Targeting Translation in Hypoxic Tumors David Ron†,* and Alan G. Hinnebusch‡,* †Skirball Institute of Biomolecular Medicine and the Departments of Medicine and Cell Biology, New
York University School of Medicine, New York, New York 10016, and ‡Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, Maryland 20892
U
nlike normal tissues that are nourished by robust and wellregulated capillaries, malignant tumors have fragile and inadequate blood vessels. Tumor cells must therefore adapt to transient fluctuations in the supply of oxygen and other nutrients (1 ). Recent work has emphasized the role of translational control in coping with this stress (2 ). The ability to rapidly attenuate global protein synthesis is believed to conserve energy and other resources in the acutely stressed cell. Translational control also interfaces with other aspects of regulated gene expression to alter the internal milieu for longer-term adaptation to metabolic challenges. Two recent papers address the complex signaling pathways by which hypoxia attenuates mRNA translation (3, 4 ). These will be considered for the insight they provide on the possibility of targeting the translational apparatus to selectively inhibit hypoxic cancer cells and possibly manipulate other physiological processes. Global protein synthesis is regulated at multiple levels, but among the best understood are two aspects that modulate initiation of mRNA translation. In eukaryotes most mRNAs are capped at their 5´ ends by m7Gppp, which serves as a ligand for eukaryotic translation initiation factor 4E (eIF4E), the cap-binding subunit of a complex (eIF4F) that recruits 40S ribosomes to the mRNA. Both stimulatory and inhibitory signals converge on eIF4E to regulate eIF4F activity and thereby rates of translation initiation (5 ). The participation of the 40S ribosome in translation initiation www.acschemicalbiolog y.o rg
is also regulated, as it must be charged with a ternary complex of eIF2, GTP, and amino-acylated initiator methionyl tRNA. Formation of this ternary complex is limited by the activity of a guanine nucleotide exchange factor for eIF2, eIF2B. The latter is inhibited, in trans, by phosphorylation of its substrate, eIF2 on serine 51 of its α subunit. Specific kinases have evolved to couple eIF2α phosphorylation and attenuated translation initiation to stressful events, and eIF2(αP)-specific phosphatases have evolved to counter-regulate and finetune translational repression (6, 7 ). It has long been known that hypoxia actively attenuates protein synthesis, but the mechanisms involved have remained obscure (8 ). A clue was provided by the finding that ischemia and the attendant hypoxia and nutrient deprivation activate PERK (9, 10 ), an eIF2α kinase that responds specifically to the stress of unfolded and misfolded proteins in the endoplasmic reticulum (ER stress) (11 ). The mechanism(s) by which hypoxia elicits ER stress and PERK activation remain obscure; however, the importance of PERK activation and eIF2(αP) to translational control in hypoxic cells is well documented by analysis of PERK–/– cells and cells bearing a Ser51 to Ala mutation in the regulatory residue of eIF2α (12 ). This adaptation to hypoxia has proven to promote survival of cancer cells in ischemic animal tumor models, as cancer cells compromised in their ability to effect eIF2α phosphorylation in response to hypoxia were compromised in their ability to proliferate and form large,
A B S T R A C T Recent insight into how mammalian cells adapt their translational machinery to hypoxic conditions raises the possibility of targeting components of the regulatory networks involved to selectively inhibit metabolically compromised tumor cells and possibly manipulate a broad range of other physiological processes.
*To whom correspondence should be addressed. Email:
[email protected]. Email:
[email protected].
Published online April 21, 2006 10.1021/cb600125y CCC: $33.50 © 2006 by American Chemical Society
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STRESS RESISTANCE Figure 1. eIF2-Mediated translation regulation in hypoxic tumors. Hypoxia and nutrient deprivation, consequences of an unstable blood supply, promote endoplasmic reticulum stress in tumor cells. The eIF2α kinase PERK is activated. eIF2(αP) inhibits the GTP exchange factor, eIF2B, and reduces levels of eIF2-GTP-charged initiator methionyl tRNA ternary complexes (see inset). Global protein synthesis is inhibited at the initiation step, conserving ATP, but some proteins, such as the transcription factor ATF4, are translationally induced as eIF2(αP) levels rise and these activate a downstream gene expression program that promotes stress resistance. ATF4 also upregulates a phosphatase, GADD34, to finely regulate levels of eIF2(αP) in the stressed cell. A specific inhibitor of PERK, or even a more general inhibitor of the entire class of eIF2α kinases, would deprive tumor cells of this adaptation to hypoxia and thereby compromise their survival.
