Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes. Basit F,Cristofanon S,Fulda S Cell death and differentiation Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death. 10.1038/cdd.2013.45
    Necroptosis promotes autophagy-dependent upregulation of DAMP and results in immunosurveillance. Lin Sheng-Yen,Hsieh Sung-Yuan,Fan Yi-Ting,Wei Wen-Chi,Hsiao Pei-Wen,Tsai Dai-Hua,Wu Tzong-Shoon,Yang Ning-Sun Autophagy Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced macroautophagic/autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin can instigate RIPK1 (receptor [TNFRSF]-interacting serine-threonine kinase 1)- and RIPK3 (receptor-interacting serine-threonine kinase 3)-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy can directly contribute to DAMP upregulation. Counterintuitively, among the released and ectoDAMPs, only the latter were shown to be able to activate the cocultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ectoDAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pretreated with chloroquine and shikonin further enhanced the antimetastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine cotreatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment. 10.1080/15548627.2017.1386359
    MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis. Wu Xiaoqin,Poulsen Kyle L,Sanz-Garcia Carlos,Huang Emily,McMullen Megan R,Roychowdhury Sanjoy,Dasarathy Srinivasan,Nagy Laura E Journal of hepatology BACKGROUND & AIMS:Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. METHODS:Rip3, Rip3, Mlkl and Mlkl mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). RESULTS:The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl but not Mlkl mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes. CONCLUSIONS:Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy. LAY SUMMARY:Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease. 10.1016/j.jhep.2020.03.023
    Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway, which is counterregulated by autophagy. Radonjic-Hoesli Susanne,Wang Xiaoliang,de Graauw Elisabeth,Stoeckle Christina,Styp-Rekowska Beata,Hlushchuk Ruslan,Simon Dagmar,Spaeth Peter J,Yousefi Shida,Simon Hans-Uwe The Journal of allergy and clinical immunology BACKGROUND:Eosinophils are a subset of granulocytes that can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. Release takes place through several different mechanisms, one of which is cytolysis, which is associated with release of intact granules, so-called clusters of free eosinophil granules. The mechanism underlying this activation-induced form of cell death in eosinophils has remained unclear. OBJECTIVE:We aimed to elucidate the molecular mechanism of eosinophil cytolysis. METHODS:Isolated blood eosinophils were incubated on glass coverslips coated with intravenous immunoglobulin and inactive complement component 3b. A morphologic characterization of the distinct stages of the proposed cascade was addressed by means of time-lapse automated fluorescence microscopy, electron microscopy, and immunohistochemistry. Experiments with pharmacologic inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients with eosinophilic skin diseases or eosinophilic esophagitis were used for in vivo analyses. RESULTS:After eosinophil adhesion, we observed reactive oxygen species production, early degranulation, and granule fusion processes, leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, cytolysis. Using a pharmacologic approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, after its activation, leads to the production of high levels of reactive oxygen species in a p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase-dependent manner. All these steps are required for cytoplasmic vacuolization and subsequent cytolysis to occur. Interestingly, triggering cytolysis is associated with an induction of autophagy in eosinophils, and additional stimulation of autophagy by means of pharmacologic inhibition of the mechanistic target of rapamycin counterregulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under in vivo inflammatory conditions. CONCLUSION:We report that adhesion-induced eosinophil cytolysis takes place through RIPK3-MLKL-dependent necroptosis, which can be counterregulated by autophagy. 10.1016/j.jaci.2017.01.044
    Gut epithelial TSC1/mTOR controls RIPK3-dependent necroptosis in intestinal inflammation and cancer. Xie Yadong,Zhao Yifan,Shi Lei,Li Wei,Chen Kun,Li Min,Chen Xia,Zhang Haiwei,Li Tiantian,Matsuzawa-Ishimoto Yu,Yao Xiaomin,Shao Dianhui,Ke Zunfu,Li Jian,Chen Yan,Zhang Xiaoming,Cui Jun,Cui Shuzhong,Leng Qibin,Cadwell Ken,Li Xiaoxia,Wei Hong,Zhang Haibing,Li Huabin,Xiao Hui The Journal of clinical investigation Although Western diet and dysbiosis are the most prominent environmental factors associated with inflammatory bowel diseases (IBDs), the corresponding host factors and cellular mechanisms remain poorly defined. Here we report that the TSC1/mTOR pathway in the gut epithelium represents a metabolic and innate immune checkpoint for intestinal dysfunction and inflammation. mTOR hyperactivation triggered by Western diet or Tsc1 ablation led to epithelium necroptosis, barrier