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. 2016 Jul 7;14(7):e1002507.
doi: 10.1371/journal.pbio.1002507. eCollection 2016 Jul.

The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis

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Free PMC article

The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis

Jae Ho Seo et al. PLoS Biol. .
Free PMC article

Abstract

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Survivin-ClpXP interaction.
(A) Heatmap of proteomics identification of mitochondrial proteins co-precipitated with an antibody to survivin (SVV) or control IgG from PC3 mitochondrial extracts. Two independent replicates per condition are shown. A fold enrichment in spectra counts (SVV/IgG) is shown. (B–D) Mitochondrial extracts from PC3 cells were immunoprecipitated (IP) with antibodies to survivin (SVV) (B), ClpP (C), ClpP or ClpX (D), or non-binding IgG and analyzed by western blotting. TCE, total cell extracts. MTE, mitochondrial extracts. CS, citrate synthase. (E) Submitochondrial fractions representative of outer membrane (OM), inter-membrane space (IMS), inner membrane (IM), or matrix were isolated from PC3 cells and analyzed by western blotting. (F) PC3 cells were treated with vehicle (Veh) or small molecule survivin (SVV) suppressant YM155 and analyzed by western blotting. (G) PC3 cells in normoxia (N) or hypoxia (H) were transfected with control non-targeting siRNA (Ctrl) or survivin (SVV)-directed siRNA and analyzed by western blotting. (H) MCF-7 cells were transfected with vector or mitochondrial-targeted (mt) survivin (SVV) cDNA and analyzed by western blotting. Expo, exposure.
Fig 2
Fig 2. ClpXP regulates mitochondrial SDHB folding.
(A) PC3 cells were transfected with control siRNA (Ctrl) or ClpP-directed siRNA and solubilized in the indicated concentrations of detergent (CHAPS), and insoluble fractions were analyzed by western blotting. The position of oxidative phosphorylation complex (C) subunits are indicated. (B) PC3 cells were transfected with control siRNA (Ctrl) or ClpP-directed siRNA and mixed with increasing concentrations of detergent (CHAPS), and insoluble fractions were analyzed by western blotting. Three independent experiments per condition are shown. (C) Densitometric quantification of protein bands in control or ClpP siRNA transfectants, as in (B). Data are the mean ± SEM of four (SDHB) or three (SDHA, COX-IV) independent experiments. (D) PC3 cells transfected with control siRNA (Ctrl) or ClpP-or ClpX-directed siRNA were mixed with the indicated increasing concentrations of CHAPS, and insoluble material was analyzed by western blotting. Raw data for this figure can be found in S1 Data.
Fig 3
Fig 3. ClpXP regulation of mitochondrial bioenergetics.
(A and B) PC3 cells were transfected with control siRNA (Ctrl), ClpX- or ClpP-directed siRNA, and mitochondrial extracts were analyzed for Complex I (A) or Complex II (B) activity. Right, Quantification of citrate synthase-normalized mitochondrial complex activity. ***, p < 0.0001. (C–E) The indicated prostate cancer cell types were transfected with control siRNA (Ctrl), ClpX- or ClpP-directed siRNA, and analyzed for changes in oxygen consumption rate (OCR, C), NAD/NADH ratio (PC3 cells, D), or ATP production (E). **, p < 0.006; ***, p < 0.0001. (F and G) PC3 cells transfected with the indicated siRNAs were analyzed by western blotting. (H and I) siRNA-silenced PC3 cells and transfected with GFP-LC3 were analyzed by fluorescence microscopy (H) and the number of LC3 puncta per cell were quantified (I). ***, p < 0.0001. (J) PC3 cells were transfected with control siRNA (Ctrl), CLpX- or ClpP-directed siRNA, and analyzed by western blotting. Raw data for this figure can be found in S2 Data.
Fig 4
Fig 4. Mitochondrial ROS modulation by ClpXP.
(A and B) PC3 cells transfected with control siRNA (Ctrl) or ClpX- or ClpP-directed siRNA were labeled with CellROX Green Reagent by flow cytometry (A), and staining intensity was quantified (B). H2O2 was a control oxidative stimulus. **, p = 0.004; ***, p < 0.0001. (C) The indicated prostate cancer cell types were analyzed for total ROS production as in (A). *, p = 0.03–0.04; **, p = 0.001; ***, p = 0.0008–<0.0001. (D) PC3 cells transfected with the indicated siRNAs as in (A) were analyzed for MitoSOX red mitochondrial superoxide reactivity by fluorescence microscopy and quantified. H2O2 was a control oxidative stimulus. **, p = 0.001; ***, p = 0.0004–0.0001. (E and F) PC3 cells stably transduced with control pLKO or shRNA to ClpX or ClpP were analyzed for mitochondrial superoxide production by fluorescence microscopy (E), and staining intensity was quantified (F). ***, p = 0.0004–0.0001. (G) PC3 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were analyzed by western blotting. (H) PC3 cells transfected with control siRNA or ClpX- or ClpP-directed siRNA were incubated with vehicle (Veh) or the combination of antioxidants NAC (N) plus mitochondrial-directed MitoTempo (MT) and analyzed by western blotting in the absence or presence of the autophagic flux inhibitor hydroxychloroquine (HCQ). (I) Densitometric quantification of LC3-II/I ratio in vehicle (Veh) or antioxidant-treated PC3 cells in (H). Raw data for this figure can be found in S3 Data.
