| Background and Objective:Non-Small Cell Lung Cancer（NSCLC）is the most common histological type of lung cancer,accounting for about 80%-85%of all lung cancers.Almost two-thirds of NSCLC patients harbor an oncogene,including epidermal growth factor receptor（EGFR）,KRAS,anaplastic lymphoma kinase（ALK）,ROS1,BRAF,mesenchymal to epidermal transformation Factors（MET）and RET,et al.These oncogenes activate their tyrosine kinase domains through gene mutation,thereby continuously activating downstream MAPK/ERK,PI3K/AKT and JAK/STAT3 signaling pathways,which in turn drives the occurrence and progression of NSCLC.In the past 20 years,small molecule tyrosine kinase inhibitors（TKI）have achieved breakthroughs in the clinical treatment of NSCLC patients harboring oncogenes,which greatly improved the quality of life and prognosis of patients with advanced NSCLC.Unfortunately,most patients will develop resistance within 0.5 to2 years after TKIs treatment,and the tumors will progress again after TKI resistance,which makes it difficult for patients to achieve long-term benefit from TKIs treatment.The occurrence of resistance to TKI has greatly limited the further improvement of the survival of NSCLC patients.And the in-depth study of TKI resistance will help provide new insights for overcoming or reversing resistance to TKI,and then provide rationales for the achievement of individualized precision treatment.The mechanisms of resistance to TKI in NSCLC patients mainly consist of primary and acquired resistance.Approximately 4%-10%of NSCLC patients have no response to the initial TKI treatment（primary resistance）,and more than 90%of patients resistant to TKI treatment via acquired resistance.The mechanisms of acquired resistance to TKI treatment include secondary mutations of targeted genes,bypass signaling activation,and histological changes,et al.Among them,the secondary mutations of targeted genes can hinder the combination of TKI and kinases via changing the conformation of tyrosine kinases and/or changing the affinity between the kinases and ATP,thereby making tumors cells escape the killing of TKI.The next-generation of TKIs are usually designed to target the secondary mutation sites,so that patients with secondary mutations of the targeted gene can continue to benefit from TKI treatment.Bypass signaling activation is to reactivate key downstream effectors required for cell survival and proliferation through parallel signaling pathways of other receptor tyrosine kinases（RTK）,so that tumors can continue to survival and growth despite the suppression of oncogene.With the clinical application of next-generation of TKIs（such as osimertinib and loratinib）,more and more NSCLC patients have acquired resistance to the next-generation of TKI treatment.However,the specific molecular mechanisms of acquired resistance to the next-generation TKIs are mostly elusive,and there are lack of effective solutions for acquired resistance to the next-generation TKIs.Therefore,the in-depth interpretation of the mechanisms of acquired resistance to the next-generation TKIs is urgently warranted,which will provide scientific rationales for profiling new strategies to overcome or reverse the acquired resistance to next-generation TKIs.Towards this goal,in this study,we generated three TKI-resistant NSCLC cell lines with corresponding TKIs,and then we performed whole-exome sequencing and transcriptomic sequencing on parental and TKI-resistant cells.Next,we characterized the changes in gene variations and gene expression profiles in NSCLC cells before and after TKI-acquired resistance,and identified GAS6/AXL as the common factors that may mediate acquired resistance to TKIs in NSCLC cells.Furthermore,through in vitro,in vivo,and clinical samples analysis,we clarified the role of GAS6/AXL in the process of acquired resistance to TKIs in NSCLC,and initially revealed the molecular mechanisms of GAS6/AXL that mediate acquired resistance to TKIs in NSCLC cells.Materials and Methods:Chapter Ⅰ.Generation of different TKI-resistant NSCLC cells1.We used the method of continuously increased drug concentration to generate EGFR-mutant and osimertinib-resistant PC-9OR cells,ALK fusion-positive and loratinib-resistant H3122LR cells,and ROS1 fusion-positive and crizotinib-resistant