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. 2015 Jul 14;112(28):8638-43.
doi: 10.1073/pnas.1500722112. Epub 2015 Jun 29.

PI3K Therapy Reprograms Mitochondrial Trafficking to Fuel Tumor Cell Invasion

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Free PMC article

PI3K Therapy Reprograms Mitochondrial Trafficking to Fuel Tumor Cell Invasion

M Cecilia Caino et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.

Keywords: PI3K; cell invasion; cytoskeleton; mitochondria; molecular therapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PI3K therapy-induced tumor transcriptional reprogramming. (A) Heat map of changes in kinome functions in patient-derived GBM organotypic cultures treated with vehicle or PX-866 (10 μM for 48 h). N, number of genes; %, percentage of genes changed for any given function. (B) Extracts from GBM organotypic cultures treated with vehicle (Veh) or PX-866 (10 μM for 48 h) were incubated with a human phospho-RTK array followed by enhanced chemiluminescence detection. The position and identity of phosphorylated proteins are indicated. M, markers. (C and D) PC3 cells were treated with vehicle or the indicated PI3K inhibitors for 48 h and analyzed for changes in Akt (C) or mTOR (D) activation by Western blotting. p, phosphorylated. (E and F) Primary GBM spheroids treated with vehicle or 10 μM PX-866 for 48 h were imaged by phase-contrast and fluorescence microscopy for phosphorylated Akt (Ser473) (E) or mTOR (Ser2448) (F). DNA was counterstained with DAPI. Nestin is a GBM marker. (G) Heat map of changes in kinome pathways in GBM organotypic cultures treated with vehicle or PX-866 (10 μM) for 48 h. N, number of changed genes; Z, z score of the estimated function state: positive (red) indicates overall function is likely increased; negative (blue) indicates it is decreased.
Fig. S1.
Fig. S1.
Molecular therapy-induced tumor reprogramming. (A) Extracts from vehicle (Veh)- or PX-866–treated (10 μM for 48 h) LN229 cells were incubated with a human phospho-RTK array and immunoreactive spots were visualized by enhanced chemiluminescence. The position and identity of phosphorylated proteins are indicated. M, markers. (B and C) PC3 cells were treated with the indicated PI3K inhibitors, and changes in the levels of the indicated phosphorylated proteins were quantified by densitometry and normalized to total levels. (D) PC3 cells were treated with vehicle or the indicated PI3K inhibitors for 48 h and analyzed for invasion across Matrigel. Representative images of invaded cells are shown. Magnification, 10×. (E and F) The experimental conditions are as in D, and the area occupied by invasive cells (E) or the number of cells before seeding (F) was quantified. Mean ± SEM (n = 3). ***P < 0.0001. (G and H) Patient-derived GBM spheroids were treated with vehicle or PX-866 (0–10 μM) for 48 h, stained with the vital dye PKH26, and the number of spheres per field (G) and sphere diameter (H) were quantified after 48 h. Mean ± SEM (n = 5 patients). *P = 0.01; **P = 0.0048; ***P = 0.0004.
Fig. 2.
Fig. 2.
PI3K therapy induces adaptive tumor cell motility and invasion. (A) Tumor cells treated with vehicle or 10 μM PX-866 for 48 h were analyzed for invasion across Matrigel-coated Transwell inserts (Top) or in 3D spheroids (Bottom). Red, invasive edge; green, core. Representative images. Magnification, 10×. (B) PC3 (Top) or LN229 (Bottom) cells were treated with the indicated increasing concentrations of PX-866 and quantified for invasion across Matrigel (Top) or in 3D spheroids (Bottom). The distance between the core and edge of 3D spheroids was determined. Mean ± SEM of replicates from a representative experiment. *P = 0.02; ***P < 0.0001. (C) Patient-derived GBM spheroids were treated with vehicle or PX-866 (0–10 μM) for 48 h and analyzed by phase-contrast (Top) or fluorescence microscopy (Bottom). The vital dye PKH26 was used to counterstain live GBM neurospheres. (Scale bar, 20 μm.) (D) Membrane ruffling was quantified in PC3 cells treated with vehicle or PI3K inhibitors for 48 h by SACED microscopy. Average values from at least 330 ruffles per treatment are shown for ruffle size (Top) and time of ruffle persistence (Bottom). Mean ± SEM (n = 15). ***P < 0.0001. (E) Representative stroboscopic images from time-lapse video microscopy of PC3 cells treated with vehicle or PI3K inhibitors. Four SACED regions corresponding to the top (1), right (2), bottom (3), and left (4) of each cell are shown. The ruffling activity (broken yellow lines) is restricted to one main region (1) on the vehicle cell but is distributed equally between three regions (1–3) on cells treated with PI3K inhibitors. See also Movie S1. (F) PC3 cells were treated with vehicle or PI3K inhibitors, and membrane dynamics at lagging areas were quantified. Ruffle size (Left) or time of ruffle persistence (Right) from at least 205 individual lagging ruffles are shown. Mean ± SEM (n = 15). **P = 0.0047; ***P < 0.0001. (G) PC3 cells were treated with vehicle or PX-866 for 48 h and quantified for directional versus random cell migration by time-lapse video microscopy (8 h). Rose plots show the distribution of cells migrating along each position interval (range interval 10°, internal angle 60°). Arrows indicate the direction of chemotactic gradient.
Fig. S2.
Fig. S2.
PI3K therapy stimulates membrane ruffle dynamics. (A) PC3 cells treated with vehicle or the indicated PI3K inhibitors for 48 h were analyzed by time-lapse video microscopy. Four discrete regions per cell were followed over time to derive stroboscopic images by SACED. (Left) Still images at the beginning of the sequence. Yellow lines indicate the discrete areas analyzed for a cell. (Right) Zoomed-in micrographs of the ruffles being analyzed (arrows). (B) PC3 cells were treated with vehicle or the indicated PI3K inhibitors and analyzed by stroboscopic microscopy. Images representing the dynamics of the cell border over 300 s are shown. The positions of membrane ruffles and cell bodies are indicated. (CE) PC3 cells (n = 15) as in B were analyzed for membrane ruffle dynamics by SACED with quantification of average ruffle size (C), time of ruffle persistence (D), and ruffle frequency (E). Each bar corresponds to an individual cell. Mean ± SEM (n = 4).
Fig. S3.
Fig. S3.
Adaptation-induced random tumor cell migration. (A) PC3 cells were treated with vehicle or the indicated PI3K inhibitors for 48 h, seeded onto 2D chemotaxis chambers, and analyzed by time-lapse video microscopy. The sequences were started immediately after setting up a gradient of NIH 3T3 conditioned media used as a chemoattractant. Videos were analyzed with WimTaxis software (Wimasis) and tracking data were exported into the Chemotaxis and Migration tool (Ibidi) for representation. Still images from the time lapse with trajectories overlaid at the indicated time intervals are shown. (Scale bars, 50 μm.) (B) PC3 cells treated with vehicle or PI3K inhibitors were analyzed for 2D chemotaxis. The trajectory (Top) and end points (Bottom) represented for all analyzed cells are shown; n ≥ 103. A circular region that splits the Euclidean distances 50:50 is indicated (radius). (C and D) PC3 cells were treated as in B, and speed of migration (C) or distance of migration (D) were quantitated by 2D chemotaxis analysis. ***P < 0.0001.
Fig. 3.
Fig. 3.
Mitochondria fuel focal adhesion dynamics. (A) PC3 cells treated with vehicle or PI3K inhibitors for 48 h were stained with MitoTracker Red, phalloidin Alexa488, and DAPI, and full cell stacks were used to generate 3D max projection images that were scored for mitochondrial morphology (polarized, perinuclear, infiltrating). (B) Representative confocal 3D max projection images of PC3 cells treated with vehicle or the indicated PI3K inhibitors and stained as in A. (Bottom) Models for quantification of mitochondrial trafficking. Mito, mitochondria. White lines indicate the distance from nuclei to the cell border. Yellow lines indicate the length of mitochondrial infiltration into membrane lamellipodia. Magnification, 63×. (C) PC3 cells treated with vehicle or the indicated PI3K inhibitors were labeled as in A and quantified for mitochondrial infiltration into lamellipodia. At least 18 cells were analyzed at two independent lamellipodia, and data were normalized to total lamellipodia length. Mean ± SEM (n = 36). ***P < 0.0001. (D) Lung adenocarcinoma A549 or glioblastoma LN229 cells were labeled as in A and scored for mitochondrial infiltration into membrane lamellipodia by fluorescence microscopy. Mean ± SEM. **P = 0.0056; ***P < 0.0001. (E) PC3 cells were transfected with control (Ctrl) or MFN1-directed siRNA, labeled as in A, and quantified for mitochondrial infiltration in the cortical cytoskeleton in the presence of vehicle or PX-866. Mean ± SEM. ***P < 0.0001. (F) PC3 cells transfected with control or MFN1-directed siRNA were treated with vehicle or PX-866 and analyzed for Matrigel invasion after 48 h. Mean ± SEM. ***P = 0.0002. (G) PC3 cells treated with vehicle or PI3K inhibitors for 48 h were replated onto fibronectin-coated slides for 5 h and labeled with an antibody to phosphorylated FAK (pY925) Alexa488, MitoTracker Red, and DAPI. Representative 1-µm extended-focus confocal images with localization of mitochondria near FA complexes are shown. Magnification, 63×. (Scale bar, 10 μm.) (H) PC3 cells expressing Talin-GFP to label FA were treated as indicated and quantified for decay, formation, and stability of FA complexes per cell over 78 min; n = 631. See also Movie S2.
Fig. S4.
Fig. S4.
Mitochondrial infiltration to the cortical cytoskeleton. (A) PC3 cells labeled with MitoTracker Red, phalloidin Alexa488, and DAPI were analyzed by fluorescence microscopy. Representative pseudocolored fluorescence microscopy images of different mitochondrial morphology (polarized, perinuclear, infiltrating) are shown. Magnification, 60×. (B) PC3 cells were treated with vehicle or PI3K inhibitors for 48 h, stained with MitoTracker Red, phalloidin Alexa488, and DAPI, and full cell stacks were used to generate 3D max projection images. Magnification, 63×. (Scale bars, 10 μm.) (C) The indicated tumor cell types were treated with vehicle or PX-866 for 48 h, stained as in B, and analyzed by fluorescence microscopy. (D) PC3 cells were transfected with control siRNA (Ctrl) or mitofusin (MFN)1- or MFN2-directed siRNA and analyzed by Western blotting. (E and F) PC3 cells transfected as in D were analyzed for cell viability by Trypan blue exclusion (E) or normalized ATP production (F). U, units. Mean ± SEM. (G) PC3 cells transfected with control siRNA or MFN1-directed siRNA were stained as in B and analyzed by confocal microscopy for changes in mitochondrial redistribution. Magnification, 63×. Insets were zoomed digitally at 20×. (Scale bar, 5 μm.) Full cell stacks were postprocessed for noise reduction using a median filter in LAS AF. (H) For cortical mitochondrial quantification, masks were manually created around the periphery of the cell based on the F-actin channel and subsequently applied to the mitochondrial channel to measure intensity at the cortical region. The intensity was normalized to total mitochondrial intensity per cell and background-subtracted. ROI, region of interest.
Fig. S5.
Fig. S5.
PI3K therapy regulation of focal adhesion dynamics. (A) PC3 cells treated with the PI3K inhibitor GDC0941 for 48 h were replated onto fibronectin-coated slides for 5 h and labeled with an antibody to phosphorylated FAK (pY925) Alexa488, MitoTracker Red, or DAPI. Representative 1-µm extended-focus confocal images with localization of mitochondria near pFAK+ focal adhesion complexes are shown. Magnification, 63×. Inset was zoomed digitally at 10×. (Scale bar, 10 μm.) (B) LN229 cells were treated with vehicle or PX-866 for 48 h and protein extracts were analyzed by Western blotting. p, phosphorylated. (C) LN229 cells expressing Talin-GFP to label FA complexes were treated with vehicle or PX-866 for 48 h and analyzed by time-lapse microscopy. Representative images at initial (0 min) and final time points (78 min) were merged to visualize FA turnover. Arrows indicate representative decayed (D) or new (N) FAs. Magnification, 40×. (DF) Individual FA complexes in LN229 cells expressing Talin-GFP as in C were manually tracked to determine FA dynamics. (D) Number of dynamic FA complexes per cell classified into disappearing (decayed) and newly formed (new). Mean ± SEM (n = 10). *P = 0.0353; ***P < 0.0001. (E) Turnover rates (number of FA complexes per h) of FA decay and formation. Mean ± SEM (n = 10). *P = 0.0235; ***P < 0.0001. (F) Stable FA complexes classified as sliding (moving) or mature (>50% overlay between initial and final localization). Mean ± SEM (n = 10). *P = 0.0393.
