MitoQ Inhibits Human Breast Cancer Cell Migration, Invasion and Clonogenicity

Abstract

To successfully generate distant metastases, metastatic progenitor cells must simultaneously possess mesenchymal characteristics, resist to anoïkis, migrate and invade directionally, resist to redox and shear stresses in the systemic circulation, and possess stem cell characteristics. These cells primarily originate from metabolically hostile areas of the primary tumor, where oxygen and nutrient deprivation, together with metabolic waste accumulation, exert a strong selection pressure promoting evasion. Here, we followed the hypothesis according to which metastasis as a whole implies the existence of metabolic sensors. Among others, mitochondria are singled out as a major source of superoxide that supports the metastatic phenotype. Molecularly, stressed cancer cells increase mitochondrial superoxide production, which activates the transforming growth factor-β pathway through src directly within mitochondria, ultimately activating focal adhesion kinase Pyk2. The existence of mitochondria-targeted antioxidants constitutes an opportunity to interfere with the metastatic process. Here, using aggressive triple-negative and HER2-positive human breast cancer cell lines as models, we report that MitoQ inhibits all the metastatic traits that we tested in vitro. Compared to other mitochondria-targeted antioxidants, MitoQ already successfully passed Phase I safety clinical trials, which provides an important incentive for future preclinical and clinical evaluations of this drug for the prevention of breast cancer metastasis.

Keywords: MitoQ; breast cancer; clonogenicity; invasion; metastasis; migration; mitochondria; mitochondria-targeted antioxidant; mitochondrial superoxide; spheroids.

Figure 1

Determination of MitoQ levels in mouse plasma following per os administration. (a) Chemical formula of MitoQ showing the antioxidant coenzyme Q10 moiety, the linker and the positively charged triphenylphosphonium (TPP⁺) group that addresses the drug to mitochondria. (b) Female BALB/c mice received increasing doses of MitoQ per os, and blood was collected 4 h later for analysis using LC/MS/MS. The graph shows the plasma concentration of MitoQ in function of the administered dose (n = 9–14). All data are shown as means ± SEM. * p < 0.05; ns: p > 0.05 compared to control; by one-way ANOVA followed by Dunnett’s post hoc test (b).

Figure 2

MitoQ selectively represses mitochondrial superoxide production by human breast cancer cells. Cells were treated ± MitoQ 100 nM for 48 h. (a) The oxygen consumption rate (OCR) of MDA-MB-231 cells was measured using Seahorse oximetry. The graph represents OCR measurements over time with the sequential addition of oligomycin, FCCP, and rotenone (Rot) together with antimycin A (AA). From Seahorse traces, basal, maximal and ATP-linked mitochondrial oxygen consumption rates (mtOCRs) were calculated (n = 8–18). (b) The mitochondrial potential (Δψ) of MDA-MB-231 cells was measured using JC-10 (n = 16). (c) Mitochondrial superoxide (mtO₂⁻) levels were measured using electron paramagnetic resonance (EPR) with MitoTEMPO-H as a selective mtO₂⁻ sensor ± PEG-SOD2 (n = 6). (d) Seahorse oximetry as in a, but using human SkBr3 breast cancer cells (n = 29–41). (e) Δψ measurement as in b, but using SkBr3 cells (n = 16). (f) Determination of mtO₂⁻ levels as in c, but using SkBr3 cells (n = 4). (g) Seahorse oximetry as in a, but using human MDA-MB-436 breast cancer cells (n = 16). Note that SEMs are smaller than symbols in the left graph showing oximetry traces. (h) Δψ measurement as in b, but using MDA-MB-436 cells (n = 8). (i) Determination of mtO₂⁻ levels as in c, but using MDA-MB-436 cells. (n = 4). (j) Seahorse oximetry as in a, but using nonmalignant MCF10A human breast epithelial cells (n = 18–24). (k) Δψ measurement as in b, but using MCF10A cells (n = 8). (l) Determination of mtO₂⁻ levels as in c, but using MCF10A cells (n = 3). All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control; ns: p > 0.05 compared to control; by Student t-test (a–l).