HCC78CR cells.2.Half maximal inhibitory concentration(IC50)of corresponding TKIs in parental and TKI-resistant cells were measured via CCK-8 assay.3.Cell viability of parental and TKI-resistant cells were measured through CCK-8assay.4.Cell apoptosis of parental and TKI-resistant cells were detected by flow cytometry.Chapter Ⅱ.Gene variations and gene expression profiles in different TKI-resistant NSCLC cells1.Whole-exome sequencing and transcriptomic sequencing were performed on PC-9and PC-9OR,H3122 and H3122LR,and HCC78 and HCC78CR cells,respectively,via NGS technology.2.Genome Analysis Toolkit（GATK）was used to analyze the gene variations of TKI-resistant cells.3.Hisat2-Stringtie-DESeq2 software was used to analyze the differential expression of genes between parental and TKI-resistant cells.4.Cluster Profiler software was used for KEGG pathway enrichment analysis.5.Based on the analysis of KEGG pathway,the Pearson correlation coefficient was used to analyze the gene regulatory network.Chapter Ⅲ.The role of GAS6/AXL in acquired resistance to TKIs in NSCLC cells1.qPCR and western blotting experiments were performed to measure the m RNA and protein expression levels of GAS6 and AXL in PC-9 and PC-9OR,H3122 and H3122LR,and HCC78 and HCC78CR cells,respectively.2.Immunohistochemistry staining were performed to detect the protein expression levels of GAS6 and AXL in tissue samples before and after TKI resistance.3.Molecular cloning technology were used to generate sh RNA viruses targeting GAS6and AXL,and then stable cell lines that knock down GAS6 and AXL in each TKI-resistant cell line were established with these sh RNA viruses.4.CCK-8 assays were used to determine the IC50s to corresponding TKIs of each TKI-resistant cell line after knocking down GAS6 and AXL,respectively.5.CCK-8 assays were used to determine the IC50s to corresponding TKIs of each TKI-resistant cell line after treated with AXL inhibitor R428.6.Cell-line derived xenograft（CDX）models were generated in nude mice to investigate the therapeutic role of osimertinib in PC-9OR tumors with AXL knockdown.Chapter Ⅳ.Molecular mechanisms of GAS6/AXL mediated acquired resistance to TKI in NSCLC cells1.The expression of transcription factor c-JUN was knocked down by use of siRNA in HCC78CR cells,and then the changes of GAS6 and AXL m RNA and protein expression were measured by q PCR and western blotting,respectively.2.Western blotting experiments were performed to measure the expression levels of AKT and p-AKT protein in parental and TKI-resistant cells treated with titrated doses of TKIs.3.Western blotting experiments were performed to measure the expression levels of AKT and p-AKT protein in TKI-resistant cells treated with TKIs±R428.Results:Chapter Ⅰ.Generation of different TKI-resistant NSCLC cells1.Generation of EGFR-mutant and osimertinib-resistant NSCLC cellsCompared with PC-9 cells,PC-9OR cells showed slight changes in morphology,such as rounded cell outlines.The IC50 to osimertinib in PC-9OR cells was higher than that in PC-9 cells.With the treatment of osimertinib at 0.1μM,the proliferation of PC-9 cells was significantly inhibited（P<0.0001）,while the proliferation of PC-9OR cells was not affected（P>0.05）.In addition,osimertinib at 0.1μM induced significant cell apoptosis in PC-9 cells（P<0.0001）,but had no obvious effect on PC-9OR cells（P>0.05）.2.Generation of ALK fusion-positive and lorlatinib-resistant NSCLC cellsSimilar to PC-9OR cells,the cell outlines of H3122LR cells were more rounded than H3122 cells.The IC50 to loratinib in H3122LR cells was higher than that in H3122cells.Loratinib at 0.1μM significantly inhibited the proliferation of H3122 cells（P<0.0001）,rather than H3122LR cells（P>0.05）.In addition,loratinib at 0.1μM induced significant cell apoptosis in H3122 cells（P<0.0001）,but had no obvious impact on H3122LR cells（P>0.05）.3.Generation of ROS1 fusion-positive and crizotinib-resistant NSCLC cellsSimilarly,when compared with crizotinib-sensitive HCC78 cells,HCC78CR cells showed a more rounded cell outline.The IC50 to crizotinib in HCC78CR cells was higher than that in HCC78 cells.Crizotinib at 1μM not only significantly inhibited the proliferation of HCC78 cells（P<0.0001）,but also significantly induced cell apoptosis in HCC78 cells（P<0.0001）;While