Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 13 (2), 367-78

Downregulation of the Werner Syndrome Protein Induces a Metabolic Shift That Compromises Redox Homeostasis and Limits Proliferation of Cancer Cells

Downregulation of the Werner Syndrome Protein Induces a Metabolic Shift That Compromises Redox Homeostasis and Limits Proliferation of Cancer Cells

Baomin Li et al. Aging Cell.

Abstract

The Werner syndrome protein (WRN) is a nuclear protein required for cell growth and proliferation. Loss-of-function mutations in the Werner syndrome gene are associated with the premature onset of age-related diseases. How loss of WRN limits cell proliferation and induces replicative senescence is poorly understood. Here, we show that WRN depletion leads to a striking metabolic shift that coordinately weakens the pathways that generate reducing equivalents for detoxification of reactive oxygen species and increases mitochondrial respiration. In cancer cells, this metabolic shift counteracts the Warburg effect, a defining characteristic of many malignant cells, resulting in altered redox balance and accumulation of oxidative DNA damage that inhibits cell proliferation and induces a senescence-like phenotype. Consistent with these findings, supplementation with antioxidant rescues at least in part cell proliferation and decreases senescence in WRN-knockdown cancer cells. These results demonstrate that WRN plays a critical role in cancer cell proliferation by contributing to the Warburg effect and preventing metabolic stress.

