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Theranostics 2021; 11(12):5650-5674. doi:10.7150/thno.55482 This issue Cite

Research Paper

Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma

Xiao Zhang^1,2#, Keke Yu^3#, Lifang Ma^1,2#, Zijun Qian^4#, Xiaoting Tian², Yayou Miao², Yongjie Niu⁴, Xin Xu⁴, Susu Guo⁵, Yueyue Yang⁵, Zhixian Wang⁴, Xiangfei Xue⁵, Chuanjia Gu^6,7, Wentao Fang¹, Jiayuan Sun^6,7✉, Yongchun Yu^2✉, Jiayi Wang^1,2,5✉

1. Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, 200030, China.
2. Shanghai Institute of Thoracic Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, 200030, China.
3. Department of Bio-bank, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, 200030, China.
4. Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200071, China.
5. Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital of Tongji University, Shanghai, 200072, China.
6. Department of Respiratory Endoscopy, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, 200030, China.
7. Department of Respiratory and Critical Care Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China.
#These authors contributed equally to the work.

✉ Corresponding authors: Jiayuan Sun, Department of Respiratory Endoscopy, Department of Respiratory and Critical Care Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China; E-mail: jysun1976com. Yongchun Yu, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 Huaihai West Road, Shanghai, 200030, China; E-mail: yyc2166edu.cn. Jiayi Wang, Department of Thoracic Surgery, Shanghai Institute of Thoracic Tumors, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 Huaihai West Road, Shanghai, 200030, China; E-mail: karajan2com. More

Abstract

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 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.

Keywords: XBP1 splicing, Ferritin, Hippo pathway, GFPT1, HBP-dependent O-GlcNAcylation, NCOA4

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