Ferroptosis, a form of oxidative cell death, represents a therapeutic vulnerability for treating apoptosis-resistant cancers. Here, we identify leucine zipper transcription factor-like 1 (LZTFL1) as a key regulator of ferroptosis that rewires glutathione (GSH) metabolism. Mechanistically, LZTFL1 promotes oxidation of glucose-6-phosphate dehydrogenase (G6PD), thereby limiting NADPH production and impairing GSH regeneration. GSH depletion in turn enhances LZTFL1 translation via an AKT-mammalian target of rapamycin (mTOR)-eukaryotic initiation factor 4E (eIF4E) pathway, establishing a feedforward loop that amplifies ferroptosis. In vivo, the LZTFL1-formin homology 2 domain-containing 1 (FHOD1)-G6PD axis sensitizes multiple tumor models, including patient-derived xenografts, to ferroptosis, leading to enhanced lipid peroxidation, reduced GSH levels, suppressed tumor growth, and prolonged survival. LZTFL1 expression restores cisplatin sensitivity in resistant lung and ovarian cancer cells and predicts improved survival outcomes in patients with lung adenocarcinoma. Moreover, FDA-approved agents upregulate LZTFL1 and re-sensitize resistant tumors to cisplatin. These findings highlight LZTFL1 as a potential biomarker and a therapeutic target for enhancing ferroptosis-based cancer therapy.

Protein myristoylation, catalyzed by N-myristoyltransferases (NMT1 and NMT2), is a key co- and posttranslational modification involved in cellular signaling, yet its role in ferroptosis remains poorly defined. Here, we developed a novel myristoylproteomics workflow leveraging click chemistry to comprehensively profile N-myristoylated proteins within NSCLC cells. We found that NMT1 and NMT2 expression positively correlates with ferroptosis sensitivity. Genetic or pharmacological inhibition of NMT attenuated ferroptosis, whereas their overexpression enhanced it. Using our optimized quantitative myristoylproteomics platform, we identified GLIPR2 as a novel myristoylated protein with elevated modification levels in ferroptosis-sensitive cells. Functional studies confirmed that GLIPR2 promotes ferroptosis, and this pro-ferroptotic activity of GLIPR2 requires its N-myristoylation, as a myristoylation-deficient mutant (G2A) failed to restore ferroptosis sensitivity. Our work establishes a robust proteomic methodology for mapping protein myristoylation and uncovers the NMT1/ NMT2-GLIPR2 axis as a pivotal regulator of ferroptosis. These findings propose new therapeutic strategies for NSCLC.

Dynamic changes occurring in the lung microbiota can impact the initiation, progression, and prognosis of lung cancer (LC). Consequently, the development of suitable intratumoral microbiota analysis methods is crucial. Although matrix‐assisted laser desorption/ionization mass spectrometry (MALDI MS) involves straightforward operations and provides precise results, the “direct smear method” limits the identification of bacterial subspecies. Furthermore, the issue of inadequate quantification with MALDI MS renders it unsuitable for direct analysis of intratumoral bacteria. To address these challenges, a novel ionic liquid in this study is employed, called norharmane conjugated to 2,5‐dihydroxybenzoic acid (Nor@DHB) for the direct detection of intratumoral bacteria using MALDI MS. Because gram‐negative bacteria are dominant within cancer cells, lipid A is selected as the chemical fingerprint for bacterial identification. The results demonstrated that using Nor@DHB can enhance the lipid A signal by an order of magnitude and achieved a good linear relationship within a concentration range of 0.01–80 ng mL −1 . Here, this method is successfully applied to the direct analysis of lipid A in actual clinical samples. Subsequent machine learning and nomogram models further confirmed the correlation between characteristic lipid A ions and LC patient clinicopathological features, which are further validated through both in vitro and in vivo experiments.

