Role Of Rab25 In Acquired Resistance Of Non-small Cell Lung Cancer To Erlotinib

BackgroundIn recent years,the incidence of lung cancer has continued to rise globally,and this trend is particularly prominent in China.In fact,it is reported that lung cancer has the highest incidence and mortality among all malignant tumors in China,which poses a serious threat to the public health.Among all new lung cancer cases,non-small cell lung cancer(NSCLC)accounts for an overwhelming majority.Unfortunately,most NSCLC patients,approximately80%,were diagnosed in advanced stages.Unlike patients in early stage who can obtain high long-term survival rates after surgical resection,cure for advanced NSCLC patients is awfully limited.Erlotinib,an epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor(TKI),brings hope to advanced NSCLC patients,especially for those harboring EGFR activating mutations.However,almost all patients undergo acquired resistance to erlotinib after 6-12 months’ treatment.Although substantial acquired resistance mechanisms have been discovered,almost 30% of the required resistant cases remain unexplainable.Therefore,further exploration of the mechanism of resistance to erlotinib is a hot issue for research.Rab25 is a kind of small GTP enzyme located in epithelial cells.The aberrant expression of Rab25 correlated with many malignant tumors including lung cancer.It is reported that Rab25 expression is related to three kinds of TKIs resistance including erlotinib according to a gene chip.However,the correlation of Rab25 expression with acquired resistance of NSCLC to erlotinib remains to be confirmed.Canonical Wnt/β-catenin pathway is involved in the regulation of a variety of activities including tumors tumors proliferation,differentiation,and metastasis.Studies have shown that EGFR mutations,especially T790 M mutations,phosphorylated and activated β-catenin,which mediated the nuclear transactivation,leading to decreased sensitivity of erlotinib in lung adenocarcinoma NCI-H1650 cells.In addition,knockdown of β-catenin expression could significantly improve the sensitivity of TKI(BIBW2992)in non sensitive cells of lungadenocarcinoma H1975.All these evidences suggests that β-catenin may play an important role in the primary and acquired TKI resistance.Therefore,we just wonder whether β-catenin is the target mechanism of Rab25 induced resistance to erlotinib.ObjectiveWe investigated the differences in expression between erlotinib resistance cells(PC9/ER)and parental PC9 cells.By means of gene knockdown technology,the role of Rab25 in erlotinib acquired resistance to erlotinib was clarified.Our research provides new ideas and theories for understanding the resistance to erlotinib.Methods1.On the basis of erlotinib-resistance cell model induced by our research team,PC9 and PC9/ER cells were subcultured to compare morphological differences and evaluate erlotinib resistance index using a Cell Counting Kit(CCK)8 kit.2.Differential Rab25 expression between PC9/ER and PC9 cells were measured using qPCR and Western-Blot.After stimulation by gradient concentrations of erlotinib,the dose dependent effect of Rab25 protein level was compared in two strings of cells.Using immunofluorescence,we also localized Rab25 protein in the cells.3.Using siRNA and lentivirus gene knockdown technology to silence Rab25 expression in PC9/ER cells,we observed the role of resistance to erlotinib induce by Rab25 by a CCK-8kit and cell cycle directed by flow cytometry.4.Wnt signal protein levels were explored by Western-Blot after knockdown of Rab25 in PC9/ER cells,and the rescue experiment was performed using LiCl.Results1.PC9 cells were shaped fusiform,while PC9/ER cells,were round under low magnification;PC9 grew in a scattered manner,while PC9/ER grew in a manner of continuous sheets under high magnification.The CCK8 measurement showed that the IC50 of PC9/ER cells was much higher than PC9 cells(P < 0.01),and the resistance index of PC9/ER cells was 68.21 + 21.42,suggesting that the PC9/ER cells met experimental requirements.2.Rab25 gene and protein expression was higher in the PC9/ER cells than in PC9 cells(P < 0.05).The heaped Rab25 protein was mainly expressed in the cell membrane.The dose dependent effect of Rab25 protein level on erlotinib concentration was just presentbetween 0-1μM in PC9 and PC9/ER cells.3.After siRab25 was successfully screened and transfected,the IC50 and proliferation index(PI)were lower in the transfected group than in the control group.[(0.59±0.33)vs(6.14±0.92)μM,P<0.05] and [(0.53±0.10)vs(0.89±0.06),P<0.05].4.After PC9/ER cells were stimulated by erlotinib,the protein levels of β-catenin were much higher than those of PC9 cells(P < 0.05)and the heaped β-catenin was mainly expressed in nucleus.The protein levels of β-catenin and p-GSK-3β were lower in LV-Rab25 group than in LV-control group.In addition,Li Cl(10mM)could rescue the depressedβ-catenin level in cytoplasm and nucleolus.Conclusion1.Rab25 expression is elevated in PC9/ER cells,which contributes to resisitence to resistance to erlotinib.2.Activation of Wnt signaling pathway and β-catenin nucleolus aberrant expression play an important role in Rab25-mediated erlotinib resistance...