The biological functions of N6-methyladenosine (m 6 A) RNA methylation are mainly dependent on the reader; however, its role in lung tumorigenesis remains unclear. Here, we have demonstrated that the m 6 A reader YT521-B homology domain containing 2 (YTHDC2) is frequently suppressed in lung adenocarcinoma (LUAD). Downregulation of YTHDC2 was associated with poor clinical outcome of LUAD. YTHDC2 decreased tumorigenesis in a spontaneous LUAD mouse model. Moreover, YTHDC2 exhibited antitumor activity in human LUAD cells. Mechanistically, YTHDC2, via its m 6 A-recognizing YTH domain, suppressed cystine uptake and blocked the downstream antioxidant program. Administration of cystine downstream antioxidants to pulmonary YTHDC2-overexpressing mice rescued lung tumorigenesis. Furthermore, solute carrier 7A11 (SLC7A11), the catalytic subunit of system X C − , was identified to be the direct target of YTHDC2. YTHDC2 destabilized SLC7A1 1 mRNA in an m 6 A-dependent manner because YTHDC2 preferentially bound to m 6 A-modified SLC7A1 1 mRNA and thereafter promoted its decay. Clinically, a large proportion of acinar LUAD subtype cases exhibited simultaneous YTHDC2 downregulation and SLC7A11 elevation. Patient-derived xenograft (PDX) mouse models generated from acinar LUAD showed sensitivity to system X C − inhibitors. Collectively, the promotion of cystine uptake via the suppression of YTHDC2 is critical for LUAD tumorigenesis, and blocking this process may benefit future treatment.

Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system X C - because of the impaired uptake of cystine. However, the outcome following the accumulation of endogenous glutamate in lung adenocarcinoma (LUAD) has not yet been determined. Yes-associated protein (YAP) is sustained by the hexosamine biosynthesis pathway (HBP)-dependent O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation), and glutamine-fructose-6-phosphate transaminase (GFPT1), the rate-limiting enzyme of the HBP, can be phosphorylated and inhibited by adenylyl cyclase (ADCY)-mediated activation of protein kinase A (PKA). However, whether accumulated endogenous glutamate determines ferroptosis sensitivity by influencing the ADCY/PKA/HBP/YAP axis in LUAD cells is not understood. Methods: Cell viability, cell death and the generation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to evaluate the responses to the induction of ferroptosis following the inhibition of system X C - . Tandem mass tags (TMTs) were employed to explore potential factors critical for the ferroptosis sensitivity of LUAD cells. Immunoblotting (IB) and quantitative RT-PCR (qPCR) were used to analyze protein and mRNA expression. Co-immunoprecipitation (co-IP) assays were performed to identify protein-protein interactions and posttranslational modifications. Metabolite levels were measured using the appropriate kits. Transcriptional regulation was evaluated using a luciferase reporter assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). Drug administration and limiting dilution cell transplantation were performed with cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. The associations among clinical outcome, drug efficacy and ADCY10 expression were determined based on data from patients who underwent curative surgery and evaluated with patient-derived primary LUAD cells and tissues. Results: The accumulation of endogenous glutamate following system X C - inhibition has been shown to determine ferroptosis sensitivity by suppressing YAP in LUAD cells. YAP O-GlcNAcylation and expression cannot be sustained in LUAD cells upon impairment of GFPT1. Thus, Hippo pathway-like phosphorylation and ubiquitination of YAP are enhanced. ADCY10 acts as a key downstream target and diversifies the effects of glutamate on the PKA-dependent suppression of GFPT1. We also discovered that the protumorigenic and proferroptotic effects of ADCY10 are mediated separately. Advanced-stage LUADs with high ADCY10 expression are sensitive to ferroptosis. Moreover, LUAD cells with acquired therapy resistance are also prone to higher ADCY10 expression and are more likely to respond to ferroptosis. Finally, a varying degree of secondary labile iron ...

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Background Resistance to ferroptosis, a regulated cell death caused by iron‐dependent 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 study was 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 generation were 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. Cell‐derived 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 ferroptosis‐associated 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.

Lung cancer is one of the most common and deadly malignancies worldwide, in spite of advances in targeted therapy in recent years. An effective strategy for lung cancer prevention remains a major problem. Advances in next-generation sequencing have helped in understanding the RNA and identifying novel circular RNAs (circRNAs) that may have a broad impact on the early diagnosis and treatment of lung cancer. The circRNAs, exhibiting spatiotemporal-specific expression, are stable and conserved and present diverse biological functions in the normal and diseased states, including cancer. In this review, we summarize the recent advances in elucidating the functional role of circRNAs in lung cancer pathogenesis and discuss their potential mechanisms.