The Expression Of Ferroptosis Related Factors In Lung Adenocarcinoma And The Exploration Of Ferroptosis As A Therapy Target After TKI Resistance

Backgrounds and Objectives Lung cancer has the second rate of incidence and the highest mortality rate worldwide.According to the cancer statistics,the incidence rate of lung cancer is 11.4% with a mortality rate of 18% in 2020.The main pathological subtypes of lung cancer include squamous cell cancer,adenocarcinoma,small cell cancer,large cell carcinoma.Due to the increasing aging population and the increase of female patients,lung adenocarcinoma has replaced lung squamous cell carcinoma as the main type of lung cancer.In the past few decades,the development of research on the biological behavior and molecular progression mechanisms of lung adenocarcinoma has promoted important progress in the clinical treatment of lung adenocarcinoma.Clinically,it has been formed a comprehensive treatment model including surgical resection,chemoradiotherapy,TKI targeted therapy,immunotherapy and other systematic treatment strategies for lung adenocarcinoma,among which TKI targeted therapy is the main method of clinical drug treatment of lung adenocarcinoma due to its extensive coverage of population-specific and significant curative effect.Patients with earlystage lung adenocarcinoma usually have a good clinical prognosis after systemic treatment.Early stage of lung cancer can acquire a good prognosis with survival rate of 80%-90%.However,the 5-year survival rate of patients with locally advanced lung adenocarcinoma is still only 15%-20%.Although the widespread application of TKI targeted therapy can improve the survival rate of patients with locally advanced lung adenocarcinoma to a certain extent,the widespread occurrence of drug resistance limits the long-term benefits of patients.The research on the drug resistance mechanism of TKI targeted therapy and the discovery of new therapeutic targets after drug resistance have become major scientific issues that urgently need to be resolved in the field of lung adenocarcinoma treatment.Malignant proliferation is the key to the persistent progression of tumors.Therefore,the key issue of overcoming the resistance of TKI targeted therapy is to inhibit tumor cell proliferation and induce cell death.It is known that the major methods of regulated cell death include apoptosis,necroptosis,pyroptosis,ferroptosis,etc.Among them,the most studied tumor-regulated death is apoptosis,but so far there is no effective drug targeting apoptosis of cancer cells.Ferroptosis is caused by the accumulation of irondependent lipid peroxidation,which is a newly discovered regulatory cell death method that is different from apoptosis and necroptosis.Ferroptosis were demonstrated to involve in the occurrence and development of a variety of malignant tumors,and is closely related to the prognosis of cancer patients.Because malignant tumor cells have the potential for mesenchymal transformation and dedifferentiation,they have certain resistance to apoptosis-inducing drugs and cytotoxic drugs and exhibit persistent treatment tolerance.However,studies have revealed that tumor cells in this state shows high sensitivity to ferroptosis inducers(FINs).Whether targeted ferroptosis can become a new treatment direction for lung adenocarcinoma after TKI therapy has not been revealed yet.In addition,the rapid proliferation of tumor cells have the characteristics of active metabolism,high reactive oxygen species(ROS)load,and excess iron supply.These characteristics are highly overlapped with the process of ferroptosis,which also makes the induction of ferroptosis of tumor cells as the treatment strategy after TKI resistance of lung adenocarcinoma feasibility.In summary,around the scientific question of whether targeted ferroptosis can become a new treatment direction for lung adenocarcinoma of TKI treatment after drug resistance,in this study we will conduct the following research: 1)Determine the expression characteristics of ferroptosis related factors based on multi-omics data and screening of ferroptosis regulatory factors that are significantly differently expressed in lung adenocarcinoma compared to normal lung tissue;2)Clarify the correlation between ferroptosis related