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. 2018 Aug 6;28(15):2388-2399.e5.
doi: 10.1016/j.cub.2018.05.094. Epub 2018 Jul 26.

AMPK-Mediated BECN1 Phosphorylation Promotes Ferroptosis by Directly Blocking System Xc- Activity

Xinxin Song  1 , Shan Zhu  2 , Pan Chen  3 , Wen Hou  3 , Qirong Wen  2 , Jiao Liu  2 , Yangchun Xie  4 , Jinbao Liu  2 , Daniel J Klionsky  5 , Guido Kroemer  6 , Michael T Lotze  3 , Herbert J Zeh  3 , Rui Kang  7 , Daolin Tang  8
Affiliations

AMPK-Mediated BECN1 Phosphorylation Promotes Ferroptosis by Directly Blocking System Xc- Activity

Xinxin Song et al. Curr Biol. .

Abstract

Ferroptosis is a form of regulated cell death triggered by lipid peroxidation after inhibition of the cystine/glutamate antiporter system Xc-. However, key regulators of system Xc- 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 Xc- 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.

Keywords: AMPK; BECN1; SLC7A11; autophagy; autosis; cancer therapy; ferroptosis; lipid peroxidation; necroptosis; phosphorylation.

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Conflict of interest statement

Declaration of Interests

The authors declare no conflicts of interest or financial interests.

