The Role of C-Myc in Regulation of Translation Initiation

The role of c-myc in regulation of translation initiation

Emmett V Schmidt*,1,2

1Cancer Research Center at Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA; 2MassGeneral Hospital for Children, 55 Fruit Street; Boston, MA 02114, USA

Translation initiation is important for the regulation of grow to aminimum size threshold, this property is both cell growth and cell division. It is uniquely poised to fundamental to both normal and malignant growth coordinate overall cell proliferation by its effects on both (Baserga, 1985). When the minimally perturbing set of growth and division. A number of translation initiation genes used to fully transform human cells was more factors are transcriptional targets of c-myc in a variety of carefully analysed, it became apparent that resistance assays. In particular, the mRNA cap-binding protein to nutrient deprivation was an equal partner to all of eIF4E has a myc-binding sequence in its promoter that is the other genetic changes required in malignant cells myc responsive in reporter assays and contains a high- (Hahn et al., 2002). Despite the simplicity of the logic affinity myc-binding site in chromosome immunoprecipi- that cells must grow in order to proliferate, relatively tation experiments. Several differential expression screens less attention has been given to studies of the genetic have demonstrated altered levels of eIF4E, along with mechanisms by which cell growth is perturbed several other translation initiation factors, in response to compared to other aspects of cellular proliferation. alterations of c-myc levels. The potential for eIF4E and Interestingly, examination of the c-myc oncogene led other translational control elements to mediate myc’s the way into these studies, just as c-myc has been a transforming functions is particularly important because paradigm for so many aspects of tumor biology. In eIF4E is itself a known oncogenic factor. The ability of reviewing c-myc’s functions, it is always important to translation initiation factors to affect both cell division remember that its deregulation in Burkitt’s lympho- control and cell growth control coincides with myc’s mas results in the most rapidly proliferating known remarkable effects on both cell growth and cell division. human tumor (Iversen et al., 1974). Thus, it is no Oncogene (2004) 23, 3217–3221. doi:10.1038/sj.onc.1207548 surprise to find that myc can deregulate many aspects of cell growth. Keywords: translational; control; eIF4E; ribosomal The broad consensus of work on c-myc places it synthesis; rDNA transcription squarely in a group of oncogenes that regulate transcription (Amati et al., 2001). Overwhelming evidence has shown that c-myc both positively and negatively regulates distinct sets of genes. Furthermore, Malignant cells develop as the result of an accumulation its interactions with protein components of the chro- of a finite set of genetic mutations that deregulate matin remodeling complexes and with histone-acetyl normal cellular proliferation. Disruption of intracellular transferases firmly establish that role. On the other pathways by as few as three genetic manipulations seems hand, the relative weakness of c-myc in standard to define alower limit of perturbationsrequired for a transactivation assays remains a conundrum in the face malignancy to develop (Hahn et al., 1999). Recent meta- of its potent oncogenic effects (Kretzner et al., 1992a). summaries hint that a minimum of six processes must be Moreover, the known binding sequence for c-myc, altered in every cancer cell before they become CACGTG, occurs frequently in the genome and the list malignant. Those changes include development of of experimentally confirmed myc-regulated genes is resistance to growth inhibition, evasion of apoptosis, startling in its breadth (Zeller et al., 2003). In general, immortalization, development of mitogenic indepen- c-myc appears to be a relatively weak transactivator of a dence, acquisition of autologous angiogenic regulation, broad array of genes that are rate-limiting steps in and development of intrinsic invasive properties nearly all aspects of normal cellular proliferation. In this (Hanahan and Weinberg, 2000; Hahn and Weinberg, context, its effects on growth regulation are to be 2002). Yet, these literature summaries have often expected. neglected a more basic property of all cancerous cells, Rigorous definitions of cell growth remain elusive. the ability to add cellular mass without being limited The earliest definitions of cell growth centered on by the normal growth mechanisms of the cell. Since cell size, cell mass, net protein content, and ribosomal cell division is completely dependent on the ability to rRNA (Killander and Zetterberg, 1965; Baserga, 1985). Myc-regulated genes now include those involved in *Correspondence: EV Schmidt, Cancer Research Center at Massachu- so many aspects of cell growth that it seems best to setts General Hospital, 55 Fruit Street, Boston, MA 02114, USA; use the loosest possible definition of growth to E-mail: [email protected] understand just how important myc is to the whole MYC and translation initiation EV Schmidt 3218 physiology of the growing cell. If there is apartof poorer fidelity. These parallels fit with the longtime the cell that needs to increase from one cell to the next, view that increased cell growth precedes, and is myc seems to regulate a rate-limiting step in its required for, DNA synthesis to occur in dividing metabolism. cells (Killander and Zetterberg, 1965; Hartwell and The first hint that myc can regulate cell growth Unger, 1977; Johnston et al., 1977). Nuclear came from myc-ER cells that express conditionally