Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

Abstract

Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin-2 (Mfn-2), which promotes fusion, and dynamin-related protein (Drp-1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp-1/Mfn-2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp-1 and decrease in Mfn-2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn-2, Drp-1 inhibition, or Drp-1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp-1 inhibition (P<0.001). In a xenotransplantation model, Mfn-2 gene therapy or Drp-1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm(3) (P<0.05) with Mfn-2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm(3) (P<0.01) with therapeutic Drp-1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.

Figure 1.

Mitochondrial fragmentation in human lung cancer cells. A) Cells were loaded with mitochondrial red fluorescent dye TMRM and imaged with confocal microscopy to assess the mitochondrial network structure. The acquired images were background subtracted, filtered (median), thresholded, and binarized to identify mitochondrial segments using ImageJ. Continuous mitochondrial structures were counted with the particle counting subroutine, and the number was normalized to the total mitochondrial area (in pixels) to obtain the MFC for each imaged cell. For every cell line or intervention, ≥n = 25 randomly selected cells were imaged to calculate the respective MFC values. Scale bar = 10 μm (all images). B) Representative images of the mitochondrial imaging from cultured human lung cancer cell lines (A549, H358, H1993, HCC827) or human lung epithelial and vascular cells (hSAECs, hBECs, hPAECs, hPASMCs) shows a marked predominance of mitochondrial network fragmentation in the cancer cells. C) Quantification of the MFC confirms that all the lung cancer cells lines have a significantly higher level of mitochondrial network fragmentation that any of the nonmalignant human lung epithelial or vascular cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. nonmalignant cells; ANOVA with post hoc test.

Figure 2.

Impaired mitochondrial networking in human lung cancer cells. A) Green and red fluorescence images of cells transfected with mito-PA-GFP and the mitochondrial red fluorescent protein (mito-DS-Red) were acquired by confocal microscopy. Following regional photoactivation, activated GFP distributes within fused mitochondria and the extent of distribution indicates the degree of mitochondrial networking. Green fluorescence outside this activation box is defined as the MNF. The first image following photoactivation is used for image analysis. B) Representative images for control human pulmonary cell types (left) and lung cancer cells (right): A549, H1993, and H358. Quantification of MNF demonstrates higher levels of mitochondrial networking in control cells when compared to all lung cancer cells (means±se, n≥6). *P < 0.05, **P < 0.01, ***P<0.001. C) Quantitative PCR of some key mitochondrial fusion and fission mediators and the mitochondrial mass marker TOM20 in A549 cells and control hSAECs shows a reduction in the expression of Mfn-2 and an increase in Drp-1 in A549 cells (means±se, n≥3). NS, not significant. ***P < 0.001; t test.

Figure 3.

Decreased Mfn-2 and increased Drp-1 in human lung cancer tissue and evidence of Drp-1 activation in lung cancer cells. A) Hematoxylin-eosin and immunofluorescence staining in representative human lung and adjacent adenocarcinoma samples. Scale bars = 100 μm. Left panels are stained for Mfn-2 (red) and the proliferation marker PCNA (green). Right panels are stained for Drp-1 (red), the proliferation marker PCNA (green) and the nuclear stain DAPI (blue). B) Quantification of immunofluorescence staining (n=5 patients) in relative fluorescence units (RFU, y axis) demonstrates lower levels of Mfn-2 and markedly higher levels of Drp-1 in human lung adenocarcinoma samples (means±se, t test). NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001). C) Representative immunoblot to assess Drp-1 and Mfn-2 expression in multiple cancer cell lines and control cell lines. Separate gels were run for Drp-1 and Mfn-2 immunoblot analysis, due to their proximity in size and to avoid artifacts related to stripping membranes. Only one representative actin loading control is shown, but actin loading of all lanes was similar on the Mfn-2 and Drp-1 gels. D) Immunoblot analysis of Drp-1 phosphorylation was determined with phospho-specific antibodies directed against either phospho-Ser-616 (enhances Drp-1 activity) or phospho-Ser-637 (suppresses Drp-1 activity) in healthy epithelial cells (hBECs) and two distinct human lung adenocarcinoma cell lines (A549 and H1993). Mean intensity for each mitochondrial protein (n≥3, mean intensity normalized to hBEC levels) was quantified and statistical analysis was performed comparing each cancer cell line to hBECs (mean±se, n≥5). *P < 0.05, **P < 0.01, ***P < 0.001 vs. hBECs.

Figure 4.

Restoration of mitochondrial networking in A549 cancer cells. A) Reversal of the mitochondrial fragmentation in cancer cells was achieved by either using siRNA mediated knockdown of the fission mediator Drp-1 or inhibition of Drp-1 with mdivi-1 or by overexpression of Mfn-2. Images are representative of A549 cells undergoing these interventions or the scramble siRNA control transfection. Scale bar = 10 μm. All 3 interventions achieve a marked reduction in the MFC. ***P < 0.001 vs. control A549 or scramble siRNA-transfected A549 cells; ANOVA with post hoc test. B) Confocal microscopy time-lapse images of human lung adenocarcinoma cells expressing the mitochondrial-targeted fluorescent proteins mito-DS-Red (red) and photoactivable GFP (green). Representative confocal microscopy time-lapse images of human lung adenocarcinoma cells treated with the control vehicle (top panels), Ad-Mfn-2 (middle panels, 24 h post-transfection), or mdivi-1, an inhibitor of the mitochondrial fission mediator (bottom panels, 4–6 h following 30 μM mdivi-1 treatment). Quantification of the MNF (y axis) shows marked increases in the MNF (means±se, n≥6). *P < 0.05, **P < 0.01; t test.

