Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 11;8(389):ra80.
doi: 10.1126/scisignal.aab1624.

Survivin Promotes Oxidative Phosphorylation, Subcellular Mitochondrial Repositioning, and Tumor Cell Invasion

Affiliations
Free PMC article

Survivin Promotes Oxidative Phosphorylation, Subcellular Mitochondrial Repositioning, and Tumor Cell Invasion

Dayana B Rivadeneira et al. Sci Signal. .
Free PMC article

Abstract

Survivin promotes cell division and suppresses apoptosis in many human cancers, and increased abundance correlates with metastasis and poor prognosis. We showed that a pool of survivin that localized to the mitochondria of certain tumor cell lines enhanced the stability of oxidative phosphorylation complex II, which promoted cellular respiration. Survivin also supported the subcellular trafficking of mitochondria to the cortical cytoskeleton of tumor cells, which was associated with increased membrane ruffling, increased focal adhesion complex turnover, and increased tumor cell migration and invasion in cultured cells, and enhanced metastatic dissemination in vivo. Therefore, we found that mitochondrial respiration enhanced by survivin contributes to cancer metabolism, and relocalized mitochondria may provide a "regional" energy source to fuel tumor cell invasion and metastasis.

Conflict of interest statement

COMPETING INTERESTS: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. Survivin targeting impairs mitochondrial metabolism
(A–F) Global metabolomics screening of PC3 cells transfected with control or survivin-directed (SVV) siRNA (n=5 biological replicates; see also table S1). Changes in the concentrations of metabolites implicated in oxidative phosphorylation (A and B), fatty acid β oxidation (C and D), or glutathione metabolism (E and F) are shown. For the heatmap (D), red indicates increased concentration; green indicates decreased concentration. Only significant changes (p<0.05) are shown. For box plots (B and F), relative metabolite abundance is represented. The limit of upper and lower quartiles, median values (straight line), and maximum and minimum distribution are shown. Cross, mean value; circle, extreme data point.
Fig. 2
Fig. 2. Survivin regulation of mitochondrial bioenergetics
(A) Prostate cancer cell lines PC3 or DU145 or glioblastoma LN229 cells transfected with control (Ctrl) or survivin-directed (SVV) siRNA were analyzed for oxygen (O2) consumption. RFU, relative fluorescence units. The results of two independent experiments are shown. (B) siRNA silenced cells as in (A) were analyzed for O2 consumption rates (OCR) under basal conditions or in response to oligomicyin (O), FCCP (F), or antimycin (A). Arrows, time of drug addition. Extra-mitochondrial respiration after addition of antimycin was subtracted as background. The profiles of two independent experiments (Expt.) are shown. (C) PC3 cells were treated with vehicle (Veh) or small molecule survivin suppressant YM155 and analyzed for O2 consumption. RFU, relative fluorescence units. Two independent experiments (Expt.) are shown. (D) PC3 cells transfected with the indicated siRNA or treated with vehicle (Veh) or YM155 were analyzed for ATP production. Graph shows means±SEM from three independent experiments. ***, p<0.0001; **, p=0.0011. (E) PC3 cells transfected with control siRNA (Ctrl) or a second independent siRNA to survivin (SVV2) were analyzed for ATP production. Two independent experiments (Expt.) are shown. (F) PC3 cells transfected with control (Ctrl) or survivin-directed siRNA were reconstituted with vector or mt-SVV cDNA and analyzed for ATP production. Graph shows means±SEM from three independent experiments. ***, p<0.0001. (G) PC3 cells were transfected with control siRNA (Ctrl) or survivin (SVV)-directed siRNA, reconstituted with pAd-mitochondrial-targeted GFP (pAd-mt-GFP) or pAd-mitochondrial-targeted survivin (pAd-mt-SVV), and analyzed for O2 consumption. RFU, relative fluorescence units. Two independent experiments (Expt.) are shown. (H and I) PC3 cells transfected with control or survivin-directed siRNA (H) or treated with vehicle or YM155 (I) were analyzed by Western blotting. d, days. p, phosphorylated. Blots are representative of two independent experiments. (J) PC3 cells transfected with control (Ctrl) or survivin-directed siRNA (SVV) plus GFP-LC3 were analyzed by fluorescence microscopy. Right, the number of GFP-LC3 puncta (autophagosomes) per cell was quantified (n=50 cells imaged/transfection condition in two independent experiments). Scale bar, 10 μm. (K) PC3 cells treated with vehicle (Veh), Rapamycin (Rap) or YM155 were analyzed by Western blotting after 24 h. p, phosphorylated. Blots are representative of two independent experiments.
Fig. 3
Fig. 3. Survivin regulation of mitochondrial Complex II
