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. 2019 Jul 21;11(7):1027.
doi: 10.3390/cancers11071027.

Mutations in the ND2 Subunit of Mitochondrial Complex I Are Sufficient to Confer Increased Tumorigenic and Metastatic Potential to Cancer Cells

Joaquín Marco-Brualla  1 , Sameer Al-Wasaby  1 , Ruth Soler  1 , Eduardo Romanos  2 , Blanca Conde  3 , Raquel Justo-Méndez  4 , José A Enríquez  4 , Patricio Fernández-Silva  5 , Luis Martínez-Lostao  6 , Martín Villalba  7   8 , Raquel Moreno-Loshuertos  9 , Alberto Anel  10
Affiliations

Mutations in the ND2 Subunit of Mitochondrial Complex I Are Sufficient to Confer Increased Tumorigenic and Metastatic Potential to Cancer Cells

Joaquín Marco-Brualla et al. Cancers (Basel). .

Abstract

Multiprotein complexes of the mitochondrial electron transport chain form associations to generate supercomplexes. The relationship between tumor cell ability to assemble mitochondrial supercomplexes, tumorigenesis and metastasis has not been studied thoroughly. The mitochondrial and metabolic differences between L929dt cells, which lost matrix attachment and MHC-I expression, and their parental cell line L929, were analyzed. L929dt cells have lower capacity to generate energy through OXPHOS and lower respiratory capacity than parental L929 cells. Most importantly, L929dt cells show defects in mitochondrial supercomplex assembly, especially in those that contain complex I. These defects correlate with mtDNA mutations in L929dt cells at the ND2 subunit of complex I and are accompanied by a glycolytic shift. In addition, L929dt cells show higher in vivo tumorigenic and metastatic potential than the parental cell line. Cybrids with L929dt mitochondria in L929 nuclear background reproduce all L929dt properties, demonstrating that mitochondrial mutations are responsible for the aggressive tumor phenotype. In spite of their higher tumorigenic potential, L929dt or mitochondrial L929dt cybrid cells are sensitive both in vitro and in vivo to the PDK1 inhibitor dichloroacetate, which favors OXPHOS, suggesting benefits for the use of metabolic inhibitors in the treatment of especially aggressive tumors.

