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Seminars in Cancer Biology 16 (2006) 275–287

Review

The Oscar-worthy role of Myc in apoptosis

Natalie Meyer, Sam S. Kim, Linda Z. Penn ∗Division of Cancer Genomics and Proteomics, Ontario Cancer Institute/Princess Margaret Hospital,

Department of Medical Biophysics, University of Toronto, 610 University Avenue, Rm 9-628,Toronto, Ont. M5G 2M9, Canada

bstract

The discovery that the Myc oncoprotein could drive cells to undergo apoptosis in addition to its well-established role in cellular proliferationame in the early 1990s, at the beginning of a period of explosive research on cell death. Experimental evidence revealed that Myc sensitises cellso a wide range of death stimuli and abrogating this biological activity plays a profound role in tumorigenesis. Our understanding of the molecular

echanism and genetic programme of Myc-induced apoptosis remains shrouded in mystery and the focus of much attention. In this review, weill discuss established data, recent advances and future objectives regarding the regulatory processes and the functional cooperators that effect

nd abrogate apoptosis induced by Myc.2006 Elsevier Ltd. All rights reserved.

eywords: Apoptosis; Oncogene cooperation; Cancer progression; p53; Signalling

ontents

1. Proliferation versus apoptosis: a good story requires a conflict . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2752. Background to the apoptosis story . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2773. Myc structure and signalling add suspense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2784. Myc and p53: co-starring roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2795. Myc cooperation, the climax of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

6. Hints at the sequel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

.. .. .

7. Denouement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Proliferation versus apoptosis: a good story requiresa conflict

Researchers studying the Myc oncogene are celebrating itssilver anniversary in 2006. For 25 years, this enigmatic moleculehas been the subject of much scrutiny. Ten years of research hadshown that Myc certainly was a driving force in the cell cycle andmalignant transformation. However, in the early 1990s, some

urprising findings arose. In addition to its established roles inrowth and proliferation, the Myc oncogene was also a potentnducer of programmed cell death [1,2]. Research into the molec-lar basis of apoptosis was itself in its infancy, and scientists

∗ Corresponding author. Tel.: +1 416 946 2276; fax: +1 416 946 2840.E-mail address: lpenn@uhnres.utoronto.ca (L.Z. Penn).

044-579X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2006.07.011

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

struggled to explain the new paradox. How could this proteindrive such disparate functions? Why would it? How does thecancer programme overcome this obvious hurdle? It was evenmore puzzling when it was shown that Myc-induced apoptosisrequired the same regions of the protein as the transformation,proliferation and autosuppression functions [2]. Moreover, thesame interaction with its dimerisation partner Max was required[3], ruling out the possibility of an entirely separate mechanismof action.

Two seminal reports in different cell systems first revealedMyc had pro-apoptotic capabilities. When cytokines were with-

drawn from 32D myeloid cells with exogenous Myc expression,they underwent rapid cell death, while the normal response wasa rapid down-regulation of Myc, G1 arrest and delayed death [1].It was also discovered that although Rat1 fibroblasts expressing

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onstitutive Myc were unable to exit the cell cycle upon growthactor withdrawal, the number of cells in culture did not increaseignificantly over control. The cell number failed to increase overime due to a concomitant increase in cell death, and this deathas classified as apoptosis [2].It was not long before similar reports began surfacing for

he viral oncogene E1A [4,5], and the pRB target E2F1 [6–8].he original observations with Myc were extended and furtherupported, and a new hypothesis was born: that the prolifera-ion of cells is essentially tied to apoptosis as a built-in safetyechanism to defend against inappropriate proliferation [2].A “dual-signal” model began to emerge, where it was pro-

osed that sensitisation to apoptosis was a normal function ofyc expression, and this suicide signal was rescued by specific

urvival factors such as insulin growth factor or platelet-derivedrowth factor [9]. Apoptosis results if deregulated Myc expres-ion is present in conjunction with an anti-proliferative signal.ctopic expression of Myc in non-transformed cells sensitises

hem to apoptosis. Myc was capable of driving cells to die at anytage of the cell cycle, including post-mitotic commitment steps,nd in the absence of de novo protein synthesis, indicating thathe machinery for sensitisation was already present in a Myc-xpressing cell [9]. Furthermore, in the two cell systems where-myc has been inactivated, the cells are remarkably resistant toeath induced by various stimuli [10,11].

The Myc protein exerts its functions as a transcription factory both activating and repressing target genes. It is a memberf a large family of basic-helix–loop–helix-leucine zipperbHLH-LZ) transcription factors, which dimerise through theLH-LZ domains and contact DNA in the basic region [12].

he protein contains four additional domains shared by allyc proteins, known as Myc homology boxes I, II, III and

V. MBI is thought to play a role in protein stability, whileBII encodes both transcriptional activation and repression

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Fig. 1. Myc-sensitised apoptosis, a timeline of events. The major advan

r Biology 16 (2006) 275–287

apabilities essential for transformation and apoptosis [12].BIII and MBIV have only been recently characterised

13,14]. The gene regulatory functions require Myc to dimeriseith its partner protein Max, a smaller, ubiquitous bHLH-LZrotein that also mates with several other bHLH-LZ proteins,ncluding the Mxd family (formerly known as Mad/Mxi). It ishought that Myc activates genes at least in part by direct DNAinding and recruitment of transcriptional co-activators, whilet represses transcription by binding activators such as Miz-1,nterfering with the assembly of active transcription complexes12,15]. Members of the Mxd family of transcription factorsre mainly thought to antagonise Myc function. The details ofhis transcriptional regulation by Myc are discussed elsewheren this issue (doi:10.1016/j.semcancer.2006.08.001).