crizotinib at 0.1μM has no significant effect on the proliferation and apoptosis in HCC78CR cells（P>0.05）.Chapter Ⅱ.Gene variations and gene expression profiles in different TKI-resistant NSCLC cells1.Gene variations in different TKI-resistant NSCLC cellsBy analyzing the whole-exome sequencing data of parental and drug-resistant cells,we found that a total of 536 non-synonymous mutations in the exon region were identified in PC-9OR cells.The EGFR C797S mutation was not detected in PC-9OR cells.There were 221 non-synonymous mutations in the exon region in H3122LR cells,and no mutations were identified in the ALK tyrosine kinase domain.Besides,a total of 154 non-synonymous mutations in the exon region were detected in HCC78CR cells,but no mutations were identified in the ROS1 tyrosine kinase domain.No mutations in driver genes such as BRAF,KRAS,NRAS and PIK3CA were detected in PC-9OR,H3122LR,or HCC78CR cells,suggesting that the acquired resistance to TKIs in these cells was not caused by secondary mutations of the targeted genes,nor by gene mutations of other common drivers.2.Gene expression profiles in different TKI-resistant NSCLC cellsBy analyzing the transcriptomic sequencing data of parental and TKI-resistant cells,we found that there were a total of 3929 differentially expressed genes between PC-9 and PC-9OR cells,with 1281 up-regulated genes and 2648 down-regulated genes in PC-9OR cells.A total of 1909 differentially expressed genes were identified between H3122 and H3122LR cells,with 807 up-regulated genes and 1102 down-regulated genes in H3122LR cells.Besides,there were a total of 1337 differentially expressed genes between HCC78 and HCC78CR cells,with 580 up-regulated genes and 757 down-regulated genes in HCC78CR cells.There were 9 common up-regulated genes and 28 common down-regulated genes among PC-9OR,H3122LR and HCC78CR cells.3.KEGG signaling pathway analysis in different TKI-resistant NSCLC cellsBy performing KEGG signaling pathway analysis of differential genes between parental and TKI-resistant cells,we found that the differentially expressed genes in PC-9OR cells,H3122LR and HCC78CR cells were significantly enriched in PI3K/AKT,MAPK and Ras signaling pathways,among which PI3K/AKT signaling pathway was the most enriched one.These results suggested that these three TKI-resistant cell lines may have similar regulatory mechanisms of acquired resistance to TKI treatment.4.Gene regulatory network analysis in different TKI-resistant NSCLC cellsBy analyzing the transcriptional regulatory network specific to drug resistance,we found that the transcriptional regulatory module of GAS6/AXL was significantly different between HCC78 and HCC78CR cells,and AXL was significantly up-regulated in both PC-9OR and H3122LR cells,which indicated that GAS6/AXL may be the key factor that mediates the acquired resistance to TKIs in these cell lines.Chapter Ⅲ.The role of GAS6/AXL in acquired resistance to TKIs in NSCLC cells1.The expression of GAS6 and AXL in different TKI-resistant NSCLC cellsBy performing q PCR and Western blotting experiments in parental and TKI-resistant cells,we found that both m RNA and protein levels of GAS6 and AXL were significantly increased in PC-9OR cells,when compared with PC-9 cells（both P<0.0001）.And compared with H3122 cells,the m RNA levels of GAS6 and AXL in H3122LR cells were significantly increased（P<0.0001 and P<0.001,respectively）,and the protein level of AXL was also obviously up-regulated.Similarly,when compared with HCC78 cells,both m RNA and protein levels of GAS6 and AXL were significantly increased in HCC78CR cells（P<0.001 and P<0.01,respectively）.2.The expression of GAS6 and AXL in NSCLC patients resistant to EGFR TKIsBy performing immunohistochemistry staining with tissue samples before and after EGFR TKI resistance,we found that the protein expression of GAS6 and AXL were significantly increased in tumor tissues after osimertinib,gefitinib and erlotinib resistance（P<0.001 and P<0.05,respectively）.3.The role of GAS6 in acquired resistance to TKIs in NSCLC cellsBy using molecular cloning technology,we generated sh RNA viruses targeting GAS6,and then established stable cell lines that knock down GAS6 in each TKI-resistant cell line with these sh RNA viruses.Compared with PC-9OR-vector