Glutathione (GSH), a non-enzymatic antioxidant in mammalian cells, plays an essential role in maintaining redox balance, mitigating oxidative stress, and preserving cellular homeostasis. Beyond its well-established function in detoxifying reactive oxygen species (ROS), GSH serves as a critical regulator of ferroptosis-an iron-dependent form of cell death marked by excessive lipid peroxidation. Serving as a cofactor for glutathione peroxidase 4 (GPX4), GSH catalyzes the conversion of lipid peroxides into non-toxic lipid alcohols, thereby preventing the accumulation of deleterious lipid oxidation products and halting the spread of oxidative damage. In cancer cells, upregulated GSH synthesis and GPX4 activity contribute to an enhanced antioxidant defense, countering oxidative stress provoked by increased metabolic demands and exposure to therapeutic agents such as chemotherapy, radiotherapy, and immunotherapy. This ability of cancer cells to modulate their ferroptosis susceptibility through GSH metabolism underscores its potential as a therapeutic target. Additionally, GSH influences several key oncogenic and tumor-suppressive signaling pathways, including NFE2L2/NRF2, TP53/p53, NF-κB, Hippo, and mTOR, which collectively regulate responses to oxidative stress, affect metabolic processes, and modulate sensitivity to ferroptosis in cancer cells. This review explores recent advancements in understanding GSH's multifaceted role in ferroptosis, emphasizing its implications for cancer biology and therapeutic interventions.

Cytokines link inflammation to tumorigenesis, but the role of post-translational modifications in regulating their function within the extra-tumoral environment remains poorly defined. Here, we identify tumor-derived tumor necrosis factor (TNF) receptor superfamily member 11B (TR11B) as a key driver of lung adenocarcinoma (LUAD) progression and therapeutic resistance. Mechanistically, O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation at serine 151 stabilizes TR11B and facilitates its interaction with the membrane protein EPS15 homology domain-containing protein 1 (EHD1), promoting cyclin dependent kinase 2 (CDK2) phosphorylation and cell cycle progression. Clinically, elevated O-GlcNAcylated TR11B correlates with advanced LUAD. Genetic deletion of Ogt suppresses tumor development in LUAD mouse models. Importantly, celecoxib, an U.S. Food and Drug Administration (FDA)-approved drug, inhibits O-GlcNAcylation and exerts antitumor effects. These findings reveal a pathological role for cytokine O-GlcNAcylation in LUAD and identify this axis as a potential therapeutic target.

N6-Methyladenosine (m6A) is the most prevalent mRNA modification. Its biological function primarily relies on its "Reader" protein, such as YTHDC2. Previous studies have shown that YTHDC2 downregulation is a pro-carcinogenic phenomenon in lung adenocarcinoma (LUAD). However, further investigation is needed to understand the molecular mechanisms of downstream genes and the associated biological phenomena following YTHDC2 downregulation. Here, we found that YTHDC2 knockout upregulated exosome content in LUAD. Following YTHDC2 knockout, the mRNA levels of OAS family members (OASs) and IFIT family members (IFITs) also decreased; and inhibition of OASs and IFITs could promote exosome content. Several m6A modification sites on the NT domain of OASs and the TPR12 domain of IFITs were found to increase the stability of OASs and IFITs in a YTHDC2-dependent manner. OASs and IFITs affected exosome content through target genes including RAB5A, RAB7 and RAB11A, and three arginine (R) amino acids on IFITs were critical for combination IFITs with targeted RAB mRNAs and subsequent degradation. Simultaneously, OASs degraded targeted RABs through RNAseL. Additionally, mutual bindings between OASs and IFITs were critical for their target gene degradation. Collectively, the above findings might provide a theoretical basis for the treatment of LUAD patients with low YTHDC2 expression.