Deregulation of Myc is known to be a common event in cel-ular transformation, but it is difficult to discern what fractionf human cancers harbour deregulated Myc [12]. This is due toifferences in defining the term, and the reality that Myc can beeregulated by several means, not all of them easily detectable.or our purposes, “deregulated Myc” specifies any situation thatauses the molecule to be inappropriately active. This includes,ut is not limited to, chromosomal translocations and ampli-cations, activation of upstream growth stimulatory signallingascades, and increased protein stability.

The E�-Myc mouse is a model for the human disease Burkittymphoma, where a reciprocal chromosomal translocation tohe immunoglobulin locus leads to inappropriate expressionf Myc in the B-cell compartment. In the E�-Myc mouse, the-myc transgene is coupled to the immunoglobulin heavy chain

enhancer, leading to B-cell specific over-expression [16,17].

he lymphomas that develop are consistently clonal, indicating

hat additional mutations are absolutely necessary to produceumours. Researchers were given a clue as to why this might behen it was discovered that Myc could drive apoptosis in addi-

ces in understanding apoptosis sensitised by Myc are highlighted.

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ion to proliferation. The BCL2 protein had been characterisedy the early 1990s as a potent inhibitor of cell death, although theechanism was still very much a mystery. A completely novelethod of oncogene cooperation was proposed when it was

hown that mice doubly transgenic for E�-Myc and E�-BCL2utations displayed a marked decrease in latency of disease,

nd the disease was a leukaemia of early progenitor cells [18],ather than the lymphoma that develops with E�-Myc alone17]. This was consistent with the identification of BCL2 as anncoprotein that cooperates with Myc by providing a survivalignal in retrovirally transduced cytokine-dependent B cells19]. The cooperation was further investigated in cell culture,here it was shown that BCL2 could increase cell viability

nd block apoptosis driven by ectopic Myc in serum-starvedhinese hamster ovary cells [20], Rat1/MycER fibroblasts [21]nd Rat1a/Myc fibroblasts [22]. The latter two reports alsoemonstrated that the inhibition of apoptosis left the prolifer-tion functions of Myc intact. This new method of oncogenicooperation was markedly different from classical cooperationuch as that between Myc and Ras, as the cells typically didot form foci in culture [21]. Enforced BCL2 expression waslso capable of blocking Myc-induced cell death followingreatment with thymidine or the chemotherapeutic agent etopo-ide [21], demonstrating a probable mechanism of resistance toancer treatments. More recently, Letai et al. demonstrated thatustained BCL2 expression is required for maintenance of the�-Myc/BCL2 leukemia by using E�-Myc mice harbouring aCL2 transgene that shuts off expression when doxycycline isdministered. When the tetracycline analogue was added to therinking water, BCL2 expression was down-regulated, massive

poptosis resulted and the tumours regressed [23].

Thus, the initial flurry of research activity clearly showed thathe ability of Myc to sensitise cells to apoptosis was an intrinsicroperty, dependent on Myc:Max interaction, and was somehow

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r Biology 16 (2006) 275–287 277

ontrolled by Myc as a regulator of gene transcription. Abro-ation of this pro-apoptotic property profoundly contributes toancer progression. To further define the role of Myc in celleath, a basic understanding of the molecular mechanism ofpoptosis is required and follows here (Fig. 1).

. Background to the apoptosis story

Programmed cell death is an evolutionarily conserved methodulticellular organisms use to dispose of cells in an orderly

ashion. It is characterised by several morphological features,amely nuclear condensation and fragmentation, membranelebbing and non-inflammatory phagocytosis of the cell frag-ents [24]. This is in contrast to cell necrosis, where the cell

ursts and an inflammatory response follows [25]. Apoptosistudies have obvious applications in cancer research, but they arelso important in many other human pathologies, including sep-is, ischemia, neurodegenerative and autoimmune diseases [26].he importance of apoptosis research was properly recognised in002, when Drs. Brenner, Horvitz, and Sulston were awarded theobel Prize in Physiology or Medicine for their pioneering workn the genes involved in organ development and programmedell death. There are two main recognised pathways of apoptosis,he intrinsic and extrinsic pathways, although there is evidenceor extensive cross-talk between the two. We will introduce herehe features of each pathway that are specifically important to

yc-induced apoptosis; readers are referred to more completepoptosis-specific reviews for more information [27–30].

The intrinsic pathway of apoptosis can be triggered by variousntracellular cues, such as DNA damage, oncogene activation,

ypoxia or limited growth factors. The tumour suppressor p53s a key manager of intracellular death signals, and just how itccomplishes this task is discussed later. Intrinsic pathway sig-als converge on the mitochondria, where the balance between

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ro- and anti-apoptotic molecules determines whether the cellill survive or proceed inexorably to death. This concept of

he “apoptotic threshold” is a recurrent theme, since evidencendicates that healthy cells contain all the necessary moleculesor programmed cell death kept in check by survival factors.he BCL2 family of proteins ultimately control the decisionn mitochondrial cell death. This family can be broadly clas-ified into two groups. Anti-apoptotic family members such asCL2 and BCL-XL contain all four BCL2 homology domains

BH1–4). Pro-apoptotic members can be either multi-domainBH1–3) pore-forming proteins, such as BAX and BAK, ormaller BH3-only proteins, such as BID, PUMA, NOXA andIM. It is the balance between these pro- and anti-apoptoticroteins that determines if cell death will proceed [29]. “Acti-ator” BH3-only domains such as BID and BIM are thought toe normally sequestered by anti-apoptotic proteins. The “sensi-iser” domains, such as those from BAD and BIK, can displacehe activators, freeing them to bind and activate the multi-domainro-apoptotics BAK and BAX [31]. These molecules are thoughto homo-oligomerise, forming pores leading to mitochondrialuter membrane permeabilisation (MOMP), loss of membraneotential and respiration, and the escape of various pro-apoptoticactors [32]. Much of the mechanistic evidence discussed in thiseview shows Myc having profound effects on mitochondrialntegrity through the BCL2 family of proteins.

