Effect And Mechanism Of BCL-2 In Resistance To Third Generation EGFR-TKI In Lung Adenocarcinoma

Objective:Lung cancer is the malignant tumor with the highest morbidity and mortality worldwide.In China,the incidence of lung cancer is also increasing year by year.Lung cancer is divided into two tissue types:Small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),of which NSCLC accounts for 85%of lung cancer,while lung adenocarcinoma is the main component of NSCLC.Early and partial Ⅲ stage lung cancer patients can be treated by surgery and achieve good prognosis.But for locally advanced and metastatic lung cancer patients,treatment is limited in addition to conventional radiation and chemotherapy.In recent years,the continuous emergence of small molecule targeted therapy drugs has provided a promising treatment for patients with incurable lung cancer.Its therapeutic effect for patients with locally advanced and advanced metastatic lung cancer with specific gene mutations is far superior to traditional radiotherapy and chemotherapy.Although targeted drugs can effectively delay tumor progression and improve patient prognosis,the inevitable drug resistance after treatment has not been fundamentally solved,which also makes it difficult for patients with advanced lung cancer to benefit from targeted therapy in the long term.EGFR mutation is the most common type of mutation in lung adenocarcinoma,accounting for about 28%.Osimertinib,as a third representative epithelial growth factor receptor tyrosine kinase inhibitor(EGFR-TKIs),has been widely used clinically in the first-line treatment for patients with advanced lung adenocarcinoma with EGFR mutation and in the second-line treatment for patients with disease progression following the first two generations of TKI therapy and associated with EFGR T790M mutation.Although osimertinib is able to reach a broader population than the previous two generations of targeted drugs,and has been effective in improving patient outcomes,the common resistance problem in the late treatment period still exists,and the mechanism of its evolution is not fully understood by researchers.Therefore,an in-depth study on the mechanism of drug resistance caused by the targeted therapy of osimertinib for lung adenocarcinoma with EGFR mutation,as well as a detailed explanation of its principle and development rules,can provide strong guidance for the improvement of its targeted treatment plan.Apoptosis,as a gene-coded programmed cell death mechanism,plays an important role in the development of tumors and their response to therapeutic drugs.Studies have shown that the abnormal regulation mechanism of apoptosis often leads to tumor cells antagonizing the apoptotic effect induced by small molecule targeted drugs,resulting in drug resistance.As the main regulator of endogenous apoptosis,the BCL-2 family plays an important role in maintaining the balance between apoptosis and survival of cells.In recent years,studies on the formation of drug resistance after the involvement of the BCL-2 family in the EGFR-TKI targeting therapy of the first and second generation of lung adenocarcinoma have been reported from time to time.However,in-depth and comprehensive studies on the association between the BCL-2 family and the drug resistance after the third-generation TKI treatment of osimertinib are relatively scarce.BCL-2 family members are diverse and have different functions.It is of great significance for the selection of clinical application strategy and the formulation of treatment plan after drug resistance to clarify the specific mechanism and pathway influence of key members on osimertinib resistance.In this study,osimertinib resistant stable cell line was established in human EGFR mutant lung adenocarcinoma HCC827 cell line,and the cytological morphology and osimertinib tolerance before and after HCC827 resistance were explored and compared.Then,the expression of BCL-2 family related genes and the differences of transcription protein products before and after osimertinib resistance in HCC827 cells were analyzed,in order to find out the key genes that may be involved in the formation of osimertinib resistance in HCC827 cells.Further,we explored the expression characteristics and function of these key genes in HCC827 osimertinib resistant cell line and use specific inhibitors by means of molecular,biochemical,cellular and in vitro animal experiments,so as to elaborate its association with osimertinib resistance in lung adenocarcinoma.Finally,we will further explore the characteristics of BCL-2 gene and its protein products in HCC827OR,and link the differences in the signal pathways after osimertinib resistance of HCC827,so as to preliminarily explore the specific molecular mechanism of the participation of this key gene in the formation of drug resistance.Materials and