The Role And Mechanism Of SHP2 In EGFR-TKI Resistance In Non-small Cell Lung Cancer

Objective:Epidermal growth factor receptor（EGFR）mutation is one of the most common driver gene in non-small cell lung cancer（NSCLC）,mainly seen in lung adenocarcinoma.EGFR-tyrosine kinase inhibitors（EGFR-TKIs）targeting EGFR can prolong the survival of advanced NSCLC patients with EGFR sensitive mutations,and are now the first-line treatment option for these patients.However,EGFR-TKIs commonly develop acquired drug resistance after use and have complex and diverse mechanisms.The main mechanisms of resistance to first and second generation EGFR-TKIs include secondary mutation of EGFR,MET amplification,FGFR overexpression,JAK/STAT bypass activation,and epithelial mesenchymal transition（EMT）.Osimertinib,a third generation EGFR-TKI targeting EGFR secondary mutation,has shown satisfactory clinical efficacy,but studies have found that it is still resistant to development.Therefore,it is of great scientific significance to find new targets that can overcome acquired resistance to EGFR-TKI in EGFR-mutated NSCLC patients.SHP2,a cytoplasmic tyrosine phosphatase widely expressed in various tissues of the body,is encoded by the PTPN11 gene and is involved in most receptor-tyrosine kinase（RTK）signaling pathways,playing an important role in tumorigenesis,inflammation,transcriptional regulation and cell migration.In past studies,SHP2 was also found to be able to be associated with multiple drug resistance by regulating MAPK pathway activation phosphorylation,and the SHP2-specific inhibitor SHP099 was able to enhance drug sensitivity of resistant cells in multiple targeted drug resistance models.In addition,activation of ERK1/2,a downstream pathway of MAPK,has been suggested to be an important mechanism of EGFR-TKI resistance.In summary,we hypothesize that SHP2 may be involved in the process of EGFR-TKI acquired resistance in non-small cell lung cancer through the MAPK-ERK pathway and plays a key role in the resistance mechanism,and the study of the role and mechanism of SHP2 in EGFR-TKI resistant cells is expected to suggest a new pathway to overcome drug resistance.However,studies on the mechanism of SHP2 involvement in EGFR-TKI resistance are still relatively limited.Previous studies have suggested that SHP2 may be involved in the development of acquired resistance to gefitinib in EGFR-mutated NSCLC cells,but the mechanisms have not been explored in depth.In addition,no studies have addressed the role and mechanism of SHP2 in acquired resistance to osimertinib in NSCLC.Therefore,the present study was proposed to establish osimertinib-acquired resistant cell lines with gefitinib-acquired resistant cell lines using EGFR-mutated lung adenocarcinoma cell line PC9 cells.Then,the correlation between SHP2 and EGFR-TKI resistance was clarified by multiple aspects such as transcriptomic analysis of parental and resistant cells,pathway enrichment analysis of SHP2,and prognostic correlation analysis of SHP2 and NSCLC.Further,in this study,gene functional studies and pharmacological intervention studies of SHP2 expression were performed by in vitro and in vivo experiments to clarify its relationship with the development of gefitinib resistance and osimertinib resistance in NSCLC.Finally,protein expression analysis of the major EGFR-TKI resistance pathways was used to clarify the pathway mechanisms involved in drug resistance by SHP2.Materials and Methods:The PC9 osimertinib-resistant cell line PC9OR and gefitinib-resistant cell line PC9GR were constructed by continuous incremental drug concentration induction;the apoptosis,the proliferation ability and the change of drug tolerance of PC9 cells before and after drug resistance were detected by flow cytometry and CCK-8 method.The expression differences between PC9 and PC9OR and PC9GR cells were clarified by transcriptome sequencing analysis and verified by q PCR and WB experiments.Bioinformatics approach was applied to analyze the cellular signaling pathways involved in SHP2 and correlate SHP2 with the prognosis of NSCLC.The lentivirus was constructed by short hairpin RNA and transfected with drug-resistant cell lines to knock down the expression of SHP2,which was verified