GBP1 Promotes Erlotinib Resistance Via PGK1-activated EMT Signaling In Non-small Cell Lung Cancer

Background:Lung cancer is the most common malignant tumor with highest incidence in China.Although there are many treatment methods,the mortality rate remains higher.At present,non-small cell lung cancer(NSCLC)is the main pathological type of lung cancer.Erlotinib,an epidermal growth factor recepter tyrosine inhibitor(EGFR-TKI),is one of the first-line treatment for the patients with advanced and EGFR-positive NSCLC,and greatly improves clinical outcome of those patients.After a median progression-free survival(PFS)of about 10 months,evolved resistance to EGFR-TKI remains a major clinical challenge.Currently,multiple mechanisms such as MET amplification,ERBB2 amplification,K-RAS mutation or PTEN deletion have been proved to be involved in EGFR-TKI resistance,but some mechanisms are still unknown.Therefore,further exploration of erlotinib resistance is an important issue that needs to be resolved.Here we predicted that guanylate binding protein 1(GBP1)may be an oncogene associated with erlotinib resistance through the Gene Expression Omnibus(GEO)database.The univariate and multivariate regression analysis confirmed that GBP1 was a factor of poor prognosis for NSCLC’s patients.Then,we induced erlotinib-resistant PC9ER and HCC827ER cells by increasing the concentration of erlotinib in erlotinib-sensitive cells,and confirmed that the expression of GBP1 regulated erlotinib resistance using these cells.In order to analyze the specific mechanism on how GBP1 contributes to erlotinib resistance,we conducted immunoprecipitation experiments and immunofluorescence experiments to confirm that GBP1 and phosphoglycerol kinase 1(PGK1)interact with each other.Finally,we analyzed which signaling pathway activated by PGK1 were involved in erlotinib resistance.Our experiments verified that GBP1 promoted erlotinib resistance through PGK1-activated EMT pathway in NSCLC through rescue experiments and IHC experiments.Objective:1.To explore whether GBP1 is related to erlotinib resistance in vivo and in vitro.2.To analyze the molecular mechanism in how GBP1 regulates erlotinib resistance3.To explore the relationship between the expression of GBP1 and the survival,clinical characteristics in the patients with NSCLC.Methods:First chapter The study in vitroThe experiments to confirm that GBP1 mediates erlotinib resistance1.Cell counting kit-8(CCK-8)experiments were used to compare the half inhibitory concentration(IC50)value of EGFR-TKI between erlotinib-resistant and erlotinib-sensitive cells.2.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)experiments were used to compare GBPl’s mRNA level between erlotinib-resistant and erlotinib-sensitive cells.3.Mann-Whitney test were used to analyze the correlation between GBP1’s mRNA expression and erlotinib resistance4.T790M site mutation detection kit were used to detect whether there were T790M site mutation in erlotinib-resistant cells.5.The univariate and multivariate Cox regression were used to analyze the correlation between overall survival and clinical features in the patients with NSCLCThe experiments to confirm that GBP1 regulates erlotinib resistance in vitro1.CCK-8 experiments were used to compare the IC50 value between knockdowning GBP1 in resistant cells and the control,or between overexpressing GBP1 in sensitive cells and the control.2.Western blot(WB)experiments were used to compare GBP1’s protein expression between knockdowning GBP1 in resistant cells and the control,or between overexpressing GBP1 in sensitive cells and the control3.qRT-PCR experiments were used to compare GBP1’s mRNA level between knockdowning GBP1 in resistant cells and the control,or between overexpressing GBP 1 in sensitive cells and the control.4.Immunofluorescence tests were used to explore the fluorescence value of GBP1 and PCNA in PC9ER-NC+erlotinib group,compared with PC9ER-shGBP1+erlotinib group.5.CCK-8 experiments were used to analyze the IC50 value in PC9ER-NC+erlotinib group,compared with PC9ER-shGBP1+erlotinib group.6.Flow cytometric experiments were used to compare the ratio of late apoptotic cells in GBP1 knockdown group with the control,or GBP1 overexpression group with the control.7.WB experiments were used to detect the protein expression of Bax,Bcl-2 and cleaved PARP1 in GBP1 knockdown group with the control,or GBP1 overexpression group with the control.8.Flow cytometry experiments were used to compare the ratio of G1 phase in GBP1 knockdown