Abstract Detecting exosomal markers using laser desorption/ionization time‐of‐flight mass spectrometry (LDI‐TOF MS) is a novel approach for examining liquid biopsies of non‐small cell lung cancer (NSCLC) samples. However, LDI‐TOF MS is limited by low sensitivity and poor reproducibility when analyzing intact proteins directly. In this report, gold nanoparticles/cellulose nanocrystals (AuNPs/CNC) is introduced as the matrix for direct analysis of intact proteins in NSCLC serum exosomes. AuNPs/CNC with “dual dispersion” effects dispersed and stabilized AuNPs and improved ion inhibition effects caused by protein aggregation. These features increased the signal‐to‐noise ratio of [M+H]+ peaks by two orders of magnitude and lowered the detection limit of intact proteins to 0.01 mg mL–1. The coefficient of variation with or without AuNPs/CNC is measured as 10.2% and 32.5%, respectively. The excellent reproducibility yielded a linear relationship (y = 15.41x – 7.983, R2 = 0.989) over the protein concentration range of 0.01 to 20 mg mL–1. Finally, AuNPs/CNC‐assisted LDI‐TOF MS provides clinically relevant fingerprint information of exosomal proteins in NSCLC serum, and characteristic proteins S100 calcium‐binding protein A10, Urokinase plasminogen activator surface receptor, Plasma protease C1 inhibitor, Tyrosine‐protein kinase Fgr and Mannose‐binding lectin associated serine protease 2 represented excellent predictive biomarkers of NSCLC risk.

背景与目的 铁死亡是一种以铁依赖方式由脂质过氧化物过度积累引起的调节性细胞死亡,近年来发现铁死亡抵抗与肺腺癌(lung adenocarcinoma,LUAD)发生发展有关.细胞内抗氧化系统是抗铁死亡所必需的.本研究旨在探讨细胞外系统是否以及如何使LUAD细胞对铁死亡失去敏感性.方法 在本研究中,使用已建立的人肺成纤维细胞(MRC-5、WI38)、人肺腺癌细胞(H1650、PC9、H1975、H358、A549和H1299细胞系)、LUAD肿瘤及相匹配的癌旁正常组织、健康个体及LUAD患者的血浆,应用免疫组化和免疫印迹方法分析蛋白表达,实时荧光定量PCR(quantitative reverse transcription-PCR,qPCR)法分析mRNA表达.通过测定细胞活力、细胞死亡和脂质活性氧(reactive oxygen species,ROS)生成评估细胞对铁死亡的反应.通过透射电镜观察外泌体,用点击化学法配合共聚焦显微镜检测花生四烯酸(arachidonic acid,AA)的定位.采用RNA pull down、RNA免疫沉淀(RNA immunoprecipitation,RIP)、光激活核糖核酸苷增强交联和免疫沉淀(photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation,CAR-CLIP)等方法检测RNA和蛋白质之间的相互作用.用蛋白质组学方法来分析RNA调控蛋白,用代谢组学方法分析代谢物.细胞源性异种移植物(cell derived xenograft,CDX)模型、患者源性异种移植物(patient-derived xenograft,PDX)模型、细胞植入肺内LUAD小鼠模型和来自LUAD患者的血浆/组织标本被用于分子机制的验证.结果 LUAD患者血浆外泌体特异地降低了脂质过氧化,使LUAD细胞对铁死亡敏感性下降.可能的机制是在LUAD中,外泌体circRNA_101093(cir93)中维持了细胞内cir93的升高,调节AA,一种对铁死亡相关的质膜过氧化增加至关重要的多不饱和脂肪酸.对具体机制而言,cir93与脂肪酸结合蛋白3(acid-binding protein 3,FABP3)相互作用增加其表达,后者转运AA并促进其与牛磺酸的反应.因此,总体AA被降低,而N-花生四烯酰基牛磺酸(N-arachidonoyl taurine,NAT;AA和牛磺酸的产物)被诱导产生.值得注意的是,NAT在抑制AA合并到质膜中的作用也被揭示.在临床前的体内模型中,减少外泌体提高了以铁死亡为基础疗法的疗效.结论 外泌体和cir93是抑制LUAD细胞铁死亡敏感性的关键,阻断外泌体可能有助于未来LUAD的治疗.

Ferroptosis is a regulatory cell death process that is accompanied by large amounts of iron ion accumulation and lipid peroxidation. Photoactivated ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) is a method used to identify the binding sites of RNA-binding proteins (RBPs) on target RNAs with high resolution at the nucleotide level. By inserting photosensitive ribonucleoside analogs into new RNA transcripts of living cells, characteristic mutations can be generated during reverse transcription and be used to accurately locate the crosslinking position of RNAs and RBPs. The use of PAR-CLIP to detect interactions and determine precise crosslinking sites between RNAs and RBPs, or to search for RNAs upstream or downstream of ferroptosis pathways genes through known proteins, can help to clarify and verify the occurrence and regulation mechanisms of the various signaling pathways of ferroptosis. Furthermore, it may reveal new targets for ferroptosis detection and improve the treatment efficiency of ferroptosis-related diseases such as cancer and neurodegenerative diseases. Here, we introduce a specific PAR-CLIP protocol for monitoring the ferroptosis process.

