Endoplasmic reticulum membrane remodeling by targeting reticulon-4 induces pyroptosis to facilitate antitumor immune

Abstract

Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the “bubble” structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to pyruvate kinase M2 (PKM2)-dependent conventional caspase-3/gasdermin E (GSDME) cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knock-down significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-programmed death-1 (anti-PD-1). In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.

Keywords

pyroptosis / ER membrane / antitumor immune / osteosarcoma / chemical degrader / reticulon-4 (RTN4)

References

[1] Adachi S, Nagao T, Ingolfsson HI et al. The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in HT29 colon cancer cells. Cancer Res 2007;67:6493–6501.

[2] Breeze E, Dzimitrowicz N, Kriechbaumer V et al. A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon. Proc Natl Acad Sci U S A 2016;113:10902–10907.

[3] Carlson SM, White FM. Using small molecules and chemical genetics to interrogate signaling networks. ACS Chem Biol 2011;6:75–85.

[4] Carter RJ, Milani M, Beckett AJ et al. Novel roles of RTN4 and CLIMP-63 in regulating mitochondrial structure, bioenergetics and apoptosis. Cell Death Dis 2022;13:436.

[5] Chen X, He WT, Hu L et al. Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis. Cell Res 2016;26:1007–1020.

[6] Chen G, Li Y, Wang W et al. Bioactivity and pharmacological properties of α-mangostin from the mangosteen fruit: a review. Expert Opin Ther Pat 2018;28:415–427.

[7] Chen X, Guo X, Ge Q et al. ER stress activates the NLRP3 inflammasome: a novel mechanism of atherosclerosis. Oxid Med Cell Longev 2019;2019:3462530.

[8] Collins RN. How the ER stays in shape. Cell 2006;124:464–466.

[9] Diekmann H, Klinger M, Oertle T et al. Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish. Mol Biol Evol 2005;22:1635–1648.

[10] Gangadhar NM, Stockwell BR. Chemical genetic approaches to probing cell death. Curr Opin Chem Biol 2007;11:83–87.

[11] Hetz C, Papa FR. The unfolded protein response and cell fate control. Mol Cell 2018;69:169–181.

[12] Hu L, Chen M, Chen X et al. Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate. Cell Death Dis 2020;11:281.

[13] Joshi AS, Huang X, Choudhary V et al. A family of membrane-shaping proteins at ER subdomains regulates pre-peroxisomal vesicle biogenesis. J Cell Biol 2016;215:515–529.

[14] Kamoshima Y, Terasaka S, Kuroda S et al. Morphological and histological changes of glioma cells immediately after 5-aminolevulinic acid mediated photodynamic therapy. Neurol Res 2011;33:739–746.

[15] Kayagaki N, Kornfeld OS, Lee BL et al. NINJ1 mediates plasma membrane rupture during lytic cell death. Nature 2021;591:131–136.

[16] Ke R, Wang Y, Hong S et al. Endoplasmic reticulum stress related factor IRe1α regulates TXNIP/NLRP3-mediated pyroptosis in diabetic nephropathy. Exp Cell Res 2020;396:112293.

[17] Kim HJ, Fei X, Cho SC et al. Discovery of α-mangostin as a novel competitive inhibitor against mutant isocitrate dehydrogenase-1. Bioorg Med Chem Lett 2015;25:5625–5631.

[18] Kiseleva E, Morozova KN, Voeltz GK et al. Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth. J Struct Biol 2007;160:224–235.

[19] Kuang E, Wan Q, Li X et al. ER stress triggers apoptosis induced by Nogo-B/ASY overexpression. Exp Cell Res 2006;312:1983–1988.

[20] Kumar D, Lak B, Suntio T et al. RTN4B interacting protein FAM134C promotes ER membrane curvature and has a functional role in autophagy. Mol Biol Cell 2021;32:1158–1170.

[21] Lebeaupin C, Proics E, de Bieville CH et al. ER stress induces NLRP3 inflammasome activation and hepatocyte death. Cell Death Dis 2015;6:1879.

[22] Li W, Deng M, Loughran PA et al. LPS induces active HMGB1 release from hepatocytes into exosomes through the coordinated activities of TLR4 and caspase-11/GSDMD signaling. Front Immunol 2020;11:229.