ischemic tumors (12 ). The adaptive role of eIF2α phosphorylation is likely played out both at the level of its global affects on protein synthesis in hypoxic cells and through the induction of a cytoprotective gene expression program, known as the integrated stress response, which is activated by translationally controlled eIF2(αP)‑dependent transcription factor(s) such as ATF4 (13 ) (Figure 1). The two new papers from Liu (4 ), Korintzinsky (3 ), and colleagues confirm the contribution of eIF2α phosphorylation to translational control in hypoxic cells but also suggest the importance of eIF4F regulation in the process. The cap-binding subunit eIF4E partitions between nonproductive associations with a family of inhibitory eIF4E-binding proteins (4EBPs) and productive associations that form eIF4F. This equilibrium is regulated by the phosphorylation of the 4EBPs, which 146
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reduces their affinity for eIF4E and thereby de-represses translation initiation. Not surprisingly, a variety of growth pathways contribute to 4EBP phosphorylation, whereas growth-factor or nutrient deprivation increases the levels of dephosphoryl ated, inhibitory 4EBPs (5 ) (Figure 2). Hypoxia, as shown in both papers, also promotes higher levels of dephosphoryl ated 4EBPs and inhibits eIF4F formation. The paper by Liu and colleagues (4 ) examines in detail the signaling pathways involved in 4EBPs dephosphorylation. They focus attention on a recently-identified pathway that gauges energy insufficiency by monitoring the AMP/ATP ratio and in which the AMP-activated protein kinase (AMPK) serves as a major node. AMPK indirectly represses the TOR kinase and thereby leads to lower levels of phosphorylated 4EBPs and inhibits translation initiation. Liu and colleagues (4 ) call attention to redunRon and Hinnebusc h
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dancy in pathways that couple hypoxia to TOR repression by demonstrating that interference with single components does not completely block 4EBPs dephosphorylation. The paper by Koritzinsky and colleagues (3 ) contains an interesting twist concerning regulation of eIF4F; 4EBP dephosphorylation does not account for the entire disruptive effect of hypoxia on eIF4F formation. As expected, they report that an mRNA cap analog affinity matrix recovers eIF4E in complex with other components of eIF4F in lysates of unstressed cells. Hypoxia rapidly disrupts this complex, but not by the expected mechanism of replacing eIF4F components with dephosphorylated 4EBP as eIF4E’s binding partners. Rather, early in the course of hypoxia, a fraction of eIF4E is whisked to the nucleus by a shuttling factor, eIF4T. The upstream signals remain obscure, but the nuclear sequestration of eIF4E, away w w w. a c s c h e m i ca l biology.org
from eIF4F (and other components of the for linking AMPK to TOR repression are to specific inhibitors. Compounds that translational apparatus), correlates with the encoded by anti-oncogenes, and some inhibit AMPK (for which iodotubercidin is dephosphorylation of eIF4T. Hours later, measure of constitutive deregulation of a relatively non-selective prototype) could they observe the expected association of mRNA translation may be a common be tested for their ability to inhibit survival dephosphorylated 4EBPs with eIF4E. By feature of cancer cells (5 ). This suggests of hypoxic tumor cells, and the findings of contrast eIF2α phosphorylation is a rapid that de-repression of eIF4F, rather than its Liu (4 ), Koritzinsky (3 ), and their colleagues and transient response to hypoxia. regulation by hypoxia, might be favored in suggest the presence of other nodes Interestingly, Koritzinsky and colleagues cancer cells. It is notable in this regard that that could be explored by more specific (3 ) note that abolishing eIF2α phosphoryl rapamycin, a drug that inhibits activation inhibitors, were they available. The relative ation eliminates most detectable changes signals from TOR to eIF4F is being tested as simplicity of signaling through phosphoryl in global translation in hypoxic cells. Thus, an anti-tumor agent (15 ). ated eIF2(αP) suggests a pathway that the physiological significance of the draChemical biology can help sort through might be especially amenable to study with matic regulation of eIF4F in hypoxic cells, the pathophysiological significance of the chemical inhibitors. The crystal structures documented by both groups, remains to be myriad pathways regulating translation in of two eIF2α kinases, PKR and GCN2 have established. Furthermore, abolishing eIF2α hypoxic tumor cells. As these rely on kinase been