run-off assays in the conditional mycer cells demon- active c-myc that require the addition of estradiol strated that myc could activate transcription of eIF4E. for myc to function (Eilers et al., 1989). The initial Cloning of the eIF4E promoter provided the strong description of those cells demonstrated myc’s ability supportive evidence that it contains two canonical to directly induce entry into S phase. However, CACGTG motifs known to be the c-myc-binding site additional experiments revealed that net protein (Blackwell et al., 1990; Jones et al., 1996). One synthesis markedly increased 8 h after the activation of problematic feature of c-myc is its relatively weak the conditional allele of myc, many hours before DNA activity in reporter gene assays (Kretzner et al., synthesis increased. Indeed, DNA synthesis did 1992b). Importantly, myc could transactivate the eIF4E not occur until myc had been active for nearly promoter in standard reporter assays but, more 24 h (Rosenwald et al., 1993b; Rosenwald, 1996; importantly, dominant negative forms of c-myc down- Schmidt, 1999). This finding has provided the regulated eIF4E promoter activity (Jones et al., 1996). background for many subsequent experiments confirm- These formal connections between myc’s transactivating ing arole for myc in cell growth. activity and a gene involved in protein synthesis revealed Just as cell growth is an elusive concept, the idea of an important connection between c-myc and growth net protein synthesis is also elusive. The rate at which a control. A connection between myc and translation radioactive amino-acid precursor will be incorporated initiation makes particular sense because translation into all of the proteins being synthesized at any given initiation elements have particular effects on translation time point is the sum of multiple components of of cell cycle regulators (Polymenis and Schmidt, 1997; translation, balanced by ongoing degradation, summed Polymenis and Schmidt, 1999). These ideas provided a over all the proteins of the cell. Components of the biochemical outline for how cell growth might regulate translational apparatus that will affect these sums cell division. include the number of ribosomes, the rate at which Identification of the Drosophila homologue of human ribosomes are recruited to the mRNA to initiate myc, dMyc, allowed formal testing of the role of c-myc translation, the rate at which the ribosomes elongate in cell growth control (Gallant et al., 1996). This formal the nascent peptide, the rate at which ribosomes are testing showed that either loss or gain of dMyc had terminated in order to recycle, and the rate at which mis- primary effects on cell size in developing embryos with folded proteins might be immediately degraded only secondary effects on cell division rates (Johnston (Matthews et al., 2000). All of these steps can be rate et al., 1999). These findings were strongly supported by limiting for synthesis of individual proteins since the development of somatic rat fibroblast cells lacking c- examples of translational control can be found for all myc that also exhibited a primary growth defect of these steps. However, in aggregate, the cell apparently (Mateyak et al., 1997). Protein synthesis was markedly regulates translation primarily at its initial step, the rate impaired in these cells and their cell division rate of initiation of ribosomal recruitment to the 50 end of precisely paralleled the degree of the growth defect. mRNAs. Evidence for translation initiation as a key This interesting cell line was then used to identify regulatory step in protein synthesis has centered on the mRNA transcripts that required the continuous pre- fact that most actively translating mRNAs have a sence of c-myc for full expression (Bush et al., 1998). maximum number of ribosomes loaded while under- Expression of asurprisingly smallnumber of genes going translation, as if elongation components were decreases in the total absence of c-myc, although one present in excess (Matthews et al., 2000). The cellular must consider that many of the genes proposed as content of translation initiation factors further supports candidate myc targets are themselves critical to all this view since translation initiation factors are the least aspects of cell metabolism. This set of critical genes is abundant components of the translational apparatus likely to require multiple redundant transcriptional (Duncan and Hershey, 1983; Duncan and Hershey, controls that might easily substitute for c-myc in its 1985). absence, yet be critical targets during the myc upregula- Translation initiation factor eIF4E binds the 7-methyl tion that occurs in cancer cells. In the opposite direction, guanosine cap structure at the 50 end of all mRNAs upregulation of myc in transforming cells in transgenic as the initial step in translation. It is a particularly mice has profound effects on cell size and protein interesting component of the translational apparatus synthesis (Iritani and Eisenman, 1999). A plethora of because overexpression of eIF4E transforms cells myc targets have now been identified. On average one (Lazaris-Karatzas et al., 1990). In evaluating might expect that CACGTG will occur three times in the potential for transcriptional regulation of eIF4E, each gene, so it is not surprising to find a long list of myc parallel regulation of eIF4E mRNA and myc candidates. The most interesting element of this search levels became apparent (Rosenwald et al., 1993b). has been the result that candidate myc targets largely Levels of eIF2a, the methionyl tRNA recruitment function in normal cellular growth and metabolism factor, similarly paralleled myc levels, although with (Figure 1).

Oncogene MYC and translation initiation EV Schmidt 3219

Figure 1 Points of involvement of c-myc with cell growth and metabolism. Identified are general areas of cell growth control. The protein synthetic cycle is outlined on the left half of the cell schematic, while other portions of cell growth are illustrated on the right half of the schematic. Genes regulated by c-myc are identified under each major heading. (1) Glucose metabolism is perturbed by antisense c-myc and myc increases glycolysis (Valera et al., 1995; Shim et al., 1997; Shim et al., 1998; Lewis et al., 2000; Osthus et al., 2000; Collier et al., 2003). (2) Mitochondrial homeostasis is influenced by mycer and in myc-null cells (Wonsey et al., 2002; Haggerty et al., 2003). (3) Ribosomal content and ribosomal genes are increased by c-myc (as well as N-myc), and RNA polymerase III transcription is activated by c-myc (Coller et al., 2000; Greasley et al., 2000; Kim et al., 2000; Boon et al., 2001; Zeller et al., 2001; Watson et al., 2002; Gomez-Roman et al., 2003). (4) Amino-acid metabolism carbon sources are altered in myc-null cells (Nikiforov et al., 2002). (5) Translation initiation factors have been upregulated targets in several screens (Bello-Fernandez et al., 1993; Rosenwald et al., 1993b; Coller et al., 2000; Kim et al., 2000). Ornithine decarboxylase is involved in both translation initiation through its role in generating the hypusine required for eIF5A function, and in polyamine synthesis and is one of the more convincing myc-targets in multiple screens (Bello-Fernandez et al., 1993; Pena et al., 1993; Wagner et al., 1993; Packham and Cleveland, 1994; Tobias et al., 1995; Haggerty et al., 2003). (6) Iron metabolism can be regulated by myc, although in this case myc works as a repressor (Wu et al., 1999). Chromosome immunoprecipitation assays demonstrate that myc is present in vivo at the promoters of eIF4E, eIF5A, and the glucose transporter (highlighted in bold, red italics) (Fernandez et al., 2003)

The fascinating array of cell growth genes whose elements usually act as translational repressors, slowing mRNAs can be altered by c-myc is confounded by some progress of the scanning ribosomal assembly to the inconsistencies between the various screens that have ATG initiation codon. They are thought to be over- identified those genes. The development of quantitative represented in the least abundant mRNAs that are most immunoprecipitation assays allowed a recent screen to critical to cell proliferation controls. Yeast mutants identify promoters that are genuinely occupied in vivo by whose eIF3Z is temperature sensitive (cdc63) most c-myc, which has helped to address these inconsistencies effectively demonstrate the importance of these ele- (Fernandez et al., 2003; Orian et al., 2003). These CHIP ments. When these mutants are switched to the assays were carried out in actively proliferating myelo- restrictive temperature to limit translation initiation, blastic cells known to overexpress c-myc, as well as in they continue to grow in size but cease entering the Drosophila. The mammalian screen preselected for S phase of the cell cycle (Hanic-Joyce et al., 1987). Both genes whose promoters contained CACGTG motifs. mutational and functional studies identified the major The translation initiation factors that contained high- G1 regulatory control cyclin, cln3, as the major affinity myc-binding sites included eIF4E, eIF4A, and translational target of the cdc63 mutation (Polymenis eIF5A. Ribosomal protein promoters L13A, L19, L22, and Schmidt, 1997). A parallel control may exist in L27A, S19, and S6 also demonstrated high-affinity myc- mammalian cells because the cyclin D1 homologue of binding sites. For the moment, this approach provides cln3 has been repeatedly shown to be translationally the most compelling evidence that myc is a direct affected by factors regulating eIF4E (Rosenwald et al., transcriptional regulator of these proteins because myc 1993a; Rosenwald et al., 1995; Rousseau et al., 1996; is caught in the act, binding directly to the promoter. Brewer et al., 1999; Hidalgo and Rowinsky, 2000; The functional significance of myc’s potential activa- Nelsen et al., 2003). This direct effect of translation tion of translation initiation goes beyond its effects on initiation factors on mRNAs that regulate cell division cell growth. Complex translational control elements are may be especially important for mammalian myc’s role present in a broad variety of mRNAs. These can often in cell proliferation because myc’s predominant role in be identified as long 50 untranslated leader sequences normal mouse cell proliferation is to regulate cell containing high GC content (Kozak, 1991). These division (Trumpp et al., 2001).

Oncogene MYC and translation initiation EV Schmidt 3220 We recently developed aconstitutively activeinhibitor why does loss of c-myc seemingly fail to lower of translation initiation to better examine its global role expression of translation initiation factors? Are there in cell proliferation (Lynch et al., 2004). The 4E-binding compensating transcriptional elements that support protein (4EBP) inhibitor of eIF4E antagonizes its eIF4E and eIF5A levels in myc’s absence? Second, what functions and is highly regulated by signal transduction are the translational target’s of myc’s effects? It is pathways that phosphorylate and inactivate the 4EBP particularly intriguing to note that ornithine decarbox- (Lin et al., 1994). By mutating all of these inactivation ylase is a prominent transcriptional target of c-myc and sites in the 4EBP, we developed aconstitutive inhibitor a translational target of eIF4E (Bello-Fernandez et al., of eIF4E function. Importantly, constitutive expression 1993; Pyronnet et al., 1996; Rousseau et al., 1996). This of this eIF4E inhibitor blocked transformation by c- suggests that control of translation initiation by c-myc myc, underlining the potential importance of eIF4E as a could amplify its transcriptional effects on particular myc regulatory target in cancer. Moreover, the main proteins. The current state of knowledge about myc’s effects of the constitutively active 4EBP turned out to be effects on translation initiation would certainly encou- on cell division rather than cell growth. This study rage further studies in this area. provides a physiological conﬁrmation that myc transcriptional effects on translation initiation are important to the overall control of cell proliferation. Acknowledgements A number of features of myc’s role in regulating This work was supported by a grant from the National Cancer translation initiation remain puzzling at this point. First, Institute of the National Institutes of Health, RO1-CA63117.

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