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Journal of Experimental & Clinical Cancer Research

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01-12-2021 | Breast Cancer | Research

Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer

Authors: Jingjing Yang, Yulu Zhou, Shuduo Xie, Ji Wang, Zhaoqing Li, Lini Chen, Misha Mao, Cong Chen, Aihua Huang, Yongxia Chen, Xun Zhang, Noor Ul Hassan Khan, Linbo Wang, Jichun Zhou

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2021

Abstract

Background

Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid peroxidation and is involved in various pathophysiological conditions, including cancer. Targeting ferroptosis is considered to be a novel anti-cancer strategy. The identification of FDA-approved drugs as ferroptosis inducers is proposed to be a new promising approach for cancer treatment. Despite a growing body of evidence indicating the potential efficacy of the anti-diabetic metformin as an anti-cancer agent, the exact mechanism underlying this efficacy has not yet been fully elucidated.

Methods

The UFMylation of SLC7A11 is detected by immunoprecipitation and the expression of UFM1 and SLC7A11 in tumor tissues was detected by immunohistochemical staining. The level of ferroptosis is determined by the level of free iron, total/lipid Ros and GSH in the cells and the morphological changes of mitochondria are observed by transmission electron microscope. The mechanism in vivo was verified by in situ implantation tumor model in nude mice.

Results

Metformin induces ferroptosis in an AMPK-independent manner to suppress tumor growth. Mechanistically, we demonstrate that metformin increases the intracellular Fe²⁺ and lipid ROS levels. Specifically, metformin reduces the protein stability of SLC7A11, which is a critical ferroptosis regulator, by inhibiting its UFMylation process. Furthermore, metformin combined with sulfasalazine, the system x_c⁻ inhibitor, can work in a synergistic manner to induce ferroptosis and inhibit the proliferation of breast cancer cells.

Conclusions

This study is the first to demonstrate that the ability of metformin to induce ferroptosis may be a novel mechanism underlying its anti-cancer effect. In addition, we identified SLC7A11 as a new UFMylation substrate and found that targeting the UFM1/SLC7A11 pathway could be a promising cancer treatment strategy.

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Pernicova I, Korbonits M. Metformin-mode of action and clinical implications for diabetes and cancer. Nat Rev Endocrinol. 2014;10(3):143–56. https://doi.org/10.1038/nrendo.2013.256.CrossRefPubMed

Barzilai N, Crandall JP, Kritchevsky SB, Espeland MA. Metformin as a tool to target aging. Cell Metab. 2016;23(6):1060–5. https://doi.org/10.1016/j.cmet.2016.05.011.CrossRefPubMedPubMedCentral

Soberanes S, Misharin AV, Jairaman A, Morales-Nebreda L, McQuattie-Pimentel AC, Cho T, et al. Metformin targets mitochondrial electron transport to reduce air-pollution-induced thrombosis. Cell Metabol. 2019;29(2):335–347.e5.CrossRef

Zhang CS, Li M, Ma T, Zong Y, Cui J, Feng JW, et al. Metformin activates AMPK through the lysosomal pathway. Cell Metab. 2016;24(4):521–2. https://doi.org/10.1016/j.cmet.2016.09.003.CrossRefPubMed

Stynen B, Abd-Rabbo D, Kowarzyk J, Miller-Fleming L, Aulakh SK, Garneau P, et al. Changes of cell biochemical states are revealed in protein homomeric complex dynamics. Cell. 2018;175(5):1418–29. https://doi.org/10.1016/j.cell.2018.09.050.CrossRefPubMedPubMedCentral

Queiroz EAIF, Puukila S, Eichler R, Sampaio SC, Forsyth HL, Lees SJ, et al. Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells. PLoS ONE. 2014;9(5):e98207.CrossRefPubMedPubMedCentral

Xiao Z, Gaertner S, Morresi-Hauf A, Genzel R, Duell T, Ullrich A, et al. Metformin triggers autophagy to attenuate drug-induced apoptosis in NSCLC cells, with minor effects on tumors of diabetic patients. Neoplasia. 2017;19(5):385–95. https://doi.org/10.1016/j.neo.2017.02.011.CrossRefPubMedPubMedCentral

Hassannia B, Vandenabeele P, Vanden BT. Targeting Ferroptosis to Iron out Cancer. Cancer Cell. 2019;35(6):830–49. https://doi.org/10.1016/j.ccell.2019.04.002.CrossRefPubMed

Xie Y, Hou W, Song X, Yu Y, Huang J, Sun X, et al. Ferroptosis: process and function. Cell Death Differ. 2016;23(3):369–79. https://doi.org/10.1038/cdd.2015.158.CrossRefPubMedPubMedCentral

10.