Protein modifications have significant effects on tumorigenesis. N-Myristoylation is one of the most important lipidation modifications, and N-myristoyltransferase 1 (NMT1) is the main enzyme required for this process. However, the mechanism underlying how NMT1 modulates tumorigenesis remains largely unclear. Here, we found that NMT1 sustains cell adhesion and suppresses tumor cell migration. Intracellular adhesion molecule 1 (ICAM-1) was a potential functional downstream effector of NMT1, and its N-terminus could be N-myristoylated. NMT1 prevented ubiquitination and proteasome degradation of ICAM-1 by inhibiting Ub E3 ligase F-box protein 4, which prolonged the half-life of ICAM-1 protein. Correlations between NMT1 and ICAM-1 were observed in liver and lung cancers, which were associated with metastasis and overall survival. Therefore, carefully designed strategies focusing on NMT1 and its downstream effectors might be helpful to treat tumors.

Background Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m 6 A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m 6 A modification in LUAD tumorigenesis is unknown. Methods Global m 6 A levels and expressions of m 6 A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m 6 A on LUAD. The RNA-protein interactions, translation, putative m 6 A sites and glycolysis were explored in the investigation of the mechanism underlying how m 6 A stimulates tumorigenesis. Results The elevation of global m 6 A level in most human LUAD specimens resulted from the combined upregulation of m 6 A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m 6 A level was associated with a poor overall survival in LUAD patients. Reducing m 6 A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m 6 A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m 6 A methylated at 359 A, which facilitated it’s binding with the m 6 A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m 6 A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m 6 A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m 6 A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD. Conclusions The m 6 A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m 6 A-dependent LUAD.

A lack of promising targets leads to poor prognosis in patients with lung adenocarcinoma (LUAD). Therefore, it is urgent to identify novel therapeutic targets. The importance of the N6-methyladenosine (m6A) RNA modification has been demonstrated in various types of tumors; however, knowledge of m6A-related proteins in LUAD is still limited. Here, we found that insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), an m6A reader protein, is highly expressed in LUAD and associated with poor prognosis. IGF2BP3 desensitizes ferroptosis (a new form of regulated cell death) in a manner dependent on its m6A reading domain and binding capacity to m6A-methylated mRNAs encoding anti-ferroptotic factors, including but not limited to glutathione peroxidase 4 (GPX4), solute carrier family 3 member 2 (SLC3A2), acyl-CoA synthetase long chain family member 3 (ACSL3), and ferritin heavy chain 1 (FTH1). After IGF2BP3 overexpression, expression levels and mRNA stabilities of these anti-ferroptotic factors were successfully sustained. Notably, significant correlations between SLC3A2, ACSL3, and IGF2BP3 were revealed in clinical LUAD specimens, further establishing the essential role of IGF2BP3 in desensitizing ferroptosis. Inducing ferroptosis has been gradually accepted as an alternative strategy to treat tumors. Thus, IGF2BP3 could be a potential target for the future development of new biomaterial-associated therapeutic anti-tumor drugs.