One of the important molecules released from the mitochon-ria is cytochrome c, which can bind to Apaf-1 and activateaspase-9, which in turn cleaves downstream caspases-3 and7, for review see [33]. Caspases are a family of proteaseshat cleave substrates at consensus sites containing asparagineesidues. They must be cleaved from the pro-form to becomectivated; upstream “activator” caspases cleave downstreamexecutioners” in a cascade. Caspase activation is antagonisedy the inhibitor of apoptosis proteins, IAPs. The executionersleave a wide variety of cellular substrates, including poly(ADP-ibose)polymerases (PARP), actin and the pRB cell cycle regu-ator. The fragmentation of the cell that takes place after caspasectivation leads to the morphological hallmarks originally usedo classify apoptosis [33].

Executioner caspases can also be activated without loss ofitochondrial potential through the death receptor pathway, also

nown as the extrinsic pathway of apoptosis. Death receptorsmplicated in Myc-induced apoptosis include CD95/Fas, tumourecrosis factor receptor (TNFR) and death receptor DR5, whichesponds to TNF-related apoptosis-inducing ligand (TRAIL).pon death ligand stimulation, receptors oligomerise, bringing

ntracellular death domains (DD) together so they can interactith adaptor molecules such as FADD and TRADD [28]. Adap-

or proteins contain both DD and death effector domains (DED),hich in turn interact with the DED domain on procaspase-8,

riggering its cleavage and activation. The complex of deatheceptor, adaptor protein and procaspase-8 is known as theeath-inducing signalling complex. Activated caspase-8 can

irectly activate executioner caspases-3, and -7, triggering celleath without input from the mitochondria. However, the pro-poptotic BH3-only molecule BID is also a substrate of activatedaspase-8. Its cleavage leads to myristoylation and targeting to

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r Biology 16 (2006) 275–287

he mitochondria where it can activate BAX and BAK, ampli-ying the death receptor signal [34]. This cross-talk betweenxtrinsic and intrinsic pathways provides enough death signal toasily overcome the apoptotic threshold, committing the cell toeath. The molecule cFLIP antagonises caspase-8 activation. Itas a similar structure to caspase-8, but it lacks the catalytic pro-ease domain and can therefore not cleave executioner caspasesr BID [33].

. Myc structure and signalling add suspense

A number of studies have indicated that Myc repression ofarget genes is correlated with its apoptosis functions, whileransactivation activities tend to segregate with proliferation androwth [35,36]. This is not absolute, however, as mutants showno be capable of driving transactivation and defective in Miz-1-ependent repression still cause apoptosis [37]. Repression ofiz-1 activity by Myc is important in Myc-induced apoptosis

s Miz-1 activates the cell cycle inhibitor p21, leading to growthrrest instead of apoptosis [38]. In addition, MBIV deletionutants were reported to have reduced function in both transac-

ivation and repression activities, while being largely resistant toerum withdrawal-induced apoptosis. This mutant was capablef transforming Rat1a cells in a soft agar assay, but was defectiven Rat1a focus formation [14]. It has also been next to impossi-le to unlink the structure–function data on Myc-induced deathrom protein stability studies. Much attention has been paido mutations found in human Burkitt lymphoma samples andhe super-transforming isoforms of the avian v-Myc protein39]. Many of these mutations centre on the apparent phos-horylation site T58 in MBI, which when mutated to alanine,oth increases protein stability [39–41] and leads to reducedpoptosis [36]. It is difficult to discern then, if one or both ofhese results leads to the increased transforming ability of the

utants. In fact, Tansey and co-workers have proposed a modelhereby transcriptional activity is dependent on degradation sig-als, and suggest a mechanism whereby Myc is “licensed” toegulate transcription for only a limited time [42,43]. Muta-ions at the T58 phosphorylation site, thought to be a signalor degradation, were shown to have slightly reduced apoptosisnd repression at one target reporter gene, and increased colonyorming potential [36]. The recent characterisation of the third

yc-homology domain, MBIII, seems to support the repressionheory. An MBIII deletion mutant reduced somewhat, but did notbolish, transactivation of four reporter genes; however it wasompletely incapable of down-regulating any of the repressioneporters tested and led to a greater number of colonies in softgar [13]. The MycS isoform results from an alternative transla-ion start site located at amino acid 100, and thus entirely lacks

BI. When human c-Myc isoforms were expressed in dmyc-nullrosophila, MycS-expressing flies displayed normal prolifera-

ion, but reduced caspase-3 activation and reaper expression,ndicating that regions missing from this protein are important

or apoptosis induction [44]. More details of the structure andunction of Myc are reviewed in [12].

