Intracellular NAD(H) levels control motility and invasion of glioma cells

Abstract

Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD(+)-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD(+) or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.

Fig. 1

Partial NAMPT inhibition generated cells with graded NAD(H) levels. a Northern blot, showing that downregulation of NAMPT by shRNA in U251 glioma cells results in 90 % knockdown of mRNA transcripts. b Western blotting, confirming that shRNA knockdown also results in 90 % decrease of NAMPT protein levels. c NAD(H) and NADP(H) levels in U251-C and U251-shNAMPT cells. NAD(H) levels in shNAMPT cells are decreased to 40 % of that in controls, while NADP(H) levels remain unchanged. d Pharmacological inhibition of NAMPT by FK866 in U251-C cells results in a stronger inhibitory effect [NAD(H)] decreased to 14 %, and [NADP] decreased to 50 % of the concentration in untreated control cells. e Combined inhibition of NAMPT, by treatment of U251-shNAMPT cells with FK866 resulted in very low [NAD(H)]: 6 % of the level in U251-C controls cells. Also NADP(H) levels appear affected under these conditions. f Cell viability, measured by MTT mitochondrial activity, remains at normal level upon NAMPT knockdown. g Cell proliferation, determined as increase in total protein mass over time, does not differ between U251-C and shNAMPT cells. h Treatment of U251-C cells with FK866 (10 nM) causes no obvious differences in proliferation (grey bars), while high concentration (100 μM) resulted in a stop of cell proliferation (black bars). i The sensitivity of U251-shNAMPT cells for FK866 is increased at least 10,000-fold. At low concentration (grey bars) the viability of U251-shNAMPT cells appeared already compromised, but more pronounced cell death was observed at high concentration of FK866. All experiments were repeated three times and shown as average ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Effects of NAMPT inhibition can be rescued by addition of NAD⁺ or re-expression of NAMPT. Cell proliferation assays of U251-C and U251-shNAMPT cells treated with FK866 in the absence (a) or presence (b–d) of exogenous NAD⁺; 10 μM extracellular NAD⁺ elicits a partial rescue of the proliferation of treated U251-shNAMPT cells. Higher concentrations of NAD⁺ resulted in complete rescue (black bars). e Northern-blot analysis, showing efficacy of re-expression of NAMPT mRNAs. Complementation with an expression vector with NAMPT cDNA insert resulted in 4–5 overexpression of NAMPT transcripts in both control and shNAMPT cells. Asterisk points to a CMV-NAMPT fusion transcript from the re-expression plasmid (not transcribed). f Western-blot analysis, showing that complementation with NAMPT resulted in protein expression in U251-C and U251-shNAMPT that is 1.5–2 times higher than that of endogenous NAMPT in U251 control cells. g Comparison of NAD(H) and NADP(H) levels between complemented and non-complemented shNAMPT U251 cells. Re-expression of NAMPT completely rescued the drop in NAD(H) levels seen upon NAMPT knockdown but overexpression to higher than endogenous NAMPT levels did not result in higher NAD(H) levels. h–j Comparison of cell proliferation capacity between complemented and non-complemented cells in the absence (h) and presence of low (10 nM, i) and high (100 μM, j) concentrations of FK866. Note, that in the absence of FK866, no differences in proliferative capacity are observed (i) and that the high sensitivity for FK866 in U251-shNAMPT cells is completely relieved when NAMPT expression is restored. All experiments were repeated three times and shown as average ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Cell motility is inhibited by low NAD(H) levels. a–c Comparison of 2D cell motility in barrier migration assays. Quantification of migratory parameters shows that U251-shNAMPT cells migrate over smaller distances than control cells (b), but that the directionality of cell movement was comparable (c). Images in a are stills at 24 h from migration movies (Supplementary Movie 1); dotted lines indicate cell front at t = 0 h, bar, 100 μm. d, e Gap closure assays of U251-C (upper row) and U251-shNAMPT cells (lower row) that were left untreated, cultivated in presence of FK866 (10 nM) or co-treated with NAD⁺ (50 μM). Results confirm the slower migration capacity of shNAMPT cells and show that FK866 further inhibits cell motility. The effect of FK866 was rescued by NAD⁺. Images are stills from migration movies (Supplementary Movie 2, 3). Quantification of migration in gap closure assays (e). Bars in the diagram indicate the time it takes to close 50 % of the initial gap. f, g Images and quantification of gap closure of complemented U251-shNAMPT cells. Image in f is derived from migration movies (Supplementary Movie 4) and should be compared to d. Bars in the diagram (g) indicate that gap-closing ability is compromised by knockdown of NAMPT expression, but restored by complementation. h Listing of migratory parameters. Migration is compromised by lower NAD⁺ levels, even when salvage synthesis is only moderately affected as in U251-shNAMPT cells. *n.c.t. no closing time. All experiments were repeated at least three times and values given are averages ± SEM, *p < 0.05, ***p < 0.001