Fig 5
Fig 5. ClpP expression in human cancer.
(A and B) Breast adenocarcinoma MCF-7 cells, non-tumorigenic breast epithelial MCF-10A cells (A), or primary human tissue samples representative of normal prostate or prostatic adenocarcinoma (B) were analyzed by western blotting. (C) Immunohistochemistry (IHC) score of ClpP expression in the indicated primary human tumors arrayed in a universal cancer tissue microarray. LN, lymph nodes; CNS, central nervous system. (D) IHC staining and Aperio quantification of ClpP expression in the indicated human tumor types. AdCa, adenocarcinoma; HCC, hepatocellular carcinoma; Endom, endometrial AdCa. (E) Aperio quantification of ClpP IHC expression in primary tissue samples of non-small cell lung cancer (NSCLC) that developed (met) or did not develop (no met) distant metastases or brain metastases (brain met) during a 5-y follow-up. (F) Quantification of ClpP staining intensity in the NSCLC cases in (E). (G) Quantification of ClpP-positive cells in primary and brain metastatic NSCLC. (H–J) Kaplan-Meier curves of metastasis-free survival (H and I) or relapse-free survival (J) in the indicated patient series with high or low expression of ClpP. AdCa, adenocarcinoma. Raw data for this figure can be found in S4 Data.
Fig 6
Fig 6. ClpXP regulation of tumor cell proliferation.
(A) PC3 cells were transfected with control siRNA (Ctrl) or ClpX- or ClpP-directed siRNA and analyzed for cell proliferation at the indicated time intervals by direct cell counting. (B and C) PC3 or DU145 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were analyzed for colony formation by crystal violet staining after 7 d (B, PC3) and quantified (C). *, p = 0.01; **, p = 0.006. (D) PC3 cells transfected with the indicated siRNAs as in (A) were analyzed by western blotting. (E and F) PC3 cells transfected with the indicated siRNAs (E) or shRNAs (F) were incubated with the combination of antioxidants NAC (N) plus mitochondrial-directed MitoTempo (MT) and analyzed for cell proliferation by direct cell counting (E) or colony formation (F). *, p = 0.028; **, p = 0.0026. (G) PC3 cells treated with control siRNA (Ctrl) or ClpP-directed siRNA were transfected with vector or MnSOD cDNA, and analyzed for cell proliferation by direct cell counting. **, p = 0.001. (H) PC3 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were incubated with the indicated increasing concentrations of H2O2 (mM) and analyzed for inhibition of cell proliferation by direct cell counting. (I and J) PC3 cells transfected with the indicated siRNAs were incubated with increasing galactose:glucose ratios (I) or amino acid-deprived medium (50% amino acids, J) and analyzed for cell proliferation by direct cell counting. FU, fluorescence units. (K) PC3 cells transfected with ClpP-directed siRNA were reconstituted with ClpP cDNA and analyzed by western blotting. Raw data for this figure can be found in S5 Data.
Fig 7
Fig 7. Requirement of ClpXP for tumor cell invasion and metastasis.
(A and B) PC3 cells transfected with control siRNA (Ctrl) or ClpX- or ClpP-directed siRNA were analyzed for directional cell migration in a wound closure assay by light microscopy (ClpP, A), and wound surface area was quantified after 18 h (B). ***, p < 0.0001. (C) PC3 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were analyzed for cell invasion across Matrigel-coated inserts. ***, p < 0.0001. (D) PC3 cells transfected with ClpP-directed siRNA were reconstituted with ClpP cDNA and analyzed for cell migration in a wound closure assay as in (A and B). (E) PC3 cells transfected with control siRNA (Ctrl) or ClpX- or ClpP-directed siRNA were analyzed for Matrigel invasion in the presence of vehicle (Veh) or the combination of antioxidants NAC (N) plus MitoTempo (MT). ***, p < 0.0001. (F) PC3 cells transfected with control siRNA (Ctrl) or ClpP-directed siRNA were analyzed for Caveolin-1 (Cav1) mRNA levels by quantitative PCR. (G and H) PC3 cells were transfected with the indicated siRNAs, reconstituted with vector or Cav1 cDNA, and analyzed by western blotting (G) or Matrigel invasion (H). ***, p < 0.0001. (I) PC3 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were injected in the spleen of immunocompromised mice and livers collected after 11 d, which were analyzed by hematoxylin-eosin staining and light microscopy. (J) Quantification of metastatic burden (% metastasis, top), number of metastatic foci (middle), and size of metastatic foci (bottom) in mice intrasplenically injected with shRNA-transduced PC3 cells as in (I). **, p = 0.002; ***, p < 0.0001. Raw data for this figure can be found in S6 Data.

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