Ras signalling has been found to play a dual role in Myc-nduced apoptosis. On the one hand, when the signal from

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as flows primarily through the PI3K and PKB/AKT pathway,yc-potentiated apoptosis is abrogated. However, when Ras

timulates the Raf, MAPK and ERK pathway, the net effects to increase the level of apoptosis [45,46]. A number of paral-el observations have been made for the stress-activated proteininase p38/c-fos pathway [47–49]. More light was shed on thisubject when it was shown that phosphorylation of the protein-tabilising site S62 was likely controlled by ERK, leading to

yc stabilisation and apoptosis; while PKB/AKT downstreamarget GSK3 was likely phosphorylating Myc at T58, leadingo protein degradation [50,51]. The small GTPase RhoB haslso recently been shown to shorten Myc half-life by increasinguclear accumulation of GSK3 [52]. Another group found thatells transformed with Myc and Ras required MEK activity tobrogate apoptosis when growth factors were present, consistentith the observation that the MEK pathway can confer survival

n certain cell types [53].Curiously, one report showed that the most common muta-

ion in Burkitt lymphoma, T58I, showed comparable apoptosisevels to wild-type in reconstituted Myc-null rat cells, and aeduced transformation ability in a Ras/Myc rat embryo fibrob-ast co-transformation assay [54]. This indicates that abrogationf apoptosis through mutation of T58 may not be a major con-ributor to cancer progression. Further support is found in humaneoplastic disease, which commonly shows deregulation of Mycy various methods, and rarely reveals changes to the proteinequence [55]. More often seen are genetic changes in cooper-ting pathways, some of which are the focus of this review.

. Myc and p53: co-starring roles

If Myc can cooperate with the over-expression of a strongnti-apoptotic molecule, it follows that cooperation would alsoe seen with loss of a pro-apoptotic molecule. The p53 tumouruppressor is widely known as the “guardian of the genome”56]. p53 acts to sense and integrate intrinsic cellular damage sig-als and leads the cell down a path towards either growth arrest,here the damage can be repaired, or into apoptosis [57,58].

ndeed, when E�-Myc mice were crossed with p53+/− mice, aecreased latency of disease was evident [59,60].

Myc has been reported to potentiate apoptosis through both53-dependent and -independent mechanisms [61]. The mecha-ism by which Myc drives apoptosis in a directly p53-dependentanner has been well characterised and involves Myc acti-

ating the ARF-Mdm2-p53 axis. The p53 tumour suppressorene is suspected to be mutated in about 50% of all tumours62,63]. It becomes activated by various stress signals, such asNA damage, hypoxia, limited growth factors, and activationf oncogenes such as Myc [64]. The activation of p53 leads toither growth arrest or apoptosis, and p53-mediated apoptosisas been shown to consist of both transcription-dependent andindependent mechanisms [65].

p53 has been classically characterised as a transcription fac-

or that binds to DNA in a consensus sequence-specific man-er, to transcribe either growth arrest-inducing genes such as21, GADD45 and 14-3-3, or apoptosis-inducing genes such asAX, PUMA, Fas, Pidd, DR5, Noxa, and BIM [58]. However,

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number of recent reports have demonstrated that p53 can alsoromote apoptosis in a transcription-independent manner, withhe p53 protein itself translocating from the nucleus to directlyunction at the mitochondria. It has been reported that p53 canind to either BCL2/BCL-XL, BAK, or promote BAX activity65]. Recent evidence suggests that transcription-dependent andindependent mechanisms of p53-induced apoptosis are linked,s p53 at the mitochondria can cooperate with its target generoduct PUMA to induce apoptosis [66].

Although p53 protein is present in cells at all times to acts a sentinel against damage, protein levels must be kept lown unstressed cells since it can so potently induce cell death.

dm2 is the best characterised negative regulator of p53. Mdm2inds to and mono-ubiquitinates p53, leading to its translocationrom the nucleus to the cytoplasm where it can be then furtherolyubiquitinated and degraded by the proteosome [67]. Thembryonic lethality of Mdm2−/− mice can be rescued whenrossbred to p53−/− mice, demonstrating that the Mdm2−/−ice die in utero due to high levels of p53 [68]. Mdm2 and

ts human homolog Hdm2 are commonly found to be amplifiedr over-expressed in tumours, suggesting that evasion of p53-ediated apoptosis can be achieved through increased Mdm2

xpression [69]. Mdm2+/−/E�-Myc mice live twice as longs their Mdm2+/+/E�-Myc littermates [70]. This phenotype isescued in the triple mutant Mdm2+/−/ARF+/−/E�-Myc mouse71].

Expression of ARF inhibits Mdm2 function. ARF is a tumouruppressor, whose transcript is formed from the alternative read-ng frame of the Ink4a locus [72]. The locus encodes two impor-ant tumour suppressors, ARF and p16INK4a, and is deleted in aariety of human cancers. p16INK4a functions as a tumour sup-ressor by inhibiting cell cycle progression through binding toyclin-dependent kinases-4 and -6 [73]. The second gene prod-ct identified from the Ink4a locus was ARF. This protein wasuspected to be in a common genetic pathway as p53 when ARFoss abrogated the requirement for p53 inactivation to immor-alise mouse embryo fibroblasts [74]. Furthermore, ARF wasble to inhibit transformation of MEFs by Mdm2 and Ras, butad no effect on transformation in cells lacking p53 [75]. Mech-nistically, ARF inhibits Mdm2 in two ways. ARF can bind todm2 directly and inhibit its p53 ubiquitination function [76],

nd it can sequester Mdm2 in the nucleolus and prevent it frominding to p53 [77,78].

ARF is sensitive to the activation of many oncogenes, includ-ng Myc, E2F1, E1A, v-abl, and Ras [73]. Myc activation leads toncreased ARF expression [79], although the exact mechanism isnknown. So far we are not aware of any evidence that Myc canranscriptionally regulate ARF by binding to Ink4a locus. The

issing link might be E2F1, since Myc induces E2F1 expressionn primary cells [80,81], and E2F1 can induce ARF directly [82],lthough this was not evident in a mouse model [83]. It remainshough, that Myc can induce p53-dependent apoptosis throughctivation of ARF, which inactivates Mdm2, in turn leading to

ctivation of p53.

p53 mutations have been found in at least 30% of Burkittymphoma biopsies [84,85] and 60–70% of Burkitt lymphomaell lines [86–89]. The great majority of Burkitt lymphoma

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ell lines and E�-Myc mice have either Ink4a locus deletions,dm2 over-expression, or p53 mutations [90,91]. Both suggest

hat inhibition of the ARF-Mdm2-p53 pathway is necessary forumours to evade Myc-induced apoptosis and maintain tumori-enicity.

Although there is strong evidence supporting the Myc-ARF-dm2-p53 axis, emerging evidence proposes more complex

egulation of this pathway. It has long been known that Mdm2nd p53 have a dynamic feedback loop where Mdm2 nega-ively regulates p53, while p53 transcriptionally up-regulates

dm2. Recently though, two independent groups have reportednovel feedback mechanism involving Myc and ARF. Although

he details differ, both groups demonstrated that when ARFnd Myc are over-expressed, they interact, translocate to theucleolus, and Myc activity is reduced [92,93]. These find-ngs advocate that ARF can function as tumour suppressor andnhibit cell proliferation through two distinct mechanisms, inac-ivation of Mdm2 and interference with Myc transcriptionalctivity.

p53 activation leads to either growth arrest or apoptosis,nd evidence shows the decision between these two possibleutcomes might be critically regulated by Myc. p53 mediatesell cycle arrest partly through direct transcription of the cellycle inhibitor p21 [94], which is also a transcriptional targetf Miz-1 [95]. Myc mediates repression of p21 [96] throughiz-1 [37,95], and the outcome of daunorubicin-mediated p53

ctivation in HCT116 colorectal cancer cells is dependent onxpression of Myc [38]. Surprisingly, ectopic expression of Mycid not alter the expression of p53 pro-apoptotic target genesUMA, PIG3, and BAX. Myc not only activates p53, but itlso causes p53 to induce apoptosis instead of growth arrest byepressing p21, while leaving pro-apoptotic targets unaffected38]. Another related feedback mechanism that has been char-cterised is between Myc and p53, with the recent report thatctivated p53 can repress c-myc in mouse tissues and humanumour cell lines [97]. The authors demonstrated p53 binding tohe c-myc promoter in vivo through chromatin immunoprecipita-ion. Ectopic expression of Myc reduced p53-mediated G1 arrestnd differentiation and increased the ability of p53 to promotepoptosis. These data suggest that for the p53 growth arrest pro-ram it is essential to not only transactivate p21, but also repress-myc. Together, the Myc and p53 feedback loop is fascinating.hese findings suggest it is the state and level of both Myc and53 that are important in determining whether cells will undergopoptosis or cell growth arrest.

. Myc cooperation, the climax of apoptosis

Despite almost heroic efforts, we are still uncertain about thepecific pathways by which Myc sensitises cells to apoptosis.he target genes activated and suppressed by Myc are offeringlues, but in many cases the wide range of targets mainly cloudshe issue [98–101]. Powerful new technologies, discussed in the

ext section, will hopefully shed light on the genetic programmeey to the apoptotic pathway. In the meantime, strong functionalvidence has emerged from the discovery of proteins, and moreecently non-coding RNAs, that cooperate with Myc in cellular

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ransformation. This is helping to unravel the molecular mech-nism by which Myc can drive apoptosis.

Because Myc can sensitise cells to such a broad range ofpoptotic stimuli, it is very likely that it affects a common noden the apoptosis pathway. Much of the evidence, whether in53-dependent or -independent systems, points to the signalmplification step at the mitochondria, where Myc seems to playrole in altering the balance between pro- and anti-apoptoticCL2 family members, moving the cell closer to the so-calledpoptotic threshold. A summary of the molecules discussed inhis section is shown in Fig. 2.

An early report from Juin et al. indicated that a robust releasef cytochrome c from the mitochondria lies downstream ofyc activation [102], and many articles have followed to try

o determine how Myc exerts this effect. Cytochrome c has beenroposed to be a direct transcriptional target of Myc [103]. Aumber of studies performed in the Cleveland lab have demon-trated Myc-dependent, yet likely indirect, suppression of BCL2nd BCL-XL [104–106], and indicate that suppression of thesenti-apoptotic family members must be averted for lymphomaevelopment in the E�-Myc mouse [104]. Reports reveal thatuppression of BCL-XL, rather than BCL2, is especially impor-ant in the p53-independent apoptosis pathways driven by Myc104,106], but that this preference might be cell-type specific10,105].