by q PCR and WB assay;the apoptosis,proliferation ability and the change of drug tolerance of drug-resistant cells before and after SHP2 knockdown were detected by flow cytometry and CCK-8 assay.The changes of drug resistance of drug-resistant cell lines to the corresponding drugs after the use of SHP099 were detected by CCK-8method.The role of SHP2 in the acquired resistance of PC9 cells to osimertinib was verified in vivo by constructing a subcutaneous transplantation tumor model in nude mice.The molecular mechanisms of drug resistance in PC9OR and PC9GR cells were clarified by WB assays.Then,the molecular mechanisms of SHP2 involvement in PC9 cell osimertinib resistance and gefitinib resistance were clarified by WB assay to dectect the changes of related pathways and their phosphorylation expression levels after the action of SHP2 knockdown or the use of SHP099 in drug-resistant lines.Based on the above detected changes in the expression and phosphorylation levels of resistance-related pathways,the molecular mechanisms of SHP2 involvement in acquired resistance to oseltinib in EGFR mutant NSCLC were verified in vitro and in vivo using the corresponding pathway inhibitors in combination with SHP099.Results:1.Histological analysis of SHP2 and EGFR-TKIs resistance in non-small cell lung cancer and prognostic correlation analysis1.1 Construction of PC9OR and PC9GR cell lines.Comparing with parental PC9 cells,both PC9OR and PC9GR cells showed partial cell morphological changes,and PC9OR was significantly more resistant to osimertinib and PC9GR was significantly more resistant to gefitinib;in the absence of drug effects,the cell proliferation and apoptosis abilities between parental cells and the two drug-resistant cells no significant difference was observed.1.2 Histological analysis of SHP2 between parental cells and drug-resistant cells.There was no notable difference in the expression of PTPN11 gene in paraneoplastic tissues and cancer tissues of NSCLC.Transcriptome sequencing showed that PTPN11 gene expression was higher in PC9OR and PC9GR cells than in PC9 cells.The q PCR results showed that SHP2 m RNA expression was increased in PC9OR and PC9GR cells compared with parental PC9 cells.The WB results showed that both PC9OR and PC9GR cells showed high expression of SHP2 protein compared with parental PC9 cells.1.3 Pathway enrichment analysis of SHP2 gene PTPN11The GO and KEGG annotation analysis of SHP2 showed that SHP2 was highly associated with metabolic processes,bioregulatory processes,and cellular processes in the GO database,and with spliceosomes,cell cycle,and MAPK pathway in the KEGG database.Continuing the enrichment analysis of SHP2 expression and MAPK pathway,the results showed that PTPN11 was highly correlated with MAPKAPK5,TAOK1,MAP3K2,PAK2 and CHUK genes in MAPK-ERK1/2 pathway,suggesting that SHP2 is highly correlated with MAPK-ERK1/2 pathway and may have a regulatory role in the expression of this pathway.1.4 Prognostic correlation analysis of SHP2 and NSCLCThe results of K-M curve analysis showed that OS（HR=1.64,P=0.00036）and PFS（HR=1.85,P=0.00029）were worse in the SHP2 high expression group than in the SHP2 low expression group,indicating that high SHP2 expression was associated with a poorer prognosis in non-small cell lung cancer.The results of univariate analysis showed that high SHP2 expression was associated with poorer prognosis（HR=1.451,P=0.017）;the results of multivariate analysis after including age,sex,ethnicity,and tumor stage showed that high SHP2 expression was an independent risk factor for poor prognosis（HR=1.434,P=0.024）.2.Study of the relationship between SHP2 and EGFR mutant NSCLC producing resistance to osimertinib and gefitinib2.1 Construction of PC9OR,PC9GR cell line SHP2 stable knockdown cell lineBoth sh1 and sh2 of PC9OR expressed significantly lower SHP2 m RNA compared to PC9OR-VECTOR cells transfected with empty plasmid,SHP2 protein expression was down-regulated,while there was no significant difference in proliferation and apoptotic capacity of each cell;sh1 and sh2 of PC9GR were similarly more significant than those of PC9GR-VECTOR cells transfected with empty plasmid Expression of SHP2 m RNA was significantly lower in PC9GR sh1and