(A) PC3 cells treated with vehicle (Veh) or YM155 were permeabilized with digitonin and analyzed for Complex II activity in the presence of succinate and rotenone. Oligo, oligomycin. Graphs shows means±SEM from three independent experiments. **, p<0.01. (B) PC3 cells transfected with control (Ctrl) or survivin-directed (SVV) siRNA were analyzed by Western blotting. Blots are representative of two independent experiments. (C) Mitochondrial Complex II was immunoprecipitated from siRNA-transfected PC3 cells as in (B) and analyzed for enzymatic activity. Right, quantification. Graph shows means±SEM from three independent experiments. *, p=0.037. (D) Mitochondria from PC3 cells treated with vehicle (Veh) or YM155 were analyzed by Western blotting. The position of oxidative phosphorylation complex subunits is indicated. d, days. Blots are representative of two independent experiments. (E and F) PC3 cells treated with vehicle (Veh) or YM155 (E), or MCF-7 cells transfected with vector or mt-SVV cDNA (F) were analyzed by Western blotting. In (F), the position of endogenous (SVV) or transfected (HA) survivin is indicated. Blots in (E) and (F) are representative of two independent experiments. (G) PC3 cells were transfected with the indicated siRNAs and proteins remaining insoluble at increasing detergent concentrations (CHAPS) were analyzed by Western blotting. The extra band in the SDHC lane corresponds to non-specific reactivity with a molecular weight marker. Blots are representative of two independent experiments. (H) Mitochondrial extracts from PC3 cells were immunoprecipitated (IP) with IgG or an antibody to survivin, and pellets were analyzed by Western blotting. Blots are representative of two independent experiments. (I) Aliquots of GST-TRAP-1 (top) or GST (bottom) were incubated with recombinant survivin (SVV), and bound proteins were analyzed by Western blotting. The molar ratio of survivin to TRAP-1 was 0, 0.1, 0.5 and 1. Blots are representative of two independent experiments. (J) PC3 mitochondrial extracts were immunoprecipitated (IP) with IgG or an antibody to survivin, and pellets were analyzed by Western blotting. Blots are representative of two independent experiments. (K and L) PC3 cells were transfected with control (Ctrl) or TRAP-1-directed siRNA (K), or treated with mitochondrial-targeted small molecule Hsp90 inhibitor, Gamitrinib (Gam) (L), and extracts were analyzed after 48 h (K) or at the indicated time intervals (L), by Western blotting. Blots are representative of two independent experiments for (K) and (L). (M and N) PC3 cells were treated with vehicle (Veh) or Gamitrinib (Gam), then cycloheximide, and analyzed by Western blotting (M), with quantification of survivin half-life by densitometry (N). The quantification from two independent experiments (Expt.) is shown.
Fig. 4
Fig. 4. Survivin-mediated regulation of subcellular mitochondrial trafficking
(A) PC3 cells were treated with vehicle (Veh) or YM155, labeled for mitochondria (MitoTracker, red), focal adhesion (FA) complexes (Paxillin, green) and DNA (DAPI, blue), and analyzed by confocal microscopy. 20 cells/treatment condition imaged in two independent experiments. Scale bar, 10 μm; insets, 5 μm. (B) PC3 cells treated as in (A) were labeled for mitochondria (MitoTracker, red), actin (phalloidin, green) and DNA (DAPI, blue), and analyzed by confocal microscopy (left). Right, masked images used to quantify mitochondrial localization to phalloidin-positive protrusions. 20 cells/treatment condition imaged in two independent experiments. Scale bar, 10 μm. (C and D) The conditions are as in (B), and the amount of cortical mitochondria (C) or total mitochondria (D) was quantified in the presence of vehicle or YM155. Each point corresponds to an individual measurement. 45 cells/treatment condition imaged in two independent experiments. ***, p<0.0001; n.s., not significant. (E and F) MCF-7 cells were transduced with vector or mt-SVV, labeled for mitochondria (MitoTracker, red) and paxillin (green) and analyzed by fluorescence microscopy (E). Right, masked (right panels) images were used to quantify mitochondrial localization to paxillin-containing regions (F). Data are expressed as percent of mitochondria localization to paxillin-containing focal adhesion (FA) complexes per cell. Mean±SEM. Vector, n=26 cells; mt-SVV, n=27; imaged in two independent experiments. *, p=0.0013. Scale bar, 10 μm. (G–I) The condition are as in (B) except that PC3 cells were treated with the mitochondrial uncoupler CCCP (G), the small molecule Complex II inhibitor TTFA (H), or Gamitrinib (I) and analyzed by confocal microscopy (top). Bottom, masked images used to quantify mitochondria localization to phalloidin-positive cellular protrusions. 45 cells/treatment condition imaged in two independent experiments. Scale bar, 10 μm. (J and K) Treated PC3 cells were analyzed by confocal microscopy as in (G–I), and the amount of cortical mitochondria (J) or total mitochondria (K) was quantified. Each point corresponds to an individual measurement. ***, p<0.0001; **, p=0.0013. 45 cells/treatment condition imaged in two independent experiments.
Fig. 5
Fig. 5. Mitochondrial bioenergetics requirements of membrane dynamics