The pro-apoptotic molecule BAX is thought to follow a multi-tep path to become fully activated, form a pore, and mediatehe release of cytochrome c and other apoptotic factors from the

itochondria [107]. The presence of BAX increased annexin-Vtaining in serum-starved MEFs expressing ectopic Myc [108].AX loss was clearly shown to cooperate with Myc in driv-

ng lymphomagenesis in the E�-Myc model, and interestingly,9% of tumours analysed did not have detectable mutations inhe ARF-Mdm2-p53 pathway [109], indicating that this path-ay might operate independently or downstream of p53. Further

vidence for this hypothesis has been found in the observationhat Myc can functionally cooperate with BAX in fibroblastsn vitro [110]. Although BAX was found to be capable ofranslocating to the mitochondria in the absence of Myc, it didot undergo the conformational change thought to be requiredor oligomerisation and pore formation [10,107]. This Myc-ependent conformational change might operate through BID111]. It is clear that BAX expression levels are not affectedy the presence or absence of Myc [10,38,104,109]. Despitehe functional overlap seen in most cases for BAX and BAK,ooperative data on Myc and BAK loss has been conspicu-usly absent. This issue was addressed definitively in a studyhowing that Myc-induced apoptosis is abrogated in pancreaticcells of Bax−/−Bak+/+ animals, while Bax+/+Bak−/− animals

ndergo apoptosis normally when Myc is driven by the insulinromoter [112]. Further strengthening the argument for BAXnvolvement in Myc-sensitised apoptosis is the glycoproteinlusterin, which was shown to block Myc-induced apopto-

is by binding to conformation-altered BAX and inhibiting itsligomerisation. Over-expression and knock-down experimentsemonstrated that Clusterin cooperates with Myc in the presencef BAX to form colonies in soft agar and tumours in nude mice

N. Meyer et al. / Seminars in Cancer Biology 16 (2006) 275–287 281

Fig. 2. Deregulated Myc can sensitise cells to apoptosis through many different pathways. Many pro-apoptotic tumour suppressor proteins, the “Ambassadors”, havebeen shown to act downstream of deregulated Myc to induce the apoptotic safety mechanism. Much of the evidence is from functional data, with no suggestion ofdirect transcriptional effects by Myc. Shown below are oncogenes that have demonstrated cooperation with deregulated Myc, the “Abrogators”. These oncogenesb o be rc

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lock Myc-induced apoptosis, allowing the full proliferative potential of Myc tross-talk exists.

113]. To be clear, none of these studies is indicating a mitochon-rial role for Myc, rather that Myc is transcriptionally up- andown-regulating other factors that control BAX oligomerisationt the mitochondria.

PUMA (p53-up-regulated modulator of apoptosis) is anotherro-apoptotic BH3-only protein linked to Myc and p53. PUMAocalises to the mitochondria to induce cell death, and experi-

ents in Puma−/− mouse thymocytes and myeloid stem cellsuggest it is essential for p53-mediated apoptosis as its lossenders cells resistant to death induced by gamma irradiationr deregulated Myc [114]. These findings imply that PUMAunctions as a pro-apoptotic molecule downstream of both Mycnd p53. The mechanism as to how PUMA can function as a

ro-apoptotic molecule at the mitochondria has been recentlylucidated. PUMA was found to be important in releasing mito-hondrial p53 from its complex with Bcl-xL presumably allow-ng p53 to activate Bax [66].

ahir

ealised. Pathways in the image are linear for simplicity, but much evidence for

It is always prudent to remember that while experimentsn culture can reveal details of molecular mechanism, they

ay not always be representative of conditions in animals. Fornstance, Soengas et al. clearly demonstrated that loss of Apaf-

and caspase-9, which are thought to operate downstream ofOMP, reduced p53-dependent, Myc-induced apoptosis in cul-

ured fibroblasts, and led to colony formation in soft agar andumours in nude mice [115]. However, when Apaf-1−/− andaspase-9−/− mice were crossed to the E�-Myc mouse, no coop-rative effect was seen in transplanted foetal liver cells [116].ther reports hint that Apaf-1 and caspase-9 might not be obli-ate mediators of apoptosis in mouse models [117,118]. Sim-larly, NF-�B activation was shown to abrogate Myc-induced

poptosis in cell lines [119–121], while its loss was not found toave any effect on E�-Myc lymphomas [122]. The in vitro exper-ments reveal important relationships, while the animal workequires us to dig deeper into functionally redundant pathways

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82 N. Meyer et al. / Seminars in C

r tumour- or cell-type specificities before we can move ournowledge into the clinic.

It has long been known that Myc can drive apoptosis throught least one mechanism that is entirely p53-independent, buthe molecular mechanism has not yet been solved. Some excit-ng research into this problem has been reported in the last twoears, beginning with the observation that haploinsufficiency oross of the BIM protein, which promotes apoptosis by bindingnd sequestering BCL2 [31], decreased the latency of disease in�-Myc transgenic mice, and that this loss actually relieved the

ransformed cells of the necessity to disable the p53 pathway123]. It was also recently shown that the common Burkitt lym-homa alleles T58A and P57S were not capable of up-regulatingim and thus showed a decreased latency of disease [124].

Functional cloning is a powerful method of identifying novelooperators, and two recent examples demonstrate how fre-uently researchers are led back to the p53 pathway. In the paperlassifying the bHLH-LZ protein Twist as a potential oncogene,he authors showed that Twist could block Myc-induced apop-osis and cooperate with E1a and Ras to drive colony growthn soft agar, and that Twist expression interfered with p53 tran-criptional activity by down-regulating ARF expression [125].iRNA-mediated knock-down of Twist in human neuroblastomaells led to increased Myc- and p53-dependent apoptosis. Inter-stingly, Ink4a-null fibroblasts were still transformed by N-Mycnd Twist, indicating that the effect on ARF is not the onlyethod of cooperation [126]. Similarly, we recently identifiedUL7, a member of the Cullin family of E3 ubiquitin ligases, asfunctional inhibitor of Myc-induced apoptosis. We have shown

hat CUL7 can cooperate with Myc to drive colony formationn soft agar. CUL7 appears to exert its anti-apoptotic functiony binding directly to p53 to promote polyubiquitination, andiRNA-mediated knock-down in human neuroblastoma cells ledo the increase in p53 protein levels and p53-dependent apoptosis127].

The ATM and ATR kinase pathways have also been shown toooperate with deregulated Myc in driving p53-mediated apop-osis. The kinases have been shown to act independently andn concert with one another following various types of DNAamage. Both kinases phosphorylate p53, stabilising the proteiny blocking interaction with Mdm2 [128]. p53 accumulationn response to deregulated Myc was abrogated by treatment ofbroblasts with caffeine, which inhibits ATM and ATR [129], anding that was strengthened with the in vivo observation thatTM deletion abrogated Myc-induced p53-dependent apopto-is [130]. The authors also suggest that DNA damage is a resultf deregulated Myc expression, and ATM kinase mediates the53 response [130]. Although ATM/R can phosphorylate andctivate p53 upon deregulated Myc expression in the absence ofRF, the presence of both ARF and ATM/R can activate p53ore strongly than when only either ARF or ATM/R is present

131]. This finding suggests that both ARF and ATM/R kinasesooperate in activating p53 in response to Myc.

Myc was linked to the extrinsic pathway of apoptosis when itas observed that serum-starved Myc-expressing cells did notie when antagonists to CD95/Fas were present. The authors alsohowed that Rat1 cells, normally refractory to CD95-induced

mwiE

r Biology 16 (2006) 275–287

eath, could be made sensitive with ectopic expression of Myc132]. A similar cooperation was observed with N-Myc in neu-oblastoma cells [133]. As with the intrinsic pathway, much ofhe data indicated that Myc was sensitising the cells to apoptosisy amplifying the death signal at the mitochondria [134]. A moreirect role in death receptor-mediated apoptosis was discoveredhen the observation was made that Myc up-regulates expres-

ion of the TRAIL receptor DR5 at the cell surface and increasesaspase-8 processing [135], and siRNA to GSK3� and FBW7imic this effect [136]. GSK3 and FBW7 have been recently

hown to act in the same pathway to target Myc for degradationt T58 [137,138]. Similarly, N-Myc over-expression sensitisedHEP neuroblastoma cells to TRAIL-induced killing by up-egulating DR5 receptors [139]. In helping to promote the sameathway, Myc has also been shown to bind the promoter regionf the TRAIL-signalling inhibitory protein c-FLIP by chromatinmmunoprecipitation in U2OS cells, leading to down-regulationf c-FLIP, and increased apoptosis as determined by caspase-3ctivation and cell cycle analysis [140].

Myc-induced apoptosis has also been linked to the proteinranslation machinery. Myc has been shown to bind to the pro-

oter of translation initiation factor eIF4E, and to activate theromoter in reporter assays [141]. Constitutive expression ofIF4E is sufficient to block Myc-induced apoptosis in rat embryobroblasts and cause an increase in transformation [142,143].he signal from the activated PI3K pathway flows throughKB/AKT and mTOR such that when PKB and mTOR arective, eIF4E is also active [144]. Data indicates that eIF4Et least partially blocks cytochrome c release from the mito-hondria, and that Myc repression of BCL-XL is abrogatedn REFs, possibly by increased translation of BCL-XL mRNA145]. eIF4E-binding protein 1 (4EBP1) binds and inactivatesIF4e, and an activated form of 4EBP1 was shown to mod-stly increase apoptosis caused by Myc [146]. Finally, a recenteport in which E�-Myc mice were crossed to eIF4E transgenicice had a reduced apoptotic index and clearly demonstrated

ooperation in vivo [147]. The cooperation was also tested inhe adoptive stem cell lymphoma model, where it was con-rmed that over-expression of PKB/AKT or eIF4e could coop-rate with Myc to reduce the latency of lymphoma onset [148].hese authors also demonstrated that rapamycin, which inhibitsTOR, markedly sensitised cells to undergo Myc-induced apop-

osis when also treated with doxorubicin, an observation also evi-ent with serum withdrawal-induced apoptosis of murine fibrob-asts expressing deregulated Myc [149]. Interestingly, althoughIF4E-dependent protein translation is shut down during apop-osis, translation of Myc continues through use of an internalibosome entry segment [150].

Many other over-expressed or inactivated proteins have beenhown to cooperate with Myc, affect Myc-induced apoptosis andrive transformation. These include, but are not limited to, Met151], the guanidine-nucleotide exchange factor Tiam1 [152],ranscription factors TEL2 [153] and Runx2 [154], macrophage

igration inhibitory factor [155], and Bin1 [156–159]. Earlyork using retroviral insertion in the E�-Myc mouse lead to the

dentification of the cooperating oncogenes Pim-1 and -2, andmi-1 and Bmi-1 [160]. The cooperation between Bmi-1 and

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yc has been further characterised, and it appears that Bmi-is responsible for repressing the INK4a locus [161,162], a

imilar function perhaps to Twist. Clearly, it will be importanto definitively determine what role these and other coopera-ors play in human neoplastic disease, their cell- and tumour-ype specificities, and how this knowledge can be exploited toevelop novel therapeutic strategies to target Myc-deregulatedumours.