Fig. 4.
Fig. 4.
Control of tumor cell invasion by spatiotemporal mitochondrial bioenergetics. (A) PC3 cells were labeled with MitoTracker Red, phalloidin Alexa488, and DAPI, treated with PX-866, and analyzed for mitochondrial infiltration into the peripheral cytoskeleton in the presence of vehicle or the mitochondrial-targeted ROS scavenger mitoTEMPO (mT; 50 μM). (B) PC3 cells were incubated with the indicated agents alone or in combination (PX-866+mT) and analyzed for tumor cell invasion across Matrigel. Mean ± SEM. P (ANOVA) < 0.0001. (C and D) Mitochondrial (mt)DNA-depleted LN229 (ρ0) cells were stimulated with NIH 3T3 conditioned media for 2 h, labeled with MitoTracker Red, DAPI, and either phalloidin Alexa488 (C) or an antibody to FA-associated paxillin (D), and analyzed by fluorescence microscopy. Representative pseudocolored images are shown. Magnification, 60×. (E) WT or ρ0 LN229 cells were analyzed for invasion across Matrigel-coated Transwell inserts. Representative images of invasive cells stained with DAPI are shown. Magnification, 20×. (F) PC3 cells treated with vehicle or PI3K inhibitors in combination with the mitochondrial-targeted small-molecule Hsp90 inhibitor Gamitrinib (Gam) were labeled with anti–pY925-FAK Alexa488 followed by fluorescence microscopy. Representative 1-µm extended-focus confocal images are shown. Magnification, 63×. (Scale bar, 10 μm.) (G) PC3 cells treated with vehicle or PI3K inhibitors with or without Gamitrinib (1 μM) were labeled with MitoTracker Red, phalloidin Alexa488, and DAPI and quantified after 48 h for mitochondrial infiltration into lamellipodia by fluorescence microscopy; n = 48. Mean ± SEM. **P = 0.0044; ***P < 0.0009. (H) PC3 cells were treated with vehicle or PX-866 (5 μM) with or without Gamitrinib and quantified for invasion across Matrigel. Mean ± SEM of replicates (n = 2). ***P < 0.0001. ns, not significant. (I) PC3 cells were incubated with vehicle or PX-866 alone or in combination with the various mitochondrial respiratory chain inhibitors and analyzed for Matrigel invasion. Ant A, Antimycin A; Oligo, Oligomycin; Roten, Rotenone. Mean ± SEM. **P = 0.006. (J) PC3 cells transfected with control siRNA or siRNA to Akt1/2, mTOR, or FAK were labeled as in C, treated with PX-866, and quantified for mitochondrial infiltration into lamellipodia; n = 44. Mean ± SEM. ***P < 0.0001. (K) siRNA-transfected PC3 cells labeled as in C were treated with PX-866 (5 μM) and analyzed for mitochondrial morphology (polarized, perinuclear, infiltrating) by fluorescence microscopy; n = 21. (L) PC3 cells transfected with the indicated siRNAs were quantified for invasion across Matrigel in the presence of vehicle or PX-866. Mean ± SEM (n = 4). ***P < 0.0001.
Fig. S6.
Fig. S6.
Role of mitochondrial ROS in organelle trafficking and tumor cell invasion. (A and B) PC3 cells were treated with vehicle or the indicated PI3K inhibitors, stained with MitoTracker Green FM and MitoSOX Red mitochondrial superoxide probes, and analyzed by fluorescence microscopy (A) and quantified (B). Menadione (Menad, 100 μM, 1 h) was used as a control stimulus for ROS production. Mean ± SEM. ***P < 0.001; P (ANOVA) = 0.0002. AU, arbitrary units of fluorescence intensity. (C) PC3 cells were treated with vehicle or PX-866 for 48 h with or without the mitochondrial-targeted ROS scavenger mitoTEMPO (mT) and analyzed for ROS levels. Mean ± SEM. **P (ANOVA) = 0.0015. ns, not significant. AU, arbitrary units of fluorescence intensity. (D and E) PC3 cells were incubated with vehicle, mitoTEMPO alone, or in combination with the indicated PI3K inhibitors (E) and analyzed for mitochondrial repositioning to the cortical cytoskeleton by fluorescence microscopy (D) and quantified (E). Mean ± SEM. P (ANOVA) < 0.0001. U, units of fluorescence intensity in the cortical area, normalized to total cell intensity. (F) PC3 cells were incubated with vehicle or PX-866 for 48 h with or without the indicated concentrations (μM) of the pan-ROS scavenger NAC and analyzed for Matrigel invasion. Mean ± SEM. P (ANOVA) < 0.0001.
