AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest

Abstract

Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells.

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Figure 1: MCD is modulated by the intrinsic apoptotic pathway.
Figure 2: Autophagy modulates cell death during mitotic arrest.
Figure 3: Reduced mitochondrial mass as a consequence of mitophagy during mitotic arrest.
Figure 4: Metabolic switch from OXPHOS to glycolysis is mediated by AMPK.
Figure 5: Glycolysis determines survival during mitotic arrest.
Figure 6: PFKFB3 regulation during mitotic arrest.
Figure 7: Taxol cooperates with different inhibitors of survival pathways to promote MCD.
Figure 8: Anti-tumoral cooperation between microtubule poisons and PFKFB3 inhibitors in vivo.

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Acknowledgements

We thank M. Soengas (Spanish National Cancer Research Centre, Spain), J. P. Bolaños (Institute of Functional Biology and Genomics, Spain) and N. Mizushima (The University of Tokyo, Japan) for reagents. We also thank S. Velasco for help with the Seahorse apparatus, J. Muñoz and M. P. Ximénez for their support in phospho-proteomics experiments, and F. Escobar and B. Pou for help with electron microscopy. E.D., C.M. and M.S.-R. were supported by the Spanish Fondo de Investigaciones Sanitarias (Madrid), MINECO (Juan de la Cierva programme) and Asociación Española contra el Cáncer (AECC), respectively. L.E.-M. is a recipient of a JAE predoctoral fellowship from the CSIC. A.K.S. was supported by USPHS grants RO1DK19514, RO1DK67509. G.V. was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) and Fondo Europeo de Desarrollo Regional (FEDER) (PI12/02248), Fundació La Marató de TV3 (m12 20134031), and Fundación Mutua Madrileña (AP101042012). M.L. was supported by the European Community’s Seventh Framework Programme under grant agreement no. 281854—the ObERStress (European Research Council project). E.R. was financially supported by a MINECO grant (SAF 2010-20256). Work in the R.M. laboratory was supported by the Fundación Botín, Banco Santander and MINECO (BFU2011-30121, BFU2014-52125-REDT and Consolider RNAREG CSD2009-00080). Work in the P.B. laboratory is supported by a grant from the Spanish Ministry for Economy and Competitiveness (MINECO; SAF2012-36079). Work in the M.M. laboratory was supported by grants from the MINECO (SAF2012-38215), Consolider-Ingenio 2010 Programme (SAF2014-57791-REDC), Excellence Network CellSYS (BFU2014-52125-REDT), the OncoCycle Programme (S2010/BMD-2470) from the Comunidad de Madrid, Worldwide Cancer Research (WCR no. 15-0278), and the European Union Seventh Framework Programme (MitoSys project; HEALTH-F5-2010-241548).

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Contributions

E.D. and M.S.-R. performed most of the cellular and biochemical assays with the help of C.M. and D.P. L.E.-M., E.S. and P.B. collaborated in the analysis of mitophagy, and G.F.-M., R.P. and R.M. collaborated in the analysis of translation. R.C.-O. generated the NMR data. M.P. and D.M. helped with microscopy analysis. A.K.S. performed the AMP/ATP measurements and M.L. contributed to the analysis of AMPK. E.R. helped with metabolic measurements. E.D., C.M., G.V., E.R., P.B., M.S.-R. and M.M. analysed the data. M.M. conceived the project, M.S.R. and M.M., supervised the experiments, and M.M. wrote the manuscript with the help of all co-authors.

Corresponding authors

Correspondence to María Salazar-Roa or Marcos Malumbres.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 Analysis of apoptosis, necrosis and necroptosis.

