| Background and Objective:PIM1 is a calcium/calmodulin kinase(calcium/calmodulin-regulated kinase CAMK)belongs to serine/threonine protein kinase family.PIM1 involved in cell proliferation,invasion,metastasis,apoptosis,cell cycle and chemotherapy resistance in hematologic malignancies,prostate cancer,gastrointestinal cancer,head and neck cancer,lung cancer and other malignant tumors.However,the mechanism of PIM1 in lung cancer is not yet clear.Whether there exist other downstream molecules of PIM1 in lung cancer,besides those classical ones such as p21,p27,cdc25A/C,BAD,FOXO3 a and ASK1,is not elucidated.In prostate cancer,studies have shown that PIM1 can regulate the expression of c-MET,suggesting that PIM1 may play a key role in the development of lung cancer by regulating c-MET and its downstream pathways.Effective inhibition of PIM1 pathway may become a new treatment modality for non-small cell lung cancer.Methods:1.In this study,we retrospectively analyzed data and samples collected from 215 radically resected adenocarcinoma patients between June 2010 and June 2012 at Tianjin Medical University Cancer Institute & Hospital.Immunohistochemical staining method was used to detect the expression of PIM1 and c-MET in lung adenocarcinoma tissues.Correlation analysis was used to anlyze the relation between expression of PIM1 and c-MET.Chi-squared test was used to analyze and the correlation between PIM1 expression with age,sex,smoking history,clinical stage,tumor size,lymph node metastasis and EGFR mutation status.Cox proportional hazards regression analyses were performed to assess the predictive and prognostic value of PIM1 and c-MET.2.To access the role of PIM1 in regulation of c-MET and its downstream pathways including PI3K/AKT pathway,RAS/ERK pathway and STAT3 pathway,we used PIM1 small molecule inhibitors(SGI-1776 and AZD1208),PIM1 siRNA and CRISPR/CAS9 nuclease system targeting PIM1 to block the expression or weaken the activity of PIM1 in HCC827 cells and A549 cells.Furthermore,PIM1 was overexpressed with PIM1 plasmid in H1299 cells and BEAS-2B cells.To investigate the mechanism by which c-MET was regulated,western blot was used to detect the expression of P-eIF4 B,the key translation initiation factor of c-MET.3.To investigate the impact of PIM1/ c-MET pathway in cell biological function,we used CRISPR/Cas9 to generate PIM1 knockout A549 SgRNA-1 cells and HCC827 SgRNA-1 cells and restoring c-MET expression with a c-MET plasmid.MTT assay was used to detect cell proliferation,cell colony formation assay was used to detect cell survival,wound healing assay and transwell assay was used to detect cell migration and invasion.Results:1.In our 215 cases of lung adenocarcinoma,the positive rate of PIM1 expression was 48.8% and the positive rate of c-MET expression was 51.2%.Correlation analysis showed that the expression of PIM1 and c-MET protein was positively correlated(correlation coefficient,R = 0.4375).In addition,the relationship between PIM1 and c-MET and clinical parameters were analyzed.The results showed that PIM1 expression was closely related to local lymph node metastasis(P = 0.012).Univariate survival analysis showed that PIM1 was closely correlated with disease recurrence(P = 0.004,hazard ratio 1.613 [HR],95% CI 1.160-2.241)and prognosis(P = 0.003,HR 1.806,95% CI 1.219-2.676),c-MET was significantly associated with DFS(P = 0.037,HR 1.416,95% CI 1.021-1.964).Multivariate analysis showed that high expression of PIM1(P = 0.059,HR 1.389,95% CI 0.988-1.953),smoking index(P = 0.019,HR 1.506,95% CI 1.068-2.124)and clinical stage(P =0.003,1.681 HR,95% CI 1.190-2.374)were independent risk factors for predicting DFS while high expression of PIM1(P = 0.014,HR 1.674,95% CI 1.110-2.525),clinical stage(P = 0.001,HR 2.046,95% CI 1.341-3.120)and adjuvant chemotherapy(P = 0.001,HR 0.448,95% CI 0.280-0.719)were independent predictors of OS in adenocarcinoma patients.2.The expression of PIM1 and c-MET were positively correlated in lung adenocarcinoma cell lines and human lung bronchial epithelial cells.When using PIM1 small molecule inhibitors(SGI-1776 and AZD1208),PIM1 siRNA and CRISPR/CAS9 nuclease system targeting PIM1 to block the expression or weaken the activity of PIM1 in HCC827 cells and A549 cells,c-MET expression was downregulated accordingly.Expression of c-MET was significantly increased when PIM1 was overexpressed with PIM1 plasmid in H1299 cells and BEAS-2B cells.In addition,some downstream pathways including RAS/ERK pathway,PI3K/AKT pathway,STAT3 pathway and the key translation initiation factor eIF4 B were changed accordingly.3.When PIM1 was knockout by CRISPR/Cas9 in HCC827 cells and A549 cells,cell proliferation capacity,colony formation capacity,wound healing capacity and transwell capacity were all decreased,while when c-MET expression was restored,the above cell biological functions were also restored part or whole.Conclusion:There is a positive correlation between the expression of PIM1 and the expression of c-MET in lung adenocarcinoma tissues.High expression of PIM1 can predict disease recurrence and prognosis while c-MET expression can predict disease recurrence.In the lung adenocarcinoma cell lines,PIM1 plays an important role in the regulation of c-MET protein,and this regulation requires the activity of PIM1 kinase.In addition,PIM1/c-MET pathway can regulate cell proliferation,survival,migration and invasion of lung adenocarcinoma cells. |
PDF Full Text Request