Fig. S7.
Fig. S7.
Requirement of mitochondrial respiration for FA turnover. (A and B) Respiration-competent (WT) LN229 cells were incubated with Mitotracker Red, phalloidin Alexa488, and DAPI (A) or labeled for FA complexes with an antibody to paxillin (B) and analyzed by fluorescence microscopy. Magnification, 60×. CM, NIH3T3 conditioned media. (C) WT LN229 or mtDNA-depleted LN229 (ρ0) cells expressing Talin-GFP to label FA complexes were analyzed by time-lapse microscopy. Representative images at initial (0 min) and final (45 min) time points of the analysis were merged to visualize FA turnover. Representative images of the different stages of FA complexes (decayed, sliding, stable, or new) are indicated. (D) WT or mtDNA-depleted LN229 (ρ0) cells expressing Talin-GFP were analyzed by time-lapse microscopy and quantitated for decay, formation, and stability of FA complexes per cell over 45 min; n ≥ 431. See also Movie S3. (E) WT or ρ0 LN229 cells were analyzed for invasion across Matrigel-coated Transwell inserts. Mean ± SEM (n = 2). ***P < 0.0001.
Fig. S8.
Fig. S8.
Requirement of oxidative phosphorylation for mitochondrial infiltration to the cortical cytoskeleton. (A) PC3 cells treated with vehicle or PI3K inhibitors in combination with the mitochondrial-targeted small-molecule Hsp90 inhibitor Gamitrinib (Gam) were labeled with MitoTracker Red, phalloidin Alexa488, and DAPI and scored for mitochondrial morphology (polarized, perinuclear, infiltrating) by fluorescence microscopy; n ≥ 42. (B and C) Patient-derived GBM spheroids were treated with increasing concentrations of PX-866 (0–10 μM) for 48 h and imaged by phase-contrast microscopy in the presence or absence of Gamitrinib (5 μM plus PX-866 at 10 μM). The size (B) or number (C) of neurospheres was quantified and normalized on initial size and number per each patient. Mean ± SEM (n = 4 patients). *P = 0.017; ***P < 0.0001. ns, not significant. (D) PC3 cells were transfected with control nontargeting siRNA or TRAP-1–directed siRNA and analyzed by Western blotting. (E) siRNA-transfected PC3 cells as in D were labeled with MitoTracker Red, phalloidin Alexa488, and DAPI with quantification of mitochondrial morphology (polarized, perinuclear, infiltrating) by fluorescence microscopy; n = 32. (F) PC3 cells were labeled as in A, treated with the indicated ETC inhibitors in the presence of PX-866, and analyzed by fluorescence microscopy. Ant. A, Antimycin A. Magnification, 60×.
Fig. S9.
Fig. S9.
Requirement of Akt/mTOR signaling for mitochondrial redistribution to the cortical cytoskeleton. (AC) PC3 cells were transfected with control nontargeting siRNA or siRNA targeting Akt1/2 (A), mTOR (B), or FAK (C) and analyzed by Western blotting. (D) PC3 cells transfected with control siRNA or siRNA directed to Akt1/2, mTOR, or FAK were treated with vehicle or PX-866, labeled with MitoTracker Red, phalloidin Alexa488, and DAPI and analyzed by fluorescence microscopy. Representative pseudocolored images are shown. Magnification, 60×. (E and F) siRNA-transfected PC3 cells were labeled with MitoTracker Red, phalloidin Alexa488, and DAPI, treated with vehicle, and scored for mitochondrial infiltration into lamellipodia (n = 44) (E) or changes in mitochondrial morphology (polarized, perinuclear, infiltrating) (n = 25) (F) by fluorescence microscopy. Mean ± SEM. (G) PC3 cells transfected with the indicated siRNAs were analyzed for invasion across Matrigel in the presence of vehicle (Veh) or PX-866. Representative images are shown. Magnification, 10×.

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