group with the control,or GBP1 overexpression group with the control.9.WB experiments were used to detect the protein expression of P21,Cycilin D1,CDK4 and CDK6 in GBP1 knockdown group with the control,or GBP1 overexpression group with the control.Second chapter The study in vivoAnimal experiments when overexpressing GBP11.The tumor formation experiments in nude mice were used to compare the size and weight of transplanted tumors between PC9ER-NC+erlotinib group and PC9ER-shGBPl+erlotinib group.2.The tumor formation experiments in nude mice were used to compare the size and weight of transplanted tumors among PC9-NC+PBS group,PC9-NC+erlotinib group,PC9-GBP1+PBS group,and PC9-GBP1+erlotinib group.3.IHC experiments were used to detect the expression of GBP1,PCNA and apoptotic proteins between PC9-NC+erlotinib group and PC9-GBP1+erlotinib group.WB and qRT-PCR experiments when overexpressing GBP1 in nude tumors1.qRT-PCR experiments were used to detect the mRNA level of GBP1 in nude tumors between PC9-NC+erlotinib group and PC9-GBP1+erlotinib group.2.WB experiments were used to detect the protein expression of GBP1 in nude tumors between PC9-NC+erlotinib group and PC9-GBP1+erlotinib group3.IHC experiments were performed to detect the protein expression of P21 and Cyclin D1 in nude tumors between PC9-NC+erlotinib group and PC9-GBP1+erlotinib group.Third chapter The exploration on mechanismThe interaction experiments between GBP1 and PGK11.Mass spectrometry analysis predicted possible interacting proteins of GBP1.2.Co-immunoprecipitation(CoIP)experiments were used to analyze the relationship between GBP1 and PGK1.3.Immunofluorescence experiments were used to detect the co-localization relationship between GBP 1 and PGK1.4.WB experiments were used to analyze the relationship between GBP1 and PGK1.The experiments in which GBP1 and PGK1 activated EMT pathway1.WB experiments were used to detect the change of EMT-related proteins between GBP 1 knockdown group,GBP 1 knockdown and PGK1 overexpression group.2.CCK-8 experiments were used to detect the IC50 value between PGK1 knockdown in resistant cells and the control.3.qRT-PCR experiments were used to detect the mRNA level of E-cadherin.4.CCK-8 experiments were used to detect the IC50 value between E-cadherin knockdown in resistant cells and the control.5.WB experiments were used to analyze the relationship between PGK1 and Twist.6.IHC experiments were performed to detect the expression of EMT protein among the control,PC9-NC+erlotinib group and PC9-GBP1+erlotinib group.Fourth chapter The clinical significanceThe relationship between GBP1 expression and the prognosis or clinical data in the patients with NSCLC1.Kaplan-Meier analysis were used to analyze the relationship between the expression of GBP1 or PGK1 and overall survival time.2.The cBioportal database were used to analyze the correlation between GBP1’s mRNA level and PGK1’s mRNA level.3.The Oncomine data were used to analysis the correlation between GBP1 expression and clinical data.Results:First chapter The expression and significance of GBP1 in erlotinib-resistant cells in NSCLC cellsGBP1 mediated erlotinib resistance and predited the prognosis of the patients1.Compared with sensitive cells,the IC50 value were increased in PC9ER,PC9ER1,PC9ER2,HCC827ER,NCIH1650,NCIH1975 cells.2.Compared with sensitive cells,the mRNA level of GBP1 were up-regulated in PC9ER,PC9ER1,PC9ER2,HCC827ER,NCIH1650,NCIH1975 cells.3.The Mann-Whitney test found that higher protein expression of GBP1 were closely related to the enhancement of erlotinib resistance.4.No T790M mutation were found in PC9ER and HCC827ER cells.5.The factors related to overall survival in the univariate and multivariate analysis were the tumor burden after treatment and the expression of GBP 1.The experiments confirmed that GBP1 expression regulated erlotinib resistance1.Compared with the control,the IC50 value were reduced when knockdowning GBP1 in resistant cells2.Compared with the control,GBP1’s protein expression were reduced when knockdowning GBP1 in resistant cells.3.Compared with the control,GBP1’s mRNA level were reduced when knockdowning GBP1 in resistant cells.4.When compared with PC9ER-NC+erlotinib group,GBP1 and PCNA in PC9ER-shGBP1+erlotinib group had weaker fluorescence value.5.Under the same concentration of erlotinib,PC9ER-shGBP1+erlotinib group had lower cell survival rate than PC9ER-NC+erlotinib group.6.Compared with the control,the proportion of late apoptotic cells were increased when knockdowning