Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression - PubMed (original) (raw)

. 2013 Mar;123(3):1068-81.

doi: 10.1172/JCI64264. Epub 2013 Feb 15.

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Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression

Antonio F Santidrian et al. J Clin Invest. 2013 Mar.

Abstract

Despite advances in clinical therapy, metastasis remains the leading cause of death in breast cancer patients. Mutations in mitochondrial DNA, including those affecting complex I and oxidative phosphorylation, are found in breast tumors and could facilitate metastasis. This study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Specific enhancement of mitochondrial complex I activity inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, nonlethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression rendered tumor cells more aggressive and increased metastasis. The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression.

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Figures

Figure 1

Figure 1. Enhancement of mitochondrial complex I activity by integration of Ndi1: metabolic characterization.

(A) Ndi1 expressed in MDA-MB-435 and MDA-MB-231 human cancer cells upon lentiviral transduction localized to mitochondria, as shown by dual label immunocytochemistry. Control cells (Ctrl) were transduced with empty vector. Shown are Ndi1 (green), mitochondrial complex V (red), and merged (yellow) staining patterns of the same representative fields. Original magnification, ×20. (B) Ndi1 was integrated into the mitochondrial respiratory chain and enhanced tumor cell respiration. Respiration of MDA-MB-435 and MDA-MB-231 (denoted “435” and “231,” respectively) control and Ndi1-expressing cells, measured by oxygen consumption. R, routine respiration; Rot, rotenone (mammalian complex I inhibitor); AA, antimycin A (complex III inhibitor). All parameters were measured by high-resolution respirometry in intact cells. (C) Effects of Ndi1 expression on cellular metabolism in MDA-MB-435 and MDA-MB-231 cells. mtDNA content was analyzed by quantitative real-time PCR and referenced to nuclear genomic DNA. Mitochondrial membrane potential was analyzed by flow cytometry and expressed as geometric mean of the signal. ATP levels were measured by ATP-dependent luciferase activity. Lactate production was measured by fluorometry. Data are mean ± SEM (n = 3). *P < 0.05, unpaired 2-tailed Student’s t test.

Figure 2

Figure 2. Mitochondrial complex I activity modulates tumor growth and metastasis.

(A) Ndi1 expression inhibited mammary fat pad tumor growth of MDA-MB-435 and MDA-MB-231 cells. Control cells were transduced with empty vector (n = 6). (B) Ndi1 expression inhibited lung colonization (experimental metastasis) by MDA-MB-435 or MDA-MB-231 cells after i.v. injection. Control cells were transduced with empty vector (n = 6). (C) Ndi1 expression inhibited multiorgan experimental metastasis, as indicated by noninvasive bioluminescence imaging 7 weeks after i.v. injection of 2.5 × 105 MDA-MB-435 control or Ndi1-expressing cells (n = 5). (D) Knockdown of complex I subunit NFUFV1 expression inhibited complex I activity and respiratory capacity in MDA-MB-435 cells. NDUFV1-knockdown (shV1) and control (shCT) cells were compared. Complex I was immunocaptured from cell lysates, analyzed based on oxidation of NADH to NAD+, expressed as mean OD/min/mg protein (n = 3). Routine mitochondrial respiration, corrected for residual oxygen consumption due to oxidative side reactions, was measured in intact MDA-MB-435 control and NDUFV1-knockdown cells by high-resolution respirometry (n = 3). (E) NDUFV1 knockdown increased lung colonization activity in MDA-MB-435 cells. NDUFV1-knockdown and control cells were compared (n = 8). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, nonparametric Mann-Whitney test (A, B, and E) or unpaired 2-tailed Student’s t test (D).

Figure 3

Figure 3. Mitochondrial complex I activity regulates mTORC1 and autophagy.