Keywords: ND2; complex I; cybrids; dichloroacetate; metastasis; mitochondria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mitochondrial supercomplex assembly and mitochondrial electron transport chain (mETC) complexes activity. (A) Mitochondria from L929 and L929dt cells were isolated, permeabilized using digitonin and mtETC complexes and supercomplexes were separated using blue native polyacrylamide gel electrophoresis (BNGE). Afterwards, proteins were transferred to a membrane and probed by immunoblot with monoclonal antibodies against complex I (anti-NDUFB6), II (anti-SDHA), III (anti-Core2) and IV (anti-Co1). The different supercomplexes (SC) and other associations are indicated on the blots: CI SC, supercomplexes that contain complex I: I + III or I + III + IV; CIII SC, supercomplexes that contain complex III; CIV SC, supercomplexes that contain complex IV. Data are representative of 6 different determinations. The amount of complex II in the same samples was used as loading control. (B) Left panel, BNGE followed by complex I in gel activity of the mitochondrial preparations solubilized with digitonin from L929 and L929dt cultured cells. Right panel, specific activities of mtETC complexes measured by spectrophotometry in mitochondria isolated from L929 and L929dt cells. All values are given as mean ± SD of the mean (n ≥ 3 in all cases). Asterisks indicate significant differences respect to L929 cells. *, p < 0.05; **, p < 0.02; ***, p < 0.01.
Figure 2
Figure 2
Analysis of mitochondrial respiration and glycolysis. (A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) measurement upon sequential addition of oligomycin, FCCP, and rotenone+antimycin A in L929 (black dots) and L929dt (grey dots) cells using the Seahorse XF96 Extracellular Flux Analyzer. (C) ECAR to OCR ratio at basal respiration in both cell lines. Data are given as mean ± SEM with 8 independent assays per cell line. Asterisks indicate significant differences respect to L929 cells p < 0.0001 using non parametric Mann Whitney test.
Figure 3
Figure 3
ROS formation, catalase and SOD activity in L929 and L929dt cells at the basal level. (A) Detection of superoxide anion by oxidation of dihydroethidium (2HE) and (B) oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) at the basal level in L929 (solid histogram) or L929dt cells (dashed histogram) were analyzed by flow cytometry. (C) Catalase specific activity was measured in cell homogenates as described in Materials and Methods. n = 3, ***, p < 0.001. (D) Mn-superoxide dismutase specific activity of L929 and L929dt cells in basal state was measured by spectroscopy, as indicated in Material and Methods. n = 3. ** p < 0.01.
Figure 4
Figure 4
In vivo tumor development of L929 and L929dt cells in athymic mice. (A,B) 1 × 106 L929 or L929dt living cells were injected s.c., respectively, in groups of 4 mice each. After three weeks, mice were sacrificed and tumors surgically excised. (A) Examples of the appearance and size of L929 or L929dt-derived tumors; (B) Mean ± SD of the tumor weights, expressed in grams. *, p < 0.05. (C,D) 125,000 L929 or L929dt living cells were injected intraesplenically, respectively, in groups of 5 mice each. After three weeks, mice were sacrificed and spleens surgically excised. (C) Examples of the appearance and size of L929 and L929dt-derived spleen tumors (squared areas); (D) Mean ± SD of tumor volumes, expressed in mm3. *, p < 0.05.
Figure 5
Figure 5
Hematoxilin/eosin staining in histological sections of L929 or L929dt-derived tumors in athymic mice. Paraffin tissue sections were stained with standard H&E coloration, as indicated in Material and Methods. Magnification was 100× in all images. N, necrotic areas; V, vessel-like structures; T, trabecular structures, all detected in L929dt-derived tumors, but not in L929-derived ones.
Figure 6
Figure 6
Characterization of complex I mtDNA mutations. Chromatograms showing the m.4206C > T and the m.4859C > T mutations within the mt-Nd2 gene in L929dt cells.
Figure 7
Figure 7
Properties of cybrid cells. L929dt cybrid cells contain L929dt mitochondria on a L929 nuclear background and L929dtL929 cells contain L929 mitochondria on an L929dt nuclear background. (A) Phase contrast micrographs of cultures of L929, L929dt, or cybrid cells. (B) MHC-I expression on the surface of the indicated cells, determined by flow cytometry using a specific anti-H-2Kk antibody conjugated with FITC. Grey histograms, labeling of the cells alone; blue histograms, labeling with an irrelevant antibody of the same isotype and conjugated with FITC; orange histograms, labeling with the anti-H2-Kk mAb conjugated with FITC. (C) Supercomplex formation involving complex I, complex III or complex IV in the different cell types, determined by immunoblot in the same way as indicated in the legend of Figure 3. The amount of complex II in the same samples was used as loading control. (D) Oxygen consumption rate (OCR) and (E) extracellular acidification rate (ECAR) measurement upon sequential addition of oligomycin, FCCP, and rotenone + antimycin A in L929 (black dots and solid black lines), L929dt (grey dots and solid grey lines), L929dt (grey dots and dashed lines) and dtL929 (black dots and dashed lines) cells using the Seahorse XF96 Extracellular Flux Analyzer. (F) ECAR to OCR ratio at basal respiration in the four cell lines, as indicated. Data are expressed as in Figure 2. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 8
Figure 8
Effect of DCA on cell growth and cell death of the different cell lines studied. (A) Cells were supplemented or not (Control) during 72 h with the indicated concentrations of DCA, and every 24 h, cell growth was determined by the MTT reduction method and results expressed as percentage of cell growth relative to control cells. Results are the mean ± SD of three different experiments. *, p < 0.05; **, p < 0.02; ***, p < 0.01. (B) Cells were supplemented or not (Control) during 72 h with the indicated concentrations of DCA. Then, cells were stained with annexin-V-FITC and PS exposure, as a marker of apoptosis induction, was analyzed by flow cytometry. Results are the mean ± SD of three different experiments. *, p < 0.05; **, p < 0.02; ***, p < 0.01.
Figure 9
Figure 9
Effect of DCA on MHC-I expression. L929 or L929dt (A), and L929dtL929 or L929dt cells (B) were supplemented or not (-) during 72 h with the indicated concentrations of DCA and MHC-I expression determined by flow cytometry using a specific anti-H-2Kk antibody conjugated with FITC. Dashed or grey histograms, labeling with an irrelevant antibody of the same isotype and conjugated with FITC; solid histograms, labeling with the anti-H2-Kk mAb conjugated with FITC. Data are representative of at least three different experiments.
Figure 10
Figure 10
In vivo effect of DCA. 1 × 106 L929, L929dt, L929dtL929 or L929dt living cells were injected s.c., respectively, in groups of 6 mice each. When the tumor began to be detectable (20 mm3), each experimental group was divided in two, and three mice received intratumoral injections of 50 µL PBS every day during 10 days (Control, CTRL; black triangles) while the other three mice received injections of 25 mM DCA in 50 µL PBS with the same time schedule (DCA; grey circles). Data show tumor size during the 10 days of the treatments for each individual mouse. † indicates the sacrifice of mice due to ethical reasons before the end of the 10-day period.
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