Novel role of NOX in supporting aerobic glycolysis in cancer cells with mitochondrial dysfunction and as a potential target for cancer therapy

Abstract

Elevated aerobic glycolysis in cancer cells (the Warburg effect) may be attributed to respiration injury or mitochondrial dysfunction, but the underlying mechanisms and therapeutic significance remain elusive. Here we report that induction of mitochondrial respiratory defect by tetracycline-controlled expression of a dominant negative form of DNA polymerase γ causes a metabolic shift from oxidative phosphorylation to glycolysis and increases ROS generation. We show that upregulation of NOX is critical to support the elevated glycolysis by providing additional NAD+. The upregulation of NOX is also consistently observed in cancer cells with compromised mitochondria due to the activation of oncogenic Ras or loss of p53, and in primary pancreatic cancer tissues. Suppression of NOX by chemical inhibition or genetic knockdown of gene expression selectively impacts cancer cells with mitochondrial dysfunction, leading to a decrease in cellular glycolysis, a loss of cell viability, and inhibition of cancer growth in vivo. Our study reveals a previously unrecognized function of NOX in cancer metabolism and suggests that NOX is a potential novel target for cancer treatment.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. POLGdn expression led to the depletion of mtDNA-encoded respiratory chain components.

(A) POLGdn-pcDNA4/TO construct and nucleotide sequencing analysis confirming the D1135A mutation. (B) Induction of POLGdn expression by doxycycline. T-Rex293 cells carrying POLGdn construction were incubated with doxycycline at an indicated time point. POLGdn expression was detected by anti-FLAG antibody, while both the endogenous POLG and POLGdn proteins were detected by anti-POLG antibody using Western blot assay. (C) Dramatic decrease of mtDNA by expression of POLGdn. Southern blot assay was used to measure mtDNA content. 10 µg total cellular DNA (including genomic DNA and mtDNA) from each sample was digested with SphI to linealize the circular mtDNA, followed by gel electrophoresis. 32P-labeled mitochondrial COII DNA fragment was used as a probe to detect mtDNA. (D) Assay of mtDNA-encoded COII RNA expression by northern blot analysis. (E) Detection of mitochondrial DNA-encoded COII protein by Western blot assay.

Figure 2. Suppression of mitochondrial respiration by POLGdn expression led to an elevation of glycolysis.

(A) Time-dependent decrease in cellular oxygen consumption following POLGdn expression. Reduction of oxygen consumption was observed as early as 2 d after POLGdn expression, and the cells dramatically decreased their ability to consume oxygen with prolonged POLGdn expression. (B) Increased glucose uptake in POLGdn-expressing cells (Tet/on, d12). Cells (2×106) were incubated in 5 ml glucose-free RPMI1640 medium for 2 h, followed by incubation with 0.2 µCi/mL 3H-2-deoxyglucose for 1 h. Cellular uptake of 3H-2-deoxyglucose was determined by liquid scintillation counting after the cells were washed two times with PBS. Error bars, ±SD. p<0.01 (n = 3). (C) Increased lactate generation in Tet/on cells. Lactate in Tet/off and Tet/on (day 12) cells was measured at the indicated time points after changing to fresh culture medium. (D) Increased protein level of hexokinase II (HKII) following POLGdn expression. Upper panels show representative HKII protein by Western blotting assay at the indicated days after POLGdn induction by doxycycline. Lower panels show quantification of Western blot results using scanning and ImageJ software. Results are expressed as integrated optical density. Each sample was normalized to β-actin content. Each bar represents the mean ± SEM of three independent experiments. * p<0.05; ** p<0.01. (E) Comparison of cellular ATP levels in cells with or without POLGdn expression. Cellular ATP contents in Tet/on cells (days 8 and 12) were measured and compared with the Tet/off cells. Error bars, ±SD (** p<0.01 and n = 3).

Figure 3. Alterations in ROS generation and SOD expression in cells with mitochondrial defect induced by POLGdn.

(A) Lower cellular O2 − in POLGdn-expressing cells (Tet/on, day 12). O2 − was detected by flow cytometry using 200 ng/ml HET as fluorescent dye (p<0.001, Tet/off versus Tet/on and n = 3). (B) Comparison of mitochondrial O2 − in cells with POLGdn expression (Tet/on day 12) or without POLGdn (Tet/off). Mitochondrial O2 − was detected by flow cytometry using 5 µm MitoSox Red as fluorescent dye (p<0.001, Tet/off versus Tet/on and n = 3). (C) Increase in cellular H2O2 level in POLGdn expressing Tet/on (day 12) cells. Cellular H2O2 was measured by flow cytometry using 4 µm DCF-DA as a fluorescent dye (p<0.001, Tet/off versus Tet/on, and n = 3). (D) Protein level of mitochondrial superoxide dismutase (SOD2) in cells at the indicated time points after POLGdn induction. SOD2 was assayed by Western blot analysis. β-actin was used as a loading control. (E) Protein level of cytosolic superoxide dismutase (SOD1) in cells at the indicated time points after POLGdn induction. Left panels show representative Western blots, and right panels show quantification of normalized SOD1 levels to β-actin controls from three independent experiments. Data are shown in mean ± SEM. * p<0.05. (F) Changes in SOD1 activity in cells at the indicated time points after POLGdn induction. Error bars, ±SD. *** p<0.001 (n = 3).

