Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are...Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are required for protection against ferroptosis.The purpose of the present studywas to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis.Methods:Established human lung fibroblasts MRC-5,WI38,and human LUAD H1650,PC9,H1975,H358,A549,and H1299 cell lines,tumor and matched normal adjacent tissues of LUAD,and plasma from healthy individuals and LUAD patients were used in this study.Immunohistochemistry and immunoblotting were used to analyze protein expression,and quantitative reverse transcription-PCR was used to analyze mRNA expression.Cell viability,cell death,and the lipid reactive oxygen species generationwere measured to evaluate the responses to ferroptosis.Exosomes were observed using transmission electron microscope.The localization of arachidonic acid(AA)was detected using click chemistry labeling followed by confocal microscopy.Interactions between RNAs and proteins were detected using RNA pull-down,RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation methods.Proteomic analysis was used to investigate RNA-regulated proteins,and metabolomic analysis was performed to analyze metabolites.Cellderived xenograft,patient-derived xenograft,cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism.Results:Plasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis.A potential explanation is that exosomal circRNA_101093(cir93)maintained an elevation in intracellular cir93 in LUAD to modulate AA,a poly-unsaturated fatty acid critical for ferroptosisassociated increased peroxidation in the plasma membrane.Mechanistically,cir93 interacted with and increased fatty acid-binding protein 3(FABP3),which transported AA and facilitated its reaction with taurine.Thus,global AA was reduced,whereas N-arachidonoyl taurine(NAT,the product of AA and taurine)was induced.Notably,the role of NAT in suppressing AA incorporation into the plasma membrane was also revealed.In pre-clinical in vivo models,reducing exosome improved ferroptosis-based treatment.Conclusion:Exosome and cir93 are essential for desensitizing LUAD cells to ferroptosis,and blocking exosome may be helpful for future LUAD treatment.展开更多
基金National Natural Science Foundation of China,Grant/Award Numbers:81871907,81822029,81872288,82173015,81902315,81902869,81774291ShanghaiMunicipal Education Commission-Gaofeng Clinical Medicine,Grant/Award Number:20191834+5 种基金Project of Clinical Research Supporting SystemClinical Medicine First-class DisciplineShanghai Municipal Education Commission and Shanghai Education Development Foundation,Grant/Award Number:18CG16Shanghai Sailing Program,Grant/Award Number:19YF1444800Science and technology commission of Shanghai municipality project,Grant/Award Numbers:19140902600,21140902800Shanghai ChestHospital,Grant/Award Numbers:2018YNJCM01,2019YNJCM06,2021YNZYJ01,2021YNZYY01,2021YNZYY02。
文摘Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are required for protection against ferroptosis.The purpose of the present studywas to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis.Methods:Established human lung fibroblasts MRC-5,WI38,and human LUAD H1650,PC9,H1975,H358,A549,and H1299 cell lines,tumor and matched normal adjacent tissues of LUAD,and plasma from healthy individuals and LUAD patients were used in this study.Immunohistochemistry and immunoblotting were used to analyze protein expression,and quantitative reverse transcription-PCR was used to analyze mRNA expression.Cell viability,cell death,and the lipid reactive oxygen species generationwere measured to evaluate the responses to ferroptosis.Exosomes were observed using transmission electron microscope.The localization of arachidonic acid(AA)was detected using click chemistry labeling followed by confocal microscopy.Interactions between RNAs and proteins were detected using RNA pull-down,RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation methods.Proteomic analysis was used to investigate RNA-regulated proteins,and metabolomic analysis was performed to analyze metabolites.Cellderived xenograft,patient-derived xenograft,cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism.Results:Plasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis.A potential explanation is that exosomal circRNA_101093(cir93)maintained an elevation in intracellular cir93 in LUAD to modulate AA,a poly-unsaturated fatty acid critical for ferroptosisassociated increased peroxidation in the plasma membrane.Mechanistically,cir93 interacted with and increased fatty acid-binding protein 3(FABP3),which transported AA and facilitated its reaction with taurine.Thus,global AA was reduced,whereas N-arachidonoyl taurine(NAT,the product of AA and taurine)was induced.Notably,the role of NAT in suppressing AA incorporation into the plasma membrane was also revealed.In pre-clinical in vivo models,reducing exosome improved ferroptosis-based treatment.Conclusion:Exosome and cir93 are essential for desensitizing LUAD cells to ferroptosis,and blocking exosome may be helpful for future LUAD treatment.