sh2 than in PC9GR-VECTOR cells transfected with empty plasmid,SHP2protein expression was down-regulated,while there was no significant difference in the proliferation and apoptosis ability of each cell.PC9OR cells showed a leftward shift in the drug inhibition curve and a decrease in IC₅₀for osimertinib after SHP2knockdown;the pharmacological inhibition curve of PC9GR on gefitinib shifted left after SHP2 knockdown and IC₅₀decreased significantly.The IC₅₀ of PC9OR cells to osimertinib decreased after the use of SHP099（10880.00±2272.00 n M vs.4844.00±584.00 n M,P<0.01）;the IC₅₀ of PC9GR cells to gefitinib decreased significantly after the use of SHP099（6910.00±287.00 n M vs.1569.00±232.00 n M,P<0.0001）.2.2 In vivo study of the effect of SHP099 on the sensitivity of PC9OR cells to osimertinibComparing the PC9 and PC9+Osi groups,we found that the tumors in the PC9group basically grew in a straight line,while the tumors in the PC9+Osi group were continuously inhibited and finally disappeared,indicating that osimertinib had a significant inhibitory effect on PC9 cell transplantation;the tumors in the PC9OR and PC9OR+Osi groups were basically the same as the tumors in the PC9 group and grew in a straight line.The tumors in the PC9OR and PC9OR+Osi groups were basically the same as those in the PC9 group and grew in a straight line,which on the one hand indicates that in vivo PC9OR cells have similar proliferation ability to PC9cells,which is consistent with the in vitro experimental observations,and on the other hand indicates that PC9OR cells have a strong tolerance effect to osimertinib and the dose of osimertinib given in the experiment could not inhibit PC9OR tumor enlargement.tumor bodies of transplanted tumors in the PC9OR+Osi+SHP099 group of nude mice were relatively The tumors in the PC9OR-Osi-ositinib group were slowed down in growth,but did not suffer from sustained inhibition and disappearance as in the PC9+Osi group,indicating that SHP099 was able to partially increase the sensitivity of PC9OR to osimertinib.3.SHP2 mediates the molecular mechanism of osimertinib and gefitinib resistance in EGFR mutant NSCLC3.1 Changes in pathway protein expression associated with PC9 osimertinib resistance and gefitinib resistanceIn the presence of osimertinib,there were no significant changes in ERK1/2expression in PC9,but ERK1/2 phosphorylation was attenuated,while no significant changes were seen in PC9OR cells;no significant changes in AKT expression and slightly enhanced AKT phosphorylation in PC9 cells,while no significant changes were seen in PC9OR cells;no significant changes in P38 expression and its phosphorylation in both PC9 and PC9OR.In the presence of gefitinib,there was no significant change in ERK1/2 expression in PC9 cells,but ERK1/2 phosphorylation was attenuated,while no significant change was seen in PC9GR;no significant change in AKT expression in PC9 cells,AKT phosphorylation was slightly enhanced,and no significant change was seen in PC9GR cells;no significant change in P38 and its phosphorylation in both PC9 and PC9GR.3.2 Effect of SHP2 knockdown on drug resistance-related pathways in PC9OR and PC9GR cellsIn PC9OR cells,ERK1/2,AKT and P38 expression did not change significantly after SHP2 knockdown,but ERK1/2 phosphorylation was significantly weakened;AKT and P38 phosphorylation were significantly enhanced.In PC9GR cells,ERK1/2,AKT and P38 expression were not significantly changed after SHP2knockdown,but ERK1/2 phosphorylation was significantly weakened;AKT and P38phosphorylation were slightly enhanced.PC9OR cells showed no significant changes in ERK1/2,AKT and P38expression and their phosphorylation at 4h and 48h of SHP099 alone;PC9OR cells showed no significant changes in ERK1/2,AKT and P38 expression after the combination of SHP099 with osimertinib,but ERK1/2 phosphorylation was significantly attenuated,while AKT and P38 phosphorylation were PC9GR cells showed no significant changes in ERK1/2,AKT and P38 expression and their phosphorylation at 4h and 48h of SHP099 alone;after SHP099 combined with gefitinib,the expression of ERK1/2,AKT and P38 were not significantly changed,but ERK1/2 phosphorylation was significantly weakened,while AKT and P38phosphorylation were