Download Full Text - Cancer Discovery

VIEWS
IN FOCUS
Surviving Metabolic Stress: Of Mice (Squirrels) and Men
William N. Hait1, Matthias Versele1, and Jin-Ming Yang2,3
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. Collections of malignant cells would seem to be especially
vulnerable, as they manage to survive under conditions of
deprivation, such as lack of adequate blood supply, low pH,
and both oxygen and nutrient shortages. Recent evidence
provides further insights into how nature deploys a parsimonious approach to address this problem, from mice, to squirrels, to man. The PI3K pathway and autophagy are sensitive
to nutrient and growth factor availability and are fundamental mechanisms for cellular adaptation to stress. Although
several components of these pathways have been carefully
elucidated and reviewed (1, 2), the role of peptide elongation
has received less attention.
While studying Ca2+/calmodulin signaling, we identified a
previously unappreciated enzymatic activity in glioblastoma cells
and specimens (3). A 100-kDa phosphoprotein was present in
malignant glial cells but not in normal white matter, a tissue rich
in normal glia. Ultimately, we isolated both the phosphoprotein
[elongation factor-2 (eEF2)] and the kinase [elongation factor-2
kinase (eEF2K; ref. 4), also known as calmodulin-dependent protein kinase 3, originally described by Nairn and colleagues (5)],
cloned the gene, and characterized the enzyme as a unique protein kinase represented by relatively few enzymes in the kinome
(6). It had been shown that this kinase terminated protein elongation (7), one of the most avaricious consumers of cellular energy,
through phosphorylation of eEF2 at Thr56, thereby decreasing
the affinity of the elongating peptide chain for the ribosome.
eEF2, the only known substrate for eEF2K, is an elongation factor that mediates the translocation of peptidyl tRNA from the
ribosomal A site to the P site.
At the time, the conspicuously increased activity of eEF2K
in glioblastoma and other types of cancer cells compared
Authors’ Affiliations: 1Janssen R&D, LLC., Raritan, New Jersey; 2Department of Pharmacology, Penn State University College of Medicine; and
3
Penn State Hershey Cancer Institute, Milton S. Hershey Medical Center,
Hershey, Pennsylvania
Corresponding Author: William N. Hait, Janssen R&D, LLC., 920 US Highway 202 South, Raritan, NJ 08869. Phone: 908-927-3516; Fax: 908-9277716; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-14-0114
©2014 American Association for Cancer Research.
646 | CANCER DISCOVERYJUNE 2014
with adjacent normal tissue was perplexing; why was this
enzymatic inhibitor of peptide elongation increased in certain malignancies? Insights came from studies of hibernating
squirrels and weanling mice.
Hibernation, a survival strategy used by mammals to combat the threat of severe nutrient deprivation, requires marked
metabolic suppression. In hibernating ground squirrels, Chen
and colleagues (8) observed increased phosphorylation of
eEF2 in the liver and brain due to increased activity of eEF2K.
They surmised that increased phosphorylation of eEF2 was
a mechanism by which hibernating animals tolerate marked
reductions in cerebral blood flow. Since then, several studies have demonstrated that environmental stress, including
nutrient and growth factor deprivation as well as oxidative
and chemical insults, is a potent activator of this kinase (9).
The regulation of eEF2K activity provided further insights.
Proud and others (reviewed in ref. 10) found that control of
eEF2K is mediated by key enzymatic activities linked to metabolic homeostasis (Fig. 1). For example, eEF2K is completely
dependent on Ca2+/calmodulin, and is activated by AMP
kinase (AMPK) through phosphorylation at Ser398. AMPK
is a sensor of cellular energy, sensitive to changes in the ratio
of AMP to ATP. When the ratio is high, that is, when energy
is depleted, eEF2K is activated by AMPK and peptide elongation is terminated. Conversely, when the ratio is low, that is,
when energy stores are replete, AMPK is less active, eEF2K is
inhibited, and elongation is restored. Furthermore, eEF2K is
also regulated by insulin and mTORC1 through phosphorylation at Ser78 and Ser361, respectively (11, 12). Thus, in the
presence of adequate nutrients, protein synthesis is favored
through inactivation of AMPK and activation of mTORC1,
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
activity of eEF2K inhibits peptide chain elongation.
