Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming

Abstract

Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer.

Keywords: Akt; PDK1; hypoxia; metabolism; mitochondria; tumor cell proliferation.

Figure 1. Mitochondrial phosphoproteome in hypoxia

(A) Phosphoproteome of prostate adenocarcinoma PC3 cells in hypoxia versus normoxia. Identified phosphosites met a minimum MaxQuant localization probability of 0.75 and a score difference of 5. Fold changes were calculated from the normalized Heavy/Light SILAC ratio. Six Akt target proteins showing increased phosphorylation in hypoxia are indicated. Grey, not significant; red, upregulated; blue, downregulated; yellow squares, Akt targets. (B) Ingenuity pathway analysis of mitochondrial phospho- and global proteome in hypoxia. (C) Kinases for which at least 5 known targets showed significant changes in phosphorylation in hypoxic versus normoxic conditions as in (A). Up, upregulation; Dn, downregulation. *, The modulated genes are: ARID1A; HIST1H1E; HMGA1; LARP1; LIG1; LIG3; LMNB2; LRCH3; LRWD1; MARCKS; MED1; MKI67; NCL; NPM1; NUCKS1; PDS5B; PTPN2; RB1; RBL1; RBL2; SAMHD1; SETDB1; TERF2; VIM. **, The modulated genes are: DUT; EEF1D; HIST1H1E; HMGA1; IRS2; LIG1; LIG3; LMNA; LMNB1; MAP4; NOLC1; NPM1; NUCKS1; PDS5B; PTPN2; RB1; SAMHD1; TCOF1; TOP2A; TPX2; VIM. (D) PC3 cells in normoxia (N) or hypoxia (H) were fractionated in cytosol (Cyto) or mitochondrial (Mito) extracts and analyzed by Western blotting. pAkt, phosphorylated Akt (Ser473). TCE, total cell extracts. (E) PC3 cells in hypoxia (H) were exposed to reoxygenation (O₂) for the indicated time intervals and analyzed by Western blotting. N, normoxia. (F) The indicated subcellular fractions isolated from normoxic (N) or hypoxic (H) PC3 cells were analyzed with an antibody to the Akt consensus phosphorylation site RxRxxS/T (Akt cons Ab) by Western blotting. Mito Sup, supernatant of mitochondrial extracts after preclearing with Akt cons Ab. (G) PC3 cells in normoxia (N) or hypoxia (H) were treated with vehicle (Veh) or Hsp90 small molecule inhibitor 17-AAG (5 µM for 6 hr), and cytosolic (Cyto) or mitochondrial (Mito) extracts were analyzed by Western blotting. (H) PC3 cells in normoxia (N) or hypoxia (H) were treated with vehicle (Veh), the antioxidant N-acetyl cysteine (NAC, 1 mM) or mitochondria-specific ROS scavenger, MitoTempo (MT, 25 µM), and subcellular fractions were analyzed by Western blotting. See also Figure S1.

Figure 2. Mitochondrial Akt phosphorylation of PDK1

(A) Schematic diagram for the identification of a mitochondrial Akt phosphoproteome in hypoxic versus normoxic PC3 cells. (B) Mitochondrial proteins reacting with Akt cons Ab showing differential expression in hypoxic versus normoxic PC3 cells. (C) Recombinant PDK1 or GSK3β was mixed in a kinase assay with active Akt1 or Akt2, and phosphorylated bands were detected with Akt cons Ab by Western blotting. (D) The indicated PDK isoforms were mixed in the presence or absence of active Akt2 in a kinase assay and phosphorylated bands were detected with Akt cons Ab, by Western blotting. (E) PC3 cells in normoxia (N) or hypoxia (H) were immunoprecipitated (IP) with an antibody to PDK1 followed by Western blotting. HIF1α reactivity (bottom) was used as a marker of hypoxia. TCE, total cell extracts. Bottom, densitometric quantification of phosphorylated (p) PDK1 bands. U, arbitrary units. (F) Extracted ion chromatogram of the PDK1 phosphorylated T346 chymotryptic peptide (STAPRPRVEpTSRAVPL, m/z 908.9751) resulting from incubation with or without active Akt1 or Akt2 in a kinase assay. (G) PC3 cells were transfected with vector or Flag-tagged wild type (WT) PDK1 or T346A PDK1 mutant, immunoprecipitated with an antibody to Flag and immune complexes were mixed with active Akt2 in a kinase assay followed by Western blotting with Akt cons Ab. Bottom, densitometric quantification of phosphorylated (p) PDK1 bands. U, arbitrary units. (H) Molecular dynamics simulation of the structure of PDK1 (ribbon) with stick representation of residues 336–356 comprising the “ATP lid”. The ATP molecule is derived from the structure of PDK3-L2-ATP (PDB code 1Y8P) superimposed onto the structure of PDK1. The predicted location of Thr346 as well as Arg343 and Arg348 is shown. (I) The experimental conditions are as in (G) except that Flag-PDK1 immune complexes mixed with active Akt2 in a kinase assay were analyzed with phospho-specific pT346 Ab by Western blotting. Exp., exposure. Bottom, densitometric quantification of phosphorylated (p) PDK1 bands. U, arbitrary units. (J) Flag-PDK1 immune complexes as in (G) were precipitated from PC3 cells in normoxia (N) or hypoxia (H) and analyzed with pT346 Ab by Western blotting. p, phosphorylated. Bottom, densitometric quantification of pPDK1 bands. U, arbitrary units. See also Figure S2 and Table S1.

Figure 3. A mitochondrial Akt-PDK1-PDHE1 phosphorylation axis in hypoxia

(A) PC3 cells in normoxia (N) or hypoxia (H) were transfected with vector, WT PDK1 or T346A PDK1 mutant and analyzed by Western blotting. Bottom, densitometric quantification of phosphorylated (p) PDHE1 bands. U, arbitrary units. (B) The indicated recombinant proteins were mixed in a kinase assay and analyzed by Western blotting. (C) PC3 cells transfected with vector or the indicated Flag-tagged WT PDK1 or T346A PDK1 mutant were immunoprecipitated (IP) with an antibody to Flag, and immune complexes were mixed in a kinase assay with recombinant Akt2 and PDHE1 followed by Western blotting. (D) PC3 cells in normoxia (N) or hypoxia (H) were transfected with control siRNA (Ctrl) or siRNA to Akt1 or Akt2, and analyzed by Western blotting. (E) PC3 cells in normoxia (N) or hypoxia (H) were treated with vehicle control (Veh) or a small molecule Akt inhibitor, MK2206 (1 µM), and analyzed by Western blotting. (F) PC3 cells in normoxia (N) or hypoxia (H) were transfected with vector, Akt-kinase dead (Akt-KD) or mitochondrial-targeted Akt-KD (mtAkt-KD) mutant, and mitochondrial extracts (Mito) were analyzed by Western blotting. (G) PC3 cells in normoxia (N) or hypoxia (H) were transduced with pLKO or PDK1-directed shRNA, reconstituted with vector, WT PDK1 or T346A PDK1 mutant cDNA and analyzed by Western blotting. Bottom, densitometric quantification of phosphorylated (p) PDHE1 bands. U, arbitrary units. (H) PC3 cells transduced with pLKO or PDK1-directed shRNA were analyzed for PDH activity in normoxia (N) or hypoxia (H) conditions. Left, representative tracings (n=4). Right, quantification of PDH activity. ns, not significant. Mean±SEM. *, p=0.03. (I) PC3 cells in hypoxia were transduced with PDK1-directed shRNA, reconstituted with vector, WT PDK1 or T346A PDK1 mutant cDNA and analyzed for PDH activity. Left, representative tracings (n=3). Right, quantification of PDH activity. Mean±SEM. **, p=0.009. (J) PC3 cells transduced with pLKO or PDK1-directed shRNA were reconstituted with vector, WT PDK1 or T346A PDK1 cDNA and analyzed for glucose consumption (n=4). Mean±SEM. ***, p<0.0002. (K) PC3 cells in normoxia (N) or hypoxia (H) were treated with vehicle control (Veh) or Akt inhibitor, MK2206 (1 µM), and analyzed for lactate production (n=3). Mean±SEM. **, p=0.001–0.004; ***, p=0.0005–0.0009. (L) PC3 cells in normoxia (N) or hypoxia (H) were transfected with control siRNA (Ctrl) or siRNA to Akt1 or Akt2 and analyzed for lactate production (n=2). Mean±SD. **, p=0.004; ***, p=0.0005. (M) PC3 cells stably silenced for PDK1 were transfected with vector (Vec), WT PDK1 or T346A PDK1 mutant, and analyzed for oxygen (O₂) consumption (n=3). Mean±SEM. For all panels, data were analyzed using the two-sided unpaired Student’s t tests. See also Figure S3.