(A) Representative stroboscopic images of FBS-stimulated PC3 cells treated with vehicle, YM155 or the Complex II inhibitor TTFA. Inset, SACED parameters of cell protrusion dynamics. (B) Quantification of membrane ruffle frequency in PC3 cells treated with vehicle (Veh), YM155 or TTFA. Each bar corresponds to an individual cell. Cells were imaged in two independent experiments. Broken lines, average values. The p values in YM155- or TTFA-treated cultures compared to Veh are indicated. (C–E) PC3 cells treated with vehicle (Veh), YM155 or TTFA were analyzed by SACED stroboscopic microscopy for average frequency of membrane ruffling (C), ruffle distance traveled (D), or ruffle persistence time (E). The quantified values are as follows (number of cells examined in parentheses): frequency of membrane ruffling (C), Veh, 1.44±0.14 (n=18); YM155, 0.61±0.14 (n=22), p=0.0002; TTFA, 0.68±0.14 (n=21), p=0.0008; ruffle distance traveled (D), Veh, 35±3.9 (n=18); YM155, 14.2±3.2 (n=22), p=0.0002; TTFA, 16±3.89 (n=21), p=0.0016; time of ruffle persistence (E), Veh, 243.3±24.3 (n=18); YM155, 136.4±26.9 (n=22), p=0.006; TTFA, 120.3±24.1 (n=21), p=0.001. Cells were imaged in two independent experiments. (F) PC3 cells treated with vehicle (Veh) or YM155 were analyzed by Western blotting. p, phosphorylated. Blots are representative of two independent experiments. (G) PC3 cells were treated with vehicle (Veh) or YM155, labeled with an antibody to phosphorylated FAK (Tyr397, pFAK) and analyzed by confocal microscopy. DNA was stained with DAPI. Right, quantification of phosphorylated FAK clusters. Mean±SEM. ***, p<0.0001. 20 cells/treatment condition imaged in two independent experiments. Scale bar, 10 μm. (H) PC3 cells treated as in (G) were transfected with α-actinin-GFP cDNA and analyzed by confocal microscopy. 10 cells (10 movies)/treatment condition imaged in two independent experiments. ***, p<0.0001. Scale bar, 10 μm. Right, FA dynamics quantified by time-lapse videomicroscopy. FA complexes examined: Veh, n=42; YM155, n=47.
Fig. 6
Fig. 6. Regulation of tumor cell motility, invasion, and metastasis by mitochondrial survivin
(A) siRNA-transfected PC3 cells were analyzed in a scratch closure assay and individual cell movements were tracked by time-lapse videomicroscopy. Ctrl siRNA, number of cells examined (n) =29; SVV siRNA, n=30; imaged in two independent determinations. (B) Average cell velocity (top) or total distance traveled (bottom) of the cells in (A) was quantified per each condition. Mean±SEM. p<0.0001. (C–E) PC3 cells treated with vehicle (Veh) or YM155 or transfected with survivin-directed siRNA (SVV2) were analyzed for Matrigel invasion. DAPI-stained nuclei of invaded cells (C) after YM155 treatment (D) or survivin knockdown (E) were quantified. Mean±SEM. ***, p<0.0001. 100–250 cells/treatment or transfection condition imaged in two independent experiments. Scale bar, 200 μm. (F) PC3 cells silenced for survivin by siRNA were reconstituted with vector or mt-SVV cDNA and analyzed for Matrigel invasion. Mean±SEM. **, p=0.0026; ***, p<0.0001. 100–250 cells/treatment or transfection condition imaged in two independent experiments. (G) INS-1 or MCF-7 cells transfected with vector or mt-SVV cDNA were analyzed for Matrigel invasion. Mean±SEM. **, p=0.003; ***, p<0.0001. 100–250 cells/treatment or transfection condition imaged in two independent experiments. (H) MCF-7 cells transfected with vector or mt-SVV cDNA were injected in the spleen of immunocompetent mice (three mice per transfection condition). Representative images of hematoxylin-eosin stained liver sections (n=15 per each transfection condition). Scale bar, 500 μm. (I and J) Morphometric quantification of total number of metastatic foci (I) and metastatic surface area (J) in serial liver sections (n=15 per each transfection condition). Mean±SEM. ***, p<0.0001.
Fig. 7
Fig. 7. Requirements of mitochondrial regulation in tumor cell invasion
(A–C) PC3 cells were incubated with vehicle or the Complex I inhibitor Rotenone (Rot) (A), the Complex II inhibitor TTFA (B), or the Complex III inhibitor antimycin A (C) and analyzed for Matrigel invasion. Right panels, quantification of DAPI-stained nuclei of invaded cells in the presence of the various inhibitors. Mean±SEM. **, p=0.0053; ***, p<0.0001. 100–250 cells/treatment condition imaged in two independent experiments. Scale bar, 200 μm. (D and E) PC3 or MDA-231 cells were transfected with control (Ctrl) or SDHB-directed siRNA, and analyzed for Matrigel invasion (D) or cell migration (E). Mean±SEM. **, p=0.001–0.0028; ***, p<0.0001. 100–250 cells/treatment condition imaged in two independent experiments. (F and G) PC3 cells transfected with vector or FAK cDNA were treated with vehicle (Veh) or YM155 (F), or silenced for SDHB by siRNA (G) and quantified for Matrigel invasion. Mean±SEM. *, p=0.03; **, p=0.0032; ***, p<0.0001. 100–250 cells/treatment condition imaged in two independent experiments.

Similar articles

See all similar articles

Cited by 31 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback