Surviving Metabolic Stress: of Mice (Squirrels) and Men William N

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IN FOCUS Surviving Metabolic Stress: Of Mice (Squirrels) and Men William N. Hait 1 , Matthias Versele 1 , and Jin-Ming Yang 2 , 3 Summary: Understanding how cancer cells survive harsh environmental conditions may be fundamental to eradicating malignancies proven to be impervious to treatment. Nutrient and growth factor deprivation, hypoxia, and low pH create metabolic demands that require cellular adaptations to sustain energy levels. Protein synthesis is one of the most notable consumers of energy. Mounting evidence implicates exquisite control of protein synthesis as a survival mechanism for both normal and malignant cells. In this commentary, we discuss the role of protein synthesis in energy conservation in cancer and focus on elongation factor-2 kinase, a downstream component of the PI3K–AKT pathway that behaves as a critical checkpoint in energy consumption. Cancer Discov; 4(6); 646–9. ©2014 AACR.

Harsh conditions create stress for all living things. Col- with adjacent normal tissue was perplexing; why was this lections of malignant cells would seem to be especially enzymatic inhibitor of peptide elongation increased in cer- vulnerable, as they manage to survive under conditions of tain malignancies? Insights came from studies of hibernating deprivation, such as lack of adequate blood supply, low pH, squirrels and weanling mice. and both oxygen and nutrient shortages. Recent evidence Hibernation, a survival strategy used by mammals to com- provides further insights into how nature deploys a parsimo- bat the threat of severe nutrient deprivation, requires marked nious approach to address this problem, from mice, to squir- metabolic suppression. In hibernating ground squirrels, Chen rels, to man. The PI3K pathway and autophagy are sensitive and colleagues (8 ) observed increased phosphorylation of to nutrient and growth factor availability and are fundamen- eEF2 in the liver and brain due to increased activity of eEF2K. tal mechanisms for cellular adaptation to stress. Although They surmised that increased phosphorylation of eEF2 was several components of these pathways have been carefully a mechanism by which hibernating animals tolerate marked elucidated and reviewed ( 1, 2 ), the role of peptide elongation reductions in cerebral blood fl ow. Since then, several stud- has received less attention. ies have demonstrated that environmental stress, including While studying Ca2+ /calmodulin signaling, we identifi ed a nutrient and growth factor deprivation as well as oxidative previously unappreciated enzymatic activity in glioblastoma cells and chemical insults, is a potent activator of this kinase ( 9 ). and specimens ( 3 ). A 100-kDa phosphoprotein was present in The regulation of eEF2K activity provided further insights. malignant glial cells but not in normal white matter, a tissue rich Proud and others (reviewed in ref. 10 ) found that control of in normal glia. Ultimately, we isolated both the phosphoprotein eEF2K is mediated by key enzymatic activities linked to meta- [elongation factor-2 (eEF2)] and the kinase [elongation factor-2 bolic homeostasis (Fig. 1). For example, eEF2K is completely kinase (eEF2K; ref. 4 ), also known as calmodulin-dependent pro- dependent on Ca2+ /calmodulin, and is activated by AMP tein kinase 3, originally described by Nairn and colleagues ( 5 )], kinase (AMPK) through phosphorylation at Ser398. AMPK cloned the gene, and characterized the enzyme as a unique pro- is a sensor of cellular energy, sensitive to changes in the ratio tein kinase represented by relatively few enzymes in the kinome of AMP to ATP. When the ratio is high, that is, when energy ( 6 ). It had been shown that this kinase terminated protein elonga- is depleted, eEF2K is activated by AMPK and peptide elongation ( 7 ), one of the most avaricious consumers of cellular energy, tion is terminated. Conversely, when the ratio is low, that is, through phosphorylation of eEF2 at Thr56, thereby decreasing when energy stores are replete, AMPK is less active, eEF2K is the affi nity of the elongating peptide chain for the ribosome. inhibited, and elongation is restored. Furthermore, eEF2K is eEF2, the only known substrate for eEF2K, is an elongation fac- also regulated by insulin and mTORC1 through phosphor- tor that mediates the translocation of peptidyl tRNA from the ylation at Ser78 and Ser361, respectively (11, 12). Thus, in the ribosomal A site to the P site. presence of adequate nutrients, protein synthesis is favored At the time, the conspicuously increased activity of eEF2K through inactivation of AMPK and activation of mTORC1, in glioblastoma and other types of cancer cells compared both converging to inhibit eEF2K. In contrast, under conditions of metabolic stress, protein synthesis is undesirable, AMPK is activated, mTORC1 is inhibited, and the increased 1 2 Authors’ Affi liations: Janssen R&D, LLC., Raritan, New Jersey; Depart- activity of eEF2K inhibits peptide chain elongation. ment of Pharmacology, Penn State University College of Medicine; and 3Penn State Hershey Cancer Institute, Milton S. Hershey Medical Center, It is now appreciated that cancer cells use these same Hershey, Pennsylvania pathways to rapidly terminate protein synthesis to conserve Corresponding Author: William N. Hait, Janssen R&D, LLC., 920 US High- energy. As mentioned above, a variety of stressful condi- way 202 South, Raritan, NJ 08869. Phone: 908-927-3516; Fax: 908-927- tions increased the activity of eEF2K including hypoxia and 7716; E-mail: [email protected] nutrient and growth factor deprivation (9 ). Furthermore, doi: 10.1158/2159-8290.CD-14-0114 when the kinase was depleted by RNA interference (RNAi) or ©2014 American Association for Cancer Research. blocked by chemical inhibition, the ability to survive stress