Yes-associated protein (YAP) activation is crucial for tumor formation and development, and its stability is regulated by ubiquitination. ISGylation is a type of ubiquitination like post-translational modification, whereas whether YAP is ISGylated and how ISGylation influences YAP ubiquitination-related function remains uncovered. In addition, YAP can activate glucose metabolism by activating the hexosamine biosynthesis pathway (HBP) and glycolysis, and generate a large number of intermediates to promote tumor proliferation. However, whether YAP stimulates the pentose phosphate pathway (PPP), another tumor-promoting glucose metabolism pathway, and the relationship between this stimulation and ISGylation needs further investigation. Here, we found that YAP was ISGylated and this ISGylation inhibited YAP ubiquitination, proteasome degradation, interaction with-beta-transducin repeat containing E3 ubiquitin-protein ligase (βTrCP) to promote YAP stability. However, ISGylation-induced pro-YAP effects were abolished by YAP K497R (K, lysine; R, arginine) mutation, suggesting K497 could be the major YAP ISGylation site. In addition, YAP ISGylation promoted cell viability, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) tumor formation. YAP ISGylation also increased downstream genes transcription, including one of the key enzymes of PPP, 6-phosphogluconolactonase (6PGL). Mechanistically, YAP promoted 6PGL transcription by simultaneously recruiting SMAD family member 2 (SMAD2) and TEA domain transcription factor 4 (TEAD4) binding to the 6PGL promoter to activate PPP. In clinical lung adenocarcinoma (LUAD) specimens, we found that YAP ISGylation degree was positively associated with 6PGL mRNA level, especially in high glucose LUAD tissues compared to low glucose LUAD tissues. Collectively, this study suggested that YAP ISGylation is critical for maintaining its stability and further activation of PPP. Targeting ISGylated YAP might be a new choice for hyperglycemia cancer treatment.

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.

Abstract Background Ferroptosis, a form of regulated cell death, is an important topic in the field of cancer research. However, the signalling pathways and factors that sensitise tumour cells to ferroptosis remain elusive. Methods We determined the level of ferroptosis in cells by measuring cell death and lipid reactive oxygen species (ROS) production. The expression of RB1‐inducible coiled‐coil 1 (RB1CC1) and related proteins was analyzed by immunoblotting and immunohistochemistry. Immunofluorescence was used to determine the subcellular localization of RB1CC1. We investigated the mechanism of RB1CC1 nuclear translocation by constructing a series of RB1CC1 variants. To examine the ferroptosis‐ and RB1CC1‐dependent transcriptional program in tumour cells, chromatin immunoprecipitation sequencing was performed. To assess the effect of c‐Jun N‐terminal kinase (JNK) agonists on strenthening imidazole ketone erastin (IKE) therapy, we constructed cell‐derived xenograft mouse models. Mouse models of hepatocellular carcinoma to elucidate the importance of Rb1cc1 in IKE‐based therapy of liver tumourigenesis. Results RB1CC1 is upregulated by lipid ROS and that nuclear translocation of phosphorylation of RB1CC1 at Ser537 was essential for sensitising ferroptosis in tumour cells. Upon ferroptosis induction, nuclear RB1CC1 sharing forkhead box (FOX)‐binding motifs recruits elongator acetyltransferase complex subunit 3 (ELP3) to strengthen H4K12Ac histone modifications within enhancers linked to ferroptosis. This also stimulated transcription of ferroptosis‐associated genes, such as coiled‐coil–helix–coiled‐coil–helix domain containing 3 (CHCHD3), which enhanced mitochondrial function to elevate mitochondrial ROS early following induction of ferroptosis. FDA‐approved JNK activators reinforced RB1CC1 nuclear translocation and sensitised cells to ferroptosis, which strongly suggested that JNK is upstream of RB1CC1. Nuclear localisation of RB1CC1 correlated with lipid peroxidation in clinical lung cancer specimens. Rb1cc1 was essential for ferroptosis agonists to suppress liver tumourigenesis in mice. Conclusions Our findings indicate that RB1CC1‐associated signalling sensitises tumour cells to ferroptosis and that targeting RB1CC1 may be beneficial for tumour treatment.