(a) Depletion of Cdc20 after treatment of Cdc20(lox/lox); CreERT2 cells with 4-hydroxytamoxifen (4-OHT) (left top panel). Immunodetection of the indicated proteins after treatment with the indicated siRNAs (rest of panels). Vinculin or α-tubulin were used as a loading control. (b) Efficiency of the indicated siRNAs by quantification of mRNA levels 48 h after siRNA nucleofection. Data were normalized against the levels of β-actin transcripts. Data are mean ± SD and represent one out of at three independent experiments. (c) Immunodetection of caspase 3 cleavage in Cdc20-null cells treated with either ZVAD, Okadaic Acid (OA) or both, at the time points indicated. α-tubulin was used as loading control. (d) Representative electron microscopy images of Bak/Bax-null and wild-type cells arrested in mitosis by the nocodazole-MG132 treatment. Scale bars, 2 μm. (e) Percentage of cells positive for Annexin V or DAPI in the indicated cultures. The left panel represents Cdc20-null or control cells expressing CreERT2 in the presence of 4-hydroxytamoxifen (4OHT). Right panel represents human cells arrested in mitosis with Taxol and proTAME (Tx + pT). Values are represented as mean ± SD (n = 3 independent experiments). For a,c unprocessed original scans of blots are shown in Supplementary Fig. 7. Source data can be found in Supplementary Table 4.

Supplementary Figure 5 Induction of autophagy during mitotic arrest.

(a) Mitotic entry in Cdc20-null cells after release from RO-3306. Cdc20(lox/lox) cells were treated with 4-OHT to eliminate Cdc20 12 h before the addition of the Cdk1 inhibitor RO-3306 to trigger G2 arrest. This compound was washed-out 18 h later allowing the accumulation of mitotic cells over time (left panel). Representative images are shown in the right panel. Scale bars, 50 μm. (b) LC3 lipidation was analyzed in MDA-MB 231 cells arrested by treatment with taxol + pro-TAME, in the presence or absence of E64D/PA. Two different time exposures for LC3 are shown. (c) Colocalization of LC3 and Annexin V (mean ± SD; n = 3 independent experiments) in Cdc20-null cells at the time points of mitotic arrest indicated in the graph. Images are representative for the double staining at 24 h. Scale bar, 10 μm. (d) mRNA levels (mean ± SD; n = 3 independent experiments) of the indicated transcripts 48 h after siRNA nucleofection. Data were normalized against the levels of β-actin transcripts. (e) Duration of mitosis (from metaphase until cell death) in Cdc20-null cells treated with ZVAD, the indicated inducers of autophagy (mTOR inhibitors PP242, temsirolimus, BEZ235), or transfected with siRNAs against Raptor (Rap.). (f) Duration of mitosis in Hela and MDA-MB 231 cells synchronized by the taxol + pro-TAME protocol and treated with the indicated inhibitors. (g) Duration of mitosis in Cdc20-null cells treated with the indicated PI3K/Akt inhibitors. (h) Immunoblot of LC3 after mTOR inhibition with PP242 or BEZ235 in Cdc20-null cells. Cdc27 band-shift (indicating phosphorylation) was used to confirm mitotic arrest. The samples were processed simultaneously and run in the same blot. (i) Immunoblot of LC3 after mTOR inhibition with PP242 in MDA-MB-231 cells synchronized by the taxol + pro-TAME protocol. (j) Duration of mitosis in Atg5-null cells treated or not with the caspase inhibitor ZVAD (left) and Bax/Bak double mutant cells treated or not with the autophagy inhibitor 3MA (right), in both cases using the taxol + pro-TAME protocol. (k) LC3 lipidation and PARP cleavage after treatment with caspase (ZVAD) and autophagy (E64d/PA) inhibitors in Cdc20-null cells. Cdc27 phosphorylation (retarded motility) indicates mitotic cells. In b,h,i,k, α-tubulin is included as a loading control. In eg; j dots represent individual cells and the mean are indicated by red lines. The number of cells analyzed (n) is indicated in each condition, and data are representative of three (e), two (f), six (g) or one (j) separate experiments. Green or red backgrounds indicate a significant delay or premature cell death in mitosis, respectively. , P < 0.05; , P < 0.01; , P < 0.001; Student’s t-test. For b,h,i,k Unprocessed original scans of blots are shown in Supplementary Fig. 7, and α-tubulin is included as a loading control. Source data can be found in Supplementary Table 4.