It is now appreciated that cancer cells use these same
pathways to rapidly terminate protein synthesis to conserve
energy. As mentioned above, a variety of stressful conditions increased the activity of eEF2K including hypoxia and
nutrient and growth factor deprivation (9). Furthermore,
when the kinase was depleted by RNA interference (RNAi) or
blocked by chemical inhibition, the ability to survive stress
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2014 American Association for Cancer Research.
VIEWS
KEY CONCEPTS
• Malignant cells have high energy requirements and
are compromised by inefficient utilization of glucose
and a variable blood supply.
• Protein synthesis is a major consumer of ATP and is
therefore tightly regulated during cell growth.
• Several important signaling pathways converge
on the protein synthetic apparatus and respond to
changes in nutrient and growth factor availability.
• Survival of malignant cells under conditions of nutrient
and growth factor depletion may rely on proper control
of the key enzymes that regulate protein synthesis.
• Although much is known about the enzymes that control the initiation of protein synthesis, far less attention has been given to the enzymes that regulate
peptide elongation, the process requiring more energy.
• This commentary focuses on the regulation of peptide elongation and discusses links to other survival pathways and ways they might be disrupted to
improve therapies.
was markedly reduced (9). Similarly, Cantley’s laboratory
found that failure to inhibit mTORC1 and block the initiation phase of protein synthesis also sensitized cells to the detrimental effects of starvation (13). An unanswered question
was, how do these kinases sense nutrient deprivation, and
what survival mechanisms does the activity induce?
A
Nutrient/growth
factors available
This leads to weanling mice. Nutrient and growth factor
deficiency is a potent inducer of autophagy (from the Greek
“self-eating”). Autophagy is stimulated in yeast through a
complex containing Atg1, a serine/threonine protein kinase
that can form a complex with Atg13 and Atg17. Autophagy
protects against severe energy depletion by digesting
organelles and cytoplasm and recycling the components for
energy production. When energy is abundant, autophagy is
suppressed by mTORC1 through direct interactions with the
Atg1/13/17 complex. Initially, differences in interpretation of
data led to uncertainty about the precise role of autophagy in
cancer; was it a mechanism of cell death or a conserved mechanism of survival, or both? An elegant set of experiments
highlighted the possibility that autophagy could serve as a
potent survival mechanism in malignancies. Like hibernating
squirrels, newborns are forced to find ways to survive during
the period of starvation between birth and the onset of lactation. Kuma and colleagues (14) studied survival in weanling
mice. At birth and before lactation begins, pups unable to use
autophagy due to lack of Atg5, an essential component of the
murine autophagosome, do not survive the abrupt lack of
nutrients preceding lactation. Survival was not impaired in
wild-type littermates or in newborn Atg5 mutants who were
force fed during the critical prelactation period.
Therefore, several laboratories considered a possible link
among nutrient deprivation, inhibition of protein elongation, and autophagy. In the face of nutrient depletion,
eEF2K was activated, autophagy increased, and cell survival
improved (9). In contrast, when eEF2K was knocked down
using RNAi, nutrient deprivation did not induce autophagy
and cell survival decreased (9, 15). In certain cells, knockdown
B
Nutrient/growth
factors depleted
AMP:ATP
AMP:ATP
mTORC1
AMPK
mTORC1
AMPK
4EBP1 S6K1
4EBP1 S6K1
eEF2K
eEF2K
eIF4E
eIF4E
Autophagy
“on”
Autophagy
“off”
Translation
initiation “on”
60S
40S
eEF2
3´
Translation
elongation “on”
Translation
initiation “off”
60S
40S
eEF2-P
3´
Translation
elongation “off”
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.
JUNE 2014CANCER DISCOVERY | 647
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2014 American Association for Cancer Research.
VIEWS
of eEF2K increased protein synthesis and decreased cellular
ATP, presumably through the inability to conserve energy by
terminating protein synthesis and activating autophagy (15,
16). Thus, in the presence of environmental stress, malignant
cells can conserve ATP by activating pathways that transiently
stall protein synthesis and, in some cases, activate autophagy.