Figure 4. Cells with mitochondrial respiratory defects exhibit elevated NOX activity and are sensitive to NOX inhibition.

(A) Increase of membrane-associated NOX activity in cells with mitochondrial respiratory defects after induction of POLGdn expression (Tet/on, 2, 8, 12 d). Error bars, ±SD. ** p<0.01 (n = 3). (B) Inhibition of NOX enzyme activity by DPI. The Tet/on cells (day 8) were treated with 10 µM DPI, 100 µM L-NAME, 20 µM rotenone, or 100 µM oxypurinol for 4 h, and the membrane-associated fractions were prepared for analysis of NOX activity. Error bars, ±SD. *** p<0.001 (n = 3). (C) Increase in mRNA expression of NOX family members in Tet/on (day 2) cells, measured by qRT-PCR analysis. Error bars, ±SD. ** p<0.01 (n = 3). (D) Comparison of changes in mitochondrial transmembrane potential in Tet/off and Tet/on cells treated with DPI. Cells were pre-induced by doxycycline for 7 d and then incubated with the indicated concentrations of DPI for 24 h. Mitochondrial transmembrane potential was measured by flow cytometry using Rhodamine-123 as a potential-sensitive dye. Cells without DPI treatment were marked as control (Cont). (E) Cells with mitochondrial respiratory defect (Tet/on, day 8) were more sensitive to DPI treatment (10 µM, 48 h) compared with the Tet/off cells. Cell viability was measured by annexin-V/PI assay. (F) Increase of NOX activity in mDNA-less HL60-C6F (C6F) cells. Mean ± SD. *** p<0.001 (n = 3). (G) Increase in NOX family mRNA expression in C6F cells, measured by qRT-PCR assay. Data are shown as mean ± SD of triplicate samples from two independent experiments. * p<0.5; *** p<0.001. (H) C6F cells were more sensitive to DPI treatment. HL60 and its derived C6F cells were treated with indicated concentration of DPI for 48 h. Cell viability was measured by annexin-V/PI assay.

Figure 5. NOX supports glycolysis in cells with mitochondrial respiratory defects induced by POLGdn.

(A) Inhibition of glucose uptake by siRNA knockdown of NOX1 or p22phox in cells with mitochondrial respiratory defects (Tet/on, day 8), but not in cells with intact mitochondria (Tet/off). p<0.05 (n = 3). Insert: Tet/off and Tet/on (at day 4) cells were transiently transfected with p22phox siRNA and the knockdown efficiency was detected by anti-p22phox antibody using Western blot. Non-targeting control siRNA (Scram or sc) was used as negative control. (B) Effect of NOX1 knockdown on ATP contents in cells with intact mitochondria (Tet/off) and cells with mitochondrial respiratory defect (Tet/on, day 8). p<0.01 (n = 4). (C) HPLC analysis of cellular NAD+ and NADH levels. Standard NAD+ and NADH were used as references, which were monitored simultaneously at 260 nm and 340 nm, respectively. A lower NAD+ content was detected in the Tet/on cells with mitochondrial respiratory dysfunction and higher glycolytic activity that consumed more of NAD+. Inhibition of NOX activity by p22phox siRNA and DPI resulted in further decrease in cellular NAD+ level. (D) Quantitation of intracellular NAD+ and NADH in triplicate experiments, using HPLC method as described above. (E) Effect of POLGdn expression on cellular NADP+/NADPH ratio. *** p<0.001. (F) Knockdown of p22phox decreased NADPH/NADH oxidase activity. Comparing to the control siRNA knockdown in Tet/on (d8) cells, p22phox knockdown significantly decreased cellular NADPH/NADH oxidase activity using either NADHP or NADH as substrate. All error bars, ±SD. ** p<0.01; *** p<0.001 and n = 3. (G) HIF-1α is not stabilized following POLGdn induction. Protein level of HIF-1α in cells at the indicated time points after POLGdn induction was assayed by Western blot analysis. Tet/off cells with 2% oxygen were used as positive control. β-actin was used as a loading control.

Figure 6. Cancer cells with loss of p53 and compromised mitochondrial respiration exhibit increased NOX and are sensitive to NOX inhibition.

(A) Comparison of membrane-associated NOX activity in human colon cancer HCT116 cells with wild-type p53 (p53+/+) or complete loss of p53 (p53−/−). Error bars, ±SD. * p<0.05 (n = 3). (B) Increased gene expression of NOX components NOX1 and p67phox in HCT116 p53−/− cells detected by qRT-PCR assay. Error bars, ±SD. * p<0.05; ** p<0.01 (n = 3). (C) Comparison of cell morphology and cell growth in HCT116 p53+/+ and HCT116 p53−/− cells treated with the NOX inhibitor DPI (10 µM, 24 h). (D) Inhibition of NOX by DPI (10 µM, 24 h) preferentially induced loss of mitochondrial transmembrane potential in HCT116 p53−/− cells.