disruption, and predisposition to dextran sulfate sodium-induced colitis and inflammation-associated colon cancer. Mechanistically, our results uncovered a critical role for TSC1/mTOR in restraining the expression and activation of RIPK3 in the gut epithelium through TRIM11-mediated ubiquitination and autophagy-dependent degradation. Notably, microbiota depletion by antibiotics or gnotobiotics attenuated RIPK3 expression and activation, thereby alleviating epithelial necroptosis and colitis driven by mTOR hyperactivation. mTOR primarily impinged on RIPK3 to potentiate necroptosis induced by TNF and by microbial pathogen-associated molecular patterns (PAMPs), and hyperactive mTOR and aberrant necroptosis were intertwined in human IBDs. Together, our data reveal a previously unsuspected link between the Western diet, microbiota, and necroptosis and identify the mTOR/RIPK3/necroptosis axis as a driving force for intestinal inflammation and cancer. 10.1172/JCI133264
    Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance. Bonapace Laura,Bornhauser Beat C,Schmitz Maike,Cario Gunnar,Ziegler Urs,Niggli Felix K,Schäfer Beat W,Schrappe Martin,Stanulla Martin,Bourquin Jean-Pierre The Journal of clinical investigation In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL. 10.1172/JCI39987
    Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation. Cosin-Roger Jesus,Simmen Simona,Melhem Hassan,Atrott Kirstin,Frey-Wagner Isabelle,Hausmann Martin,de Vallière Cheryl,Spalinger Marianne R,Spielmann Patrick,Wenger Roland H,Zeitz Jonas,Vavricka Stephan R,Rogler Gerhard,Ruiz Pedro A Nature communications Hypoxia regulates autophagy and nucleotide-binding oligomerization domain receptor, pyrin domain containing (NLRP)3, two innate immune mechanisms linked by mutual regulation and associated to IBD. Here we show that hypoxia ameliorates inflammation during the development of colitis by modulating autophagy and mammalian target of rapamycin (mTOR)/NLRP3 pathway. Hypoxia significantly reduces tumor necrosis factor α, interleukin (IL)-6 and NLRP3 expression, and increases the turnover of the autophagy protein p62 in colon biopsies of Crohn's disease patients, and in samples from dextran sulfate sodium-treated mice and Il-10 mice. In vitro, NF-κB signaling and NLRP3 expression are reduced through hypoxia-induced autophagy. We also identify NLRP3 as a novel binding partner of mTOR. Dimethyloxalylglycine-mediated hydroxylase inhibition ameliorates colitis in mice, downregulates NLRP3 and promotes autophagy. We suggest that hypoxia counteracts inflammation through the downregulation of the binding of mTOR and NLRP3 and activation of autophagy.Hypoxia and HIF-1α activation are protective in mouse models of colitis, and the latter regulates autophagy. Here Cosin-Roger et al. show that hypoxia ameliorates intestinal inflammation in Crohn's patients and murine colitis models by inhibiting mTOR/NLRP3 pathway and promoting autophagy. 10.1038/s41467-017-00213-3
    Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis. Li Ming-Yong,Zhu Xia-Lian,Zhao Bi-Xia,Shi Lei,Wang Wei,Hu Wei,Qin Song-Lin,Chen Bing-Hai,Zhou Pang-Hu,Qiu Bo,Gao Yong,Liu Bo-Long Cell death & disease Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM's effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1β, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3β-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS-AMPK-mTOR axis. 10.1038/s41419-019-1728-5
    Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Sepsis. Pu Qinqin,Gan Changpei,Li Rongpeng,Li Yi,Tan Shirui,Li Xuefeng,Wei Yuquan,Lan Lefu,Deng Xin,Liang Haihua,Ma Feng,Wu Min Journal of immunology (Baltimore, Md. : 1950) Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure, with high morbidity and mortality. The literature implies that autophagy is a crucial regulator of inflammation in sepsis. In this article, we report that autophagy-related protein 7 (Atg7) is involved in inflammasome activation in abdominal infection. Following i.p. challenge with , mice showed impaired pathogen clearance, decreased survival, and widespread dissemination of bacteria into the blood and lung tissue compared with wild-type mice. The septic mice also exhibited elevated neutrophil infiltration and severe lung injury. Loss of Atg7 resulted in increased production of IL-1β and pyroptosis, consistent with enhanced inflammasome activation. Furthermore, we demonstrated that flagellin is a chief trigger of inflammasome activation in the sepsis model. Collectively, our results provide insight into innate immunity and inflammasome activation in sepsis. 10.4049/jimmunol.1601196