cells,the IC50 to osimertinib in PC-9OR-sh GAS6-1 and PC-9OR-sh GAS6-2 cells were decreased.Similarly,the IC50 to crizotinib in HCC78CR-sh GAS6-1 and HCC78CR-sh GAS6-2 cells were also lower than that of HCC78CR-vector cells.These results indicated that reducing the expression of GAS6 in TKI-resistant cells can increase the sensitivity of these cells to TKI treatment.4.The role of AXL in acquired resistance to TKIs in NSCLC cells（1）Similarly,we established stable cell lines that knock down AXL in each TKI-resistant cell line with AXL sh RNA viruses.Compared with PC-9OR-vector cells,the IC50 to osimertinib in PC-9OR-sh AXL-1 and PC-9OR-sh AXL-2 cells were decreased.And when compared with H3122LR-vector cells,the IC50 to lorlatinib in H3122LR-sh AXL-1 and H3122LR-sh AXL-2 cells were reduced.Besides,the IC50 to crizotinib in HCC78CR-sh AXL-1 and HCC78CR-sh AXL-2 cells was also lower than that of HCC78CR-vector cells.These results suggested that reducing the expression of AXL in TKI-resistant cells can increase the sensitivity of these cells to TKI treatment.（2）In addition,we found that the IC50 to osimertinib in PC-9OR cells treated with R428 was significantly lower than that cells treated with DMSO.Similarly,the IC50 to lorlatinib in H3122LR cells treated with R428 was significantly lower than that cells treated with DMSO.Besides,in HCC78CR cells,the IC50 to crizotinib in the R428treatment group was significantly lower than that in the DMSO treatment group.These results suggested that inhibiting the activation of AXL in TKI-resistant cells can resensitize the resistant cells to TKI treatment.5.The role of AXL in PC-9OR cells in CDX modelsCDX models were generated in nude mice,and we found that in the PC-9OR tumor modes with AXL knockdown,tumor growth was significantly inhibited with osimertinib treatment（P<0.05）,suggesting that inhibiting the expression of AXL in vivo can increase the sensitivity of PC-9OR tumor to osimertinib treatment.Chapter Ⅳ.Molecular mechanisms of GAS6/AXL mediated acquired resistance to TKI in NSCLC cells1.The role of c-JUN in the expression of GAS6 and AXL in HCC78CR cellsAfter knocking down c-JUN in HCC78CR cells,we found that both m RNA and protein expression of GAS6 and AXL in HCC78CR cells were significantly decreased（P<0.001 and P<0.01,respectively）,suggesting that c-JUN regulates the expression of GAS6 and AXL at both m RNA and protein levels in HCC78CR cells.2.The status of downstream signaling pathways in TKI-resistant NSCLC cellsThe expression levels of p AKT rather than AKT protein were significantly decreased in PC-9,H3122,and HCC78 cells treated with corresponding TKI drugs;while the expression levels of both AKT and p-AKT proteins did not change in PC-9OR,H3122LR and HCC78CR cells treated with corresponding TKI drugs.These results suggested that the PI3K/AKT signaling was continuously activated in these TKI-resistant NSCLC cells.3.The effect of AXL on the activation of PI3K/AKT signaling pathway in TKI-resistant NSCLC cellsThe expression levels of both AKT and p-AKT proteins did not change in PC-9OR,H3122LR and HCC78CR cells treated with corresponding TKI drugs or R428;while the expression levels of p AKT protein were significantly decreased in PC-9OR,H3122LR,and HCC78CR cells treated with corresponding TKI drugs plus R428.And these results indicated that PI3K/AKT signaling was continuously activated by AXL bypass pathway in these TKI-resistant NSCLC cells,while inhibiting EGFR and AXL synchronously can inhibit the activation of the PI3K/AKT signaling pathway.Conclusion:In this study,we generated three TKI-resistant NSCLC cell lines with corresponding TKIs.And we identified GAS6/AXL as the key factors that mediated the acquisition of TKIs resistance in NSCLC cells.Mechanistically,the transcription factor c-JUN can regulate the expression of GAS6 and AXL,while AXL can continuously activate the PI3K/AKT signaling through the bypass pathway to mediate the acquired resistance of NSCLC cells to TKIs.In summary,this study elucidates the common molecular mechanisms in different TKI-resistant NSCLC cells,which provides a scientific rationale for the combination therapy to overcome the acquired resistance to next-generation TKIs in NSCLC patients. |
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