Mancias JD, Wang X, Gygi SP, Harper JW, Kimmelman AC. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature. 2014;509(7498):105–9. https://doi.org/10.1038/nature13148.CrossRefPubMedPubMedCentral

11.

Richardson DR, Lane DJ, Becker EM, Huang ML, Whitnall M, Rahmanto YS, et al. Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol. Proc Natl Acad Sci U S A. 2010;107(24):10775–82. https://doi.org/10.1073/pnas.0912925107.CrossRefPubMedPubMedCentral

12.

Lill R, Srinivasan V, Mühlenhoff U. The role of mitochondria in cytosolic-nuclear iron-sulfur protein biogenesis and in cellular iron regulation. Curr Opin Microbiol. 2014;22(9):111–9. https://doi.org/10.1016/j.mib.2014.09.015.CrossRefPubMed

13.

Yang WS, Stockwell BR. Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 2016;26(3):165–76. https://doi.org/10.1016/j.tcb.2015.10.014.CrossRefPubMed

14.

Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell. 2017;171(2):273–85 2017/10/07.CrossRefPubMedPubMedCentral

15.

Conrad M, Kagan VE, Bayir H, Pagnussat GC, Head B, Traber MG, et al. Regulation of lipid peroxidation and ferroptosis in diverse species. Genes Dev. 2018;32(9–10):602–19. https://doi.org/10.1101/gad.314674.118.CrossRefPubMedPubMedCentral

16.

Koppula P, Zhang Y, Zhuang L, Gan B. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer. Cancer Commun. 2018;38(1):12.CrossRef

17.

Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–72. https://doi.org/10.1016/j.cell.2012.03.042.CrossRefPubMedPubMedCentral

18.

Gout PW, Buckley AR, Simms CR, Bruchovsky N. Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the xc? cystine transporter: a new action for an old drug. Leukemia. 2001;15(10):1633–40.CrossRefPubMed

19.

Lei G, Zhang Y, Koppula P, Liu X, Zhang J, Lin SH, et al. The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression. Cell Res. 2020;30(2):146–62. https://doi.org/10.1038/s41422-019-0263-3.CrossRefPubMedPubMedCentral

20.

Komatsu M, Chiba T, Tatsumi K, Lemura SI, Tanida I, Okazaki N, et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J. 2004;23(9):1977–86. https://doi.org/10.1038/sj.emboj.7600205.CrossRefPubMedPubMedCentral

21.

Daniel J, Liebau E. The Ufm1 Cascade. Cells. 2014;3(2):627–38. https://doi.org/10.3390/cells3020627.CrossRefPubMedPubMedCentral

22.

Tatsumi K, Sou YS, Tada N, Nakamura E, Iemura SI, Natsume T, et al. A novel type of E3 ligase for the Ufm1 conjugation system. J Biol Chem. 2010;285(8):5417–27. https://doi.org/10.1074/jbc.M109.036814.CrossRefPubMed

23.

Tatsumi K, Yamamoto-Mukai H, Shimizu R, Waguri S, Sou YS, Sakamoto A, et al. The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice. Nat Commun. 2011;2(1):181. https://doi.org/10.1038/ncomms1182.CrossRefPubMed

24.

Yoo HM, Kang SH, Kim JY, Lee JE, Seong MW, Lee SW, et al. Modification of ASC1 by UFM1 is crucial for ERα transactivation and breast Cancer development. Mol Cell. 2014;56(2):261–74. https://doi.org/10.1016/j.molcel.2014.08.007.CrossRefPubMed

25.