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This leads to weanling mice. Nutrient and growth factor KEY CONCEPTS defi ciency is a potent inducer of autophagy (from the Greek “self-eating”). Autophagy is stimulated in yeast through a • Malignant cells have high energy requirements and complex containing Atg1, a serine/threonine protein kinase are compromised by ineffi cient utilization of glucose that can form a complex with Atg13 and Atg17. Autophagy and a variable blood supply. protects against severe energy depletion by digesting • Protein synthesis is a major consumer of ATP and is organelles and cytoplasm and recycling the components for therefore tightly regulated during cell growth. energy production. When energy is abundant, autophagy is suppressed by mTORC1 through direct interactions with the • Several important signaling pathways converge on the protein synthetic apparatus and respond to Atg1/13/17 complex. Initially, differences in interpretation of changes in nutrient and growth factor availability. data led to uncertainty about the precise role of autophagy in cancer; was it a mechanism of cell death or a conserved mech- • Survival of malignant cells under conditions of nutrient anism of survival, or both? An elegant set of experiments and growth factor depletion may rely on proper control highlighted the possibility that autophagy could serve as a of the key enzymes that regulate protein synthesis. potent survival mechanism in malignancies. Like hibernating • Although much is known about the enzymes that con- squirrels, newborns are forced to fi nd ways to survive during trol the initiation of protein synthesis, far less atten- the period of starvation between birth and the onset of lactation has been given to the enzymes that regulate tion. Kuma and colleagues (14 ) studied survival in weanling peptide elongation, the process requiring more energy. mice. At birth and before lactation begins, pups unable to use • This commentary focuses on the regulation of pep- autophagy due to lack of Atg5, an essential component of the tide elongation and discusses links to other sur- murine autophagosome, do not survive the abrupt lack of vival pathways and ways they might be disrupted to nutrients preceding lactation. Survival was not impaired in improve therapies. wild-type littermates or in newborn Atg5 mutants who were force fed during the critical prelactation period. Therefore, several laboratories considered a possible link was markedly reduced (9 ). Similarly, Cantley’s laboratory among nutrient deprivation, inhibition of protein elon- found that failure to inhibit mTORC1 and block the initia- gation, and autophagy. In the face of nutrient depletion, tion phase of protein synthesis also sensitized cells to the det- eEF2K was activated, autophagy increased, and cell survival rimental effects of starvation ( 13 ). An unanswered question improved (9 ). In contrast, when eEF2K was knocked down was, how do these kinases sense nutrient deprivation, and using RNAi, nutrient deprivation did not induce autophagy what survival mechanisms does the activity induce? and cell survival decreased (9 , 15 ). In certain cells, knockdown