regulatory factors and the prognosis of lung adenocarcinoma;3)Based on multiple TKI drug-resistant lung adenocarcinoma cell models to detect the expression changes of ferroptosis related regulatory factors before and after drug resistance,and target ferroptosis related regulatory factors to clarify the role of inducing cell ferroptosis in overcoming TKI drug resistance.This study can systematically explain the expression pattern of ferroptosis-related factors in lung adenocarcinoma and its relationship with prognosis,and identify new therapeutic targets for TKI resistance in lung adenocarcinoma.It has certain scientific significance and clinical application prospects.Materials and Methods Chapter 1: Research on the expression of ferroptosis-related factors in lung adenocarcinoma based on multi-omics Determine the gene set of ferroptosis related factors based on published literatures and KEGG(Kyoto Encyclopedia of Genes and Genomes)ferroptosis pathway.Using TCGA lung adenocarcinoma data,download the whole transcriptome,whole genome,and DNA methylation data of ferroptosis-related factors;analyze the differential expression,gene mutation,CSV,and promoter methylation of ferroptosis-related factors in lung adenocarcinoma tissue compared to normal paratumor lung tissue through bioinformatics technology;combined with the results of differential expression analysis and existing literatures on the function of ferroptosis related factors,the key factors that may regulate ferroptosis in lung adenocarcinoma are finally determined;RT-PCR and western blotting are used to detect the m RNA and protein expression of key ferroptosis regulated factors on clinical samples and cell lines of lung adenocarcinoma.Chapter 2 The relationship between key ferroptosis regulators and the prognosis of lung adenocarcinoma Use TCGA database to download lung adenocarcinoma gene expression data and corresponding patient clinical data,use Kaplan-Meier method to analyze the correlation between key ferroptosis regulators and lung adenocarcinoma survival;analyze whether the ferroptosis regulatory factors are independent factors for the prognosis of lung adenocarcinoma based on COX univariate and multivariate risk regression models.;the expression of ferroptosis related regulatory factors was detected by immunohistochemistry using the tissue chip of lung adenocarcinoma,to verify the relationship of ferroptosis related regulatory factors and the prognosis of lung adenocarcinoma.Chapter 3: Research on Key Ferroptosis Regulators as Targets of Treatment after TKI ResistanceIn a variety of TKI-resistant cell lines,RT-PCR and western blotting were used to detect the expression changes of key ferroptosis regulators before and after drug resistance;CCK8 proliferation experiment detected the function of targeted key ferroptosis regulators(RSL3,Erastin treatment)on the lung adenocarcinoma TKIresistant cell lines and its capacity of reversing TKI resistance.Results Chapter 1 Research on the expression of ferroptosis-related factors in lung adenocarcinoma based on multi-omics1.The expression of ferroptosis related factors in lung adenocarcinoma Through the review of Hassannia B and the KEGG ferroptosis signaling pathway,a total of 42 ferroptosis-related factors were summarized.Based on the analysis of TCGA lung adenocarcinoma transcriptome data,a total of 30 ferroptosis-related factors occurred in normal lung tissues and lung adenocarcinoma tissues including NCOA4,NFE2L2,CS,FDFT1,SLC1A5,HMOX1,NQO1,G6 PD,GPX4,PEBP1,PGD,CARS,PHKG2,KEAP1,GOT1,LPCAT3,TFRC,ACSL4,NFS1,CISD1,HMGCR,STEP3,GCLC,CBS,AKR1C1,FTH1,etc.;GO enrichment analysis of differentially expressed factors found that they are mainly enriched in biological processes such as sulfide metabolism,oxidative stress metabolism,glutathione metabolism,and iron homeostasis.2.Mutations of ferroptosis-related factors in lung adenocarcinoma Based on the analysis of TCGA lung adenocarcinoma genome data,a total of 31ferroptosis-related factors have gene mutations.Among them,factors with higher mutation rates include KEAP1,ZEB1,ACACA,GCLC,GLS2,FANCD2,NOX1,NFE2L2,NCOA4,HMGCR,AKR1C1,ACO1,TFRC,HMOX1,ACSL4,PHKG2,etc.,KEAP1 mutation frequency is as high as 17.39%;GPX4,FTH1 and LPCAT3 and other factors have not detected gene mutations.3.Copy