Figures

Figure 1
Figure 1
Werner syndrome protein (WRN) depletion affects the pathways linked to nicotinamide and glutathione metabolism. (A) Growth curves of NDF expressing shRNAs for WRN or green fluorescence protein (GFP). One microgram per millilitre doxycycline was added to the media, and the growth rate of each line was measured by counting viable cells at the indicated days. Cells were seeded at a low density, and the medium was changed every 2 days. Values represent the mean ± the standard deviation of three experiments (n = 3). (B) Western blot analysis showing the levels of WRN, p21, and p16 in normal diploid human fibroblasts (NDF) transduced with lentiviral vector for the conditional expression of shRNA targeting GFP (shCTR) and WRN (shWRN) at different days after the addition of doxycycline (1 μg mL−1). α-Tubulin was used as loading control. (C) 2D differential in-gel electrophoresis of extracts prepared from control fibroblasts expressing shCTR for 5 days and fibroblasts expressing shWRN for 3 and 5 days. Gene ontology analysis of pathways enriched in proteins that are affected in WRN-knockdown cells identifies nicotinamide and glutathione metabolism as the most significantly altered pathways in WRN-knockdown cells. Biological pathways that are significant (P < 0.05) using the Bonferroni multitest are shown.
Figure 2
Figure 2
Werner syndrome protein (WRN) depletion alters the levels of metabolic enzymes. (A) Western blot analysis showing the abundance of WRN, p21, and p16 in control and shWRN cells before and after induction with doxycycline. α-tubulin was used as normalizing factor. (B) Representative western blots showing the levels of G6PD, IDH1, and TKTL1 in HeLa cells before and at 3 and 5 days after the induction of shRNAs against WRN or green fluorescence protein (shCTR). Quantification of chemiluminescent signals from western blots of three biological replicates was carried out using the Image Analyzer LAS-4000 (Fujifilm Life Science, Stamford, CT, USA) as described in the Data S1, and mean relative values [shCTR-dox set at 100 arbitrary units (a.u.)] ± SD are shown. α-Tubulin was used as normalizing factor. ***Denotes a P value <0.00001.
Figure 3
Figure 3
Changes in the levels of metabolic enzymes in Werner syndrome protein (WRN)-knockdown cancer cells result in reduced levels of NADPH and reduced glutathione (GSH) and altered mitochondrial function. (A) NADPH levels were measured in Hela cells transduced with lentiviral vectors for the expression of shRNAs against green fluorescence protein (GFP) or WRN that were grown in the presence of doxycycline (+dox) for 3 days under 21% or 1% oxygen. (B) GSH levels were measured in Hela cells transduced with lentiviral vectors for the expression of shRNAs against GFP or WRN that were grown in the absence or presence of doxycycline (+dox) for 3 days in an atmosphere of 21% or 1% oxygen. Each data point represents the mean ± SD of three biological replicates, and P values were calculated by two-tailed Student’s t-test. (C) Mitochondrial DNA quantification by qPCR in Hela cells expressing conditional shRNAs for WRN before and after the induction or GFP controls. Mean values (n = 4) ± SDs are shown, and P values were calculated by two-tailed Student’s t-test. (D) Oxygen consumption rates in HeLa cells transduced with lentiviral vectors for the expression of siRNAs against GFP or WRN that were grown in the absence or presence of doxycycline (+dox) for 5 days. Maximal respiratory capacity was measured after the addition of the uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Each data point represents the mean ± SDs of three independent experiments, each of which was carried out using five replicates. (E) Representative transmission electron microscopy images (2500×) of Hela cells for the inducible expression of shRNAs against WRN or GFP showing swollen mitochondria in Hela cells 3 days after WRN knockdown. N, nucleus. (F) Mitochondrial membrane potential in Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or GFP grown in the absence (-dox) or in the presence of doxycycline (+dox) for 3 and 5 days. Cells were stained with 2 μm JC-1 for 15 min at 37 °C, 5% CO2 and then washed with phosphate-buffered saline and analyzed on a flow cytometer using 488-nm excitation with 530-nm and 585-nm bandpass emission filters. Cells were binned into two sectors of either red (high membrane potential) or green (low membrane potential) fluorescence. An increase in the percentage of cells with green fluorescence after WRN knockdown reflects a decrease in membrane potential. The uncoupler CCCP was added as a control for membrane depolarization: note the shift of cells to the green sector indicative of depolarized mitochondria. Representative FACS analysis and mean value ± SD of percentage of cells in FITC (green) sector from three biological replicates are shown.
Figure 4
Figure 4