    The Crohn's Disease Risk Factor IRGM Limits NLRP3 Inflammasome Activation by Impeding Its Assembly and by Mediating Its Selective Autophagy. Mehto Subhash,Jena Kautilya Kumar,Nath Parej,Chauhan Swati,Kolapalli Srinivasa Prasad,Das Saroj Kumar,Sahoo Pradyumna Kumar,Jain Ashish,Taylor Gregory A,Chauhan Santosh Molecular cell Several large-scale genome-wide association studies genetically linked IRGM to Crohn's disease and other inflammatory disorders in which the IRGM appears to have a protective function. However, the mechanism by which IRGM accomplishes this anti-inflammatory role remains unclear. Here, we reveal that IRGM/Irgm1 is a negative regulator of the NLRP3 inflammasome activation. We show that IRGM expression, which is increased by PAMPs, DAMPs, and microbes, can suppress the pro-inflammatory responses provoked by the same stimuli. IRGM/Irgm1 negatively regulates IL-1β maturation by suppressing the activation of the NLRP3 inflammasome. Mechanistically, we show that IRGM interacts with NLRP3 and ASC and hinders inflammasome assembly by blocking their oligomerization. Further, IRGM mediates selective autophagic degradation of NLRP3 and ASC. By suppressing inflammasome activation, IRGM/Irgm1 protects from pyroptosis and gut inflammation in a Crohn's disease experimental mouse model. This study for the first time identifies the mechanism by which IRGM is protective against inflammatory disorders. 10.1016/j.molcel.2018.11.018
    N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis. Karmakar Mausita,Minns Martin,Greenberg Elyse N,Diaz-Aponte Jose,Pestonjamasp Kersi,Johnson Jennifer L,Rathkey Joseph K,Abbott Derek W,Wang Kun,Shao Feng,Catz Sergio D,Dubyak George R,Pearlman Eric Nature communications Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3 autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation. 10.1038/s41467-020-16043-9
    Chaperone-mediated autophagy is involved in the execution of ferroptosis. Wu Zheming,Geng Yang,Lu Xiaojuan,Shi Yuying,Wu Guowei,Zhang Mengmeng,Shan Bing,Pan Heling,Yuan Junying Proceedings of the National Academy of Sciences of the United States of America Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis. 10.1073/pnas.1819728116
    Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Mancias Joseph D,Wang Xiaoxu,Gygi Steven P,Harper J Wade,Kimmelman Alec C Nature Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo-receptor connectivity. 10.1038/nature13148
    Ferroptosis is a type of autophagy-dependent cell death. Zhou Borong,Liu Jiao,Kang Rui,Klionsky Daniel J,Kroemer Guido,Tang Daolin Seminars in cancer biology Macroautophagy (hereafter referred to as autophagy) involves an intracellular degradation and recycling system that, in a context-dependent manner, can either promote cell survival or accelerate cellular demise. Ferroptosis was originally defined in 2012 as an iron-dependent form of cancer cell death different from apoptosis, necrosis, and autophagy. However, this latter assumption came into question because, in response to ferroptosis activators (e.g., erastin and RSL3), autophagosomes accumulate, and because components of the autophagy machinery (e.g., ATG3, ATG5, ATG4B, ATG7, ATG13, and BECN1) contribute to ferroptotic cell death. In particular, NCOA4-facilitated ferritinophagy, RAB7A-dependent lipophagy, BECN1-mediated system x inhibition, STAT3-induced lysosomal membrane permeabilization, and HSP90-associated chaperone-mediated autophagy can promote ferroptosis. In this review, we summarize current knowledge on the signaling pathways involved in ferroptosis, while focusing on the regulation of autophagy-dependent ferroptotic cell death. The molecular comprehension of these phenomena may lead to the development of novel anticancer therapies. 10.1016/j.semcancer.2019.03.002
    AMPK-Mediated BECN1 Phosphorylation Promotes Ferroptosis by Directly Blocking System X Activity. Song Xinxin,Zhu Shan,Chen Pan,Hou Wen,Wen Qirong,Liu Jiao,Xie Yangchun,Liu Jinbao,Klionsky Daniel J,Kroemer Guido,Lotze Michael T,Zeh Herbert J,Kang Rui,Tang Daolin Current biology : CB Ferroptosis is a form of regulated cell death triggered by lipid peroxidation after inhibition of the cystine/glutamate antiporter system X. However, key regulators of system X activity in ferroptosis remain undefined. Here, we show that BECN1 plays a hitherto unsuspected role in promoting ferroptosis through directly blocking system Xc activity via binding to its core component, SLC7A11 (solute carrier family 7 member 11). Knockdown of BECN1 by shRNA inhibits ferroptosis induced by system X inhibitors (e.g., erastin, sulfasalazine, and sorafenib), but not other ferroptosis inducers including RSL3, FIN56, and buthionine sulfoximine. Mechanistically, AMP-activated protein kinase (AMPK)-mediated phosphorylation of BECN1 at Ser90/93/96 is required for BECN1-SLC7A11 complex formation and lipid peroxidation. Inhibition of PRKAA/AMPKα by siRNA or compound C diminishes erastin-induced BECN1 phosphorylation at S93/96, BECN1-SLC7A11 complex formation, and subsequent ferroptosis. Accordingly, a BECN1 phosphorylation-defective mutant (S90,93,96A) reverses BECN1-induced lipid peroxidation and ferroptosis. Importantly, genetic and pharmacological activation of the BECN1 pathway by overexpression of the protein in tumor cells or by administration of the BECN1 activator peptide Tat-beclin 1, respectively, increases ferroptotic cancer cell death (but not apoptosis and necroptosis) in vitro and in vivo in subcutaneous and orthotopic tumor mouse models. Collectively, our work reveals that BECN1 plays a novel role in lipid peroxidation that could be exploited to improve anticancer therapy by the induction of ferroptosis. 10.1016/j.cub.2018.05.094