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, 34 (28), 3728-36

Deletion of Pim Kinases Elevates the Cellular Levels of Reactive Oxygen Species and Sensitizes to K-Ras-induced Cell Killing

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Deletion of Pim Kinases Elevates the Cellular Levels of Reactive Oxygen Species and Sensitizes to K-Ras-induced Cell Killing

J H Song et al. Oncogene.

Abstract

The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-Ras(G12V)) and undergo cell death. Transduction of K-Ras(G12V) into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuated ROS production and reversed the cytotoxic effects of K-Ras(G12V) in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-Ras(G12V)-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-Ras(G12V)-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases have an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth.

Conflict of interest statement

The authors disclose no potential conflicts of interest.

Figures

Fig. 1
Fig. 1. Knockout of Pim kinases switches oncogenic activity of K-Ras to cell death
(a) Fluorescence microscopy of primary WT and TKO MEF cells expressing, FUCRW empty vector (EV) or K-RasG12V (K-Ras). At day 4 post-infection, expression of red fluorescent protein (RFP) and morphological changes were visualized. (b, c) Primary WT and TKO MEF cells were infected with empty vector (-) or K-Ras. After 7 days, cell viability was evaluated by the crystal violet assay. (d) A focus-forming assay was performed at 14 days post-infection using lentiviruses carrying EV or K-Ras in immortalized WT and TKO MEF cells. The numbers of cell foci were counted under inverted microscope. Mean value and standard deviation were obtained from triplicates. (e) Pim1, 2, or 3 single knockout MEF cells were infected with lentivirus expressing K-Ras (+) or empty vector (-). Cell viability was determined by a crystal violet assay on day 4. (f) Cell viability was examined 7 days after K-Ras expression in TKO MEF cells expressing EV, Pim1, 2, or 3. The experiment was done in triplicate.
Fig. 2
Fig. 2. Lethal accumulation of reactive oxygen species (ROS) induced by K-RasG12V in absence of Pim kinases
(a) WT and TKO MEF cells were exposed to 1 μM H2DCF-DA for 30 min and cells were evaluated by real-time confocal analysis for ROS production. (b) Fluorescence microscopy for detection of ROS accumulation in WT and TKO MEF cells at day 4 post-infection of empty vector (EV) or K-Ras lentiviruses. (c) Flow cytometric analysis of ROS in WT and TKO MEF cells expressing EV or K-Ras. (d, e) Cell viability was determined by crystal violet staining. After infection for 2 days, EV or K-Ras infected WT and TKO MEF cells were treated with 5 mM NAC for 48 h (triplicates).
Fig. 3
Fig. 3. Pim kinases are required for regulation of glycolysis
(a) Metabolomics analysis reveals that key glycolytic metabolites are repressed in TKO MEFs (mean +/- SD, n=5). The metabolites shown are glucose-1-phosphate (G6P), glucose-1-phosphate (G6P), fructose-6-phosphate (F6P), 2-phosphoglycerate (2PG). Pyruvate and 3-phosphoglycerate (3PG) levels were not different between WT and TKO MEFs. * denotes a statistical significance of p<0.05. (b) GSEA reveals the most significantly altered metabolic pathway in TKO MEFs. Selected metabolic gene sets are shown. NES, normalized enrichment score; q-values, FDR-adjusted p-value. The enrichment plot of the glucose metabolism gene set is found to be the most significantly associated with downregulation in TKO MEFs. The heatmap shows the expression profile of genes significantly downregulated in TKO MEFs. (c) Quantitative real-time PCR (qPCR) analysis of mRNA expression of glycolytic enzymes and PPP enzymes. Normalized fold expression of Slc2a1 (Glut1), Hk1 (hexokinase 1), Hk2, Eno3 (enolase 3), Aldoa (Aldolase A), Pfkm (phosphofructokinase muscle), Ldha (lactate dehydrogenase A) and Pgm1 (phosphoglucomutase 1) are shown (triplicates) and β-Actin was used for normalization. (d) Immunoblot analysis of key glycolytic enzymes in WT and TKO MEF cells. Protein expression of Hk2, Glut1, Pfk1, Pdk4 (pyruvate dehydrogenase kinase 4), enolase, Pkm1/2, Ldha and β-actin are shown.