Cai Y, Pi W, Sivaprakasam S, Zhu X, Zhang M, Chen J, et al. UFBP1, a key component of the Ufm1 conjugation system, is essential for Ufmylation-mediated regulation of erythroid development. PLoS Genet. 2015;11(11):e1005643. https://doi.org/10.1371/journal.pgen.1005643.CrossRefPubMedPubMedCentral

26.

Lemaire K, Rodrigo M, Granvik M, Hohmeier H, Hendrickx N, Newgard C, et al. New players in the beta cell ER stress response: UFM1 and UFBP1. Diabetologia. 2010;53(9):S211.

27.

Jiang L, Ning K, Li T, Wang SJ, Su T, Hibshoosh H, et al. Ferroptosis as a p53-mediated activity during tumour suppression. Nature. 2015;520(7545):57–62. https://doi.org/10.1038/nature14344.CrossRefPubMedPubMedCentral

28.

Zhang Y, Shi J, Liu X, Feng L, Gong Z, Koppula P, et al. BAP1 links metabolic regulation of ferroptosis to tumour suppression. Nat Cell Biol. 2018;20(10):1181–92. https://doi.org/10.1038/s41556-018-0178-0.CrossRefPubMedPubMedCentral

29.

Fan Z, Wirth AK, Chen D, Wruck CJ, Rauh M, Buchfelder M, et al. Nrf2-keap1 pathway promotes cell proliferation and diminishes ferroptosis. Oncogenesis. 2017;6(8):e371. https://doi.org/10.1038/oncsis.2017.65.CrossRefPubMedPubMedCentral

30.

Mukhopadhyay S, Goswami D, Adiseshaiah PP, Burgan W, Yi M, Guerin TM, et al. Undermining glutaminolysis bolsters chemotherapy while NRF2 promotes chemoresistance in KRAS-driven pancreatic cancers. Cancer Res. 2020;80(8):1630–43. https://doi.org/10.1158/0008-5472.CAN-19-1363.CrossRefPubMedPubMedCentral

31.

Mukhopadhyay S, Heiden MG, Mccormick F. The metabolic landscape of RAS-driven cancers from biology to therapy. Nat Cancer. 2021;2(3):271–83. https://doi.org/10.1038/s43018-021-00184-x.CrossRefPubMedPubMedCentral

32.

Li P, Zhao M, Parris AB, Feng X, Yang X. P53 is required for metformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. Biochem Biophys Res Commun. 2015;464(4):1267–74. https://doi.org/10.1016/j.bbrc.2015.07.117.CrossRefPubMed

33.

Urpilainen E, Kangaskokko J, Puistola U, Karihtala P. Metformin diminishes the unfavourable impact of Nrf2 in breast cancer patients with type 2 diabetes. Tumor Biol. 2019;41(1):1010428318815413.CrossRef

34.

Schulten HJ. Pleiotropic effects of metformin on cancer. Int J Mol Sci. 2018;19(10):2850.CrossRefPubMedCentral

35.

Fan C, Wang Y, Liu Z, Sun Y, Wang X, Wei G, et al. Metformin exerts anticancer effects through the inhibition of the sonic hedgehog signaling pathway in breast cancer. Int J Mol Med. 2015;36(1):204–14. https://doi.org/10.3892/ijmm.2015.2217.CrossRefPubMedPubMedCentral

36.

Rothman RJ, Serroni A, Farber JL. Cellular pool of transient ferric iron, chelatable by deferoxamine and distinct from ferritin, that is involved in oxidative cell injury. Mol Pharmacol. 1992;42(4):703–10.PubMed

37.

Stockwell BR, Jiang X, Gu W. Emerging mechanisms and disease relevance of Ferroptosis. Trends Cell Biol. 2020;30(6):478–90. https://doi.org/10.1016/j.tcb.2020.02.009.CrossRefPubMedPubMedCentral

38.

Chen L, Hambright WS, Na R, Ran Q. Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. J Biol Chem. 2015;290(47):28097–106. https://doi.org/10.1074/jbc.M115.680090.CrossRefPubMedPubMedCentral

39.

Yang WS, Sriramaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, et al. Regulation of Ferroptotic Cancer cell death by GPX4. Cell. 2014;156(1–2):317–31. https://doi.org/10.1016/j.cell.2013.12.010.CrossRefPubMedPubMedCentral

40.