Recent work by Leprivier and colleagues (16) confirmed and
extended several of these observations. By studying adaptation of transformed fibroblasts to nutrient deprivation, they
found that adaptation was mediated through the activation of eEF2K by AMPK, and that the ability to adapt was
severely compromised in cells where eEF2K was knocked out
or depleted by RNAi. The importance of eEF2K activation was
also seen in nontransformed fibroblasts, and both these and
adapted transformed cells acutely blocked elongation during
starvation. These and other data suggest that by limiting peptide elongation, cells are able to decrease the rate of ATP depletion, as suggested by earlier studies (15). Interestingly, when
RASV12-transformed NIH 3T3 cells overexpressing eEF2K
were transplanted into nu/nu mice, they were resistant to the
growth-inhibitory effects of caloric restriction. Finally, this
group also found that human gliomas with increased EEF2K
transcripts were of high grade and poor prognosis.
If starvation simultaneously inhibits protein synthesis
and activates autophagy, how are these processes connected?
AMPK seems to provide a critical link. Activation of AMPK by
energy depletion promotes autophagy, activates eEF2K, and
inhibits mTORC1. Furthermore, loss of AMPK or ULK1 (the
mammalian ortholog of ATG1) led to the accumulation of
an autophagy adaptor protein, p62, which itself is degraded
through autophagy (17). AMPK promotes autophagy not only
through direct phosphorylation and activation of ULK1, but
also through phosphorylation of beclin-1, a critical adaptor
molecule for the class III lipid kinase VPS34, which is essential
for autophagosome formation. We recently discovered potent
and selective inhibitors of eEF2K and of VPS34. VPS34 inhibitors completely impaired autophagy flux induced by metabolic
stress (as indicated by accumulation of p62), whereas eEF2K
inhibitors did not, raising questions about earlier data using
RNAi of a direct involvement of eEF2K in autophagy regulation. However, the selective eEF2K inhibitors, when used as
affinity probes, bound an eEF2K–beclin-1–VPS34 complex
present in cellular extracts. The availability of VPS34- and
eEF2K-specific inhibitors should allow careful dissection of
the regulation of elongation rate and autophagy under stress.
Like other members of the PI3K pathway, inhibition of
eEF2K may represent an attractive target for depriving cancer cells of an energy-conserving mechanism. In fact, there
have been several attempts at identifying potent and selective
eEF2K inhibitors (18, 19). We hypothesized that inhibitors of
prokaryotic histidine kinases might also inhibit the activity of
eEF2K based on the enzyme’s predicted amino acid sequence
deduced from the cloned cDNA. A family of histidine kinase
inhibitors contained a small molecule that nonspecifically
blocked the kinase at low micromolar concentrations (19).
As mentioned above, we recently identified extremely potent
and selective inhibitors of both eEF2K and VPS34. Recently,
it was shown that MK-2206, the first allosteric inhibitor of
AKT to enter the clinic, increased the expression of eEF2K,
apoptosis, and autophagy. Inhibition of eEF2K decreased the
648 | CANCER DISCOVERYJUNE 2014
autophagic response and increased the apoptotic effects of
AKT inhibition (20).
In summary, several lines of evidence suggest that cancer
cells have adapted mechanisms to survive stressful conditions
by conserving energy through rapidly terminating protein
synthesis. In the presence of adequate nutrients, protein synthesis proceeds through pathways that inhibit AMPK, activate
mTORC1 to initiate protein synthesis, and inhibit eEF2K to
promote peptide elongation. In contrast, under conditions
of metabolic stress where protein synthesis is metabolically
detrimental, AMPK inactivates mTORC1 and activates eEF2K,
thereby inhibiting protein synthesis at both the initiation and
elongation stages of protein translation. Drugs that disrupt
this important survival mechanism may help produce better
results in treating previously refractory malignancies.
Disclosure of Potential Conflicts of Interest
W.N. Hait and M. Versele are employees of Janssen, the pharmaceutical companies of Johnson & Johnson. No potential conflicts of
interest were disclosed by the other author.
Grant Support
J.-M. Yang was supported by U.S. Public Health Service NCI
R01CA135038.
Published online June 2, 2014.
REFERENCES
1. Cantley LC. The role of phosphoinositide 3-kinase in human disease.
Harvey Lect 2004;100:103–22.
2. White E. Deconvoluting the context-dependent role for autophagy in
cancer. Nat Rev Cancer 2012;12:401–10.
3. Bagaglio DM, Cheng EH, Gorelick FS, Mitsui K, Nairn AC, Hait WN.
Phosphorylation of elongation factor 2 in normal and malignant rat
glial cells. Cancer Res 1993;53:2260–4.