Reactive oxygen species accumulation and oxidative damage to DNA in Werner syndrome protein (WRN)-knockdown cancer cells. (A) Superoxide anion radicals generated by the mitochondria in Hela cells transduced with lentiviral vectors for the expression of siRNAs against green fluorescence protein (GFP) or WRN that were grown in the absence (−) or presence (+) of doxycycline (dox) for 5 days were determined using MitoSOX Red and analyzed by flow cytometry. Histogram from representative experiment and mean fluorescence emission for each sample from two independent replicates are shown. (B) Measurement of intracellular oxidative status after WRN knockdown. Experiments were performed exactly as in (A), and peroxide levels (primarily H2O2) were detected using the fluorescent indicator Peroxy Fluor-6 (PF-6) by flow cytometry, as described in Experimental procedures. Histogram from representative experiment and mean fluorescence emission for each sample from two independent experiments are shown. (C) Measurement of mitochondrial oxidative status in Hela cells transduced with lentiviruses for the conditional expression of shRNAs targeting WRN or GFP grown in the absence (−) or presence (+) of doxycycline (dox) for 5 days. Cells were incubated with mitochondrial Peroxy Yellow 1 (mitoPY-1) as described in Experimental procedures and subjected to analysis by flow cytometry. Histogram from representative experiment and mean emission intensities for each sample from three independent replicates are shown. (D) Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or GFP (shCTR) were grown in the absence or presence of doxycycline (+dox), and the oxidized nucleoside 8 hydroxy-2′-deoxyguanosine was detected by immunofluorescence microscopy. Quantification of total nuclei fluorescence for each sample was carried out using Image J software (National Institute of Health, Bethesda, MD, USA). Mean values of two independent experiments (n > 100 nuclei per experiment) ± SDs are shown, and P values were calculated by two-tailed Student’s t-test. (E) Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or GFP (shCTR) were grown in the absence or presence of doxycycline (+dox), and phosphorylated histone H2AX (γH2AX) was detected by immunofluorescence microscopy and signal was quantitated as described above.
Figure 5
Figure 5
Werner syndrome protein (WRN) knockdown affects the levels of hypoxia-inducible factor 1 (HIF1α). Nuclear extracts were prepared from Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or green fluorescence protein (GFP) (CTR) that were grown in the absence or presence of doxycycline (+dox) for 3 or 5 days under atmosphere of 1% (A) or 21% (B) oxygen, and analyzed by immunoblotting using antibodies against WRN, HIF1α and α-tubulin as loading control. (C) Nuclear extracts from shCTR and shWRN Hela cells treated with or without MG-132 or dimethyloxalylglycine as indicated were analyzed by immunoblotting with antibodies specific to hydroxylated HIF1 α (hydroxyl-HIF1α) or total HIF1α (D), Reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) analysis of mRNA steady-state levels for HIF1α in Hela cells grown in 1% oxygen 3 days after WRN knockdown. mRNA expression levels were normalized to tubulin mRNA and are shown as relative to shCTR cells. RNA was isolated from at least three biological samples of Hela cells with shCTR or shWRN, and RT–qPCR assays were carried out in triplicate samples. The graph and statistics were generated using Excel. (E) Extracts were prepared from Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or GFP (CTR) that were grown in the absence or presence of doxycycline (+dox) for the indicated days, and analyzed by immunoblotting using antibodies against WRN, phosphorylated 4E binding protein 1 (4EBP1) (P-4EBP1), total 4EBP1, and α-tubulin as loading control.
Figure 6
Figure 6
Supplementation with reduced glutathione (GSH) ameliorates growth and reduces senescence of Werner syndrome protein (WRN)-depleted cells. (A) Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN were grown in normal media or media supplemented with 2 mM GSH. Cells were seeded at a low density in an atmosphere of 1% oxygen, and the medium was changed every 2 days. Values represent the mean ± the standard deviation of three experiments (n = 3). (B) GSH levels was measured in Hela cells transduced with lentiviral vectors for the expression of shRNAs against WRN or shGFP that were grown in the presence of doxycycline (+dox) for 3 days in an atmosphere of 1% oxygen. When indicated, GSH was added to the culture media. (C) Detection of senescence-associated β-galactosidase (SA-β-gal) activity. Hela cells transduced with lentiviruses for the conditional expression of shRNAs targeting WRN or green fluorescence protein (GFP) were grown for 5 days after the addition of DOX and stained for SA-β-gal activity as previously described (Li et al., 2008). Values are the mean ± the standard deviation of three independent experiments (n = 3) carried out in duplicates, in which 500 cells were scored for SA-β-galactosidase. Student’s t-test was used to evaluate the differences in means between the two groups. (D) Hypothetical model: downregulation or loss of WRN compromises the cellular antioxidant system by reducing the levels of NADPH-producing enzymes glucose 6 phosphate dehydrogenase and isocitrate dehydrogenase. Reduced levels of NADPH, a source of reducing power, will affect cellular anabolism. In cancer cells, concomitant to altered mitochondrial function, this metabolic shift leads to the accumulation of reactive oxygen species, which causes damage to macromolecules and impairs cell viability.

Similar articles

See all similar articles

Cited by 9 PubMed Central articles

See all "Cited by" articles

References

    1. Arai A, Chano T, Futami K, Furuichi Y, Ikebuchi K, Inui T, Tameno H, Ochi Y, Shimada T, Hisa Y, Okabe H. RECQL1 and WRN proteins are potential therapeutic targets in head and neck squamous cell carcinoma. Cancer Res. 2011;71:4598–4607. - PubMed
    1. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat. Rev. Cancer. 2011;11:85–95. - PubMed
    1. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat. Rev. Mol. Cell Biol. 2007;8:729–740. - PubMed
    1. Crabbe L, Verdun RE, Haggblom CI, Karlseder J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science. 2004;306:1951–1953. - PubMed
    1. Dickinson BC, Chang CJ. A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells. J. Am. Chem. Soc. 2008;130:9638–9639. - PMC - PubMed

Publication types

MeSH terms

Feedback