Glucose-6-phosphate dehydrogenase and transketolase modulate breast cancer cell metabolic reprogramming and correlate with poor patient outcome

Abstract

The pentose phosphate pathway is a fundamental metabolic pathway that provides cells with ribose and NADPH required for anabolic reactions - synthesis of nucleotides and fatty acids - and maintenance of intracellular redox homeostasis. It plays a key role in tumor metabolic reprogramming and has been reported to be deregulated in different types of tumors. Herein, we silenced the most important enzymes of this pathway - glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) - in the human breast cancer cell line MCF7. We demonstrated that inhibition of G6PD, the oxidative branch-controlling enzyme, reduced proliferation, cell survival and increased oxidative stress. At the metabolic level, silencing of both enzymes reduced ribose synthesis. G6PD silencing in particular, augmented the glycolytic flux, reduced lipid synthesis and increased glutamine uptake, whereas silencing of TKT reduced the glycolytic flux. Importantly, we showed using breast cancer patient datasets that expression of both enzymes is positively correlated and that high expression levels of G6PD and TKT are associated with decreased overall and relapse-free survival. Altogether, our results suggest that this metabolic pathway could be subjected to therapeutic intervention to treat breast tumors and warrant further investigation.

Keywords: breast cancer; glucose-6-phosphate dehydrogenase; pentose phosphate pathway; transketolase; tumor metabolism.

Figure 1. Silencing of TKT and G6PD in MCF7 cells

(A) TKT and G6PD mRNA levels three days after transfection of non-targeting siRNA (siNEG) or siRNA targeting TKT (siTKT) or G6PD (siG6PD). (B) TKT activity five days after siTKT transfection. (C) G6PD activity after siG6PD transfection. In all plots values are expressed as fold change versus siNEG and bars represent mean (n = 3) ± SD.

Figure 2. Role of PPP enzymes TKT and G6PD in cell proliferation, survival and cell cycle

(A) Effect of TKT and G6PD silencing on proliferation six days after transfection expressed as percentage of siNEG. (B) Effect of TKT and G6PD silencing on cell death six days after transfection (percentage of PI-positive cells). Bars in A and B represent mean (n = 2) ± SEM for TKT and mean (n = 5) ± SEM for G6PD. (C) Effect of TKT and G6PD silencing on cell cycle progression five days after siRNA transfection. The percentages of cells in each phase are depicted as mean (n = 3) ± SD. (D) Cyclin E and β-actin (loading control) protein levels were analyzed four days after siRNA transfection.

Figure 3. Metabolic effects of TKT and G6PD silencing

[¹³C]-assisted metabolomics experiment was performed by replacing culture media by fresh media containing 50% of [1,2-¹³C₂]-glucose four days after siRNA transfection. Cells were incubated with the tracer for 24h and extracellular fluxes and isotopologue distribution of different metabolites were determined. (A) CORE (consumption and release) metabolic profile showing rates of consumption of glucose (Glc) and glutamine (Gln) and release of lactate (Lac) and glutamate (Glu). (B) Flux of production of lactate from glucose via glycolysis. (C) Proportion of consumed glucose dedicated to either production of lactate or other uses. (D) Levels of m2 ¹³C-alanine in media. (E) Total ¹³C-labeled ribose from RNA. (F) Isotopologue distribution of ribose from RNA. (G) Ratio of m1 ¹³C-ribose to m2 ¹³C-ribose as an indicator of the oxidative vs. non-oxidative PPP activity. In all cases values represent mean (n = 3) ± SD.

Figure 4. Role of PPP enzymes in ROS levels and lipid synthesis

(A). ROS levels five days after transfection. Bars represent mean (n = 3) ± SEM. (B) Total ¹³C-labeled palmitate and (C) stearate. (D) Estimated fraction of ¹³C-acetyl-CoA depicted as percentage of the total pool. (E) Percentage of glucose dedicated to the synthesis of acetyl-CoA estimated using the fraction of ¹³C-acetyl-CoA in combination with the maximal theoretical enrichment of acetyl-CoA from glucose. Data shown in figures D and E was calculated as described in Boren et al 2003 [32]. Bars represent mean (n = 3) ± SD.

Figure 5. Survival analysis of breast cancer patients associated to the expression of TKT and G6PD

Kaplan–Meier survival analysis showing relapse-free and overall survival for breast cancer patients with high and low expression levels of G6PD or TKT in three independent datasets. p-values of log-rank test are depicted in each plot.

Figure 6. Gene expression correlation analyses

Pearson’s correlation analysis showing gene expression levels of TKT and G6PD in breast cancer patients from three independent datasets. Pearson’s correlation coefficient (r) and p-values are shown for each analysis.