Angeli JPF, Shah R, Pratt DA, Conrad M. Ferroptosis inhibition: mechanisms and opportunities. Trends Pharmacol Sci. 2017;38(5):489–98. https://doi.org/10.1016/j.tips.2017.02.005.CrossRefPubMed

41.

Song X, Zhu S, Chen P, Hou W, Wen Q, Liu J, et al. AMPK-Mediated BECN1 Phosphorylation Promotes Ferroptosis by Directly Blocking System Xc– Activity. Current Biol. 2018;28:2388–99. https://doi.org/10.1016/j.cub.2018.05.094.

42.

Cao JY, Dixon SJ. Mechanisms of ferroptosis. Cell Mol Life Sci. 2016;73(11–12):2195–209. https://doi.org/10.1007/s00018-016-2194-1.CrossRefPubMedPubMedCentral

43.

Ota S, Horigome K, Ishii T, Nakai M, Hayashi K, Kawamura T, et al. Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism. Biochem Biophys Res Commun. 2009;388(2):311–6. https://doi.org/10.1016/j.bbrc.2009.07.164.CrossRefPubMed

44.

Zhong T, Men Y, Lu L, Geng T, Zhou J, Mitsuhashi A, et al. Metformin alters DNA methylation genome-wide via the H19/SAHH axis. Oncogene. 2017;36(17):2345–54. https://doi.org/10.1038/onc.2016.391.CrossRefPubMed

45.

Zhou J, Yang L, Zhong T, Mueller M, Men Y, Zhang N, et al. H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase. Nat Commun. 2015;6(1):10221. https://doi.org/10.1038/ncomms10221.CrossRefPubMed

46.

Doll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature. 2019;575(7784):693–8. https://doi.org/10.1038/s41586-019-1707-0.CrossRefPubMed

47.

Lee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, et al. Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol. 2020;22(2):225–34. https://doi.org/10.1038/s41556-020-0461-8.CrossRefPubMedPubMedCentral

48.

Gao M, Yi J, Zhu J, Minikes AM, Monian P, Thompson CB, et al. Role of Mitochondria in Ferroptosis. Mol Cell. 2019;73(2):354–363.e3.CrossRefPubMed

49.

Mukhopadhyay S, Chatterjee A, Kogan D, Patel D, Foster DA. 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) enhances the efficacy of rapamycin in human cancer cells. Cell Cycle. 2015;14(20):3331–9. https://doi.org/10.1080/15384101.2015.1087623.CrossRefPubMedPubMedCentral

50.

Gao M, Monian P, Pan Q, Zhang W, Xiang J, Jiang X. Ferroptosis is an autophagic cell death process. Cell Res. 2016;26(9):1021–32 2016/08/16.CrossRefPubMedPubMedCentral

51.

Li Y, Wang X, Yan J, Liu Y, Yang R, Pan D, et al. Nanoparticle ferritin-bound erastin and rapamycin: a nanodrug combining autophagy and ferroptosis for anticancer therapy. Biomater Sci. 2019;7(9):3779–87. https://doi.org/10.1039/C9BM00653B.CrossRefPubMed

52.

Mukhopadhyay S, Saqcena M, Chatterjee A, Garcia A, Frias MA, Foster DA. Reciprocal regulation of AMP-activated protein kinase and phospholipase D. J Biol Chem. 2015;290(11):6986–93. https://doi.org/10.1074/jbc.M114.622571.CrossRefPubMedPubMedCentral

53.

Mukhopadhyay S, Saqcena M, Foster DA. Synthetic lethality in KRas-driven cancer cells created by glutamine deprivation. Oncoscience. 2015;2(10):807–8. https://doi.org/10.18632/oncoscience.253.CrossRefPubMedPubMedCentral

54.

Zheng J, Conrad M. The metabolic underpinnings of Ferroptosis. Cell Metab. 2020;32(6):920–37. https://doi.org/10.1016/j.cmet.2020.10.011.CrossRefPubMed

55.

Zhou B, Liu J, Kang R, Klionsky DJ, Kroemer G, Tang D. Ferroptosis is a type of autophagy-dependent cell death. Semin Cancer Biol. 2020;66:89–100. https://doi.org/10.1016/j.semcancer.2019.03.002.CrossRefPubMed

56.

