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. 2018 Nov 9;9(1):4728.
doi: 10.1038/s41467-018-07188-9.

The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance

Fei Han  1   2 , Chien-Feng Li  3   4 , Zhen Cai  1   2 , Xian Zhang  1   2 , Guoxiang Jin  1   2 , Wei-Na Zhang  1 , Chuan Xu  1 , Chi-Yun Wang  1   2 , John Morrow  5 , Shuxing Zhang  5 , Dazhi Xu  6   7 , Guihua Wang  8 , Hui-Kuan Lin  9   10   11   12
Affiliations

The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance

Fei Han et al. Nat Commun. .

Abstract

PI3K/Akt signaling is activated in cancers and governs tumor initiation and progression, but how Akt is activated under diverse stresses is poorly understood. Here we identify AMPK as an essential regulator for Akt activation by various stresses. Surprisingly, AMPK is also activated by growth factor EGF through Ca2+/Calmodulin-dependent kinase and is essential for EGF-mediated Akt activation and biological functions. AMPK phosphorylates Skp2 at S256 and promotes the integrity and E3 ligase activity of Skp2 SCF complex leading to K63-linked ubiquitination and activation of Akt and subsequent oncogenic processes. Importantly, AMPK-mediated Skp2 S256 phosphorylation promotes breast cancer progression in mouse tumor models, correlates with Akt and AMPK activation in breast cancer patients, and predicts poor survival outcomes. Finally, targeting AMPK-mediated Skp2 S256 phosphorylation sensitizes cells to anti-EGF receptor targeted therapy. Our study sheds light on how stress and EGF induce Akt activation and new mechanisms for AMPK-mediated oncogenesis and drug resistance.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
AMPK is required for Akt phosphorylation and activation under various cellular stresses. a Screening of kinases in the hypoxia-induced Akt phosphorylation using kinase inhibitor library. Immunoblotting of HEK293 cells pretreated with indicated inhibitors at 10 μΜ for 2 h and challenged with 1% O2 for another 4 h. b Immunoblotting of control (shLuc) and AMPKα1 knockdown MDA-MB-231 cells (#1, #2) treated with 1% O2 for 4 h. c–e Immunoblotting of WT and AMPKα1/α2 double knockout (AMPKα-/-) restored with vector, WT or kinase dead AMPKα under hypoxia (1% O2), 100 μΜ H2O2 or glucose deprivation for indicated time. In each restoration experiment, 5 μg of the plasmid was transfected into indicated cell lines. f, g Immunoblotting of with control (shLuc) and AMPKα1 knockdown MDA-MB-231 cells under H2O2 (100 μΜ) or glucose deprivation for indicated time
Fig. 2
Fig. 2
Ca2+/CaMKKβ-AMPK signaling is critical for EGF-induced Akt activation. a Immunoblotting of control (shLuc) and AMPKα1 knockdown (#1) MDA-MB-231 cells starved for 16 h and treated with 50 ng/ml EGF for 5 and 15 min. b Immunoblotting of AMPKα+/+ and AMPKα-/- MEFs restored with vector, WT and kinase dead AMPKα starved for 16 h and treated with 50 ng/ml EGF for 10 min. In each restoration experiment, 5 μg of the plasmid was transfected into indicated cell lines. c Immunoblotting of control (shLuc) and CaMKKβ knockdown (#1) MDA-MB-231 cells serum starved and treated with EGF for 5 and 15 min. d Immunoblotting of MDA-MB-231 cells treated with EGF (50 ng/ml) for 5 min and STO609 (10 μM) for 5 min. e Immunoblotting of control (shLuc), CaMKKβ knockdown (#1) restored with vector and CaMKKβ MDA-MB-231 cells serum starved and treated with EGF for 10 min. In each restoration experiment, 5 μg of the plasmid was transfected into indicated cell lines. f Immunoblotting of MDA-MB-231 cells pretreated with BAPTA-2AM (10 μM) for 15 min and treated with EGF (50 ng/ml) for another 5 min
Fig. 3
Fig. 3