Supplementary Figure 6 Autophagosomes and ROS accumulate during mitotic arrest.

(a) Representative profiles of mitotracker (left) and Dihydroethidium (DHE; right) in Cdc20-null (green) or control (grey) cells expressing CreERT, at the indicated time points after the release from RO-3306 (all the cells were previously treated 18 h with 4OHT). (b) Immunodetection of the indicated mitochondrial proteins at 24 h after the release from RO-3306 (all the cells were previously treated 18 h with 4OHT) in control-CreERT2 (left) and Cdc20-null cells treated with vehicle or the autophagy inhibitor 3MA. Unprocessed original scans of blots are shown in Supplementary Fig. 7. β-actin was used as a loading control. (c) Effect of 3MA and Cyclosporin A (CsA) on mitotracker levels in control-CreERT2 (left panels) and Cdc20-null cells 24 h after the release from RO-3306 (all the cells were previously treated 18 h with vehicle or 4OHT). Representative FACS profiles are shown and quantification of mitotracker signal is represented in the bottom graphs. Data are mean ± SEM (n = 3 independent experiments). (d) Quantification of ROS (mean ± SEM; n = 3 independent experiments) in Cdc20-null cells as determined by the OxiSelect TM Intracellular ROS assay kit. The effect of NAC treatment in the accumulation of ROS (mean ± SEM; n = 3 independent experiments) is also shown at the indicated times. (e) Quantification of ROS (mean ± SEM; n = 3 independent experiments) in Cdc20-null cells or Cdc20(lox/lox) cells arrested in mitosis after 24 h with taxol and pro-TAME, as determined by the OxiSelect TM Intracellular ROS assay kit. (f) Duration of mitosis until cell death in Cdc20-null and HeLa cells treated with the antioxidant NAC or the caspase inhibitor ZVAD after synchronization with nocodazole and release in MG132. The mean is indicated by red bars and data represent two separate experiments. In d,e,f the number of cells analyzed (n) is indicated in each condition. , P < 0.05; , P < 0.01; , P < 0.001 (Student’s t-test). Source data can be found in Supplementary Table 4.

Supplementary Figure 7 AMPK is reactivated during mitotic arrest.

(a) Schematic representation of the protocol followed for synchronization in human cells. Cells were treated with RO-3306 and this compound was washed-out 18 h later allowing mitotic entry in the presence of taxol and pro-TAME (an APC/C inhibitor) which mimic the arrest in metaphase imposed by Cdc20 ablation. (b) The levels of the indicated antigens were analyzed by immunoblot in HeLa or MDA-MB-231 cells. α-tubulin was included as a loading control and unprocessed original scans of blots are shown in Supplementary Fig. 7. (c) Changes in AMPK activity were monitored by using a FRET biosensor in Cdc20(lox/lox) cells in the absence of 4-OHT. Upon treatment of these control cells with 2-DG, a glucose analog, AMPK is activated giving an increase of about 2-fold in biosensor signal (indicated in red in the images after 2DG addition; bottom panel). This increase is not observed in cells treated with siRNAs against AMPK. Data are mean ± SD (n = 4 independent experiments). (d) AMPK activity was also monitored by the FRET biosensor in mitotic cells treated with vehicle (blue), 2DG (green) or Glucose (red). Data are represented as mean ± SD (n = 3 independent experiments) after scoring the indicated number of mitotic cells in each condition. (e) mRNA levels of APMKα1 and AMPKα2 48 h after nucleofection of specific siRNAs. Data are mean ± SD (n = 3 independent experiments) normalized against β-actin mRNA levels. (f) Levels of OCR (left) and ECAR (right) in Cdc20-null cells arrested in mitosis for 24 h and transfected with siRNAs against siAMPKα1 (siα1), siAMPKα2 (siα2) or the combination of both relative to Scrambled (Scr.) sequences. Data are mean ± SD (one experiment with 12 replicates). (g) Effect of siRNAs against AMPKα1 and/or AMPKα2 on mitotracker levels in Cdc20-null cells 24 h after the release from RO-3306. Representative FACS profiles are shown on the left, and the quantification of mitotracker signal mean ± SEM (n = 3 independent experiments) is represented in the histogram. In g, NS, no significant; , P < 0.05 (Student’s t-test). Source data for all the figures can be found in Supplementary Table 4.