Methods:Chapter 1.Establishment of lung adenocarcinoma HCC827 drug-resistant cell line and analysis of gene and protein expression of BCL-2 familyFirst of all,we established HCC827 drug-resistant stable strain(HCC827OR)by using the method of increasing drug concentration step by step,and explore its biological characteristics through cell morphology observation under microscope,drug gradient concentration experiment,clone formation experiment,flow cytometry apoptosis detection,etc.,to verify whether the stable strain is successfully constructed.Based on the summary of the research background on apoptosis regulation and osimertinib resistance and the flow cytometric apoptosis detection results before and after acquiring osimertinib resistance in HCC827 cells.At the same time,we will simultaneously detect the protein expression levels of each member of the BCL-2 family in HCC827 and HCC827OR by western blot.Subsequently,we summarized and comprehensively analyzed the above experimental results to find out the key members of the BCL-2 family that may be potentially involved in the antagonism of lung adenocarcinoma against the apoptotic effect induced by osimertinib and thus acquire drug resistance.Chapter 2.Study on the correlation between BCL-2 and the development of osimertinib resistance in lung adenocarcinoma HCC827 cell lineBased on gene transcriptome sequencing analysis and protein expression level detection of BCL-2 family before and after HCC827 cell acquiring osimertinib resistance,we found that both gene and protein levels of BCL-2 were activated and highly expressed.In this chapter,we will use shRNA construct HCC827OR BCL-2 gene knockdown strains,and use the BCL-2 specific inhibitor ABT-199 to suppress the BCL-2 in the state of high expression inside the cell.The correlation between BCL-2 and HCC827 osimertinib resistance was investigated and verified from various aspects by biochemical,molecular,cellular and animal in vitro experiments and other methods.Finally,Western blotting and immunocoprecipitated methods were used to detect the changes in the protein expression and function of other BCL-2 family members after ABT-199 inhibited the function of BCL-2,so as to further explain the phenomenon of HCC827OR recovering certain drug sensitivity after combined administration.Chapter 3.Study on the molecular mechanism of osimertinib drug resistance in HCC827 cell line mediated by BCL-2In this chapter,the specific molecular mechanism of BCL-2 involved in regulating the formation of osimertinib resistance in HCC827 cells will be preliminary explored.We first explored the characteristics of BCL-2 in HCC827OR from gene modification to the level of transcriptional protein products.And we analyzed the difference of methylation abundance in each region of BCL-2 gene before and after drug resistance to trace the cause of its high gene expression,then carries on the mice symbol tissue samples by immunohistochemical mirror under direct observation and extracting HCC827OR cell components(cell nuclear membrane,the cytoplasm membrane,organelles membrane,cytoplasm)protein immunoblot experiments to determine the BCL-2 protein products within HCC827OR area.In the last chapter,HCC827OR was treated with protein synthesis and decomposition inhibitors to investigate the stability of BCL-2 gene expression products.We hope to fully describe the related characteristics of BCL-2 gene and its transcripts after drug resistance through this series of experiments.Furthermore,western blotting was used to detect the expression differences of key proteins in the pathways related to BCL-2 regulation(PI3K/AKT,JAK/STAT3,p53,JNK/SAPK,ERK/MAPK)reported in domestic and foreign studies before and after HCC827 acquiring resistance to osimertinib,so as to reveal the involvement of upstream BCL-2 signaling pathway in the formation of resistance in HCC827OR.Results:Chapter 11.Establishment and cell function detection of osimertinib resistant cell lines of HCC827Microscopic observation of the constructed HCC827OR showed that it had morphologic changes compared with its parent susceptible strain.The STR homologous sequencing results indicated that HCC827OR belonged to the same cell type as the parental HCC827,which proved that our results based on HCC827 drug-resistant strains were reliable.HCC827OR was significantly more resistant to osimertinib than its parents(IC50(HCC827OR):6.528±1.093 vs.IC50(HCC827):1194±522.9nM,P=0.017)by cell growth inhibition using osimertinib.In addition,the presence of osimertinib resistance in HCC827OR was also confirmed by clonal formation experiments.Further,flow cytometry was performed on cells before and after HCC827 drug resistance,and it was found that the apoptosis rate(especially in the late stage of apoptosis)of cells treated with different concentrations of osimertinib after HCC827 drug