Figure 4. Mitochondrial Akt-PDK1 phosphorylation, in vivo

(A) GBM neurospheres (top) or differentiated GBM cultures (bottom) were stained for DNA (DAPI), HIF1α, pT346-phosphorylated PDK1, or hypoxia (hypoxia-sensitive probe). Merged images of nuclear-localized HIF1α in hypoxic neurospheres (by velocity mask) are indicated (Merge). Yellow box, Volocity analysis to identify cells with nuclear HIF1α in each single z-stack. Scale bar, 20 µm. (B and C) Immunohistochemical staining of primary, patient-derived GBM samples with high (≥2) (B) or low (0) (C) score for HIF1α and phosphorylated protein (pProt) expression. Scale bar, 100 µm. p, phosphorylated. (D–F) Quantitative immunohistochemical correlation of patient-derived GBM samples (n=24) or grade II gliomas (n=2) for HIF1α expression and pPDK1 (D), or pPDHE1 (E), or between pPDK1 and pPDHE1 (F). Four tissue microarray (TMA) cores/patient. The scoring is as follows: 0, no staining; 1, staining in at least one TMA core; 2, staining in ≥2 TMA cores. The individual p values per each analysis are indicated (Chi-Square test). See also Figure S4 and Table S2.

Figure 5. Requirement of mitochondrial Akt for tumor cell proliferation in hypoxia

(A and B) Bioluminescence imaging of immunocompromised mice carrying U251 intracranial GBMs (3 animals/group) expressing luciferase under the control of HIF1-responsive elements (Luc) and mCherry (cell viability) and exposed to a hypoxia-sensitive probe (Hypox). Scans were obtained at days 20 and 34 (A) and fluorescence signals were quantified (B). *, p=0.016–0.057 by Mann-Whitney test. (C) Tissue samples from intracranial GBMs as in (A) were harvested at day 34 and analyzed for expression of HIF1α, phosphorylated (p) PDK1 (pT346 Ab) or pPDHE1, by immunohistochemistry. Yellow lines were used to delineate the tumor mass within mice’ brain. Scale bar, 100 µm. Asterisks, mitotic cells; Insets (H&E and pPDK1 panels), high-power magnification of mitotic cells. Scale bar, 25 µm. (D and E) PC3 cells transfected with control siRNA (Ctrl) or Akt1- or Akt2-directed siRNA (D) or stably transduced with pLKO or PDK1-directed shRNA (E) were analyzed for cell proliferation in normoxia or hypoxia by direct cell counting (n=5). Mean±SEM. ***, p<0.001; **, p=0.002 (F and G) PC3 cells stably transduced with pLKO or PDK1-directed shRNA were analyzed in normoxia or hypoxia for colony formation by crystal violet staining after 10 days (F) and quantified (n=3) (G). Mean±SEM. ns, not significant. **, p=0.003. For all panels, data were analyzed using the two-sided unpaired Student's t test. See also Figure S5.

Figure 6. Mitochondrial Akt regulation of stress signaling in hypoxia

(A and B) PC3 cells in normoxia (N) or hypoxia (H) were treated with vehicle (Veh) or MK2206 (1 µM) (A) or transduced with pLKO or PDK1-directed shRNA (B) and analyzed for ROS production by CELLROX Green staining and flow cytometry. Upper panels, representative tracings. Bottom panels, quantification of ROS production under the various conditions tested (n=2). Mean±SD for both datasets. *, p=0.01–0.02; **, p=0.004; ns, not significant. (C) The experimental conditions are as in (A) and treated cells were analyzed for cell viability by direct cell counting relative to control (n=3). Mean±SEM. ***, p<0.0001. (D) PC3 cells in normoxia (N) or hypoxia (H) were incubated with vehicle (Veh) or small molecule inhibitors of Akt (MK2206, 1 µM) or PI3K (PX-866, 10 µM) and analyzed by Western blotting. (E) PC3 cells stably silenced for PDK1 were reconstituted with vector, WT PDK1 or T346A PDK1 mutant and analyzed for cell viability by direct cell counting relative to control (n=3). Mean±SEM. ***, p=0.0002. (F and G) PC3 cells in normoxia (N) or hypoxia (H) were transduced with pLKO or PDK1- directed shRNA (F) or control siRNA (Ctrl) or Akt1- or Akt2-directed siRNA (G), and analyzed by Western blotting. (H and I) PC3 cells as in (E) were analyzed for LC3 reactivity by fluorescence microscopy, Scale bars, 10 µm (H), and cells with LC3 puncta (>3) were quantified (n=250–860 cells) (I). Mean±SEM.*, p=0.014; ***, p=0.0005. ns, not significant. For all panels, data were analyzed using the two-sided unpaired Student's t test. See also Figure S6.

