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The Metabolite α-KG Induces GSDMC-Dependent Pyroptosis Through Death Receptor 6-Activated Caspase-8

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The Metabolite α-KG Induces GSDMC-Dependent Pyroptosis Through Death Receptor 6-Activated Caspase-8 www.nature.com/cr www.cell-research.com ARTICLE OPEN The metabolite α-KG induces GSDMC-dependent pyroptosis through death receptor 6-activated caspase-8 Jia-yuan Zhang1, Bo Zhou1, Ru-yue Sun1, Yuan-li Ai1, Kang Cheng1, Fu-nan Li2, Bao-rui Wang2, Fan-jian Liu1, Zhi-hong Jiang1, Wei-jia Wang1, Dawang Zhou 1, Hang-zi Chen 1 and Qiao Wu 1 Pyroptosis is a form of regulated cell death mediated by gasdermin family members, among which the function of GSDMC has not been clearly described. Herein, we demonstrate that the metabolite α-ketoglutarate (α-KG) induces pyroptosis through caspase-8- mediated cleavage of GSDMC. Treatment with DM-αKG, a cell-permeable derivative of α-KG, elevates ROS levels, which leads to oxidation of the plasma membrane-localized death receptor DR6. Oxidation of DR6 triggers its endocytosis, and then recruits both pro-caspase-8 and GSDMC to a DR6 receptosome through protein-protein interactions. The DR6 receptosome herein provides a platform for the cleavage of GSDMC by active caspase-8, thereby leading to pyroptosis. Moreover, this α-KG-induced pyroptosis could inhibit tumor growth and metastasis in mouse models. Interestingly, the efficiency of α-KG in inducing pyroptosis relies on an acidic environment in which α-KG is reduced by MDH1 and converted to L-2HG that further boosts ROS levels. Treatment with lactic acid, the end product of glycolysis, builds an improved acidic environment to facilitate more production of L-2HG, which makes the originally pyroptosis-resistant cancer cells more susceptible to α-KG-induced pyroptosis. This study not only illustrates a pyroptotic pathway linked with metabolites but also identifies an unreported principal axis extending from ROS-initiated DR6 endocytosis to caspase-8-mediated cleavage of GSDMC for potential clinical application in tumor therapy. 1234567890();,: Cell Research (2021) 31:980–997; https://doi.org/10.1038/s41422-021-00506-9 INTRODUCTION treatment with chemotherapeutic drugs, activated caspase-3 Cell death is a very complicated and important biological process cleaves GSDME to induce pyroptosis in GSDME-expressing cells. that contributes to the regulation of various physiological Unfortunately, GSDME is silenced in most tumor cells but is highly functions and maintenance of body homeostasis. Dysregulation expressed in normal cells and tissues, which leads to strong of cell death programming leads to the occurrence of various induction of pyroptosis in normal tissues upon the administration diseases, including cancers. Therefore, further understanding the of chemotherapeutic drugs.5 Therefore, specifically inducing functional mechanism and signaling pathways involved in cell pyroptosis in tumor cells while avoiding normal cell and tissue death would benefit the treatment of various diseases. Pyroptosis damage has become a key issue. We previously demonstrated is a form of regulated cell death mediated by gasdermin family that iron reinforces the effect of ROS-inducing drugs in boosting proteins.1 In humans, the gasdermin family consists of six ROS levels, leading to the activation of GSDME and pyroptosis of members: GSDM-A, -B, -C -D, -E (also called DFNA5), and DFNB59. melanoma cells. Hence, iron supplementation was used as a Mice lack GSDMB but express three GSDMAs (mGSDMA1–3) and sensitizer in combination with clinical drugs to inhibit the growth four GSDMCs (mGSDMC1–4).2 Structurally, all gasdermin family and metastasis of melanoma in a mouse model, with decreased members except DFNB59 have an N-terminal pore-forming toxicity.7 Although the currently limited research findings cannot domain, a C-terminal autoinhibitory domain, and a loop domain fundamentally explain the two outcomes of pyroptosis in normal that links the N- and C-terminal domains. Protease-mediated and tumor tissues, targeting pyroptosis for tumor therapy may be cleavage within the linker loop releases the N-terminal domain, a good strategy, because pyroptosis of tumor cells would not only which then oligomerizes to form nonselective pores at the plasma overcome the apoptosis resistance of tumor cells but also trigger membrane and causes membrane permeability changes, cell antitumor immunity.8–11 However, the specific pyroptosis inducers swelling and membrane rupture. For example, inflammasome- in tumors are largely unknown. activated caspase-1/4/5/11 cleave GSDMD to produce an N- Cell fate is closely associated with metabolic homeostasis. α- terminal fragment that triggers pyroptosis,2–4 while proapoptotic Ketoglutarate (α-KG) is an essential metabolite in the tricarboxylic stimuli activate caspase-3, which cleaves GSDME to