(A) Ndi1 expression enhanced resistance to glucose deprivation in MDA-MB-231 cells, shown after 72 hours of incubation in medium with 5 versus 1 mM glucose. Viability was measured by flow cytometry (non–sub-G0/G1 population). n = 3 independent analyses. *P < 0.05, unpaired 2-tailed Student’s t test. (B) Ndi1 expression influenced mTORC1 activity and autophagy. Western blot analysis for p62, phospho-AKT substrates, and the mTORC1 kinase–related substrates phospho-S6Ser240/244 and phospho-4EBPThr37/46 in MDA-MB-435 or MDA-MB-231 control and Ndi1-expressing cells. β-Tubulin was used as protein loading control. Signal quantification, measured by infrared imaging (total of detectable bands) and expressed relative to control, is shown below. Results are representative of 5 independent experiments. Lanes were run on the same gel but were noncontiguous (white lines). (C) H&E staining and p62, Ki67, or Ndi1 expression in mammary fat pad tumors 5 weeks after implanting 2.5 × 105 MDA-MB-435 control versus Ndi1-expressing cells into SCID mice. 2 representative tumors of 6 are shown per group. Original magnification, ×10. (D) Inhibition of complex I activity through NDUFV1 knockdown affected mTORC1 activity and p62 elimination. Western blot analysis for p62, phospho-AKT substrates, phospho-S6Ser240/244, and phospho-4EBPThr37/46, comparing NDUFV1-knockdown versus control MDA-MB-435 cells. Signal quantification, measured by infrared imaging (total of detectable bands) and expressed relative to control, is shown below. Results are representative of 3 independent experiments. Lanes were run on the same gel but were noncontiguous (white lines).

Figure 4

Figure 4. Metastasis inhibition by enhanced complex I activity depends on autophagy.

(A) ATG5 knockdown (shATG5) inhibited autophagy in MDA-MB-435 and MDA-MB-231 control and Ndi1-expressing cells, as shown by p62 and LC3BI accumulation. Signal quantification of ATG5, p62 signal, and LC3BI/II ratios, measured by infrared imaging (total of detectable bands) and expressed relative to control, is shown below. β-Tubulin served as protein loading control. Lanes were run on the same gel but were noncontiguous (white lines). (B) ATG5 knockdown blocked the antimetastatic effect of Ndi1 in MDA-MB-435 and MDA-MB-231 cells. Lung colonization was measured by ex vivo lung imaging 7 weeks after i.v. injection of 2.5 × 105 tumor cells (n = 8 per group). Boxes denote interquartile range; lines within boxes denote median; whiskers denote minima and maxima. *P < 0.05, nonparametric Mann-Whitney test. (C) ATG5 knockdown enhanced multiorgan metastasis and reversed metastasis inhibition by Ndi1 in MDA-MB-435 cells. Shown is noninvasive bioluminescence imaging of 5 representative mice per group at 7 weeks after tail vein injection of 2.5 × 105 MDA-MB-435 control or Ndi1-expressing cells, with or without ATG5 knockdown.

Figure 5

Figure 5. NAD+ level modulation by complex I and NAD+ synthesis and recycling pathways regulate AKT/mTORC1 activity, autophagy, and metastasis.

(A) Ndi1 expression enhanced NAD+/NADH balance. NAD+/NADH ratios in whole-cell or mitochondrial extracts of MDA-MB-435 or MDA-MB-231 control versus Ndi1-expressing cells. Ndi1 stabilized NAD+/NADH ratios, especially under metabolic stress induced by glucose deprivation and hypoxia. NAD+/NADH ratios under stress were measured in whole-cell extracts after 48 hours of culture. (B) Interference with NAD+ synthesis and recycling pathways reduced NAD+/NADH ratios. Knockdown of NAMPT (shNAMPT) in MDA-MB-435 and MDA-MB-231 cells decreased NAD+/NADH ratios (whole-cell extracts after 48 hours growth in 5 mM glucose and normoxia). (C) NAMPT knockdown increased lung colonization activity in MDA-MB-435 and MDA-MB-231 cells (n = 6 per group). (D) NAMPT knockdown affected mTORC1 activity and p62 elimination. Western blot analysis for p62, phospho-AKT substrates, phospho-S6Ser240/244, and phospho-4EBPThr37/46 in MDA-MB-435 and MDA-MB-231 NAMPT-knockdown versus control cells. β-Tubulin served as protein loading control. Signal quantification, measured by infrared imaging (total of detectable bands) and expressed relative to control, is shown below. Lanes were run on the same gel but were noncontiguous (white lines). Results are representative of 3 independent experiments. (A–C) Data are mean ± SEM. *P < 0.05, **P < 0.01, unpaired 2-tailed Student’s t test (A and B) or nonparametric Mann-Whitney test (C).