Skp2 and AMPK are crucial for stress-induced Akt ubiquitination and activation. a HEK293 cells in normoxia and hypoxia were subject to immunoprecipitation with Ubiquitin antibody, followed by immunoblotting. Input is 10% of whole cell lysate. b In vivo ubiquitination assay of Akt from 293T cells transfected with WT, K63R and K48R His-Ub in response of 1% O2. Input is 10% of whole cell lysate. c Control (shLuc) and AMPKα1 knockdown MDA-MB-231 cells in normoxia and hypoxia were subjected to immunoprecipitation with K63-specific Ubiquitin antibody, followed by immunoblotting. Input is 10% of whole cell lysate. d–f In vivo ubiquitination assay of Akt from WT, AMPKα-/- MEFs restored with vector and WT AMPKα together with transfection of HA-Akt and His-Ub in response of hypoxia (1% O2), glucose deprivation and H2O2 for indicated time. Input is 10% of whole cell lysate. g WT and Skp2-/- MEFs challenged with 1% O2 for 8 h were subjected to immunoprecipitation with K63-specific Ubiquitin antibody, followed by immunoblotting. h Immunoblotting of WT and Skp2-/- MEFs that were challenged with 1% O2 for 4 and 8 h. i Immunoblotting of control (shLuc) and Skp2 knockdown (#1) MDA-MB-231 cells were serum starved and treated with H2O2 (100 μM) for indicated time
Fig. 4
Fig. 4
AMPK directly phosphorylates Skp2 at S256 to promote Skp2 SCF complex formation. a HEK293 cells transfected with Skp2 ΔN, 1–200 and the indicated HA-AMPKα (CA) constructs were subjected to immunoprecipitation, followed by immunoblotting. b The sequence of optimal AMPK motif, Skp2 and known AMPK substrates are listed. The Serine (S) residues that phosphorylated by AMPK are highlighted. c HEK293 cells transfected with Skp2 WT, S256, and the indicated HA-AMPKα (CA) constructs were subjected to immunoprecipitation, followed by immunoblotting. d Constitutively active (CA) and kinase dead (KD) HA-AMPKα1 proteins purified from 293T cells and recombinant GST-Skp2 WT purified from bacteria were subjected to in vitro kinase assay, followed by Immunoblotting. e Constitutively active (CA) HA-AMPKα1 purified from 293T cells and recombinant GST-Skp2 WT, S256A purified from bacteria were subjected to in vitro kinase assay, followed by Immunoblotting. f In vitro kinase assay of recombinant GST-Skp2 and active AMPKα1β1γ1 complex purified from sf9 cells with or without Compound C (5 μM) for 30 min followed by Immunoblotting. g Immunoblotting of control (shLuc) and AMPKα1 knockdown (#1) MDA-MB-231 cells treated with EGF, hypoxia (1% O2), glucose deprivation and H2O2 using Skp2 S256 phosphor-specific antibody. h In vivo ubiquitination assay in 293T cells transfected with the indicated plasmids was performed, followed by immunoblotting. i 293T cells transfected with Flag-Skp2 WT, S256A/D, and the indicated HA-AMPKα (CA) constructs were subjected to immunoprecipitation with Flag antibody, followed by immunoblotting. j MDA-MB-231 cells treated with or without glucose free medium and compound C were subjected to immunoprecipitation with Skp2 antibody, followed by immunoblotting. k The potential binding pocket on the interface of Skp2-Cullin1. Skp2 is shown in green and Cullin1 is displayed in yellow. l 293T cells transfected with Flag-Cullin1 WT and K221R were subjected to immunoprecipitation with Flag antibody, followed by immunoblotting
Fig. 5
Fig. 5