reported recently (16 ), and these phosphorylation also eliminates most cascades, they are potentially susceptible contain design features unique to this detectable changes in mRNAspecific translation. However, it Growth signals remains unclear if this reflects O2, nutrients an ascertainment bias due to the AMPK ? limited number of genes examined or whether indeed eIF2(αP) elF4T elF4E also dominates as a signal to alter gene-specific translational Nuclear elF4T profiles in hypoxic cells. sequestration The existence of two discrete ways to regulate translation in 4EBP hypoxic cells begs the question: MET GTP How does regulation of eIF2 and P eIF4F affect survival of hypoxic elF2 cells and growth of tumors? The TOR 40S role of eIF2(αP) in promoting the elF4A survival of hypoxic tumor cells 4EBP is supported by genetic experielF3 elF4G ments (12 ); the contribution of elF4E elF4E hypoxia-mediated inhibition of eIF4F is less clear. Experiments in flies show that mutations mRNA compromising the ability to mRNA repress TOR and promote the Translation on Translation off 3′ regulated dephosphorylation 3′ of 4EBPs sensitize animals to Figure 2. eIF4F-Mediated translational regulation in hypoxic tumors. Translation initiation requires the hypoxia (14 ). Such experiments formation of an eIF4F complex (consisting of eIF4E, 4G and 4A), which binds the capped 5´ end of mRNAs via eIF4E and recruits the fully-equipped 40S ribosome to initiate scanning for an AUG start codon on the predict that stress-mediated 4EBPs dephosphorylation might mRNA. Hypoxia and nutrient deprivation activate the AMP-activated kinase and other stress-responsive pathways to inhibit the TOR kinase, which is under positive control of growth signals. Diminished TOR contribute to survival of hypoxic signaling results in hypophosphorylation of eIF4E binding proteins, the 4EBPs, and these sequester eIF4E tumor cells. At the same time, in an inactive complex, repressing translation initiation. By poorly understood mechanisms hypoxia also components of the apparatus promotes the eIF4T-mediated sequestration of eIF4E in the nucleus away from the translational apparatus. www.acschemicalbiolog y.o rg
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entire family that could be exploited by specific inhibitors. Even the phosphatases that dephosphorylate eIF2(αP) might be accessed by small molecules, as attested to by the properties of salubrinal, an indirect inhibitor that has recently been shown to promote survival of ER stressed cells (17 ). The two major pathways for general control of mRNA translation, touched on above, are very old and have arborized to influence not only survival of stressed cells but also a variety of other biological processes. These range from proliferation of immune cells required for graft rejection (pharmacological targets of the TORinhibitor rapamycin) to control of feeding behavior (18, 19 ) and memory formation (20 ). Small molecules that manipulate these pathways could find broad application as physiological probes and, possibly, valuable therapeutic agents. REFERENCES 1. Harris, A. L. (2002) Hypoxia–a key regulatory factor in tumour growth, Nat. Rev. Cancer 2, 38–47. 2. Wouters, B. G., van den Beucken, T., Magagnin, M. G., Koritzinsky, M., Fels, D., and Koumenis, C. (2005) Control of the hypoxic response through regulation of mRNA translation, Semin. Cell Dev. Biol. 16, 487–501. 3. Koritzinsky, M., Magagnin, M. G., van den Beucken, T., Seigneuric, R., Savelkouls, K., Dostie, J., Pyronnet, S., Kaufman, R. J., Weppler, S. A., Voncken, J. W., Lambin, P., Koumenis, C., Sonenberg, N., and Wouters, B. G. (2006) Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control, EMBO J. 25, 1114–1125. 4. Liu, L., Cash, T. P., Jones, R. G., Keith, B., Thompson, C. B., and Simon, M. C. (2006) HypoxiaInduced Energy Stress Regulates mRNA Translation and Cell Growth, Mol. Cell 21, 521–531. 5. Richter, J. D., and Sonenberg, N. (2005) Regulation of cap-dependent translation by eIF4E inhibitory proteins, Nature 433, 477–480. 6. Hinnebusch, A. G. (2000) Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes. In Translational Control of Gene Expression; Sonenberg, N., Hershey, J. W. B., Mathews, M. B., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor; pp 185–243. 7. Ron, D. (2002) Translational control in the endoplasmic reticulum stress response, J. Clin. Invest. 110, 1383–1388. 8. Lefebvre, V. H., Van Steenbrugge, M., Beckers, V., Roberfroid, M., and Buc-Calderon, P. (1993) Adenine nucleotides and inhibition of protein synthesis in isolated hepatocytes incubated under different pO2 levels, Arch. Biochem. Biophys. 304, 322–331.