Figure 6

Figure 6. NAD+ precursor treatment inhibits metastatic activity.

(A) NAD+ precursor treatment enhanced the NAD+/NADH ratio in cultured MDA-MB-435 and MDA-MB-231 parental cells. NAD+/NADH levels were measured after 3 days of cell treatment with 10 mM NIC or NAM in complete medium. n = 3 independent experiments. (B and C) NAD+ precursor treatment of experimental mice inhibited lung metastasis. Lung colonization by MDA-MB-435 (B) or MDA-MB-231 (C) parental cells (2.5 × 105 i.v. each) in mice treated with NIC or NAM (1% in the drinking water ad libitum throughout the experiment). Controls received no treatment (plain drinking water at same pH). Metastatic growth was measured by repeated noninvasive bioluminescence imaging. n = 6 per group. (D) NIC or NAM treatment influenced mTORC1 activity and autophagy. Western blot analysis for p62, phospho-AKT substrates, and phospho-S6Ser240/244 in MDA-MB-435 or MDA-MB-231 parental cells with or without 48 hours of treatment with 10 mM NIC or NAM. β-Tubulin served as protein loading control. Signal quantification, measured by infrared imaging (total of detectable bands) and expressed relative to control, is shown below. Results are representative of 3 independent experiments. *P < 0.05, **P < 0.01, unpaired 2-tailed Student’s t test (A) or nonparametric Mann-Whitney test (B and C).

Figure 7

Figure 7. NAD+ precursor treatment after primary breast tumor removal increases animal survival.

(A) NAM treatment enhanced the NAD+/NADH ratio in cultured 4T1 murine breast carcinoma cells. NAD+/NADH levels were measured after 2 days of cell treatment with 10 mM NAM in complete medium. **P < 0.01, unpaired 2-tailed Student’s t test. n = 2 independent experiments. (B) Weight of 4T1 mammary fat pad tumors from untreated BALB/c mice. Tumors were surgically removed when their volume reached 300 mm3. Data show tumor weight distribution at time of surgery and randomization into groups (n = 8), before treatment was initiated. Boxes denote interquartile range; lines within boxes denote median; whiskers denote minima and maxima. P = 0.7480, unpaired 2-tailed Student’s t test. (C) Kaplan-Meier curves comparing survival of NAM-treated and untreated BALB/c mice after surgical removal of 4T1 mammary fat pad tumors. Mice were untreated or treated with 1% NAM in the drinking water after tumor removal (assigned as day 0). n = 8 per group. P = 0.0386, log-rank test.

Figure 8

Figure 8. NAD+ precursor treatment inhibits spontaneous breast cancer progression in MMTV-PYMT mice.

(A) NAM treatment (1% in drinking water throughout experiment, beginning at weaning) reduced mammary tumor growth. Weights of all 10 mammary fat pads from each treated mouse (n = 10), untreated control mice (n = 11), and untreated age- and strain-matched PyMT-negative mice (Normal; n = 3). Boxes denote interquartile range; lines within boxes denote median; whiskers denote minima and maxima. ***P < 0.001, nonparametric Mann-Whitney test. (B) Fat pads of untreated versus NAM-treated PyMT mice, representative of the 10 fat pad locations. (C) NAM treatment inhibited PyMT-induced breast cancer progression. Percent area of morphological stages of 8 representative fat pads from untreated (PyMT-Ctrl) or NAM-treated (PyMT-NAM) mice. Averages from each group are shown below. Scoring of hyperplasia, adenoma, and early and advanced carcinoma was performed on whole-slide scans of H&E-stained tumor sections by morphometric measurements. (D) Representative microscopic fields of 2 H&E-stained sections from 4 tumors of untreated and NAM-treated PyMT mice. Scale bars: 200 μm (top row for each treatment group); 50 μm (bottom row). (E) Quantification of Western blot analyses of mammary tumors from control and NAM-treated PyMT mice (n = 8 tumors per group). Shown is relative protein abundance of p62, phospho-S6Ser240/244, and phospho-4EBPThr37/46. Boxes denote interquartile range; lines within boxes denote median; whiskers denote minima and maxima. ***P < 0.001, unpaired 2-tailed Student’s t test.

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