GBP1 in resistant cells.7.Compared with the control,the protein expression of Bcl-2 and PCNA were decreased,while the protein expression of Bax and cleaved PARP1 were increased when knockdowning GBP1 in resistant cells.8.Compared with the control,the ratio of G1 phase were increased when knockdowning GBP1 in resistant cells.9.Compared with the control,the protein expression of P21 were increased,the protien expression of Cyclin D1,CDK4 and CDK6 were decreased when knockdowning GBP1 in resistant cells.All of results showed that GBP1 was highly expressed in resistant cells,and elevated GBP1 regulated erlotinib resistance through inducing apoptosis and decreasing the ratio of G1 phase.Second chapter In vivo studies showed that GBP1 was involved in erlotinib reistanceOverexpressing GBP1 regulated erlotinib resistance1.Compared with PC9ER-NC+erlotinib group,the size and weight of transplanted tumors in nude mice were reduced in PC9ER-shGBP1+erlotinib group.2.Compared with PC9-NC+erlotinib group,the size and weight of the transplanted tumor were reduced in PC9-GBP1+erlotinib group.3.Compared with PC9-NC+erlotinib group,the protein expression of GBP1,PCNA and Bcl-2 were decreased,while the protein expression of cleaved PARP1 were increased in PC9-GBP1+erlotinib group.Overexpressing GBP1 decreased the ratio of G1 phase1.Compared with PC9-NC+erlotinib group in nude tumors,GBP1’s mRNA level were increased in PC9-GBP1+erlotinib group.2.Compared with PC9-NC+erlotinib group in nude tumors,GBP1’s protein expression were increased in PC9-GBP1+erlotinib group.3.Compared with PC9-NC+erlotinib group in nude tumors,the expression of P21 were decreased,and the expression of Cyclin D1 were increased in the PC9-GBP1+erlotinib group.All of results showed that overexpressed GBP1 increased erlotinib resistance in vivo,and decreased the ratio of G1 phaseThird chapter GBP1 and PGK1 interacted to promote erlotinib resistance GBP1 and PGK1 interacted with each other1.Mass spectrometry analysis predicted that GBP1 may interact with the proteins such as MYL9,ANXA2,ALDOA,PGK1,etc.2.CoIP experiments confirmed the interaction between GBP1 and PGK1.3.Immunofluorescence experiments were used to confirm co-localization relationship in the cytoplasm between GBP1 and PGK1.4.GBP1 regulated PGK1 at the protein level.GBP1 and PGK1 cooperately activated EMT pathway in erlotinib resistance1.Knockdowning GBP1 and overexpressing PGK1 in resistant cells could restore the effect of knockdowned GBP1 on EMT-related proteins2.Compared with the control,the IC50 value were decreased when knockdowning PGK1 in resistant cells3.Compared with sensitive cells,E-cadherin’s mRNA level were lower in resistant cells.4.Compared with the control,the IC50 value were increased when knockdowning E-cadherin in resistant cells.5.PGK1 increased the expression of Twist protein6.When compared with PC9-NC+erlotinib group,the protein expression of PGK1,Vimentin and N-cadherin in PC9-GBP1+erlotinib group were increased,while the protein expression of E-cadherin were decreased.All of results showed that GBP1 regulated erlotinib resistance through activating EMT pathway by PGK1.Fourth chapter The expression of GBP1 were related to the prognosis and pathological grade of the patientsHighly expressed GBP1 and PGK1 predicted poor survival in NSCLC1.Highly expressed GBP1 and PGK1 had short overall survival and first progression time.2.The mRNA level of GBP1 were positively correlated with the mRNA level of PGK1.3.There was a significant positive correlation between tumor grade and GBP1 protein expressionAll of results showed that high expression of GBP1 indicated poor prognosis and high pathological grade.Conclusions:We found that regardless of protein level or mRNA level in this study,GBP1 was significantly upregulated in erlotinib-resistant NSCLC cells when compared with erlotinib-sensitive NSCLC cells.Elevated expression of GBP1 made erlotinib-sensitive cells acquire the resistance to erlotinib in vitro and in vivo.Up-regulation of GBP1 expression decreased the expression of apoptotic proteins,and caused G1-S transition.GBP1 were involved in erlotinib resistance by PGK1-activated EMT signaling pathway.Then,high GBP 1 and PGK1 had short overall survival and first progression time by analyzing TCGA database.Overall,we confirmed that GBP1 regulated erlotinib resistance using cell models、animal models and clinical samples.Our results provide a rational basis that GBP1 can be a potential therapy intervention in erlotinib-resistant NSCLC.