Figure 7. Mitochondrial Akt-directed hypoxic reprogramming supports tumor growth in vivo

(A) PC3 cells transduced with pLKO or PDK1-directed shRNA were injected s.c. in the flanks of male NSG immunocompromised mice (3 animals/group; 2 tumors/mouse) and superficial tumor growth was quantified with a caliper at the indicated time intervals for 20 days. Data were analyzed using the two-sided unpaired Student's t test. Mean±SEM. ***, p<0.0001. (B) PC3 cells stably transduced with pLKO or PDK1-directed shRNA were reconstituted with WT PDK1 or T346A PDK1 mutant and injected s.c. in the flanks of immunocompromised mice (5 mice/group; 2 tumors/mouse). Tumor growth in the various groups was quantified at the indicated time intervals for 20 days. Data were analyzed using the two-sided unpaired Student's t test. Mean±SEM. *, p=0.01–0.02; ***, p<0.0001. (C) PC3 cells stably transduced with pLKO or PDK1-directed shRNA were reconstituted with vector, WT PDK1 or T346A PDK1 mutant and injected s.c. in immunocompromised mice with determination of tumor growth after 18 days. Each point corresponds to an individual tumor. (D and E) Tumors harvested from the animals in (C) were analyzed for histology (D) and cell proliferation (top, Ki-67), autophagy (middle, LC3-II) or apoptosis (bottom, TUNEL) was quantified (E). The statistical analysis of the various groups by ANOVA is as follows: Ki-67, p<0.0001; LC3, p=0.024; TUNEL, p=0.039. Scale bars, 100 µm. (F and G) Superficial flank tumors of PC3 cells transduced with control pLKO or PDK1-directed shRNA were harvested after 18 day and processed for immunohistochemistry (F) with quantification of reactivity for Ki-67 (top), LC3 (middle) or TUNEL (bottom) (H). Representative images per each condition are shown. (n=3, 10 images per mouse), Mean±SD. Scale bars, 100 µm. (H) Schematic model of a mitochondrial Akt-PDK1-PDHE1 phosphorylation axis in hypoxic tumor reprogramming.

Figure 8. Mitochondrial Akt phosphorylation of PDK1 is a negative prognostic marker in human gliomas

(A) Representative micrographs of immunohistochemical staining of non-neoplastic human brain parenchyma (normal) or grade II-IV gliomas (WHO classification) with PDK1 pT346 Ab. OD, oligodendroglioma; AOD, anaplastic OD; GBM, glioblastoma. Scale bar, 100 µm. (B) Quantification of pT346 staining in a series of human brain tumors (n=116) and 85 nonneoplastic brain parenchyma using a two-factor scoring system that considers the percentage of positive cells and the intensity of the staining (pPDK1 score). ***, p<0.0001; **, p=0.002 by Mann Whitney U-test. Each symbol represents an individual patient. (C–E) Differences in pPDK1 score in human brain tumors as in (B) (n=116) according to nuclear HIF1α expression (C, **, p=0.008 by Mann Whitney U-test), IDH1 mutation status (D; *, p=0.02 by Mann Whitney U-test), or MGMT promoter methylation (D; *, p=0.01 by Mann Whitney U-test). Data are presented as Tukey box-and-whisker plots. The bottom and top of the box represent the first and third quartiles, and the band inside the box represents the median (i.e. the 2^nd quartile). The bottom end of the whisker represents the lowest datum within the 1.5 interquartile range (IQR) of the lower quartile, and the top end of the whisker represents the highest datum within 1.5 IQR of the upper quartile. Outlier data, if any, are represented by single points. (F and G) Kaplan-Meier curves were generated with either the complete series of glioma patients (n=116; F) or with GBM cases only (n=61; G) sorted into “Low” or “High” groups according to pPDK1 score. Cutoffs to rank patients in these two categories were generated using ROC curves and the Youden’s J statistic. Overall survival curves were compared using the Log-Rank test. HR, Hazard Ratio; CI, Confidence Interval. See also Figure S7 and Table S3.