Up-regulation of the ATPase inhibitory factor 1 (IF1) of the mitochondrial H+-ATP synthase in human tumors mediates the metabolic shift of cancer cells to a Warburg phenotype

Abstract

The H(+)-ATP synthase is a reversible engine of mitochondria that synthesizes or hydrolyzes ATP upon changes in cell physiology. ATP synthase dysfunction is involved in the onset and progression of diverse human pathologies. During ischemia, the ATP hydrolytic activity of the enzyme is inhibited by the ATPase inhibitory factor 1 (IF1). The expression of IF1 in human tissues and its participation in the development of human pathology are unknown. Here, we have developed monoclonal antibodies against human IF1 and determined its expression in paired normal and tumor biopsies of human carcinomas. We show that the relative mitochondrial content of IF1 increases significantly in carcinomas, suggesting the participation of IF1 in oncogenesis. The expression of IF1 varies significantly in cancer cell lines. To investigate the functional activity of IF1 in cancer, we have manipulated its cellular content. Overexpression of IF1 or of its pH-insensitive H49K mutant in cells that express low levels of IF1 triggers the up-regulation of aerobic glycolysis and the inhibition of oxidative phosphorylation with concurrent mitochondrial hyperpolarization. Treatment of the cells with the H(+)-ATP synthase inhibitor oligomycin mimicked the effects of IF1 overexpression. Conversely, small interfering RNA-mediated silencing of IF1 in cells that express high levels of IF1 promotes the down-regulation of aerobic glycolysis and the increase in oxidative phosphorylation. Overall, these findings support that the mitochondrial content of IF1 controls the activity of oxidative phosphorylation mediating the shift of cancer cells to an enhanced aerobic glycolysis, thus supporting an oncogenic role for the de-regulated expression of IF1 in cancer.

FIGURE 1.

Expression of IF1 in human tumors. A, purification of recombinant IF1 (r-IF1). The gel shows protein extracts from noninduced (−) and induced (+) bacterial extracts and the purified recombinant IF1. B and C, IF1 and β-F1-ATPase (βF1) expression in different human tissues (B) and cell lines (C). The migration of the native IF 12-kDa isoform (n-IF1) is indicated. In the right panel, two different exposures of the IF1 film are presented. Cells with high and low IF1 content could be distinguished. D, immunofluorescence microscopy of HeLa cells stained with 200 nm MitoTracker (red) and with the IF1 monoclonal antibody (green) revealing the co-localization (Merge) of IF1 in mitochondria. Images are shown at ×63 magnification. Bar, 20 μm. E–H, Western blots of IF1 and β-F1-ATPase (βF1) in paired normal (N) and tumor (T) biopsies derived from three representative patients are shown. The histograms represent the fold of control of the IF1/βF1 ratio in ductal invasive breast (E, n = 9), colon (F, n = 12), and lung (G, n = 15) adenocarcinomas and squamous lung carcinomas (H, n = 7) relative to paired normal samples. *, p < 0.05 when compared with normal by Student's t test.

FIGURE 2.

Overexpression of IF1 or H49K triggers the induction of aerobic glycolysis. A–C, histograms show the changes in the rates of aerobic glycolysis (Ctr, open bars) mediated by the overexpression of IF1 (dotted bars) or H49K (hatched bars) in NRK (A), Hepa 1–6 (B), and HepG2 (C) cells, as representative examples of cell lines with negligible or low content of IF1 (Fig. 1C). For comparison, the effect of 6 μm oligomycin (OL.) treatment (closed bars) on the rates of aerobic glycolysis in the same cellular types (A–C) is shown. *, p < 0.05 when compared with control by Student's t test. Multiple comparisons by analysis of variance and post hoc Dunnett's tests confirmed the statistical significance reported except in HepG2 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant.

FIGURE 3.

Overexpression of IF1 or H49K triggers the inhibition of oxidative phosphorylation (A) and mitochondrial hyperpolarization (B–D). A, NRK cells were transfected with green fluorescent (control (Ctr), open bar), IF1 (dotted bar), or H49K (hatched bar) plasmids, and the effect on oligomycin-sensitive respiration (OSR) was determined. *, #, p < 0.05 when compared with control or IF1 by Student's t test, respectively. B–D, histograms show the relative changes in the mitochondrial membrane potential (ΔΨ_m) (open bars) mediated by the overexpression of IF1 (dotted bars) or H49K (hatched bars) in NRK (B), Hepa 1–6 (C), and HepG2 (D) cells. The effect of 6 μm oligomycin (OL) (closed bars) in ΔΨ_m (B–D) is shown. D, HepG2 Tet-On advanced cell line was transfected with a bidirectional luciferase-galactosidase (control), a luciferase-IF1 (IF1), or a luciferase-H49K (H49K) doxycycline (Dox.)-responsive plasmid. The relative changes in luciferase activity or in ΔΨ_m in induced (+Dox) and noninduced (−Dox) cells are shown. *, #, p < 0.05 when compared with control or noninduced cells by Student's t test, respectively. In all panels, multiple comparisons by analysis of variance and post hoc Dunnett's tests confirmed the statistical significance reported.

FIGURE 4.

Silencing of IF1 triggers the inhibition of aerobic glycolysis and activates oxidative phosphorylation. The rates of aerobic glycolysis (A) and oxidative phosphorylation (B) were determined in HeLa cells expressing an inefficient (control: Ctr, open bars) or IF1 siRNA to promote the silencing of IF1 (closed bars). A, effect of 6 μm oligomycin treatment (OL. +) is shown in control and IF1-silenced cells. *, and #, p < 0.05 when compared with control or oligomycin-treated cells by Student's t test, respectively.