Figure 7. Elevation of NOX in Ras transformed cells and in primary pancreatic cancer tissues.

(A) Increase of NOX activity in T-Rex 293 Tet/on cells with 1 mo K-rasG12V induction compared with control. Error bars, ±SD. ** p<0.01 (n = 3). (B) NOX activity was substantially elevated in pancreatic K-rasG12V stably transformed HPDE-kRasG12V cells compared with the parental HPDE (human pancreatic ductal epithelial) cells. Error bars, ±SD. * p<0.05 (n = 3). (C) Increased protein level of p22phox in HPDE-K-rasG12V cells and primary pancreatic cancer cells Aspc1 and Panc-1 compared to HPDE cells. (D) Increased NOX activity in the H-RASV12-transformed (T72Ras) cells compared to normal ovarian epithelial cells (T72). Error bars, ±SD. ** p<0.01 (n = 3). (E) Increased gene expression of NOX components (NOX1, NOX2, NOXA1, p22phox, and p47phox) in the H-RASV12-transformed T72Ras cells when compared to the parental T72 cells. Expression of mRNA was measured by qRT-PCR analysis. Error bars, ±SD. * p<0.05; ** p<0.01 (n = 3). (F) Preferential disruption of mitochondrial transmembrane potential by DPI (3–10 µM, 20 h) in H-RASV12-transformed T72Ras cells compared with the parental T72 cells. Mitochondrial transmembrane potential was measured by flow cytometry using rhodamine-123 as a probe. (G) Representative tissue staining showing no expression of p22phox protein in normal pancreas (a, single arrow, normal pancreatic duct; double arrows, islet cells) and chronic pancreatitis (b, arrows, benign pancreatic ducts), and a moderately differentiated pancreatic ductal carcinoma (c) and strong positive staining in a moderately differentiated pancreatic ductal carcinoma (d). The strong positive staining in the inflammatory cells served as internal positive controls for our immunohistochemical stain (original magnification, 200×). Expression of p22phox in stage II pancreatic ductal carcinoma (PDC) and benign pancreatic tissue on microarray. p22phox expression is considered to be significantly different between PDC and benign group and higher in PDC group (p<0.0001 analyzed by Fisher's exact test).

Figure 8. Inhibition of pancreatic tumor growth by NOX inhibition.

(A) Decrease of NOX activity by stable shRNA knockdown of p22phox (p22phox-shRNA) in Panc-1 cells, but not in cells with control shRNA (c-shRNA) knockdown. *** p<0.001 (n = 3). Insert, Panc-1 cells were infected with p22phox-shRNA lentiviral particles, and the knockdown efficiency was detected by anti-p22phox antibody using Western blot. Non-targeting control shRNA lentiviral particles (c-shRNA) were used as negative control. (B) p22phox knockdown significantly decreased Panc-1 cell growth. 1×105 cells were seed to six-well plates. Cell numbers were counted using Z2 coulter counter (Beckman Coulter) during 6 d of culture. (C) p22phox knockdown significantly suppressed colony formation of Panc-1 cells. 1×104 cells were seed in 0.35% of the soft agar and assayed for colony formation after 2 wk. The numbers of colonies formed on soft agar were counted. Error bars, ±SD. *** p<0.001 (n = 3). (D) p22phox knockdown decreased glucose uptake in Panc-1 cells. Cells (1×106) were incubated in 5 ml glucose-free DMEM medium for 4 h, followed by incubation with 0.2 µCi/mL 3H-2-deoxyglucose for 1 h. Cellular uptake of 3H-2-deoxyglucose was determined by liquid scintillation counting after the cells were washed 2 times with PBS and normalized by cell number. Error bars, ±SD. * p<0.05 (n = 3). (E) p22phox knockdown decreased lactate generation in Panc-1 cells. Lactate was measured 24 h after changing to fresh culture medium and normalized by cell number. Error bars, ±SD. ** p<0.01 (n = 3). (F–I) Each side of athymic nude mice (n = 7) received subcutaneously injections of 5×106 Panc-1 cells bearing p22phox-shRNA (left flank) or c-shRNA (right flank). The mice were monitored for tumor growth and body weight throughout the experiment. All the mice were sacrificed when tumor size reached about 10% of body weight as mandated by the IACUC protocol. Tumor volume was calculated using the following equation: tumor volume (mm3) = L * W *(L+W)/2 * 0.526. (F) The tumor sizes were measured throughout the experiment to evaluate p22phox knockdown effect. Data represent tumor volume: mm3±SEM. *** p<0.001 (n = 7). (G) Photographs of athymic nude mice bearing p22phox-shRNA (left flank) or c-shRNA (right flank) xenografts. (H) Photograph and comparison of excised tumor size. (I) Tumor weight derived from p22phox-shRNA knockdown or c-shRNA knockdown was measured. Error bars, ±SEM. p<0.05 (n = 7).