slightly enhanced.3.3 Effect of SHP099 combined with LY294002（AKT phosphorylation inhibitor）and SB202190（P38 phosphorylation inhibitor）on Osi resistance in PC9OR cellsThe IC₅₀ of PC9OR cells decreased on osimertinib with SHP099;the IC₅₀ of PC9OR on osimertinib did not change significantly with LY294002 alone;PC9OR had a decreased IC₅₀ on osimertinib when SHP099 was combined with LY294002and compared to SHP099 alone further decreased when SHP099 was used.The IC₅₀of PC9OR on osimertinib decreased after SB202190 alone;the IC₅₀of PC9OR on osimertinib when SHP099 was combined with SB202190 The IC₅₀ for osimertinib decreased when usedand further decreased when compared to SHP099 or SB202190alone.PC9OR cells showed a decreased IC₅₀ for osimertinib when SB202190 was used in combination with LY294002.PC9OR cells showed a significant decrease in IC₅₀ to osimertinib when SHP099 was combined with SB202190 and LY294002.Compared with the control group（PC9OR group without drug use）,PC9OR cells showed no significant changes in each pathway with osimertinib or SHP099alone;phosphorylation of AKT and P38 was attenuated in PC9OR with LY294002and SB202190 alone,and no significant changes in the remaining pathways;PC9OR cells were combined with osimertinib and SHP099,no significant changes in ERK1/2,AKT and P38 expression,but phosphorylation of ERK1/2 was diminished,while phosphorylation of AKT and P38 was enhanced;phosphorylation of AKT and P38 was diminished and phosphorylation of ERK1/2 was not significantly changed in PC9OR cells in combination of osimertitinib with LY294002-and SB202190.Phosphorylation of AKT and P38 was attenuated in PC9OR cells in combination of SHP099 with LY294002-and SB202190,while phosphorylation of ERK1/2 was not significantly changed;PC9OR cells in combination of osimertinib,SHP099,LY294002 and SB202190,ERK1/2,AKT and P38 expression were not significantly changes,but the phosphorylation of ERK1/2,AKT and P38 were significantly attenuated.3.4 In vivo validation of the effect of SHP099 combined with LY294002 and SB202190 on the resistance of PC9OR cells to OsiIn the PC9OR cell transplanted tumors constructed in this section,the tumor volume was reduced in the Osi+SHP099 group relative to the Osi alone group,indicating that SHP099 was able to increase the sensitivity of PC9OR cells to Osi;the Osi+LY294002+SB202190 group also reduced the tumor volume relative to the Osi group alone,while there was no significant difference in tumor volume with the Osi+SHP099 group,indicating that the combination of LY294002 and SB202190also increased the sensitivity of PC9OR to The tumor size in the Osi+SHP099+LY294002+SB202190 group was further reduced compared with the Osi+SHP099 group,and the tumor size in the Osi+LY294002+SB202190 group was further reduced compared with the Osi+SHP099 group.SB202190 group also had further tumor shrinkage indicating that the combination of SHP099 with LY294002and SB202190 was able to substantially increase the sensitivity of PC9OR cells to Osi in the in vivo environment;meanwhile,SHP099+LY294002+SB202190 group showed no significant difference in tumor volume from the Osi group alone,while the difference with the Osi+SHP099+LY294002+SB202190 group was significant（P<0.0001）,indicating that in the absence of Osi effect,the combination of SHP099,LY294002 and SB202190 had no inhibitory effect on PC9OR cells,and it was the combination of SHP099,LY294002 and SB202190 that increased the sensitivity of PC9OR to Osi.Conclusion:SHP2 plays an important role in the drug resistance of NSCLC generation gefitinib-resistant cell line PC9GR cells and generation III osimertinib-resistant cell line PC9OR cells.In osimertinib resistance,SHP2 is involved in resistance through activation of MAPK-ERK and PI3K-AKT pathways;SHP2 inhibition enhances the sensitivity of PC9OR cells to osimertinib,but also causes compensatory activation of PI3K-AKT and MAPK-P38 pathways;the combination of SHP2 inhibitor with AKT inhibitor and P38 inhibitor was able to further reverse PC9OR osimertinib resistance.In gefitinib resistance,SHP2 is involved in drug resistance through activation of MAPK-ERK pathway,and inhibition of SHP2 can substantially enhance the sensitivity of PC9GR cells to gefitinib.