Mechanisms Of Ferroptosis Induced By Radiotherapy In Non-Small Cell Lung Cancer

Objective: Radiotherapy is an important treatment for non-small cell lung cancer(NSCLC),but a proportion of NSCLC is resistant to radiotherapy,and the mechanisms of radiotherapy-induced cell death and radioresistance in NSCLC remain incompletely understood.Radiotherapy generates reactive oxygen species(ROS)to attack lipids and other biomolecules,thereby damaging cells.Ferroptosis is an iron-dependent form of RCD caused by lipid peroxidation,which has been identified as a natural mechanism of tumor suppression.Polyunsaturated fatty acidcontaining phospholipids(PUFA-PL)on the plasma membrane undergo peroxidation in the presence of ROS,leading to the accumulation of lipid peroxides,which underlie the ferroptosis onset.Acyl coenzyme A synthetase long-chain family member 4(ACSL4)is the key enzyme for PUFA-PL biosynthesis.In addition,cells have developed several ferroptosis defense systems to antagonize ferroptosis by neutralizing lipid peroxides,such as the solute carrier family 7 member 11(SLC7A11)-glutathione(GSH)-glutathione peroxidase 4(GPX4)axis.This study aims to provide insight into the mechanism of radiotherapy-induced ferroptosis in NSCLC and strategies for its translation.Methods:1.In vitro: Flow cytometry,real-time quantitative PCR,or western blotting was used to measure lipid peroxidation or the expression of ferroptosis marker/regulatory genes after radiotherapy.Electron microscopy was used to observe the radiotherapy-induced cellular morphological characteristics.A clonogenic assay was used to assess the effects of ferroptosis inhibition or augmentation on radiosensitivity.Western blotting and immunofluorescence were used to assess the potential interaction between ferroptosis and DNA damage response after radiotherapy.Overexpression or knockout of ferroptosis regulatory genes that were significantly altered in post-radiotherapy NSCLC cells to observe their effects on radiotherapy-induced ferroptosis and radiosensitivity.The correlation of gene expression in NSCLC patient samples was analyzed using The Cancer Genome Atlas(TCGA)database.Chromatin immunoprecipitation-sequencing or-quantitative PCR(Ch IP-seq or-q PCR)was used to analyze relevant protein-DNA interactions.2.In vivo: We established NSCLC cell line-derived and patientderived xenograft(CDX and PDX)models.In these models,we deleted relevant ferroptosis genes or combined ferroptosis inhibitors or inducers with radiotherapy to observe their effects on radiotherapy-mediated tumor suppression.These can demonstrate the role and mechanisms underlying radiotherapy-induced ferroptosis and determine whether ferroptosis inhibition causes tumor radioresistance and whether ferroptosis augmentation overcomes radioresistance.The staining levels of lipid peroxide 4-hydroxynonenal(4-HNE),DNA damage marker phosphorylated H2 A histone family member X(H2AX),and apoptosis marker cleaved caspase-3 in tumor samples were analyzed by immunohistochemistry to clarify whether such radioresistant and radiosensitizing phenotypes are specific to ferroptosis.Results:1.In vitro:(1)Radiotherapy induced accumulation of lipid peroxides,upregulated the ferroptosis marker gene PTGS2,and caused ferroptotic morphological features in NSCLC cells.Ferroptosis inhibitors(namely lipid peroxide scavengers)rescued cell survival after radiotherapy,indicating that radiotherapy triggers ferroptosis in NSCLC cells by inducing lipid peroxidation.The inhibitory effect of ferroptosis inhibitors on radiotherapy was more pronounced than that of apoptosis inhibitors and necroptosis inhibitors,and ferroptosis inhibition resulted in radioresistance without affecting radiotherapy-induced DNA damage response.