[23] Li L, Song D, Qi L et al. Photodynamic therapy induces human esophageal carcinoma cell pyroptosis by targeting the PKM2/caspase-8/caspase-3/GSDME axis. Cancer Lett 2021a;520:143–159.

[24] Li M, Lu H, Wang X et al. Pyruvate kinase M2 (PKM2) interacts with activating transcription factor 2 (ATF2) to bridge glycolysis and pyroptosis in microglia. Mol Immunol 2021b;140:250–266.

[25] Lin JH, Li H, Yasumura D et al. IRE1 signaling affects cell fate during the unfolded protein response. Science 2007;318:944–949.

[26] Liu J, Zhong Y, Liu H et al. Oncostatin M sensitizes keratinocytes to UVB-induced inflammation via GSDME-mediated pyroptosis. J Dermatol Sci 2021;104:95–103.

[27] Lorenzo AD, Manes TD, Davalos A et al. Endothelial reticulon-4B (Nogo-B) regulates ICAM-1-mediated leukocyte transmigration and acute inflammation. Blood 2011;117:2284–2295.

[28] Loveless R, Bloomquist R, Teng Y. Pyroptosis at the forefront of anticancer immunity. J Exp Clin Cancer Res 2021;40:264.

[29] Mamun AA, Shao C, Geng P et al. Pyroptosis in diabetic peripheral neuropathy and its therapeutic regulation. J Inflamm Res 2024;17:3839–3864.

[30] Oertle T, Merkler D, Schwab ME. Do cancer cells die because of Nogo-B? Oncogene 2003;22:1390–1399.

[31] Puhka M, Joensuu M, Vihinen H et al. Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells. Mol Biol Cell 2012;23:2424–2432.

[32] Rühl S, Shkarina K, Demarco B et al. ESCRT-dependent membrane repair negatively regulates pyroptosis down-stream of GSDMD activation. Science 2018;362:956–960.

[33] Shibata Y, Voeltz GK, Rapoport TA. Rough sheets and smooth tubules. Cell 2006;126:435–439.

[34] Shibata Y, Shemesh T, Prinz WA et al. Mechanisms determining the morphology of the peripheral ER. Cell 2010;143:774–788.

[35] Tall AR, Westerterp M. Inflammasomes, neutrophil extracellular traps, and cholesterol. J Lipid Res 2019;60:721–727.

[36] Voeltz GK, Prinz WA, Shibata Y et al. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 2006;124:573–586.

[37] Wang Y, Gao W, Shi X et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 2017;547:99–103.

[38] Wang W, Hu D, Wu C et al. STING promotes NLRP3 localization in ER and facilitates NLRP3 deubiquitination to activate the inflammasome upon HSV-1 infection. PLoS Pathog 2020;16:e1008335.

[39] Wang S, Liu Y, Zhang L et al. Methods for monitoring cancer cell pyroptosis. Cancer Biol Med 2021;19:398–414.

[40] Westrate LM, Lee JE, Prinz WA et al. Form follows function: the importance of endoplasmic reticulum shape. Annu Rev Biochem 2015;84:791–811.

[41] Wu H, Voeltz GK. Reticulon-3 promotes endosome maturation at ER membrane contact sites. Dev Cell 2021;56:52–66.e7.

[42] Xiao P, Gu J, Xu W et al. RTN4/Nogo-A-S1PR2 negatively regulates angiogenesis and secondary neural repair through enhancing vascular autophagy in the thalamus after cerebral cortical infarction. Autophagy 2022;18:2711–2730.

[43] Yu LM, Zhang WH, Han XX et al. Hypoxia-induced ROS contribute to myoblast pyroptosis during obstructive sleep apnea via the NF-κB/HIF-1α signaling pathway. Oxid Med Cell Longev 2019;2019:4596368.

[44] Yu P, Zhang X, Liu N et al. Pyroptosis: mechanisms and diseases. Signal Transduct Target Ther 2021;6:128.

[45] Zeng X, Zhu M, Liu X et al. Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis. Nutr Metab (Lond) 2020;17:11.

[46] Zhang R, Kang R, Tang D. The STING1 network regulates autophagy and cell death. Signal Transduct Target Ther 2021;6:208.

[47] Zheng Z, Yang S, Dai W et al. The role of pyroptosis in metabolism and metabolic disease. Biomed Pharmacother 2024;176:116863.

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