Fig. 4

Matrix degradation by invadopodia is regulated by NAD⁺-levels. a, c Representative images of fixed cells in invadopodia assays stained with phalloidin. a U251-C and shNAMPT cells were left untreated, treated with FK866 or co-treated with NAD⁺ for 24 h. c Similar images of shNAMPT and complemented shNAMPT cells. Cells were allowed to degrade the matrix for 1 h. Bar 10 μm. b, d Quantification of matrix degradation. The extent of degradation was determined and compared relative to controls. The strongest inhibition of invadopodia formation is seen in shNAMPT cells. The effect of FK866 on invadopod formation was milder, though still significant, but could not be rescued by addition of NAD⁺ (b). In contrast, complete rescue of matrix degrading ability was achieved by re-expression of the NAMPT protein in U251-shNAMPT cells (d). Experiments were repeated at least four times and data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Low NAD⁺-levels in U251 interferes with invasive growth. a Time-lapse microscopy of U251 spheroids embedded in Matrigel/agarose lattices for 72 h. Images of spheroid outgrowth are stills at t = 72 h from Supplementary Movie 5. Invasive growth is strongly inhibited in both FK866 (10 nM)-treated U251-C and U251-shNAMPT cells. Black dotted lines indicate spheroid dimensions at t = 0 h. Bar 100 μm. b Quantification of invasive outgrowth of spheroids. FK866 treatment or knockdown of NAMPT lowered the invasive potential of U251 cells. Rescue of outgrowth capacity by NAD⁺ addition or complementation by NAMPT re-expression was partial, but significant. c Inverted invasion of U251 cells against gravity. Images were taken as serial optical sections of the Matrigel-matrix-invading U251 cells. A sequence is given of images at 20 μm spacing at increasing invasion depths from left to right, as indicated at the bottom. d Quantification of inverted invasion. Both the inhibition by FK866 in control cells and in shNAMPT cells was rescued by addition of NAD⁺ and re-expression of NAMPT, respectively. Experiments were repeated at least three times, *p < 0.05, ***p < 0.001