resistance was much lower than that of parental cells.These results preliminarily confirmed the antagonistic effect of HCC827 on the apoptotic effect induced by osimertinib.2.Analysis of gene expression of BCL-2 family in HCC827 and HCC827OR cellsBy extracting total RNA of HCC827 and HCC827OR,establishing cDNA libraries,and performing high-throughput sequencing,we analyzed the differences in gene expression of 9 major members of BCL-2 family in these two cell lines,and drew a heat map of gene differential expression.Our results showed that MCL-1 and BCL-XL were highly expressed in both types of cells without significant differences,while BCL-2 was highly expressed in HCC827OR and extremely low in parental HCC827,showing significant differences.3.Expression of BCL-2 protein in HCC827 and HCC827ORThe expression of BCL-2 family related proteins in HCC827 and HCC827OR cells after treatment with different concentrations of osimertinib was detected by Western blot,and it was found that BCL-2 was almost not expressed in parental HCC827 cells and highly expressed in HCC827OR.In consideration of the transcription and sequencing results of BCL-2 family genes in the previous chapter,we believe that BCL-2 may be the key gene in the process of HCC827 antagonizing the apoptotic effect induced by osimertininb and thus forming drug resistance.Chapter 21.Establishment and verification of HCC827 BCL-2 knockdown stabilized strainsWe constructed knockdown stabilized strains of HCC827OR BCL-2 using BCL-2 shRNA,and verified the low expression of BCL-2 after knockdown by Western blot,which proved that we successfully constructed knockdown stabilized strains of BCL-2 in HCC827OR.2.Osimertinib treatment of HCC827OR BCL-2 knocked down stable strains to verify BCL-2 and the association of osimertinib resistance in HCC827We treated the established BCL-2 knockdown HCC827OR stable strain with osimertinib,and tested whether the response of HCC827OR to osimertinib after knockdown of BCL-2 gene was changed by drawing the growth inhibition curve and detecting IC50,clone formation experiment and other methods.(1)After osimertinib treatment,compared with HCC827OR,both BCL-2 knockdown HCC827OR cell lines recovered certain drug sensitivity IC50(HCC827OR BCL-2 shRNAl):657.7±423.5nM vs IC50(HCC827OR):6378±2853nM,P=0.0264;IC50(HCC827OR BCL-2 shRNA2):946.2±55.77nM vs IC50(HCC827OR):6378±2853nM,P=0.03);(2)In the clone formation experiment,HCC827OR was inhibited at osimertinib concentration greater than 500nM,while HCC827OR BCL-2 shRNAl was inhibited at osimertinib concentration more than 100nM.The results showed that HCC827OR BCL-2 gene knockdown cell line recovered certain drug sensitivity compared with HCC827OR.(3)The results of animal experiments in vitro showed that the tumor growth of mice inoculated with HCC827OR BCL-2 shRNAl cells was significantly inhibited after osimertinib treatment,indicating that the BCL-2 knockdown of HCC827OR restored its sensitivity to osimertinib to a certain extent.On the basis of the above experimental results,we believe that the knockdown of BCL-2 gene with high expression in HCC827OR did indeed restore the osimertinib sensitivity of the cells to a certain extent,and also indirectly proved that BCL-2 did indeed participate in the formation of osimertinib resistance in HCC827 cells.3.Treating HCC827OR with BCL-2 specific inhibitors to verify the association between BCL-2 and osimertinib resistance in HCC827(1)After ABT-199 single treatment of HCC827 and HCC827OR,we found that although HCC827OR cells had a certain growth limitation compared with their parental HCC827 cells,the cells in the two groups did not show significant growth inhibition by this drug after statistical analysis(IC50(HCC827):52.97±56.32μM vs IC50(HCC827OR):20.85±13.99 μM,P=0.3107),and the clone formation experiment further confirmed this view,the two groups of cells treated with gradient concentration of ABT-199 did not show significant cell growth inhibition phenomenon.In conclusion,we concluded that ABT-199 single drug could not produce significant inhibitory effect on HCC827OR.(2)ABT-199 μM combined with osimertinib could not effectively improve the drug resistance of HCC827OR(IC50(HCC827OR OSI):3552±1334nM nM vs.IC50(HCC827OR OSI+ABT 1μM):1889±900 nM,P=0.1479),and ABT-199 2.5 μM and 5 μM combined with osimertinib could restore certain drug sensitivity of HCC827OR(IC50(HCC827OR OSI):3552±1334nM vs IC50(HCC827OR OSI+ABT 2.5 μM):149±82.66 nM,P=0.0116;IC50(HCC827OR OSI):3552±1334nM vs IC50(HCC827OR OSI+ABT 5μM):800±240.4nM,P=0.0245);(3)Cell proliferation and cloning experiments also showed that ABT-199 2.5μM and 5μM combined with osimertinib could restore the drug sensitivity of HCC827OR,and the combination of 2.5μM ABT-199 and osimertinib could restore the drug sensitivity of HCC827OR best.