The Anti-tumor Effect Of The Inhibition Of CENP-E And Its Immunoregulatory Effect And Mechanism In Non-small Cell Lung Cancer

BackgroundLung cancer is one of the most common malignancies and the leading cause of cancer related death worldwide.Although progress has been made in the diagnosis and treatment of lung cancer,the 5 year survival rate of lung cancer patients,especially advanced stage patients,is still poor.Although the new era of tumor immunotherapy in 2013 have changed the treatment model for lung cancer,the 5 year survival rate of advanced stage lung cancer patients is improved to only 16%.Patients treated with traditional therapy including chemotherapy and radiotherapy often experience intolerable side effects and drug resistance.Besides,these treatment could cause immunosuppression through PD-L1up-regulation and cause disease progression.And immune checkpoint blockade PD-1/PD-L1 antibody is effective in only about 20%patients with NSCLC.Therefore,the combination of traditional therapy with immune checkpoint blockade may be a new era in anticancer treatment.However,its combination with chemotherapy and targeted therapy are still under debate.For example,EGFR-TKI combined with PD-1/PD-L1 antibody could lead to disease progression in patients with mutant EGFR.Therefore,it is necessary to find more treatment targets and strategies combined with immune therapy.CENP-E is one member of centromere proteins,which participate mainly in the regulation of cell cycle.CENP-E plays an important role in the development and progression of various malignancies.However,its function in lung caner and whether treatment targeting CENP-E combined with PD-L1 antibody could improve the anti-tumor efficacy are still unclear.Therefore,our study is aimed to determine the expression of CENP-E and its clinical significance in lung cancer,the direct anti-tumor effect of CENP-E inhibition,the regulation of inhibited CENP-E on the expression of PD-L1 in lung cancer cells and on the immune microenvironment,and the anti-tumor effect of the combination of CENP-E inhibition and PD-L1 antibody.Our results will provide evidence for targeting CENP-E and its combination with immunotherapy in anticancer treatment.Methods1.CENP-E expression and its prognostic significance were analyzed by using platform databases,and the expression of CENP-E in human lung cancer cell lines and lung cancer tissues were verified by Western blot and immunohistochemistry,respectively.Meanwhile,the relationship between the expression of CENP-E and immune molecules was also roughly analyzed.2.CCK-8 assays and Ed U assays were conducted to investigate the effect of CENP-E specific inhibitor GSK923295 on the proliferation ability of lung cancer cells.Colony formation assays were conducted to investigate the influence of GSK923295 on the colony formation ability of lung cancer cells.PI staining was utilized to test the cell cycle distribution of lung cancer cells.A549 nude mice models were established to determine the anti-tumor activity of GSK923295 in vivo.Lewis lung cancer bearing-mice model was utilized to explore the anti-tumor efficacy of CENP-E knock-down.3.RNA sequencing was utilized to analyze the changes in A549 m RNA after treatment with 100 nmol/L GSK923295 for 72 hours,and to explore the changes of immune regulating molecules including PD-L1.The expression of PD-L1 in lung cancer cells treated with GSK923295 were verified through RT-PCR,Western blot and flow cytometry.To further confirm the role of CENP-E in the stability of PD-L1 m RNA,m RNA stability assays,3'UTR luciferase reporter assays and RIP assays were conducted.4.After GSK923295 treatment,A549 was co-cultured with immune cells isolated from malignant pleural effusions of lung cancer patients.The frequencies of CD4~+T cells,CD8~+T cells,apoptotic T cells,NK cells and Tregs,as well as the expression of PD-L1 and PD-1 in T cells,were evaluated by flow cytometry.Finally,the function of CENP-E in the immunomicroenvironment in lung cancer was verified by using Lewis mice model.5.Colony formation assays were utilized to investigate the efficacy of GSK923295combined with PD-L1 antibody in vitro.And in vivo experiment,Lewis lung cancer bearing-mice model was utilized to explore the efficacy of CENP-E knock-down combined with PD-L1 antibody.Results1.Bioinformatic