. Hints at the sequel

Despite the frustration sometimes felt by researchers whoave been scrutinising Myc and its role in cancer for 25 years, it isow, more than ever, an exciting time to be studying Myc. Recentdvances in experimental model systems as well as genomicsnd proteomics provide new opportunities that should allowesearchers to discover the unexpected and answer questionshey previously could only pose. For example, all of the studies

entioned so far have demonstrated Myc driving apoptosis incell-autonomous manner. Using developing Drosophila wing

maginal discs engineered to contain clones expressing differ-nt levels of dmyc, two groups clearly demonstrated that cellsxpressing higher levels of Myc were capable of inducing celleath in neighbouring cells [163,164], although further work isequired to resolve the mechanism of signal transmission. Theesults could have far reaching implications on just how a high

yc-expressing tumour might act towards its tissue environment165].

Several new technologies have already begun to impactur understanding of Myc biology. For instance, the studyf novel cooperators in vivo has exploded recently, with thedoptive stem cell transfer technique used extensively by theowe lab and collaborators [166]. This technique was initiallysed to study cooperation of over-expressed oncogenes in theaematopoietic compartment, but has since been modified tollow for the concomitant study of an over-expressed oncogenend knock-down of a tumour suppressor by stable integrationf siRNAs [167]. This will allow many of the cooperatingutations studied in vitro to be verified in vivo without the

equirement of a viable knock-out animal. The true power ofiRNA knock-down technology is also finally being realisedith vastly improved siRNA libraries which are already yielding

esults through library screens and synthetic lethal relationships135,136,168].

One of the most exciting areas of research today is focused onicroRNAs (miRNAs), non-coding RNAs previously thought

o have no real function, but that have recently been showno act as regulatory units for mRNA expression [169,170]. Ario of reports in the summer of 2005 showed that miRNAsould act as potential oncogenes and tumour suppressors. Hend colleagues demonstrated the tumour-specific up-regulationf a cluster of miRNAs, known as the mir-17-92 polycistron.hen this cluster of miRNAs was used in the adoptive stem

ell transfer system into E�-Myc mice, a marked decrease inatency of pre-B cell disease was found. The tumours showed

uch-reduced TUNEL staining when compared to the E�-Mycumours [171]. It was also determined that two miRNAs induced

er Biology 16 (2006) 275–287 283

by Myc, one of which is encoded by the above cluster, neg-atively regulate E2F1 expression. The mechanism suggests adampening of the proliferative feedback loop generated by theinduction of E2F1 by Myc and vice versa [172]. The third paperin the set could hold clues to why cDNA expression array analy-sis might be confounded in its ability to identify Myc-regulatedgenes involved in transformation and apoptosis. This large studyrevealed miRNA expression patterns were better able to classifytumours than conventional mRNA expression patterns, and thatmiRNA alterations in cancer are at least as important as changesin mRNA when considered from a prognosis perspective [173].It seems that it will be very important in the future to considercancer and apoptosis from the point of view of miRNAs. SincemRNA expression levels do not always accurately represent pro-tein levels, quantitative proteomic approaches are improving andcould reveal which proteins are actually up- and down-regulated[174,175].

Finally, the true holy grail of Myc research is on the horizon. Itwill soon be possible to cut through the myriad of genes regulatedby Myc to determine those genes critically important in cellulartransformation and apoptosis. Chromatin immunoprecipitationhas become the gold standard to definitively assess endogenoustranscription factor binding to DNA. By combining this powerfulmethod with the technology of DNA promoter-region arrays wecan rapidly screen through many thousands of genes [176,177],including eventually non-coding regulatory RNAs. Determiningthe genetic programme by which Myc drives apoptosis, we cancombine the functional and biochemical data to develop tangi-ble strategies to attack the latent death programme abrogatedin tumour cells. This so-called “ChIP-on-chip” technology willalso help answer several key questions challenging the Myc field.Is Myc binding to a gene regulatory region sufficient for func-tion? Does the cohort of genes bound by Myc change underderegulated conditions, or are the bound genes merely regulatedinappropriately? What signals and co-factors are involved inchanging the genetic programme? By combining ChIP-on-chipwith expression array analysis, we can begin to address thesequestions.

7. Denouement

Deregulated Myc is currently thought of as a poor prognos-tic indicator for cancers, yet this hallmark may provide noveltherapeutic options in the future. Researchers hope to exploitthis tumour-specific feature to drive the death of cancer cells.By learning which molecules effect and abrogate Myc-inducedapoptosis, the goal is to develop novel therapeutic strategiesthat will overcome these anti-apoptotic mechanisms. By iden-tifying cooperating pathways, therapies targeting each can becombined to eradicate tumour cells. More precise knowledgeof the differences in the genetic programmes driven by nor-mal and deregulated Myc will lead to more specific therapies,

reducing off-target effects. If these strategies prove successful, asignificant advance will be realised against tumours harbouringderegulated Myc. We wish our fellow researchers, past, presentand future, a happy anniversary.

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cknowledgements

We apologise to our colleagues whose work we were not ableo include due to space constraints. We thank our fellow Pennab members for helpful comments. This work was supportedy an operating grant from the Canadian Institutes of Healthesearch (LZP) and studentships from the Canadian Institutes ofealth Research (NM) and National Sciences and Engineeringesearch Council (SSK).

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