Stoyanovsky DA, Tyurina YY, Shrivastava I, Bahar I, Tyurin VA, Protchenko O, et al. Iron catalysis of lipid peroxidation in ferroptosis: regulated enzymatic or random free radical reaction? Free Radic Biol Med. 2019;133:153–61. https://doi.org/10.1016/j.freeradbiomed.2018.09.008.CrossRefPubMed

57.

Asensio-López MC, Sánchez-Más J, Pascual-Figal DA, Abenza S, Pérez-Martínez MT, Valdés M, et al. Involvement of ferritin heavy chain in the preventive effect of metformin against doxorubicin-induced cardiotoxicity. Free Radic Biol Med. 2013;57:188–200. https://doi.org/10.1016/j.freeradbiomed.2012.09.009.CrossRefPubMed

58.

Lane DJR, Merlot AM, Huang MLH, Bae DH, Jansson PJ, Sahni S, et al. Cellular iron uptake, trafficking and metabolism: key molecules and mechanisms and their roles in disease. Biochim et Biophys Acta Mol Cell Res. 2015;1853(5):1130–44. https://doi.org/10.1016/j.bbamcr.2015.01.021.CrossRef

59.

Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F, et al. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Res. 2007;67(14):6745–52. https://doi.org/10.1158/0008-5472.CAN-06-4447.CrossRefPubMed

60.

Hou W, Xie Y, Song X, Sun X, Lotze MT, Zeh HJ 3rd, et al. Autophagy promotes ferroptosis by degradation of ferritin. Autophagy. 2016;12(8):1425–8. https://doi.org/10.1080/15548627.2016.1187366.CrossRefPubMedPubMedCentral

61.

Jiralerspong S, Palla SL, Giordano SH, Meric-Bernstam F, Liedtke C, Barnett CM, et al. Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. J Clin Oncol. 2009;27(20):3297–302. https://doi.org/10.1200/JCO.2009.19.6410.CrossRefPubMedPubMedCentral

62.

Sonnenblick A, Agbor-Tarh D, Bradbury I, Di Cosimo S, Azim HA, Fumagalli D, et al. Impact of diabetes, insulin, and metformin use on the outcome of patients with human epidermal growth factor receptor 2–positive primary breast Cancer: analysis from the ALTTO phase III randomized trial. J Clin Oncol. 2017;35(13):1421–9. https://doi.org/10.1200/JCO.2016.69.7722.CrossRefPubMedPubMedCentral

63.

Nanni O, Amadori D, De Censi A, Rocca A, Freschi A, Bologna A, et al. Metformin plus chemotherapy versus chemotherapy alone in the first-line treatment of HER2-negative metastatic breast cancer. The MYME randomized, phase 2 clinical trial. Breast Cancer Res Treat. 2019;174(2):433–42. https://doi.org/10.1007/s10549-018-05070-2.CrossRefPubMed

64.

Chen RS, Song YM, Zhou ZY, Tong T, Li Y, Fu M, et al. Disruption of xCT inhibits cancer cell metastasis via the caveolin-1/β-catenin pathway. Oncogene. 2009;28(4):599–609. https://doi.org/10.1038/onc.2008.414.CrossRefPubMed

65.

Lee J, Yesilkanal AE, Wynne JP, Frankenberger C, Liu J, Yan J, et al. Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism. Nature. 2019;78(13 Supplement):5497.

Title: Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer
Authors: Jingjing Yang
Yulu Zhou
Shuduo Xie
Ji Wang
Zhaoqing Li
Lini Chen
Misha Mao
Cong Chen
Aihua Huang
Yongxia Chen
Xun Zhang
Noor Ul Hassan Khan
Linbo Wang
Jichun Zhou
Publication date: 01-12-2021
Publisher: BioMed Central
Keywords: Breast Cancer
Metformin
Breast Cancer
Sulfasalazine
Published in: Journal of Experimental & Clinical Cancer Research / Issue 1/2021
Electronic ISSN: 1756-9966
DOI: https://doi.org/10.1186/s13046-021-02012-7

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