number variation of ferroptosis-related factors in lung adenocarcinoma Based on TCGA lung adenocarcinoma genome-wide data analysis,a total of 38ferroptosis-related factors have undergone copy number variation,including SQLE,FDFT1,TFRC,AKR1C1,LPCAT3,NFS1,GPX4,GLS2,ACACA,NFE2L2,MIR137,KEAP1,STEAP3,PHKG2,NQO1,NCOA4,FTH1,ACSL3,FANCD2,GOT1.The copy number variation rate is between 0.21%-10.71%,and the top five copy number variation rate factors are SQLE,FDFT1,TFRC,AKR1C1 and LPCAT3.The copy number variation of related factors is dominated by copy number amplification,accounting for 64.99% of all copy number variation.4.DNA methylation modification changes of ferroptosis-related factors in lung adenocarcinoma Based on the analysis of TCGA lung adenocarcinoma methylation data,the promoter DNA methylation of 16 ferroptosis-related factors in lung adenocarcinoma was significantly changed compared with normal lung tissue,including HSBP1,PEBP1,GLS2,ACSF2,ACSL3,and STEAP3,NCOA4,PHKG2,MIR137,TFRC,SLC7A11,ACO1,ZEB1,FANCD2,FTH1,PGD.5.Based on clinical sample experiments of lung adenocarcinoma to verify the differential expression of key ferroptosis regulators Based on the previous literatures and the bioinformatic analysis of the abovementioned TCGA data,six key ferroptosis regulators GPX4,NRF2,KEAP1,FTH1,ACSL4 and LPCAT3 were screened out.RT-PCR detection revealed that compared with the normal adjacent tissue,the m RNA expression of GPX4,NRF2,ACSL4,and LPCAT3 were down regulated(p<0.05).The expression of FTH1 in lung adenocarcinoma and adjacent normal lung tissues had no difference.KEAP1 m RNA expression was not detected in lung adenocarcinoma and normal lung tissue;Western blotting showed that GPX4,FTH1,ACSL4 and LPCAT3 were up regulated in lung adenocarcinoma tissue than in normal tissue,while NRF2 and KEAP1 were downregulated in lung adenocarcinoma.6.Based on the experiment of lung adenocarcinoma cell lines to verify the differential expression of key ferroptosis regulators RT-PCR detection revealed that the m RNA expression of GPX4,NRF2,KEAP1,ACSL4,LPCAT3 and FTH1 in PC9,HCC827,HCC78,H3122,H1975 and A549 had no consistent changes trend in comparison with HBE;Protein level detection showed that compared with normal bronchial epithelial HBE cell line,GPX4,FTH1,ACSL4,and LPCAT3 are generally up-regulated in lung adenocarcinoma cell lines,while KEAP1 and NRF2 have no consistent changes in lung adenocarcinoma cell lines.Chapter 2 The relationship between core ferroptosis regulatory factors and the prognosis of lung adenocarcinoma1.The relationship between core ferroptosis regulatory factors and the prognosis of lung adenocarcinoma based on TCGA data.Kaplan-Meier survival analysis found that GPX4,NRF2,ACSL4,and LPCAT3 are associated with the prognosis of lung adenocarcinoma patients(p<0.05),while KEAP1 and FTH1 are not associated with the prognosis of lung adenocarcinoma patients(p>0.05);Higher expression of GPX4,NRF2,and LPCAT3 predict better prognosis of lung adenocarcinoma.The median survival time of patients in the high expression group of GPX4,NRF2 and LPCAT3 is 53.3 months,49.9 months and 67.6 months,respectively,which is significantly longer than the median survival time of patients in the low expression group;Lower expression of ACSL4 predicts worse prognosis of lung adenocarcinoma patients.Multivariate COX regression analysis found that the high expression of GPX4 and NRF2 is an independent protective factor for the prognosis of lung adenocarcinoma,and the HR of GPX4 was 1.606(95%CI: 1.174-2.198,p=0.003),the HR of NRF2 was 1.944(95%CI: 1.243-3.093,p=0.004),and the corresponding TNM staging HR was 1.591(95%CI: 1.375-1.842,p=4.48e-10).2.Based on lung adenocarcinoma tissue chip to verify the expression of GPX4 and NRF2 Immunohistochemical staining showed that GPX4 and NRF2 are mainly located in the cytoplasm.Lung adenocarcinoma,normal alveolar epithelial and bronchial epithelial cells can be observed the expression of GPX4 and NRF2.GPX4 expression in lung adenocarcinoma tissues is generally strongly positive,while the expression of GPX4 in corresponding normal para-tumor alveolar epithelial and bronchial epithelial cells was negative or weakly positive,and the expression of NRF2 is strong in both tumor tissue and normal