BackgroundResistance to ferroptosis, a regulated cell death caused by iron-dependent excessive accumulation of lipid peroxides has recently been linked to lung adenocarcinoma (LUAD), the most prevalent lung cancer subtype. Despite intracellular antioxidant system is required for protection against ferroptosis, whether and how extracellular system desensitizes LUAD cells to ferroptosis is incompletely known. MethodsImmunohistochemistry (IHC) and immunoblotting (IB) were used to analyze protein expression, and quantitative RT-PCR (qPCR) was used to analyze mRNA level. Cell viability, cell death and the lipid reactive oxygen species (ROS) generation were measured to evaluate the responses to ferroptosis induction. Metabolites were measured using appropriate kits. Exosomes were observed using transmission electron microscope (TEM). The localization of arachidonic acid (AA) was detected using click chemistry labeling followed by confocal microscope observation. Interaction between RNA and protein were detected using RNA-pulldown, RNA immunoprecipitation (RIP) and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Proteomic analysis was used to investigate circRNA_101093 (cir93) regulated protein and metabolomic analysis was used to analyze metabolites that changed among LUAD tissues expressing low and high Fatty acid-binding protein 3 (FABP3) levels. Cell-derived xenograft (CDX), patient-derived xenograft (PDX) and cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the mechanism that exosome and cir93 desensitized ferroptosis in LUAD. ResultsHere, the roles of exosome and circular RNA (circRNA) to desensitize LUAD cells to ferroptosis were investigated. Plasma exosome from LUAD patients exclusively reduced lipid peroxidation and desensitized ferroptosis. This might because exosomal-cir93 sustained an elevation of intracellular-cir93 in LUAD to modulate AA, a poly-unsaturated fatty acid that is critical for ferroptosis-associated over peroxidation in the plasma membrane. Mechanistically, cir93 interacted and lifted FABP3, which transported and facilitated AA reaction with taurine. Thus, global-AA was reduced while N-Arachidonoyl taurine (NAT), the product of AA and taurine, was induced. Notably, a role of NAT to suppress AA incorporation into plasma membrane was also revealed. In pre-clinical in vivo models, reducing exosome exhibited an improvement of ferroptosis-based treatment. ConclusionExosome and cir93 are essential for desensitizing LUAD cells to ferroptosis, and blocking exosome will be helpful for future LUAD treatment.

BackgroundA tremendous amount of studies have suggested that post-translational modifications (PTMs) play pivotal roles during tumorigenesis. Compared to other PTMs, lipid modification is less studied. Recently, N-myristoylation, one type of lipid modification, has been paid attention to the field of cancer. However, whether and how N-myristoylation exerts its roles in liver tumorigenesis still remains unclear.MethodsParallel reaction monitoring (PRM) was conducted to evaluate the expression of protein modification enzymes in paired tissues. Liver conditionally knocking NMT1 out mice model was used to assess the critical roles of N-myristoylation during liver tumorigenesis. Proteomics isobaric tags for relative and absolute quantification (iTraq) was performed to identify proteins that changed while NMT1 was knocked down. The click chemistry assay was used to evaluate the N-myristoylation levels of proteins.ResultsHere, N-myristolyation and its enzyme NMT1, but not NMT2, were found to be critical in liver cancer. Two categories of proteins, i.e., N-myristolyation down-regulated proteins (NDP, including LXN, RPL29, and FAU) and N-myristolyation up-regulated proteins (NUP, including AHSG, ALB, and TF), were revealed negatively and positively regulated by NMT1, respectively. Both NDP and NUP could be N-myristolyated by NMT1 indispensable of POTEE. However, N-myristolyation decreased and increased stability of NDP and NUP, respectively. Mechanistically, NDP-specific binding protein RPL7A facilitated HIST1H4H, which has ubiquitin E3 ligase function, to ubiquitinate NDP. By contrast, NUP-specific binding protein HBB prevented NUP from ubiquitination by HIST1H4H. Notably, function of RPL7A and HBB was all NMT1-dependent. Moreover, NDP suppressed while NUP stimulated transformative phenotypes. Clinically, higher levels of NMT1 and NUP with lower levels of NDP had worse prognostic outcome.ConclusionCollectively, N-myristolyation by NMT1 suppresses anti-tumorigenic NDP, whereas it stimulates pro-tumorigenic NUP by interfering their ubiquitination to finally result in a pro-tumorigenic outcome in liver cancer. Targeting N-myristolyation and NMT1 might be helpful to treat liver cancer.