Figure 5.

Mfn-2 gene therapy promotes apoptosis and suppresses proliferation in A549 cancer cells. A) Compartment-specific immunoblot analysis was performed to analyze the mitochondrial and cytosolic distribution of cytochrome c. Left panel: representative immunoblot demonstrates that Ad-Mfn-2 therapy induces mitochondrial apoptosis, as evidenced by redistribution of mitochondrial cytochrome c to the cytosolic fraction, when compared to untreated A549 cells (Ctrl) or A549 cells treated with an adenoviral control vector (Ad-Vec). Right panel: quantification of the normalized mean intensity for cytochrome c in mitochondrial and cytosolic compartments (means±se, n=4). *P < 0.05; t test. B) Induction of spontaneous apoptosis with Ad-Mfn-2 transfection in A549 cells was assessed using TUNEL staining. Left panel: representative immunofluorescence images show an increase in TUNEL-positive cells (arrows) with Ad-Mfn-2. Scale bars = 200 μm. Right panel: quantification confirms a marked increase in the percentage of TUNEL-positive cells (means±se, n=15). ***P < 0.001 vs. other treatments. C) Proliferation of A549 cells was determined by flow cytometric assessment of BrdU incorporation at 24 h following the transfection. Quantification demonstrates a dose-dependent effect of Ad-Mfn-2 (varying MOI on x axis) on the inhibition of cancer cell proliferation when compared to control transfected cells at 24h post-transfection (mean±se percentage of BrdU-positive cells on y axis, n=3). ***P < 0.001; t test. D) Cellular redox state was assessed using a roGFP construct. The y axis of the bar graph shows the redox ratio of cells treated with a control adenoviral vector, with adenoviral Mfn-2, or with the combination of adenoviral Mfn-2 and mitochondrial-targeted catalase. A higher redox ratio indicates a higher oxidation state (means±se, n≥5). ***P < 0.001.

Figure 6.

Inhibition of mitochondrial fission with mdivi-1 increases apoptosis and reduces proliferation in A549 cancer cells. A) Flow cytometric assessment of apoptosis by annexin V staining in A549 cells treated with either DMSO or the Drp-1 inhibitor mdivi-1 (30 μM for 24 h) showed a significant increase in the percentage of annexin V⁺/PI⁻ cells (y axis) with mdivi-1 therapy (means±se, n=3). *P < 0.05; t test. B) Time course analysis of apoptosis, quantified by TUNEL staining, showed that induction of apoptosis required 16 h of treatment with mdivi-1 (means±se, n≥10 at each time point). **P < 0.01 vs. 0 h; ANOVA with post hoc testing. C) TUNEL staining was performed on A549 cells undergoing si-RNA knockdown of Drp-1. Percentage of TUNEL-positive cells markedly increased with knockdown of Drp-1 in A549 cells (means±se, n≥10). *P < 0.05 vs. scramble; ANOVA with post hoc analysis. D) Flow cytometric cell cycle analysis was performed using BrdU uptake as a marker of cell proliferation (S phase) and the DNA stain 7-AAD. E) Mdivi-1(24 h, 30 μM) markedly reduces the percentage of proliferating cells in S phase and concomitantly increases the percentage of cells in G2 phase (meas±se, n=3) **P < 0.01, ***P < 0.001; t test.

Figure 7.

Enhancing mitochondrial network formation suppresses tumor growth in vivo. A) Tumor size assessed over time in nude mice is shown as calculated volume (mm³; means±se, n=9/group). Therapeutic interventions were applied 2 wk after tumor implantation (d 0). Ad-Mfn-2 and mdivi-1 both prevented tumor progression (P<0.05 vs. control Ad-GFP and P<0.01 vs. control DMSO, respectively, by repeated measures ANOVA). B) Representative excised tumors from animals that were treated either with control therapies (Ad-GFP or DMSO) or the fusogenic therapies (Ad-Mfn-2, mdivi-1). C) Tumor volume (mm³ on y axis, n=9/group) was measured radiographically by micro-CT 2–3 wk post-therapy and demonstrated a significant decrease in tumor volume with Ad-Mfn-2 therapy or mdivi-1 treatment. *P < 0.05, **P < 0.01; t test. D) ¹⁸F-FDG uptake in implanted xenograft tumors was measured by PET; representative scans are shown for the respective treatment groups, and a color scale is attached to indicate the glucose uptake. Quantification of ¹⁸F-FDG uptake (¹⁸F-FDG counts/mm³, y axis) was normalized to tumor volume. No significant effect was found with Ad-Mfn-2 therapy on ¹⁸F-FDG uptake of the residual tumor, but an increase was found in ¹⁸F-FDG uptake of residual tumors in mdivi-1-treated tumors (means±se, n=9/group) *P < 0.05; t test.