4. Mitsui K, Brady M, Palfrey HC, Nairn AC. Purification and characterization of calmodulin-dependent protein kinase III from rabbit
reticulocytes and rat pancreas. J Biol Chem 1993;268:13422–33.
5. Nairn AC, Bhagat B, Palfrey HC. Identification of calmodulindependent protein kinase III and its major Mr 100,000 substrate in
mammalian tissues. Proc Natl Acad Sci U S A 1985;82:7939–43.
6. Ryazanov AG, Ward MD, Mendola CE, Pavur KS, Dorovkov MV,
Wiedmann M, et al. Identification of a new class of protein kinases
represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad
Sci U S A 1997;94:4884–9.
7. Carlberg U, Nilsson A, Nygard O. Functional properties of phosphorylated elongation factor 2. Eur J Biochem 1990;191:639–45.
8. Chen Y, Matsushita M, Nairn AC, Damuni Z, Cai D, Frerichs KU,
et al. Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels. Biochemistry
2001;40:11565–70.
9. Wu H, Yang JM, Jin S, Zhang H, Hait WN. Elongation factor-2
kinase regulates autophagy in human glioblastoma cells. Cancer Res
2006;66:3015–23.
10. Pigott CR, Mikolajek H, Moore CE, Finn SJ, Phippen CW, Werner JM, et
al. Insights into the regulation of eukaryotic elongation factor 2 kinase
and the interplay between its domains. Biochem J 2012;442:105–18.
11. Browne GJ, Proud CG. A novel mTOR-regulated phosphorylation site
in elongation factor 2 kinase modulates the activity of the kinase and
its binding to calmodulin. Mol Cell Biol 2004;24:2986–97.
12. Proud CG. Signalling to translation: how signal transduction pathways control the protein synthetic machinery. Biochem J 2007;403:
217–34.
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2014 American Association for Cancer Research.
VIEWS
13. Choo AY, Kim SG, Vander Heiden MG, Mahoney SJ, Vu H, Yoon
SO, et al. Glucose addiction of TSC null cells is caused by failed
mTORC1-dependent balancing of metabolic demand with supply.
Mol Cell 2010;38:487–99.
14. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori
T, et al. The role of autophagy during the early neonatal starvation
period. Nature 2004;432:1032–6.
15. Wu H, Zhu H, Liu DX, Niu TK, Ren X, Patel R, et al. Silencing of
elongation factor-2 kinase potentiates the effect of 2-deoxy-D-glucose
against human glioma cells through blunting of autophagy. Cancer
Res 2009;69:2453–60.
16. Leprivier G, Remke M, Rotblat B, Dubuc A, Mateo AR, Kool M, et al.
The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation. Cell 2013;153:1064–79.
17. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair
W, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein
kinase connects energy sensing to mitophagy. Science 2011;331:456–61.
18. Lockman JW, Reeder MD, Suzuki K, Ostanin K, Hoff R, Bhoite L,
et al. Inhibition of eEF2-K by thieno[2,3-b]pyridine analogues. Bioorg
Med Chem Lett 2010;20:2283–6.
19. Arora S, Yang JM, Kinzy TG, Utsumi R, Okamoto T, Kitayama T,
et al. Identification and characterization of an inhibitor of eukaryotic
elongation factor 2 kinase against human cancer cell lines. Cancer
Res 2003;63:6894–9.
20. Cheng Y, Ren X, Zhang Y, Patel R, Sharma A, Wu H, et al. eEF-2 kinase
dictates cross-talk between autophagy and apoptosis induced by Akt
Inhibition, thereby modulating cytotoxicity of novel Akt inhibitor
MK-2206. Cancer Res 2011;71:2654–63.
JUNE 2014CANCER DISCOVERY | 649
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2014 American Association for Cancer Research.
Surviving Metabolic Stress: Of Mice (Squirrels) and Men
William N. Hait, Matthias Versele and Jin-Ming Yang
Cancer Discovery 2014;4:646-649.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerdiscovery.aacrjournals.org/content/4/6/646
This article cites 20 articles, 12 of which you can access for free at:
http://cancerdiscovery.aacrjournals.org/content/4/6/646.full#ref-list-1
This article has been cited by 1 HighWire-hosted articles. Access the articles at:
http://cancerdiscovery.aacrjournals.org/content/4/6/646.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
[email protected].
To request permission to re-use all or part of this article, contact the AACR Publications Department at
[email protected].
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2014 American Association for Cancer Research.