Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine.

Naniye Mallı Cetinbas,Jessica Sudderth,Ralph J Deberardinis,Aysun Cebeci,Robert C Harris,Ömer H Yılmaz,Gian L Negri,Poul H Sorensen

doi:10.1038/srep32606

Abstract

Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis.

Highlights

Several studies have shown that cancer cells can be selectively killed by small molecule inducers of oxidative stress[18,19,20], underscoring the importance of fine-tuning of ROS levels for cancer cell survival
Because GLN is a precursor for GSH and NADPH antioxidant synthesis and effective ROS management is vital for cancer cell survival, we hypothesized that GLN starvation-induced cell death correlates with increased ROS
We measured ROS and cell death at an earlier time point and found that ROS is induced after 20 hours of GLN starvation, whereas no significant cell death was observed at this time point (Fig. S1), suggesting that GLN starvation induces ROS prior to cell death

Summary

Introduction

Several studies have shown that cancer cells can be selectively killed by small molecule inducers of oxidative stress[18,19,20], underscoring the importance of fine-tuning of ROS levels for cancer cell survival. NADPH is indispensible for cellular antioxidant activity Both GSH and NADPH can be synthesized from GLN22, and recent studies have implicated GLN as a key factor in ROS homeostasis in cancer cells[9,23]. Using diverse human cancer cell lines, we show that cells that do not rely on GLN for survival are independent of GLN for ROS homeostasis. We found that GLN starvation-induced ROS elevation causes increased glucose uptake in GLN addicted cells. Inhibition of the mitochondrial pyruvate transporter in GLN independent cells leads to GSH depletion and oxidative stress, rendering them GLN addicted. GLN starvation increases PDK1 expression and activity, as observed by increased levels of phosphorylated (inactivated) PDH, only in GLN addicted cells, accounting for reduced glucose oxidation via the TCA cycle. We show that GLN starvation synergizes with the pro-oxidant small molecule piperlongumine in effective killing of GLN addicted tumor cells

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Results

Conclusion