(4)Flow cytometric analysis of apoptosis of HCC827OR treated with ABT-199 2.5μM combined with osimertinib and osimertinib alone showed that the total apoptosis rate of HCC827OR in the combined treatment group was higher than that in the osimertinib alone group,and the total apoptosis rate of HCC872OR in the combined treatment group was mainly affected by promoting the increase of HCC872OR late modulated cells.(5)Western blotting results after ABT-199 treatment of HCC827OR showed that the expression levels of MCL-1 and BCL-XL increased with the increase of treatment drug concentration,and BCL-2 also maintained a stable expression level,while the expression level of pro-apoptotic family members BIM did not change.The results of immunoprecipitation showed that the function of BCL-2 was completely inhibited after ABT-199 treatment,while the functions of MCL-1 and BCL-XL were upregulated to varying degrees after ABT-199 addition.Based on this,we believe that combination therapy can restore osimertinib sensitivity of HCC827OR by inhibiting the function of BCL-2,but the up-regulation of expression and function of other anti-apoptotic family members MCL-1 and BCL-XL due to the compensatory mechanism also leads to the combination therapy of ABT-199 and Osimertinib.The drug sensitivity of HCC827OR could not be fully restored.All the above experimental results showed that the use of BCL-2 specific inhibitors restored the drug sensitivity of HCC827OR to a certain extent.However,in order to completely solve the problem of osimeritinib resistance of HCC827 from the perspective of apoptosis mechanism,a more comprehensive,in-depth and complete medication strategy to inhibit the anti-apoptotic family is still needed in the future.Chapter31.Study on BCL-2 Gene and Its Protein Related CharacteristicsWe first explored the BCL-2 gene and its protein-related characteristics in HCC827OR,and found that the increased methylation level in the gene body region of BCL-2 gene in HCC827OR might be correlated to a certain extent with the high gene expression.At the same time,through the immunohistochemical staining of EGFR-mutated PDX mice lung cancer tissue samples under microscope observation and the detection of each component of HCC827OR cell BCL-2 protein,we found that it was mainly located in the organelle membrane function.Finally,through the inhibition of protein synthesis and decomposition pathway,we revealed that the product with high BCL-2 expression is a stable protein structure that exists in the intracellular function of HCC827OR and will not be degraded and deactivated by the intracellular lysosome and ubiquitin-proteasome system.2.Difference analysis of BCL-2 related signaling pathway before and after drug resistance of HCC827Difference in expression of BCL-2 related signaling pathway proteins in HCC827 and HCC827OR detected by Western blot:Proteins related to the JAK/STAT3 and JNK/SAPK signaling pathways were up-regulated and activated in HCC827OR cells,while proteins related to the p53 signaling pathway were inhibited and down-regulated.The results of our KEGG pathway enrichment analysis showed that there was no significant abnormality in the expression of genes related to these pathways.Therefore,we believe that the abnormal changes in the protein expression levels of p53,JAK/STAT3 and JNK/SAPK signaling pathways may be correlated with the promotion of the high expression of BCL-2,thus enabling HCC827 cells to antagonize the apoptotic effect induced by osimertinib and gradually develop drug resistance.Conclusion:This study used the method of gradually increasing drug concentration to build the HCC827 drug-resistant cell line.Transcriptome analysis of BCL-2 family related genes showed that BCL-2 was significantly up-regulated during the development of osimertinib resistance in HCC827 cells.Furthermore,we comprehensively explore the features of BCL-2 gene and its protein products in the HCC827OR cell line.Then,by establishing BCL-2 knockdown stabilized strains and using BCL-2 specific inhibitors,we proved that BCL-2 was directly related to antagonism against osimertinib-induced apoptosis and thus gradually forming drug resistance in HCC827 cell line.Finally,we preliminarily explored the specific molecular mechanism of the involvement of BCL-2 in the formation of HCC827 drug resistance.We found that the high methylation of the BCL-2 gene body region may be related to its high expression of activation,and the stable structure of its protein products laid an important foundation for its continuous anti-apoptotic function in the process of drug resistance.At the same time,we found that the inhibition of p53 signaling pathway,the activation and up-regulation of JAK/STAT3 and JNK/SAPK signaling pathway may be correlated with the promotion of the high expression of BCL-2,thus mediating the formation of osimertinib resistance in HCC827 cells.