Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion

ARTICLE Received 19 Jun 2014 | Accepted 2 Aug 2014 | Published 25 Sep 2014 DOI: 10.1038/ncomms5900 OPEN Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion Naval P. Shanware1,*, Kevin Bray1,*, Christina H. Eng1, Fang Wang1, Maximillian Follettie1, Jeremy Myers1, Valeria R. Fantin2 & Robert T. Abraham2 The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents. 1 Oncology Research Unit, Pfizer Worldwide Research and Development, 401 N. Middletown Road, Pearl River, New York 10965, USA. 2 Oncology Research Unit, Pfizer Worldwide Research and Development, 10777 Science Center Drive, La Jolla, California 92121, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.T.A. (email: Robert.Abraham@pfizer.com). NATURE COMMUNICATIONS | 5:4900 | DOI: 10.1038/ncomms5900 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE R NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5900 eprogramming of molecular and metabolic pathways involved in intermediate metabolism is now recognized as a hallmark of cancer1. Oncogenic signals drive constitutive cell growth and proliferation, and place heavy demands on the pathways responsible for providing the metabolic building blocks needed for the synthesis of proteins, nucleic acids, lipids and other macromolecules. To meet the increased demand for biosynthetic precursors, cancer cells increase uptake of glucose and other nutrients, and shift overall metabolism from bioenergy (ATP) production and cell maintenance activities to anabolic processes that support cell mass accumulation and mitotic cell division2,3. The shift toward anabolic metabolism is exemplified by the altered catabolism of glucose in tumour tissues4. Normal, nonproliferating cells primarily convert glucose to pyruvate via glycolysis. Pyruvate is then imported into the mitochondria, where it is converted into acetyl CoA for entry into the tricarboxylic acid (TCA) cycle. The glucose-derived carbon is then completely oxidized to produce carbon dioxide and ATP. In contrast, tumour cells reduce pyruvate to lactate for export from the cells. The glycolytic breakdown of glucose to lactate in oxygenated tumour tissues is termed the Warburg effect5. In addition to lactate, glycolysis generates intermediates that fuel anabolic metabolism via the pentose-phosphate and serine biosynthesis pathways4. Similarly, the TCA cycle is involved in both energy production and in the generation of building blocks for protein and lipid biosynthesis. The diversion of glucosederived carbon away from the mitochondria, together with the withdrawal of TCA cycle intermediates for biosynthetic reactions, creates a carbon deficit in the TCA cycle that must be corrected by entry of carbon from other sources, a process termed anaplerosis6. These and other alterations in nutrient uptake and utilization in transformed cells have spawned considerable interest in cancer metabolism as a promising area for the discovery of novel antitumour agents7,8. The non-essential amino acid, glutamine, is a major contributor to anaplerotic replenishment of the TCA cycle, and serves as a source of carbon and nitrogen for the synthesis of proteins, lipids and amino acids9,10. Proliferating cells avidly import extracellular glutamine, and catabolize it via glutaminolysis, during which glutamine undergoes sequential deamination in the mitochondria to glutamate and further into the TCA cycle intermediate, a-ketoglutarate (a-KG)11. As a nitrogen donor, glutamine supports both nucleotide and nonessential amino acid synthesis, in addition to protein glycosylation through the hexosamine pathway12. Finally, glutamine plays a key role in oxidative stress resistance by serving as a source of glutamate for the production of glutathione9. Many cancer cells exhibit strikingly increased rates of glutamine uptake and metabolism. Notably, cells transformed by the MYC proto-oncogene or oncogenic KRAS display glutamine auxotrophy13–15. The increased sensitivity of certain transformed cells to glutamine restriction suggests that drugs interfering with glutamine catabolism might have clinically exploitable antitumour activities16,17. An actionable target for such inhibitors is the mitochondrial enzyme, glutaminase, which catalyses the conversion of glutamine to glutamate. Clearly, our understanding of the potential benefits and challenges of therapeutic targeting of glutamine metabolism in cancer patients will benefit from a more complete understanding of the cellular responses to manipulations that deprive cancer cells of glutamine or interfere with glutaminolysis. We and others have recently described an unanticipated contribution of glutaminolysis to autophagy, a cytoplasmic pathway that delivers autophagosome-encapsulated macromolecules and organelles to lysosomes for degradation and recycling 2 into metabolic processes18–21. Cells normally exhibit a basal level of autophagic flux that is strongly enhanced by certain environmental stresses, such as nutrient starvation. Under such stressful conditions, autophagy allows cells to degrade nonessential macromolecules into products that support cellular bioenergetics and viability22. A recent manuscript by Narita et al.23 also identified an essential role for autophagy in the context of a multi-component endomembrane structure termed the TOR-autophagy spatial coupling complex (TASCC). In cells undergoing oncogene-induced senescence (OIS), the TASCC is formed by the spatial colocalization of the autophagy machinery with lysosomes, and appears to facilitate the mass synthesis of secretory proteins that comprise the senescence-associated secretory phenotype (SASP). In this report, we demonstrate that short-term glutamine restriction results in a chemokine-secretory response that is depen (...truncated)