and RBM17 to nucleus.MSI2 may activate downstream CHK1 through recruitment of RBM17 and interaction with ATR to repair DNA damage and finally induce radiation resistance in lung cancer.Conclusion1.Inhibiting TBK1 could significantly facilitate prevention and treatment of radiationinduced pulmonary fibrosis by reversing EMT.2.TBK1 might promote radiation-induced EMT in alveolar epithelial cells by activating AKT-ERK pathway.3.Inhibition of TBK1 had no effect on radiosensitivity of NSCLC4.MSI2 promoted the genesis and development of NSCLC5.Inhibition of MSI2 had significant radiosensitization effect on NSCLC6.MSI2 entered the nucleus under the recruitment of RBM17,then interacted with ATR to activates ATR and its downstream of DNA damage response pathway,and exerted radioresistance.