induce acid (TCA) cycle and plays a significant role in physiological pyroptosis.5,6 processes, including lipid biosynthesis, oxidative stress reduction, The functions of pyroptosis have become a hot spot in cancer protein modification, autophagy, and cell death. Several studies research. The Shao group found that GSDME-mediated pyroptosis have shown that α-KG is a prospective antitumor agent. For is associated with the side effects of chemotherapy. Following example, α-KG suppresses breast cancer oncogenesis by switching 1State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China and 2School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, China Correspondence: Hang-zi Chen ([email protected]) or Qiao Wu ([email protected]) These authors contributed equally: Jia-yuan Zhang, Bo Zhou, Ru-yue Sun, Yuan-li Ai Received: 17 September 2020 Accepted: 2 April 2021 Published online: 19 May 2021 © The Author(s) 2021 Article 981 metabolism from glycolysis to oxidative phosphorylation.12 concluded that α-KG specifically induces GSDMC-dependent Accumulated α-KG blocks malignant progression by driving tumor pyroptosis. cell differentiation in p53-null tumors.13 Moreover, increasing the Treatment with Z-VAD, a pancaspase inhibitor, suppressed DM- α-KG/succinate ratio by disrupting mitochondrial complex I αKG-induced GSDMC cleavage, thereby ameliorating the pyropto- suppresses tumor growth.14,15 However, the role of α-KG in tic cell morphology and reducing LDH release in HeLa, SGC-7901 pyroptosis remains unreported. and B16 cells (Supplementary information, Fig. S1i, j). These results The aim of this study was to investigate the molecular strongly suggest that caspases might be involved in GSDMC mechanism and signaling pathway of α-KG in pyroptotic cell cleavage. To determine the participation of caspase subtypes, death. We demonstrated that α-KG is promiscuously reduced to different exogenous recombinant caspases were incubated with another metabolite, L-2HG, by the metabolic enzyme MDH1 in an GSDMC proteins that were isolated from GSDMC-overexpressing acidic environment, which then increases ROS levels to induce the human embryonic kidney (HEK293T) cells. The results revealed oxidation and internalization of the plasma membrane-localized that GSDMC was effectively cleaved by caspase-8 and -9 in vitro death receptor DR6. Internalized DR6 further recruits both pro- (Supplementary information, Fig. S1k). However, DM-αKG treat- caspase-8 and GSDMC to the DR6 receptosome that provides a ment activated only caspase-8 but not caspase-9 in HeLa cells in a platform for the cleavage of GSDMC by active caspase-8, thereby time- and dose-dependent manner (Supplementary information, inducing pyroptosis. This pyroptosis was sufficient for the Fig. S1l), suggesting the involvement of caspase-8 but not inhibition of tumor growth and metastasis in mouse models. caspase-9 in α-KG-induced pyroptosis. Indeed, knockdown of Collectively, this study not only identifies the key node protein caspase-8 but not caspase-9 prevented the initiation of DM-αKG- (DR6), pyroptosis initiator (caspase-8), and executor (GSDMC) in induced pyroptosis in HeLa cells (Fig. 1f, g and Supplementary the α-KG-induced pyroptotic pathway but also provides an information, Fig. S1d, m). The involvement of caspase-8 in DM- important research direction for clinical tumor treatment via αKG-induced pyroptosis was also observed in SGC-7901 and B16 pyroptosis induction. cells (Supplementary information, Fig. S1n). In caspase-8 knock- down HeLa cells, re-expression of wild-type caspase-8 (CASP8WT) but not its enzymatically dead mutant (CASP8C360S)18 promoted RESULTS DM-αKG-induced GSDMC cleavage and pyroptosis (Fig. 1h and α-KG induces pyroptosis through caspase-8-mediated cleavage of Supplementary information, Fig. S1o). Clearly, active caspase-8 is GSDMC required for the cleavage of GSDMC and the subsequent The metabolite α-ketoglutarate (α-KG), a key metabolite in the TCA pyroptosis in response to α-KG stimulation. cycle, plays important roles in both metabolic and cellular Caspase cleaves target proteins downstream of an Asp pathways.16 Since exogenously added α-KG cannot pass through residue. Given that the in-gel migration of the N-terminal the cell membrane, we used dimethyl-α-ketoglutarate (DM-αKG, a cleavage product of GSDMC indicated that its molecular weight cell-permeable analog of α-KG) to treat HeLa cervical carcinoma is approximately 35 kDa (Fig. 1c), the potential Asp site of α-KG- cells and unexpectedly found that DM-αKG significantly induced induced caspase-8-mediated cleavage in GSDMC may lie in a cell death with typical morphological features of pyroptosis, i.e., sequence of between 200 and 300 amino acid residues. To cell swelling and large areas of plasma membrane blebbing investigate this possibility, Asp231, Asp232, Asp233, Asp240 and (Fig. 1a, left;
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