tissue.Wilcox rank sum test analyzed lung adenocarcinoma tissue and normal lung tissue GPX4,NRF2.The expression of GPX4 was significantly higher in adenocarcinoma tissues compared to normal tissue(p=0.00238),while the expression of NRF2 was significantly higher in adenocarcinoma tissues compared to normal tissue(p=0.00238).3.The relationship between the expression of GPX4 and the clinical characteristics of patients with lung adenocarcinoma Age,gender,TNM stage,tumor size,and lymph node metastasis of patients with lung adenocarcinoma were not significantly related to the expression of GPX4.4.The relationship between the expression of GPX4 and the prognosis of patients with lung adenocarcinoma Kaplan-Meier survival analysis showed that the GPX4 expression in tissue chip was positively correlated with the prognosis of patients with lung adenocarcinoma,which was consistent with the results of TCGA data analysis;the median survival time of patients in the GPX4 high expression group was 57.0 months,which was significantly longer than the 38.0 patients in the low expression group Month(p=0.023);Multivariate COX regression analysis showed that GPX4 was an independent predictor of overall survival for patients with lung adenocarcinoma(HR: 2.490,1.359-4.562,p=0.003).Chapter 3: Intervention Research on Key Ferroptosis Regulators as Targets of Treatment after TKI Resistance1.The expression changes of key ferroptosis regulators before and after TKI resistance The key ferroptosis regulators GPX4,NRF2,ACSL4,FTH1,and LPCAT3 changed in RNA levels and protein levels before and after TKI resistance.Among them,the m RNA expression of GPX4 did not show a significantly change in the sensitive and resistance lung adenocarcinoma cells while its protein levels showed an upwardregulated trend.2.The effect of GPX4 inhibitor RSL3 on the proliferation of TKI-sensitive and drugresistant cells Using GPX4 inhibitors RSL3 and type I ferroptosis inducer erastin to treat PC9,HCC78,H3122 and HCC827 TKI sensitive and resistant cell lines,72 hours later CCK8 cell proliferation experiment showed RSL3 and erastin is sensitive to the above TKI sensitive and resistant cell lines.The inhibition function was dose dependent.The average IC50 of RSL3 in TKI sensitive cell lines was 290 + 431 n M,and the average IC50 of RSL3 in TKI resistant cell lines was 234 + 222 n M,significantly lower than the IC50 of Erastin(Average IC50:1871±2054 n M in TKI sensitive cell lines;Average IC50 in TKI resistant cell lines:2335±377 n M).3.Combined treatment of RSL3 and corresponding TKI-targeted drugs in TKI resistant lung adenocarcinoma cell lines showed that RSL3 could reverse the resistance to osimertinib in the osimertinib-resistant cell line PC9-OR(IC50 decreased from1850 n M to 1115 n M).In the crizotinib resistant cell line HCC78-CR,the tolerance to crizotinib was not significantly reversed.(IC50 decreased from 1370 n M to 1132 n M).Tolerance of the lorlatinib resistant cell line H3122-LR was reversed(IC50 decreased from 5974 n M to 614 n M).Resistance to gefitinib was reversed in the gefitinib resistant cell line HCC827-GR(IC50 decreased from 8777 n M to 209 n M).RSL3 had the most significant drug resistance reversal effect in the H3122 lorlatinib resistant cell line and HCC827 gefitinib resistant cell line.Conclusion1.Significant changes in expression,gene mutations,and copy number variations of large number of ferroptosis-related factors in the occurrence and development of lung adenocarcinoma;2.Based on the ferroptosis regulatory network and the expression changes of ferroptosis-related factors in lung adenocarcinoma,the key regulatory factors GPX4,NRF2,KEAP1,ACSL4,LPCAT3 and FTH1 that may be involved in the occurrence and development of lung adenocarcinoma were screened;3.Analysis of the correlation between the key ferroptosis regulators GPX4,NRF2,KEAP1,ACSL4,LPCAT3 and FTH1 and the prognosis of patients with lung adenocarcinoma and tissue microarray verification indicates that GPX4 is an independent factor and potential intervention target for the prognosis of patients with lung adenocarcinoma point;4.GPX4 changes before and after the resistance of a variety of TKI targeted therapies.Targeting GPX4 can significantly reverse the resistance of related TKI drugs.