analysis showed that CENP-E expression is significantly elevated in lung adenocarcinoma(by 5.8 folds,p<0.001)and squamous cell carcinoma(by 13.5 folds,p<0.001),and the increased CENP-E expression indicated poor survival,the median overall survival in CENP-E-low and-high groups were 96 and 45 months respectively(p<0.001).Besides,the expression of CENP-E is inversely correlated with immune checkpoints including PD-L1(CD274),Galectin-9,HVEM and cytokine including TGFB1(R=-0.482,-0.312,-0.590,-0.524,p<0.001),while positively correlated with IL2 and IL12B(R=0.368,0.368,p<0.001).2.CENP-E specific inhibitor GSK923295 inhibited the proliferation of A549 and H522,especially at the concentration of 100nmol/L,the proliferation rate of A549 was significantly decreased(0.293±0.031 Vs.0.008±0.002,p<0.001),the colonies number of A549 was also reduced(223.0±9.5 Vs.60.7±2.4,p<0.001).Meanwhile,GSK923295induced the cell cycle arrest at G2/M(p<0.001).In immune impaired A549 nude mice model,GSK923295 also resulted in the reduction of tumor volume(1359.0±266.7 mm~3 Vs.139.2±70.3mm~3,p<0.001)and tumor weight,and inhibited the tumor growth.However,after the knockdown of CENP-E,the tumor volume reduction(p=0.011)in immune intact C57/BL6 mice Lewis lung tumor model was obviously less than in A549 bearing nude mice model treated with CENP-E inhibitor.3.RNA sequence indicated elevated expression of immunosuppressive molecules including PD-L1,IL-6 and IL-33,as well as molecules regulating PD-L1 including IFNGR,IL-6 and JAK3.GSK923295 induced the expression of PD-L1 at both RNA and protein level.Besides,this effect was time-and dose-dependent.After treatment with 100nmol/L GSK923295 for 72 hours,the protein expression of PD-L1 in A549 was obviously up-regulated(1.000±0.062 Vs.2.125±0.109,p<0.001).Mechanistically,the inhibition of CENP-E by GSK923295 could stabilize PD-L1 m RNA through 3'UTR targeted by TTP,and improved the expression of PD-L1.4.After co-cultured with A549 treated with 100nmol/L GSK923295,the ratio of CD8~+T/CD4~+T cells was reduced(1.76±0.18 Vs.1.10±0.08,p=0.004),the frequencies of apoptotic CD4~+T cells and apoptotic CD8~+T cells were increased(p=0.029,p=0.012).The surface expression of PD-L1 and PD-1 in CD4~+T cells were elevated(p=0.006,p<0.001),and the surface expression of PD-L1 and PD-1 in CD8~+T cells were also elevated(p<0.001).The frequencies of NK cells were decreased(p=0.001),while the proportion of Tregs were elevated from(13.07±0.12)%in control to(21.30±1.39)%(p=0.009).Finally,the knockdown of CENP-E in mice model reduced the infiltration of CD8~+T cells and IFN-?~+CD8~+T cells(Ctrl-sh RNA Vs.CENPE-sh RNA#3:(18.82±6.26)%Vs.(11.78±2.07)%,p=0.044),while increased Tregs in lung tumor tissues(Ctrl-sh RNA Vs.CENPE-sh RNA#3:(11.12±0.67)%Vs.(14.06±1.16)%,p=0.001).5.In vitro experiment showed that GSK923295 combined with PD-L1 antibody suppressed the colony formation ability more efficiently(Colonies number of the coculture system at the 1:25 ratio:GSK923295 Vs.Combination treatment:116.00±29.34 Vs.8.00±5.00,p=0.003).In vivo experiment indicated that anti-PD-L1 treatment can better suppressed tumor growth in CENP-E knock-down mice(tumor volume at d30:Ig G+CENPE-sh RNA#1 Vs.anti-PD-L1+CENPE-sh RNA#1:1388.46±159.90mm~3Vs.572.82±210.73mm~3,p<0.01,Ig G+CENPE-sh RNA#3 Vs.anti-PD-L1+CENPE-sh RNA#3:1213.69±108.46mm~3Vs.504.89±113.80mm~3,p<0.01).Besides,the combination with PD-L1antibody recovered the frequencies of tumor infiltrating CD107~+CD8~+T cells from(1.65±0.31)%to(13.15±2.34)%(p<0.001)and Tregs were reduced(Ig G+CENPE-sh RNA#3 Vs.anti-PD-L1+CENPE-sh RNA#3:(23.40±2.06)%Vs.(7.38±1.80)%)(p<0.001).ConclusionsThis study clarified the high expression of CENP-E and its negative correlation with the prognosis of lung cancer patients.In addition,CENP-E inhibitor directly inhibited tumor growth but improved the stability of PD-L1 m RNA and its expression through 3'UTR targeted by TTP,consequently induced immunosuppression and restrained its in vivo anti-tumor function.CENP-E inhibition combined with PD-L1 antibody recovered the impaired anti-tumor immunity caused by CENP-E inhibition,and finally enhanced the anticancer efficacy in lung cancer.Therefore,our research provides evidence for targeting CENP-E and its combination with immunotherapy in anticancer treatment.