Fig. 4
Fig. 4. K-Ras requires Pim kinases for regulation of glycolysis
(a) Immunoblot analysis of glycolytic enzyme expression regulated by K-RasG12V. WT and TKO MEFs were infected with lentiviruses of EV, K-Ras or c-Myc. After transduction for 96 h, cells were lysed and subjected to immunoblottting. (b) K-RasG12V-mediated changes in gene expression were assayed using qPCR. Normalized expression of Glut1, Hk1, Hk2, Eno3, Aldoa, Pfkm, and Ldha mRNA to β-Actin mRNA is shown (Mean +/- SD, triplicates). WT and TKO MEF cells were infected with lentiviruses containing empty vector (EV) or K-RasG12V for 72 hours and then total RNA was isolated.
Fig. 5
Fig. 5. Pim kinases are required for regulation of the pentose phosphate pathway (PPP)
(a) Key metabolites within the PPP were significantly altered in TKO MEFs compared to WT MEFs. Levels of G6P, 6-phosphoglucognate (6PG), sedoheptulose 7-phosphate (S7P), F6P and ribulose 5-phosphate (Ru5p) + xylulose 5-phosphate (Xu5P) are shown (n=5). (b) qPCR analysis of mRNA expression of PPP enzymes. Measurement of key enzymes in the PPP, including G6pd (glucose-6-phosphate dehydrogenase), Pgls (6-phosphoglucono-δ-lactone), Pgd (6-phosphogluconate dehydrogenase), Rpia (ribose-5-phosphate isomeras A), Rpe (ribose-5-phosphate epimerase), Tktl2 (transketolase 2), and Tald1 (transaldolase 1). (c) K-RasG12V-mediated changes in gene expression were assayed by qPCR. Normalized expression of G6pd, Pgd, Rpe, and Rpia to β-Actin mRNA is shown (triplicates). WT and TKO MEF cells were infected with lentiviruses of EV or K-RasG12V for 72 h and then total RNA was isolated.
Fig. 6
Fig. 6. Pim kinases are involved in regulation of the TCA cycle and mitochondrial oxidative phosphorylation
(a) K-RasG12V-mediated changes in gene expression were assayed with a qPCR. Normalized expression of Pcx and Idh1 to β-Actin mRNA is shown (Mean +/- SD, triplicates). (b) OCR was evaluated in WT and TKO MEF cells. Oligomycin (2.5 μM), FCCP (2.5 μM), antimycin A (1 μM) and rotenone (1 μM) were sequentially added as indicated by an arrow (triplicates). (c) Immunoblot analysis of electron transport chain complex proteins. Two independently derived sets of WT and TKO MEF cells were subjected to subcellular fractionation to obtain mitochondria and cytosolic fractions. A cocktail of antibodies, CV-ATPA, CII-UQCR2, CII-SDHB and CI-NDUFB8, was used to probe the membranes of mitochondria of various MEF cells. β-actin protein was used as a loading control. (d) Mitochondrial DNA was quantified by qPCR by measuring the ratio of mitochondrially encoded Cox2 to an intron of the nuclear-encoded β-globin gene. The ratio of mitochondrial cytochrome b (Cyt b) to an intron of nuclear glucagon gene is shown.
Fig 7
Fig 7. NAD(P)+ coenzymes for metabolic redox reaction is modulated by loss of Pim kinases
(a) NADP+, NADPH and total NADP (NADP+ plus NADPH) levels in WT and TKO MEF cells are shown (triplicates). NADP+ concentrations were calculated by the subtraction of NADPH from total NADP (nmol per mg protein). (b) The content of reduced glutathione (GSH), oxidized glutathione (GSSG) was normalized to cellular protein content. (Mean +/- SD, n=3). WT and TKO MEFs were transduced for 72 hours using lentiviruses carrying empty vector (EV), K-RasG12V (KRAS), c-Myc (MYC) or both. (c) Focus forming assay. WT and TKO MEFs were transduced with lentiviruses carrying empty vector (EV), K-RasG12V (KRAS), c-Myc (MYC) or both. After 10 days of growth, the number of foci was documented with crystal violet staining. Cell viability was evaluated by MTT assay (Mean +/- SD, n=4). (d) Quantitation of DCF labeling from flow analysis is shown with relative ROS levels (triplicates) with the control value 1 set in the RFP cells. (e) NADP+ and NAD+ production was measured in WT and TKO MEF cells expressing empty vector (EV) or c-Myc (MYC). (f) Immunoblotting assay for Sod2 protein expression in cells transduced with HA-Myc. (g) Model demonstrating the role of Pim, Ras, and Myc in regulating cellular metabolism.

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