(2)Radiotherapy generated substantial ROS and upregulated m RNA and protein levels of ACSL4,SLC7A11,and GPX4 in NSCLC cells.ACSL4 upregulation preceded and exceeded the upregulation of SLC7A11 and GPX4.Scavenging ROS or deleting ACSL4 blocked radiotherapyinduced lipid peroxidation and ferroptosis,leading to radioresistance.(3)ROS activated interferon regulatory factor 1(IRF1)in NSCLC cells.Analysis in the TCGA database revealed a positive correlation between IRF1 expression and ACSL4 expression in NSCLC patient samples.Ch IP-seq analysis revealed the existence of binding sites for IRF1 at the promoter region of ACSL4,and Ch IP-q PCR revealed that IRF1 bound on the ACSL4 promoter under ROS stimulation.Scavenging ROS or deleting ACSL4 abolished the upregulation of ACSL4 by radiotherapy.(4)Radiotherapy moderately upregulated SLC7A11,but dramatically upregulated transcription factors promoting SLC7A11 transcription in NSCLC cells,indicating that radiotherapy may activate certain mechanisms to antagonize SLC7A11 upregulation.Analysis of the SLC7A11 regulators revealed that radiotherapy activated p53 to antagonize the SLC7A11 upregulation.p53 knockout significantly augmented the upregulation of SLC7A11 by radiotherapy without affecting ACSL4 expression.p53 knockout inhibited radiotherapy-induced lipid peroxidation and ferroptosis,and deletion of SLC7A11 in p53 knockout cells restored radiotherapy-induced lipid peroxidation and ferroptosis,resensitizing p53-knockout NSCLC cells to radiotherapy.Conversely,p53 overexpression in p53-deficient NSCLC cells inhibited radiotherapyupregulated SLC7A11 and promoted radiotherapy-induced lipid peroxidation and ferroptosis;while SLC7A11 restoration re-inhibited radiotherapy-induced lipid peroxidation and ferroptosis,allowing cells to regain radioresistance.(5)p53 knockout increased GSH levels,and knockout of SLC7A11 in p53-deficient cells decreased GSH levels.Inhibition of GSH synthesis acted similarly to knockout of SLC7A11,restoring radiotherapy-induced lipid peroxidation and ferroptosis and resensitizing p53-deficient cells to radiotherapy.Conversely,supplementation of GSH or stimulation of GSH synthesis in p53 wild-type cells inhibited radiotherapy-induced lipid peroxidation and ferroptosis,leading to radioresistance.(6)SLC7A11 inhibitors restored radiotherapy-induced lipid peroxidation and ferroptosis in p53-deficient or-inactivated NSCLC cells,resensitizing cells to radiotherapy.In addition,the radiosensitizing effect of SLC7A11 inhibitor was weaker in normal bronchial epithelial cells than in p53-deficient or-inactivated NSCLC cells.2.In vivo:(1)ACSL4 deletion blocked radiotherapy-induced lipid peroxidation and ferroptosis,resulting in NSCLC-CDX tumor radioresistance.(2)SLC7A11 inhibited radiotherapy-induced ferroptosis by neutralizing lipid peroxides,leading to radioresistance in NSCLC-CDX tumors with p53-knockout.(3)SLC7A11 inhibitors radiosensitized p53-knockout or-mutated NSCLC CDX or PDX tumors by potentiating radiotherapy-induced ferroptosis.(4)ACSL4 deletion or SLC7A11 inhibition didn’t affect the staining levels of phosphorylated H2AX(a marker of DNA damage response)and cleaved caspase-3(an apoptosis marker).SLC7A11 inhibitor combined with radiotherapy didn’t affect body weight in mice.Conclusions:1.Radiotherapy generates lipid peroxides through the ROS-IRF1-ACSL4 axis and safeguards against effective neutralization of lipid peroxides through the p53-SLC7A11-GSH axis,which together lead to the accumulation of lipid peroxides to a lethal level,thereby triggering ferroptosis in NSCLC.2.ACSL4 deletion or dramatic upregulation of SLC7A11 abolished radiotherapy-induced accumulation of lipid peroxides,thereby blocking ferroptosis and leading to radioresistance in NSCLC.3.SLC7A11 inhibitors resensitize p53-defective NSCLC to radiotherapy by boosting radiotherapy-induced ferroptosis,and SLC7A11 inhibitors combined with radiotherapy exhibited a favorable safety profile.