AMPK-Skp2-Akt axis is critical for survival under stress and glucose deprivation induced VEGF secretion and EGF-induced glycolysis and migration. a, c Cell survival analysis of MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown, AMPKα1 knockdown along with Myr-Akt, Skp2 WT, or Skp2 S256A or Skp2 S256D restoration under hypoxia (1% O2) for 72 h. b, d Cell survival analysis of MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown, AMPKα1 knockdown along with Myr-Akt, Skp2 WT, or Skp2 S256A or Skp2 S256D restoration under glucose deprivation for 16 h. e MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown and AMPKα1 knockdown with vector control, Skp2 WT and S256A or Skp2 S256D restoration were treated with a glucose-free medium for 8 h, and supernatant was collected for HUVEC tube formation assay. Each value represents the mean ± SEM in three independent experiments. **P < 0.01. f MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown (#1) and Myr-Akt restoration in AMPKα1 knockdown were starved in DMEM glucose-free medium for 4 h and added with 2-NBDG for 30 min. Cells were then subjected to FACS analysis. Each value represents the mean ± SEM (n = 4 per group) in three independent experiments. **P < 0.01. g Immunoblotting of control (shLuc) and AMPKα knockdown (#1) MDA-MB-231 cells serum starved and treated with EGF for 15 and 30 min. h Immunoblotting for MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown and AMPKα1 knockdown along with Myr-Akt restoration. i MDA-MB-231 AMPKα1 knockdown cells with Skp2 WT, S256A or S256D restoration were starved in DMEM glucose-free medium for 4 h and added with 2-NBDG for 30 min. Cells were then subjected to FACS analysis. Each value represents the mean ± SEM (n = 4 per group) in three independent experiments. **P < 0.01. j Cell migration assay of MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown and AMPKα1 knockdown along with Myr-Akt restoration. k MDA-MB-231 AMPKα1 knockdown cells with Skp2 WT, S256A or S256D restoration were subject to cell migration assay with or without EGF
Fig. 6
Fig. 6
AMPK-mediated Skp2 S256 phosphorylation and Akt activation promotes cancer progression, predicts poor survival outcome and contributes to resistance of EGFR targeting therapy. a MDA-MB-231 cells with control (shLuc) and AMPKα1 knockdown were subcutaneously injected into nude mice. Tumor size was measured by the caliper, and the result was showed as means ± SEM. (n = 5). **P < 0.01. b MDA-MB-231 cells with stable expression of the indicated pBabe-Skp2 WT, S256A and S256D were subcutaneously injected into nude mice. Tumor size was measured by the caliper, and the result is shown as means + SEM (n = 5). **P < 0.01. c pSkp2 (S256) and pAMPK are upregulated in breast cancer patients. Representative images of histological analysis of pSkp2 (S256), pAkt, pAMPK and p27 staining in early (Left panel) and late-stage human breast cancer (Right panel). Scale bar, 200 μm. d Overexpression of pSkp2 (S256) and pAMPK are detected in advanced stage of breast cancer patient. Box plot represents pSkp2 (S256), pAMPK and pAkt expression in different stages of breast cancer patient. **P < 0.01. e pAMPK and pAkt expression both correlate with pSkp2 (S256) in breast cancer patients. Scatter plots of pSkp2 (S256) expression vs. pAMPK (Upper panel) and pAkt (Lower panel) expression in breast cancer patient were presented. f Overexpression of pAMPK, pSkp2 and pAkt predicts poor survival outcome of breast cancer patients. Kaplan–Meier plots showed that high expression of pAMPK (Left panel), pSkp2 (S256) (Middle panel) and pAkt (Right panel) significantly predicted disease-specific survival. p-values are shown in the graphs. g Gefitinib-resistant H1975 NSCLC cells with stable expression of the indicated vector, pBabe-Skp2 S256A and S256D were treated with Gefitinib at indicated concentrations for 72 h, and cell viability was measured by Cell counting kit 8. Columns, mean (n = 3); bars, mean ± S.D. *P < 0.05; **P < 0.01. h Hypothetic model of the role of AMPK in diverse stresses and EGF-induced Akt activation and tumorigenesis
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