Supplementary Figure 8 Metabolic profile of mitotic arrested cells.

(a) Protocol for synchronized mitotic entry. Cdc20(lox/lox) cells were untreated or treated with 4-OHT to eliminate Cdc20 expression, 12 h prior the addition of the Cdk1 inhibitor RO-3306 to trigger G2 arrest. This compound was washed-out 18 h later, allowing cells to progress through the cell cycle (Cdc20(lox/lox); blue) or arrest in mitosis in the absence of Cdc20 (Cdc20(Δ/Δ); red). Green lines indicate asynchronous cultures and these colour codes are maintained throughout the figure to indicate the three different cell cultures. (b) Similar protocol followed for synchronization in human MDA-MB-231 cells. (c) Cell viability (TO-PRO3 staining) assessed by flow cytometry in the different cultures represented in a. (d) Differential extracellular concentration of the indicated metabolites after 24 h of adding new medium, as monitored by NMR. 34 metabolites were analyzed and only those exhibiting significant differences are represented (23 in this figure + glucose and lactate). No differences were found in carnosine, adenine, glyoxylate, cholesterol, EtOH, niacinamide, acetate, asparagine and aspartic acid. (e) Cell viability (TO-PRO3 staining) assessed by flow cytometry in the cultures represented in b. (f) Glucose uptake and concentration of extracellular lactate in asynchronous (green), normally release from G2 arrest (blue) or mitotic arrested (red) MDA-MB-231 cells, in the presence of 13C-labelled glucose. Data in d and f are not normalized for death cells and the differences in mitotic-arrested cells are likely under-estimated. (g) PFKFB1/4 protein levels in asynchronous (As.), G2-arrested or mitotic Cdc20-deficient cells in the presence or absence or cordycepin (Cord.) or cycloheximide (Chx.). GAPDH was used as a loading control. Images are representative of 2 independent experiments. (h) Immunodetection of PFKFB1 and PFKFB3 48 h after transfection. α-tubulin was included as a loading control. For panels (g,h), Unprocessed original scans of blots are shown in Supplementary Fig. 7. (i) Duration of mitosis (from mitotic entry until cell death) in Cdc20-null cells transfected with siRNAs against PFKFB1 or Scrambled sequences. Each dot indicates a single cells and red lines indicate mean (n = 50 cells per condition; one independent experiment). NS, not significant differences. In c,-f) data are mean ± SD (n = 3 independent experiments. Source data can be found in Supplementary Table 4.

Supplementary Figure 9 Cooperation between taxol and inhibitors of mitotic survival pathways.

Cell death was quantified by scoring To-Pro3 levels by high-throughput microscopy after the addition of the indicated compounds in the presence or absence of taxol in MDA-MB-468, EVSA-T or MCF7 breast cancer cells (left panels) or BJ-TERT-2 and RPE non-transformed cells (right panels). Pink columns indicate significant cooperation when compared to both single inhibitors (black dotted comparisons) and taxol (red dotted comparisons). The number of cells analyzed (n) is indicated in each condition. Data are mean ± SEM (n = 6 independent experiments). , P < 0.001; Student’s t-test, and represent one out of 6 independent experiments. Source data can be found in Supplementary Table 4.

Supplementary Table 1 Small-molecule inhibitors and other chemicals used in this work.
Supplementary Table 2 Oligonucleotides used in this work.
Supplementary Table 3 Antibodies used in this work for immunodetection of proteins.

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Doménech, E., Maestre, C., Esteban-Martínez, L. et al. AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest. Nat Cell Biol 17, 1304–1316 (2015). https://doi.org/10.1038/ncb3231

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