Fig. 6

Lactic acid addition rescues the motility and matrix degradation phenotype induced by low [NAD]⁺. a Intracellular [lactate] in U251-shNAMPT and FK866-treated (10 nM, 24 h) control cells is lower than in U251-C or NAMPT-complemented cells. b Lactate secretion in U251-shNAMPT and FK866-treated (10 nM, 24 h) control cells is not different from U251-C or NAMPT-complemented cells. c, d Reduction of NAMPT activity in U251 cells results in intra- and pericellular pH (pH_i and pH_e) changes. The bar diagrams in c show the pH_i, randomly determined, in cell bodies of U251-C, shNAMPT, and complemented cells. Knockdown or pharmacological inhibition of NAMPT resulted in higher pH_i, whereas NAMPT complementation lowered pH_i significantly, to even beyond normal values. Pericellular pH (pH_e in the glycocalyx, d) varied comparably. e Gap closure assays of U251-shNAMPT cells untreated or treated with FK866 (10 nM) in the presence or absence of lactic acid (LA, 20 mM). LA completely rescued the FK866-effect on gap closure, see also Supplementary Movie 6 (as did NAD⁺ addition, see Fig. 4d). f Quantification of gap closure assays of e. Graph shows the time it takes to close 50 % of the initial gap. g Matrix degradation of U251-C and U251-shNAMPT cells in absence and presence of LA (20 mM). LA rescued the poor capacity to degrade extracellular matrix of U251-shNAMPT cells. All experiments were performed at least three times and shown as average ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Lactic acid and pyruvate contribute to proliferation and motility differences in U251-C and U251-shNAMPT cells. Cell proliferation of U251-C (a, c) and U251-shNAMPT cells (b, d) in low glucose (a, b) and low glucose without pyruvate (c, d), without/with FK866, in the absence or presence of LA. Note that U251-C cells stop proliferating in low glucose after 48 h irrespective of treatment (a, compare with normal glucose, see Supplementary Fig. 6) and become sensitive to FK866 treatment when pyruvate is omitted. This effect could be rescued by the addition of LA for up to 48 h (c). U251-shNAMPT cells stop proliferating after 24 h (b). Without pyruvate, the U251-shNAMPT cells died 1 day earlier than U251-C cells and the effect of FK866 could be (partially) rescued by LA. Both U251-C and U251-shNAMPT cells survive on medium with LA, without pyruvate, and without FK866-treatment. e Gap closure assays in low-glucose medium with and without pyruvate. Migratory capacity of U251-shNAMPT—but not U251-C cells—was reduced by depletion of pyruvate, see also Supplementary Movie 7. f Quantification of gap closure assays of e. All experiments were performed at least three times and shown as average ± SEM, *p < 0.05 **p < 0.01, ***p < 0.001. PYR pyruvate

Fig. 8

LDH knockdown phenocopies the effects of partial NAD⁺ depletion. a Northern blot from U251 cells stably transfected with a LDH-A shRNA producing vector. Knockdown to 55 % of the normal LDH-A RNA level in control cells is achieved. b LDH isoenzyme profile of U251-C and U251-shLDHA cells. LDH-A containing isoform LDH-2 is almost completely absent in U251-shLDHA cells. c Quantification of lactate secretion by U251-C and U251-shLDHA cells. Lactate accumulation was measured in conditioned medium collected from cells after a 24-h cultivation period. d Cell proliferation of U251-C and U251-shLDHA cells with and without FK866 treatment (10 nM). Note that U251-shLDHA and U251-C cells proliferated at comparable rates, but that only U251-shLDHA cells appear sensitive to FK866 treatment. e Gap closure assays of U251-C and U251-shLDHA cells with and without FK866 (10 nM). Both with and without FK866 treatment, U251-shLDHA cells migrated slower than U251-C cells. Images are derived from Supplementary Movie 8. f Quantification of gap closure assays shown in e. Experiments were repeated at least three times, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 9

Scheme summarizing findings on differential effects of NAD⁺ levels on cell proliferation and morphodynamics. Mild NAD⁺ depletion through NAMPT inhibition reduces motility and invasive capacity of glioma cells, severe NAD⁺ depletion affects viability of glioma cells. The upper cell shows metabolites, enzymes, and processes studied in this paper. The white box indicates the NAD⁺-salvage pathway that was inhibited. Pivotal metabolic reaction steps involved in global or local steering of morphodynamic activity (lower cell to left) or coupled to viability and growth control (enzymes and pathways in dying cell to the right) are depicted in this scheme. Steady-state global NAD(H) concentrations are represented by font size and blue shading. G6P glucose-6-phosphate, Glyc3P glyceraldehyde-3-phosphate, TCA tricarboxylic acid cycle, LDH lactate dehydrogenase, NMN nicotinamide mononucleotide, Nam nicotinamide, pH _i intracellular pH, pH _e pericellular pH