PTTG1 Regulates The Invasion,metastasis And Immune Function Of Lung Adenocarcinoma Following Irradiation Through TGF-β1/SMAD3 Pathway

Background and ObjectivesLung adenocarcinoma(LAC)is the most common pathological type of lung cancer.Radiotherapy(RT)is one of the main treatment modalities in LAC.Tumor burden is reduced,and local and systemic antitumour immune response can be enhanced to a certain extent after RT.However,some studies have shown that ionizing radiation(IR)may also induce epithelial-mesenchymal transition(EMT)to increase residual tumour cell invasive capacity and elicit immunosuppression in the tumour microenvironment(TME),which contributes to local tumour recurrence and even distant metastasis in many types of cancer,including in LAC.Pituitary tumour transforming gene 1(PTTG1)is one of the key genes regulating the separation of sister chromatids.PTTG1 was upregulated in LAC tissue and cell lines and related to lymph node metastasis of LAC patients.Previous studies have shown that PTTG1 modulated LAC cell invasion and proliferation via the TGF-β1/SMAD3 signaling pathway.Transforming growth factor-Beita 1(TGF-β1)is the main member of TGF-βsuperfamily and serves many biological functions.Many evidences showed that EMT and immune regulation mediated by TGF-β1/SMAD3 pathway affected the proliferation,invasion,metastasis and immune microenvironment of tumour cells.Therefore,we aimed at exploring whether LAC cell invasion worsens and the related immune response changes following radiation,whether PTTG1 affected the biological characteristics and immune function after IR in vitro or in vivo,and whether the TGF-β1/SMAD3 pathway participated in this process.It is helpful to understand the biological behavior changes of LAC cells after IR and the relationship with immunomodulation,so as to provide theoretical basis and potential intervention target for improving RT efficacy,and ultimately benefit LAC patients.Methods1.RNA interference(RNAi)was used to silence PTTG1 and SMAD3 expression in LAC cells.A549 cells were infected with lentivirus-mediated short hairpin RNA-PTTG1(Lv-shRNA-PTTG1)to constructed stable PTTG1 knock-down cell line(A549P-).And A549-P-cells were transfected with SMAD3-specific siRNA to build A549P-S-cells.QRT-PCR and WB assay were applied to verify the RNAi efficiency,detect the invasion-related protein and proteins:E-cadherin,MMP-2,etc.and the expression of key genes and proteins in TGF-β1/SMAD3 pathway(TGF-β1,SMAD3,p-SMAD3).A Transwell assay was applied to study the invasive abilities of cells after exposure to different doses(0,4,8 Gy)of X-ray irradiation2.Fresh peripheral blood was collected from healthy volunteer,and PBMCs were isolated with density gradient centrifugation.A coculture system of tumor cells and PBMCs was constructed.The coculture system were exposed to different doses(0,4,8 Gy)of X-ray irradiation,followed by multicolor flow cytometric analysis.The related indexes of immune-function(Tregs,CD8+Tcell and MHC-I molecules)were detected 48 hours after radiation in the A549P-group coculture system.In coculture system of A549P-S-cells and PBMCs,immune function was tested by using the same method,the immune indexes were detected 24 hours after radiation following coculture with PBMCs for 24h.3.Lewis lung cancer cell line with luciferase gene(LLC-LUC)were infected with Lv-shRNA-PTTG1 to create a cell line with silenced PTTG1.QRT-PCR and WB were used to verify the silence efficiency.To establish orthotopic Lewis lung cancer(LLC)mouse tumour model,LLC-LUC cells were transpleurally orthotopically injected into lungs tissue of BALB/c mice.The growth,invasion and metastasis of LLC tumour were monitored by regular bioluminescence imaging(BLI).One week after tumorigenesis,LLC tumour was exposed to a single dose of X-ray(6MV-X-ray,15Gy)irradiation.Mice were killed 3 days after treatment with 15 Gy of IR.Blood,tumor tissue,lung tissue,spleen and lymph node samples were collected.FCM was used to detect the immune indexes as described above.WB and immune-histochemistry were used to detect the expression of TGF-β1 and SMAD3 in lung and tumour tissues.Results1.PTTG1 knockdown cell line(A549P-)and double gene knockdown cell line of A549 cells,A549(P-S-),were successfully constructed.Compared with the 0 Gy group,8Gy,not 4Gy of X-ray irradiation reduced the invasive ability of A549.The invasive ability of A549P-cells decreased with the increasing of IR dose.Under the same dose of irradiation,the A549P-group showed an obvious decrease in invaded cell number.After X-ray radiation,the expression of MMP-2,N-cadherin,Snaill and TGF-β1 mRNA decreased,and the expression of E-cadherin gene and protein was up-regulated,part of which were related to the dose of irradiation.Compared with the control group,the changes of A549P-group were more obvious,including the changes of SMAD3 and p-SMAD3 protein.Compared with negative control cell line,A549 P-S-cells had a significant decrease in the number of invaded cells with the same IR dose.2.Both PBMCs and tumour cells appeared to exhibit growth inhibition after coculture.The immune function was enhanced after different doses of X-ray irradiation on coculture system,but the response of different immune indexes to X-ray dose was different:in control group,the decrease of Tregs proportion did not change with the increasing of IR dose(there was no difference between 4Gy and 8Gy),while the increasing of CD8+T cells and MHC-I molecules was closely related to the radiation dose(only increased after 8Gy of IR).In the coculture system of A549P-cells,PTTG1 silencing enhanced the expression of MHC-I molecules and CD8+T cells at 0 or 4Gy of IR.After further interfering with SMAD3 expression in A549P-cells,the MHC-I molecule expression was up-regulated at 4Gy of IR,and the CD8+T cell percentage increased at 8Gy of X-ray irradiation.3.LLC cells with silenced PTTG1 and NC cells were successfully constructed(LLC-shRNA-PTTG1 and LLC-shRNA-NC).An orthotopic LLC mouse tumour model was established(divided into PTTG1 group and NC group).The tumour was formed in 5-7 days,the weight of tumour bearing mouse(TBM)did not increase in 8-10 days,after the tumour cells inoculated.And LAC related symptoms gradually appeared.BLI showed that there was no statistical difference in the rate of tumour formation between the two groups,but the tumour size in the NC group was larger than that in the PTTG1 group,and the mice in the NC group showed higher metastatic rate.The median survival were 20 d and 30 d in the NC group and PTTG1 group,respectively.However,the difference was not significant.In NC group,Tregs were significantly up-regulated in tumour tissues,blood and peripheral lymphoid organs,MHC-I molecules and CD8+T cells were up-regulated as well,while in PTTG1 group,Tregs were not significantly changed after radiation,and the proportion of MHC-I and/or CD8+T cells in detected tissues and immune organs were further increased.IHC and WB assay of tumour and lung tissue showed that the expression of TGF-β1 and SAMD3 in PTTG1 group were down-regulated after radiationConclusions1.PTTG1 regulates the invasive abilities and antitumour immune response of LAC cells after IR through TGF-β1/SMAD3 pathway by affecting EMT,Tregs、MHC-I molecules and CD8+T cells.2.PTTG1 knockdown ameliorates the immune-suppression state and enhances the local and systemic antitumour immune response following irradiation by affecting the activity of TGF-β1/SMAD3 pathway,and further deceases the metastasis of LAC.3.Targeting PTTG1 and TGF-β1/SMAD3 pathway may be a potential therapeutic strategy to improve the efficacy of LAC radiotherapy and combined with immunotherapy.