Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma.
Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma.
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内源性谷氨酸通过 ADCY10 依赖性 YAP 抑制决定肺腺癌铁死亡敏感性
作者：
Zhang X;Yu K;Ma L;Qian Z;Tian X;Miao Y;Niu Y;Xu X;Guo S;Yang Y;Wang Z;Xue X;Gu C;Fang W;Sun J;Yu Y;Wang J
基本原理：铁凋亡是一种新发现的调节性细胞死亡形式，由于胱氨酸摄取受损，胱氨酸-谷氨酸反向转运体系统XC-被抑制后可诱导铁凋亡。然而，肺腺癌（LUAD）内源性谷氨酸积累后的结果尚未确定。Yes相关蛋白（雅普）由己糖胺生物合成途径（HBP）依赖性O-连接β-N-乙酰葡糖胺化（O-GlcNAc化）维持，HBP的限速酶谷氨酰胺-果糖-6-磷酸转氨酶（GFPT 1）可被腺苷酸环化酶（ADCY）介导的蛋白激酶A（PKA）活化磷酸化和抑制。然而，积累的内源性谷氨酸是否通过影响LUAD细胞中的ADCY/PKA/HBP/雅普轴来决定铁凋亡敏感性尚不清楚。研究方法：测定细胞活力、细胞死亡以及脂质活性氧（ROS）和丙二醛（MDA）的产生，以评估系统XC-抑制后对诱导铁凋亡的反应。串联质量标签（TMTs）被用来探索LUAD细胞的铁凋亡敏感性的潜在关键因素。免疫印迹（IB）和定量RT-PCR（qPCR）用于分析蛋白质和mRNA表达。进行免疫共沉淀（co-IP）测定以鉴定蛋白质-蛋白质相互作用和翻译后修饰。使用适当的试剂盒测量代谢物水平。使用荧光素酶报告基因测定、染色质免疫沉淀（ChIP）和电泳迁移率变动测定（EMSA）评估转录调控。使用细胞来源的异种移植物（CDX）和患者来源的异种移植物（PDX）小鼠模型进行药物施用和有限稀释细胞移植。基于接受根治性手术的患者的数据确定临床结果、药物疗效和ADCY 10表达之间的关联，并使用患者来源的原代LUAD细胞和组织进行评价。结果如下：已显示系统XC-抑制后内源性谷氨酸的积累通过抑制LUAD细胞中的雅普来确定铁凋亡敏感性。雅普O-GlcNAc酰化和表达在GFPT 1受损后不能在LUAD细胞中持续。因此，雅普的Hippo途径样磷酸化和泛素化增强。ADCY 10作为一个关键的下游靶点，使谷氨酸对GFPT 1的PKA依赖性抑制的作用多样化。我们还发现ADCY 10的促肿瘤和促铁作用是分别介导的。ADCY 10高表达的晚期LUAD对铁凋亡敏感。此外，具有获得性治疗耐药性的LUAD细胞也容易产生更高的ADCY 10表达，并且更有可能对铁凋亡产生反应。最后，不同程度的继发性不稳定铁的增加是由于在后期阶段未能维持YAP刺激的铁蛋白转录补偿引起的，这进一步解释了为什么LUAD细胞中的铁凋亡敏感性不同。结论：内源性谷氨酸对LUAD细胞中系统XC-抑制后的铁凋亡敏感性至关重要，基于铁凋亡的治疗是患有晚期和/或治疗抗性肿瘤的LUAD患者的良好选择。
Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system XC- 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 XC- in LUAD cells, and ferroptosis-based treatment is a good choice for LUAD patients with later-stage and/or therapy-resistant tumors.
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ACSL4 通过塑造细胞脂质成分来决定铁死亡敏感性。
影响因子：
14.8
作者：
Doll S;Proneth B;Tyurina YY;Panzilius E;Kobayashi S;Ingold I;Irmler M;Beckers J;Aichler M;Walch A;Prokisch H;Trümbach D;Mao G;Qu F;Bayir H;Füllekrug J;Scheel CH;Wurst W;Schick JA;Kagan VE;Angeli JP;Conrad M
通讯作者：
Conrad M
影响因子：
14.8
作者：
Kagan VE;Mao G;Qu F;Angeli JP;Doll S;Croix CS;Dar HH;Liu B;Tyurin VA;Ritov VB;Kapralov AA;Amoscato AA;Jiang J;Anthonymuthu T;Mohammadyani D;Yang Q;Proneth B;Klein-Seetharaman J;Watkins S;Bahar I;Greenberger J;Mallampalli RK;Stockwell BR;Tyurina YY;Conrad M;Bayır H
通讯作者：
Bayır H
影响因子：
64.5
作者：
Dixon SJ;Lemberg KM;Lamprecht MR;Skouta R;Zaitsev EM;Gleason CE;Patel DN;Bauer AJ;Cantley AM;Yang WS;Morrison B 3rd;Stockwell BR
通讯作者：
Stockwell BR
影响因子：
13.5
作者：
Chang, Jung-Chin;Go, Simei;de Waart, Dirk R.;Munoz-Garrido, Patricia;Beuers, Ulrich;Paulusma, Coen C.;Elferink, Ronald Oude
通讯作者：
Elferink, Ronald Oude
影响因子：
5.6
作者：
Hsieh, Tusty-Jiuan;Lin, Tzu;Shin, Shyi-Jang
通讯作者：
Shin, Shyi-Jang