Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells

Abstract

Autophagy degrades and is thought to recycle proteins, other macromolecules, and organelles. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes malignancy. Autophagy-deficient tumor-derived cell lines are respiration-impaired and starvation-sensitive. However, to what extent their sensitivity to starvation arises from defective mitochondria or an impaired supply of metabolic substrates remains unclear. Here, we sequenced the mitochondrial genomes of wild-type or autophagy-deficient (Atg7) Kras-driven lung tumors. Although Atg7 deletion resulted in increased mitochondrial mutations, there were too few nonsynonymous mutations to cause generalized mitochondrial dysfunction. In contrast, pulse-chase studies with isotope-labeled nutrients revealed impaired mitochondrial substrate supply during starvation of the autophagy-deficient cells. This was associated with increased reactive oxygen species (ROS), lower energy charge, and a dramatic drop in total nucleotide pools. While starvation survival of the autophagy-deficient cells was not rescued by the general antioxidant N-acetyl-cysteine, it was fully rescued by glutamine or glutamate (both amino acids that feed the TCA cycle and nucleotide synthesis) or nucleosides. Thus, maintenance of nucleotide pools is a critical challenge for starving Kras-driven tumor cells. By providing bioenergetic and biosynthetic substrates, autophagy supports nucleotide pools and thereby starvation survival.

Recent insights into the function of autophagy in cancer

“…To test this hypothesis, the mitochondrial genomes of tumors from the Kras-driven GEMM for NSCLC with and without Atg7 were sequenced. While the mitochondrial variant allele frequency was higher in Atg7-deficient tumors, there were too few nonsynonymous mutations, and their frequency was below that of manifestation of mitochondrial dysfunction (Guo et al 2016). Thus, autophagy does control quality of the mitochondrial genome; however, this is not sufficient to explain the pronounced metabolic defect in tumors and tumor cells upon deletion of Atg7 (Guo et al 2016).…”

“…Perhaps, if all forms of mitophagy are inactivated, this would have a different phenotypic consequence more similar to inhibition of canonical autophagy genes. Note that autophagy supplies substrates for mitochondrial metabolism, the loss of which significantly impairs mitochondrial function beyond what would be expected for loss of mitophagy alone (Guo et al 2016). In addition, genetic autophagy inhibition produces an accumulation of oncogenic p62, whereas genetic mitophagy inhibition does not, further distinguishing the function of autophagy from mitophagy.…”

“…The label was then briefly flushed out of metabolic pathways by chasing with unlabeled medium, and then the tumor cells were challenged with starvation to test for the ability to recycle the stably labeled macromolecules. In comparison with wild type, Atg7-deficient tumor cells had a defective mitochondrial substrate supply, as indicated by less labeled glutamate, α-ketoglutarate, and aspartate and profound depletion of nucleotide pools in starvation (Guo et al 2016). This defective recycling and substrate supply in Atg7-deficient tumor cells decreased mitochondrial oxygen consumption and increased oxidative stress and energy crisis, which was rescued by glutamine, glutamate, and nucleosides.…”

“…This defective recycling and substrate supply in Atg7-deficient tumor cells decreased mitochondrial oxygen consumption and increased oxidative stress and energy crisis, which was rescued by glutamine, glutamate, and nucleosides. Thus, a primary function of autophagy in Kras-driven lung cancer is to recycle Continued macromolecules into mitochondrial metabolism to prevent AMP accumulation, energy crisis, and fatal nucleotide degradation as an attempt to remove AMP and maintain energy charge (Guo et al 2016), analogous to what happens in yeast (Walther et al 2010). Going forward, it will be of interest to determine whether other cancers and normal tissues similarly require autophagy for this purpose.…”

“…Control of the trafficking machinery then orchestrates fusion of the cargo-laden autophagosomes with lysosomes. Degradative enzymes, contributed by lysosomes, break down the cargo, and the products are exported into the cytoplasm, where they are recycled into metabolic and biosynthetic pathways (Rabinowitz and White 2010;Guo et al 2016). Under normal nutrient conditions, autophagy functions at a constitutive, low basal level to maintain protein and organelle quality, quantity, and functionality.…”