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9. Kumar, R., Azam, S., Sullivan, J., Owen, C., Cavener, D., Zhang, P., Ron, D., Harding, H., Chen, J., Han, A., White, B., Krause, G., and DeGracia, D. (2001) Brain ischemia and reperfusion activates the eukaryotic initiation factor 2α kinase, PERK, J. Neurochem. 77, 1418–1421. 10. Koumenis, C., Naczki, C., Koritzinsky, M., Rastani, S., Diehl, A., Sonenberg, N., Koromilas, A., and Wouters, B. G. (2002) Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha, Mol. Cell. Biol. 22, 7405–7416. 11. Harding, H., Zhang, Y., and Ron, D. (1999) Translation and protein folding are coupled by an endoplasmic reticulum resident kinase, Nature 397, 271–274. 12. Bi, M., Naczki, C., Koritzinsky, M., Fels, D., Hu, N., Harding, H., Novoa, I., Varia, M., Raleigh, J., Scheuner, D., Kaufman, R., Bell, J., Ron, D., Wouters, B., and Koumenis, C. (2005) ER stressregulated translation increases tolerance to extreme hypoxia and promotes tumor growth, EMBO J. 24, 3470–3481. 13. Harding, H., Zhang, Y., Zeng, H., Novoa, I., Lu, P., Calfon, M., Sadri, N., Yun, C., Popko, B., Paules, R., Stojdl, D., Bell, J., Hettmann, T., Leiden, J., and Ron, D. (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress, Mol. Cell. 11, 619–633. 14. Reiling, J. H., and Hafen, E. (2004) The hypoxiainduced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila, Genes Dev. 18, 2879–2892. 15. Sawyers, C. L. (2003) Will mTOR inhibitors make it as cancer drugs? Cancer Cell 4, 343–348. 16. Hinnebusch, A. G. (2005) eIF2alpha kinases provide a new solution to the puzzle of substrate specificity, Nat. Struct. Mol. Biol. 12, 835–838. 17. Boyce, M., Bryant, K. F., Jousse, C., Long, K., Harding, H. P., Scheuner, D., Kaufman, R. J., Ma, D., Coen, D., Ron, D., and Yuan, J. (2005) A selective inhibitor of eIF2a dephosphorylation protects cells from ER stress, Science 307, 935–939. 18. Hao, S., Sharp, J. W., Ross-Inta, C. M., McDaniel, B. J., Anthony, T. G., Wek, R. C., Cavener, D. R., McGrath, B. C., Rudell, J. B., Koehnle, T. J., and Gietzen, D. W. (2005) Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex, Science 307, 1776–1778. 19. Maurin, A., Jousse, C., Averous, J., Parry, L., A, B., Cherasse, Y., Zeng, H., Zhang, Y., Harding, H., Ron, D., and Fafournoux, P. (2005) The GCN2 kinase biases feeding behavior to maintain amino-acid homeostasis in omnivores, Cell Metab. 1, 273–277. 20. Costa-Mattioli, M., Gobert, D., Harding, H. P., Herdy, B., Azzi, M., Bruno, M., Ben Mamou, C., Marcinkiewicz, E., Yoshida, M., Imataka, H., Cuello, A. C., Seidah, N., Sossin, W., Lacaille, J.-C., Ron, D., Nader, K., and Sonenberg, N. (2005) Translational control of hippocampal synaptic plasticity and memory by an eIF2 kinase, GCN2. Nature 436, 1166–1173.
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