Figure 3

MitoQ increases glucose consumption and lactate release by human breast cancer cells, and is cytostatic at doses ≥ 250 nM. (a) MDA-MB-231 cells were treated ± MitoQ 100 nM for 48 h. Glucose consumption (left), lactate production (middle) and the lactate/glucose ratio (right) were then determined using enzymatic assays on a CMA600 analyzer (n = 3 all). (b) Viable MDA-MB-231 cells were counted on a SpectraMax i3 spectrophotometer at the indicated time points after treatment with increasing doses of MitoQ (n = 4). (c) Enzymatic measurements of glucose and lactate consumption as in a, but using SkBr3 cells (n = 10). (d) SkBr3 cell viability was determined as in b (n = 8). (e) Enzymatic measurements of glucose and lactate consumption as in a, but using MDA-MB-436 cells (n = 10). (f) MDA-MB-436 cell viability was determined as in b (n = 8). (g) Enzymatic measurements of glucose and lactate consumption as in a, but using MCF10A normal epithelial breast cells (n = 4). (h) MCF10A cell viability was determined as in b (n = 8). All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control; ns: p > 0.05 compared to control; by Student t-test (a,c,e,g) or 2-way ANOVA with Tukey’s post hoc test (b,d,f,h).

Figure 4

MitoQ has mitigated effects on the epithelial to mesenchymal transition (EMT) of human breast cancer cells. Cells were treated ± MitoQ for 48 h. (a) Representative immunocytological pictures where MDA-MB-231, SkBr3 and MDA-MB-436 cells are stained with hematoxylin and eosin. Bars = 1 mm. (b) Cells were stained with primary antibodies anti-vimentin and anti-E-cadherin (green fluorescence), and nuclei were stained with DAPI (blue). Representative pictures are shown on top, and the graphs on the bottom show the fluorescence intensity for MDA-MB-231 (n = 9–12), SkBr3 (n = 9–12) and MDA-MB-436 (n = 11-12) cells. Bars = 20 µm. (c) mRNA expression of EMT markers vimentin (VIM), SNAIL (SNAI1), SLUG (SNAI2), ZEB1 and TWIST1 in MDA-MB-231 cancer cells treated ± 500 nM MitoQ for 48 h (n = 6–9). (d) Western blots (WBs) of the corresponding proteins with β-actin as a loading control. (e) mRNA expression in c, but in SkBr3 cells (n = 3–9). (f) WBs as in d, but using SkBr3 cells. (g) mRNA expression in c, but in MDA-MB-436 cells (n = 6–9). (h) WBs as in d, but using MDA-MB-436 cells. All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control; ns: p > 0.05 compared to control; by Student t test (b,c,e,g).

Figure 5

MitoQ represses human breast cancer cell migration and invasion. Cells were treated for 48 h ± MitoQ (100 nM). (a) MDA-MB-231 (left, n = 6), SkBr3 (middle, n = 3–8) and MDA-MB-436 (right, n = 10–13) cancer cell migration over 24 h was determined using a scratch assay. Representative pictures are shown on top and quantification graphs on the bottom. Bars = 50 µm. (b) MDA-MB-231 (left, n = 3), SkBr3 (middle, n = 3) and MDA-MB-436 (right, n = 3) cancer cell invasion was quantified in a Boyden chamber assay. Representative images are shown together with overnight invasion data. All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control; by Student t-test (a,b).

Figure 6

MitoQ represses human breast cancer cell clonogenicity, sphere formation and spheroid stability. (a,b) Cells were pretreated for 48 h with the indicated doses of MitoQ. (a) Clonogenic assay using adherent MDA-MB-231 (left, n = 6) and SkBr3 (right, n = 9) cells. Bars = 1 cm. (b) Clonogenic assay on soft agar using MDA-MB-231 (n = 16), SkBr3 (n = 16) and MDA-MB-436 (n = 8) cells. (c) Shown are representative images of MDA-MB-231, SkBr3 and MDA-MB-436 spheroid formation over 4 days in the presence or not of 250 nM MitoQ. Bar = 250 µm. (d) Representative images of mature MDA-MB-231 spheroids before and after 4 days of treatment ± 250 nM MitoQ are shown on the left (Bar = 250 µm). On the right, the graph represents spheroid size determined using the bright field mode of phase contrast microscope (n = 9–11). (e) As in d, but using MDA-MB-436 cells and a treatment of 7 days (n = 6 all; bars = 50 µm for images on the top and 200 µm for images on the bottom). (f) mRNA expression of cancer stem cell markers MYC, POU5F1 (Oct4), NANOG and SOX2 in MDA-MB-231 (left, n = 3–7), SkBr3 (middle, n = 4–6) and MDA-MB-436 (right, n= 4–8) spheres treated for 48 h ± 250 nM MitoQ. All data are shown as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control; ns: p > 0.05 compared to control; by Student t test (a,d,f) or one-way ANOVA followed by Dunnett’s post hoc test (b,e).