The regulation of homeostasis in the Ubiquitin (Ub) proteasome system (UPS) is likely to be important for the development of liver cancer. Tribbles homolog 2 (TRIB2) is known to affect Ub E3 ligases (E3s) in liver cancer. However, whether TRIB2 regulates the UPS in other ways and the relevant mechanisms are still unknown. Here, we reveal that TRIB2 decreased Ub levels largely by stimulating proteasome degradation of Ub. In the proteasome, proteasome 20S subunit beta 5 (PSMB5) was critical for the function of TRIB2, although it did not directly interact with TRIB2. However, poly (rC) binding protein 2 (PCBP2), which was identified by mass spectrometry, directly interacted with both TRIB2 and PSMB5. PCBP2 was a prerequisite for the TRIB2 induction of PSMB5 activity and decreased Ub levels. A significant correlation between TRIB2 and PCBP2 was revealed in liver cancer specimens. Interestingly, TRIB2 suppressed the K48-ubiquitination of PCBP2 to increase its level. Therefore, a model showing that TRIB2 cooperates and stimulates PCBP2 to reduce Ub levels was established. Additionally, the reduction in Ub levels induced by TRIB2 and PCBP2 was dependent on K48-ubiquitination. PCBP2 was one of the possible downstream factors of TRIB2 and their interaction relied on the DQLVPD element of TRIB2 and the KH3 domain of PCBP2. This interaction was necessary to maintain the viability of the liver cancer cells and promote tumor growth. Mechanistically, glutathione peroxidase 4 functioned as one of the terminal effectors of TRIB2 and PCBP2 to protect liver cancer cells from oxidative damage. Taken together, the data indicate that, in addition to affecting E3s, TRIB2 plays a critical role in regulating UPS by modulating PSMB5 activity in proteasome to reduce Ub flux, and that targeting TRIB2 might be helpful in liver cancer treatments by enhancing the oxidative damage induced by therapeutic agents.

Tribbles homolog 2 (TRIB2) is known to boost liver tumorigenesis via regulating Ubiquitin (Ub) proteasome system (UPS). At least two ways are involved, i.e., acts as an adaptor protein to modulate ubiquitination functions of certain ubiquitin E3 ligases (E3s) and reduces global Ub levels via increasing the proteolysis activity of proteasome. Recently, we have identified the role of TRIB2 to relieve oxidative damage via reducing the availability of Ub that is essential for the ubiquitination and subsequent degradation of Glutathione peroxidase 4 (GPX4). Although GPX4 is a critical antioxidant factor to protect against ferroptosis, the exact evidence showing that TRIB2 desensitizes ferroptosis is lacking. Also, whether such function is via E3 remains unclear. Here, we demonstrated that deletion of TRIB2 sensitized ferroptosis via lifting labile iron in liver cancer cells. By contrast, overexpression of TRIB2 led to the opposite outcome. We further demonstrated that transferrin receptor (TFRC) was required for TRIB2 to desensitize the cells to ferroptosis. Without TFRC, the labile iron pool could not be reduced by overexpressing TRIB2. We also found that beta-transducin repeat containing E3 ubiqutin protein ligase (βTrCP) was a genuine E3 for the ubiquitination of TFRC, and TRIB2 was unable to decline labile iron level once upon βTrCP was knocked out. In addition, we confirmed that the opposite effects on ferroptosis and ferroptosis-associated lipid reactive oxygen species (ROS) generation resulted from knockout and overexpression of TRIB2 were all indispensible of TFRC and βTrCP. Finally, we demonstrated that TRIB2 exclusively manipulated RSL3- and erastin-induced-ferroptosis independent of GPX4 and glutathione (GSH). In conclusion, we elucidated a novel role of TRIB2 to desensitize ferroptosis via E3 βTrCP, by which facilitates TFRC ubiquitiation and finally decreases labile iron in liver cancer cells.

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 XC-. 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 XC- 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 increase is caused by the failure to sustain YAP-stimulated transcriptional compensation for ferritin at later stages further explains why ferroptosis sensitivity varies among LUAD cells. Conclusions: Endogenous glutamate is critical for ferroptosis sensitivity following the inhibition of system X-C-in LUAD cells, and ferroptosis-based treatment is a good choice for LUAD patients with later-stage and/or therapy-resistant tumors.