ABNutrient/growth Nutrient/growth factors available factors depleted

AMP:ATP AMP:ATP

mTORC1 AMPK mTORC1 AMPK

4EBP1 S6K1 4EBP1 S6K1 eEF2K eEF2K

eIF4E eIF4E

Autophagy Autophagy “off” “on”

Translation Translation Translation eEF2-P Translation 60S eEF2 60S initiation “off” elongation “off” initiation “on” 40S elongation “on” 3´ 3´ 40S

Figure 1. A, nutrient availability favors protein synthesis. Adequate supply of glucose and amino acids triggers a series of reactions that stimulate protein synthesis. When the ratio of AMP to ATP is low, AMPK is inhibited and eEF2K is activated, thereby favoring peptide chain elongation. In addition, nutrient availability activates mTORC1, leading to initiation of protein synthesis and inhibition of autophagy. B, nutrient deprivation favors energy conservation. Inadequate glucose and amino acids trigger a series of reactions that diminish protein synthesis. Energy depletion increases the ratio of AMP to ATP, thereby activating AMPK, resulting in the activation of eEF2K, thereby inhibiting elongation through ribosomal stalling. In addition, starvation blocks the activity of mTORC1, leading to inhibition of initiation of protein synthesis and activation of autophagy.

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Downloaded from cancerdiscovery.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. VIEWS of eEF2K increased protein synthesis and decreased cellular autophagic response and increased the apoptotic effects of ATP, presumably through the inability to conserve energy by AKT inhibition ( 20 ). terminating protein synthesis and activating autophagy (15, In summary, several lines of evidence suggest that cancer 16 ). Thus, in the presence of environmental stress, malignant cells have adapted mechanisms to survive stressful conditions cells can conserve ATP by activating pathways that transiently by conserving energy through rapidly terminating protein stall protein synthesis and, in some cases, activate autophagy. synthesis. In the presence of adequate nutrients, protein syn- Recent work by Leprivier and colleagues (16 ) confi rmed and thesis proceeds through pathways that inhibit AMPK, activate extended several of these observations. By studying adapta- mTORC1 to initiate protein synthesis, and inhibit eEF2K to tion of transformed fi broblasts to nutrient deprivation, they promote peptide elongation. In contrast, under conditions found that adaptation was mediated through the activa- of metabolic stress where protein synthesis is metabolically tion of eEF2K by AMPK, and that the ability to adapt was detrimental, AMPK inactivates mTORC1 and activates eEF2K, severely compromised in cells where eEF2K was knocked out thereby inhibiting protein synthesis at both the initiation and or depleted by RNAi. The importance of eEF2K activation was elongation stages of protein translation. Drugs that disrupt also seen in nontransformed fi broblasts, and both these and this important survival mechanism may help produce better adapted transformed cells acutely blocked elongation during results in treating previously refractory malignancies. starvation. These and other data suggest that by limiting peptide elongation, cells are able to decrease the rate of ATP deple- Disclosure of Potential Confl icts of Interest tion, as suggested by earlier studies ( 15 ). Interestingly, when W.N. Hait and M. Versele are employees of Janssen, the pharma- RASV12-transformed NIH 3T3 cells overexpressing eEF2K ceutical companies of Johnson & Johnson. No potential confl icts of were transplanted into nu/nu mice, they were resistant to the interest were disclosed by the other author. growth-inhibitory effects of caloric restriction. Finally, this group also found that human gliomas with increased EEF2K Grant Support transcripts were of high grade and poor prognosis. J.-M. Yang was supported by U.S. Public Health Service NCI If starvation simultaneously inhibits protein synthesis R01CA135038. and activates autophagy, how are these processes connected? AMPK seems to provide a critical link. 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William N. Hait, Matthias Versele and Jin-Ming Yang

Cancer Discovery 2014;4:646-649.

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