Figures

Figure 1
Figure 1. BECN1 is required for system Xc− inhibitor-induced ferroptosis
(A) Effects of erastin on the expression of indicated proteins in HCT116 (20 μM), CX-1 (20 μM), PANC1 (10 μM), HT1080 (5 μM), and Calu-1 (10 μM) cells. (B) Western blot analysis of BECN1 expression in BECN1-overexpressing cells. (C) Overexpression of BECN1 increased erastin (20 μM for HCT116 and CX-1 cells; 5 μM for HT1080 cells)-, sulfasalazine (“SAS”, 1 mM)-, and sorafenib (“SOR”, 10 μM)-induced cell death, but not RSL3 (1 μM)-, FIN56 (5 μM)-, and buthionine sulfoximine (“BSO”, 100 μM)-induced cell death at 24, 48, and 72 h (n=3, *, P<0.05 versus control group, t test). (D) Western blot analysis of BECN1 expression in BECN1-knockdown cells. (E) Knockdown of BECN1 inhibited erastin (20 μM for HCT116 and CX-1 cells; 5 μM for HT1080 cells)-, sulfasalazine (“SAS”, 1 mM)-, and sorafenib (“SOR”, 10 μM)-induced cell death, but not RSL3 (1 μM)-, FIN56 (5 μM)- and buthionine sulfoximine (“BSO”, 100 μM)-induced cell death at 24, 48, and 72 h (n=3, *, P<0.05 versus control group, t test). (F) Western blot analysis of BECN2 expression in BECN2-knockdown HeLa cells. (G) Indicated HeLa cells were treated with erastin (20 μM), sulfasalazine (“SAS”, 1 mM), and sorafenib (“SOR”, 10 μM) for 24 h and cell viability were assayed. See also Figures S1 and S2.
Figure 2
Figure 2. BECN1 promotes GSH depletion and lipid peroxidation in ferroptosis
(A) Indicated HCT116 and CX-1 cells were treated with erastin (20 μM) for 24 h. The protein levels of MAP1LC3B were assayed. (B) The indicated HCT116 and CX-1 cells were treated with HBSS for 6 h. The protein levels of MAP1LC3B were assayed. (C) The indicated HCT116 cells were treated with erastin (20 μM) or sulfasalazine (“SAS”, 1 mM) for 24 h. The relative levels of C11-BODIPY, MDA, Fe2+, and GSH were assayed (n=3, *, P<0.05 versus control group, t test). (D) Intracellular chelatable iron in indicated HT1080 cells treated with or without erastin (5 μM, 24 h) was determined using the fluorescent indicator Phen Green SK (green). (E) Q-PCR analysis of gene expression in indicated HT1080 cells treated with or without erastin (5 μM, 24 h). RU=relative units. (F) Administration of GSH (5 mM) restored intracellular GSH level and inhibited erastin (20 μM)- or sulfasalazine (“SAS”, 1 mM)-induced cell death at 24–72 h in wild-type and BECN1-overexpressing HCT116 cells. See also Figure S3.
Figure 3
Figure 3. BECN1 inhibits system Xc− activity through direct binding to SLC7A11
(A) Analysis of glutamate release from the indicated cells in response to erastin (20 μM) for 24 h (n=3, *, P<0.05 versus control group, t test). (B) IP analysis of BECN1-SLC7A11 formation in the indicated cells following erastin (20 μM) treatment for 24 h. (C) IP analysis of BECN1-SLC7A11 formation in the indicated cells following erastin (20 μM) treatment for 24 h. (D) IP analysis of BECN1-SLC7A11 formation in the indicated cells following erastin (10 μM for PANC1 cells; 5 μM for HT1080 cells; 10 μM for Calu-1 cells) or sulfasalazine (“SAS”, 1 mM) treatment for 24 h. (E) Image assay showed that the colocalization between BECN1 (green) and SLC7A11 (red) in cytoplasm in CX-1 cells following erastin (20 μM) treatment for 24 h. Nucleus were stain by DAPI (blue). (F) IP analysis of BECN1-SLC7A11 formation in HCT116 cells following erastin (20 μM) treatment with or without Tat-beclin 1 (10 μM) for 24 h. (G) Analysis of glutamate release from HCT116 cells in response to erastin (20 μM) or sulfasalazine (“SAS”, 1 mM) with or without Tat-beclin 1 (10 μM) and control peptides (10 μM) for 24–72 h (n=3, *, P<0.05, ANOVA test). (H) Analysis of cell viability from HCT116 cells in response to erastin (20 μM) or sulfasalazine (“SAS”, 1 mM) with or without Tat-beclin 1 (10 μM) control peptides (10 μM) for 24–72 h (n=3, *, P<0.05, ANOVA test). See also Figure S4.
Figure 4
Figure 4. BECN1 phosphorylation at S90/93/96 contributes to BECN1-SLC7A11 complex formation
(A) IP analysis of BECN1-SLC7A11 formation in HEK293T cells expressing HA-SLC7A11-cDNA and the indicated FLAG-BECN1 mutants following erastin (20 μM) treatment for 24 h. (B) The levels of cell viability, MDA, GSH, and Fe2+ were assayed in HEK293T cells expressing the indicated FLAG-BECN1 mutants following erastin (20 μM) or sulfasalazine (“SAS”, 1 mM) treatment for 24 h (n=3, *, P<0.05 versus control group, t test). (C) IP analysis of BECN1-SLC7A11 formation in HEK293T cells expressing HA-SLC7A11-cDNA and the indicated FLAG-BECN1 mutants following erastin (20 μM) treatment for 24 h. (D) The levels of cell viability, MDA, GSH, and Fe2+ were assayed in HEK293T cells expressing the indicated BECN1 mutants following erastin (20 μM) or sulfasalazine (“SAS”, 1 mM) treatment for 24 h (n=3, *, P<0.05 versus control group, t test).
Figure 5
Figure 5. AMPK is required for BECN1 phosphorylation in ferroptosis
(A) Western blot analysis of phosphorylation of PRKAA/AMPKα and BECN1 in HCT116 and CX-1 cells following erastin (20 μM) treatment for 3 to 24 h. (B) Knockdown of PRKAA/AMPKα inhibited erastin (20 μM, 24 h)-induced BECN1 phosphorylation in HCT116 and CX-1 cells. (C) Compound C (Comp C, 1 μM) inhibited erastin (20 μM, 24 h)-induced BECN1 phosphorylation in HCT116 and CX-1 cells. (D) Knockdown of PRKAA/AMPKα inhibited erastin (20 μM, 24 h)-induced BECN1-SLC7A11 complex formation in HCT116 cells. (E) Compound C (1 μM) inhibited erastin (20 μM, 24 h)-induced BECN1-SLC7A11 complex formation in HCT116 cells. (F) Analysis of the levels of cell viability, MDA, GSH, and Fe2+ in the indicated HCT116 cells following erastin (20 μM) or sulfasalazine (SAS, 1 mM) treatment for 24 h (n=3, *, P<0.05 versus control group, t test). (G) Analysis of the levels of cell viability, MDA, GSH, and Fe2+ in HCT116 cells following erastin (20 μM) or sulfasalazine (1 mM) treatment with or without compound C (1 μM) for 24 h (n=3, *, P<0.05 versus erastin or SAS group, t test). See also Figure S5.
Figure 6
Figure 6. BECN1 contributes to the anticancer activity of erastin in vivo
(A) Athymic nude mice were injected subcutaneously with the indicated HCT116 cells and treated with erastin (40 mg/kg/intraperitoneal injection, once every day) at day 7 for two weeks. Tumor volume was calculated weekly (n=5 mice/group, * p < 0.05, ANOVA LSD test). (B, C) Representative photographs of tumor-bearing mice and isolated tumors at day 14 after treatment. (D) In parallel, MDA, GSH, and Fe2+ levels in isolated tumors at day 14 after treatment were assayed (n=5 mice/group). (E) Western blot analysis of cleaved-CASP3 and p-MLKL in isolated tumors at day 14 after treatment from (A). (F) Athymic nude mice were injected subcutaneously with HCT116 or HT1080 cells for seven days and then treated with the indicated erastin (40 mg/kg/intraperitoneal [i.p.] injection, once every day), Tat-beclin 1 (20 mg/kg/i.p. injection, once every day), or liprostatin-1 (10 mg/kg/i.p. injection, once every day) at day 7 for two weeks. Tumor volume was calculated weekly (n=5 mice/group, * p < 0.05, ANOVA test). (G) Western blot analysis of cleaved-CASP3 and p-MLKL in isolated tumors at day 14 after treatment from (F). (H) KPC cells were surgically implanted into the pancreases in B6 mice for seven days and then treated with erastin (40 mg/kg/i.p. injection, once every day), Tat-beclin 1 (20 mg/kg/i.p. injection, once every day), or liprostatin-1 (10 mg/kg/i.p. injection, once every day) at day 7 for two weeks. Animal survival was calculated weekly (n=10 mice/group, Kaplan-Meier survival analysis, * p < 0.05). See also Figure S6.
See this image and copyright information in PMC

Comment in

  • BECN1 is a new driver of ferroptosis.
    Kang R, Zhu S, Zeh HJ, Klionsky DJ, Tang D. Kang R, et al. Autophagy. 2018;14(12):2173-2175. doi: 10.1080/15548627.2018.1513758. Epub 2018 Sep 6. Autophagy. 2018. PMID: 30145930 Free PMC article.

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