Tumour suppression by p53: the importance of apoptosis and cellular senescence

Journal of PathologyJ Pathol 2009; 219: 3–15Published online 1 June 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2584

Invited Review

Tumour suppression by p53: the importance of apoptosisand cellular senescenceValentina Zuckerman,1# Kamil Wolyniec,2# Ronit V Sionov,1 Sue Haupt2 and Ygal Haupt1,2*1Lautenberg Centre for General and Tumour Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel2Research Division, The Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne 3002, Victoria, Australia

*Correspondence to:Ygal Haupt, Lautenberg Centrefor General and TumourImmunology, The HebrewUniversity Hadassah MedicalSchool, Jerusalem 91120, Israel.E-mail: [email protected]

#These authors contributedequally to this review.

No conflicts of interest weredeclared.

Received: 15 April 2009Revised: 15 May 2009Accepted: 19 May 2009

Abstractp53 is regarded as a central player in tumour suppression, as it controls programmed celldeath (apoptosis) as well as cellular senescence. While apoptosis eliminates cells at high riskfor oncogenic transformation, senescence acts as a barrier to tumourigenesis by imposingirreversible cell cycle arrest. p53 can act directly or indirectly at multiple levels of the tumoursuppression network by invoking a myriad of mechanisms. p53 induces the extrinsic andintrinsic apoptotic pathways at multiple steps to ensure an efficient death response. Thisresponse involves transcriptional activation or repression of target genes, as well as therecently identified microRNAs, and transcription-independent functions. Importantly, p53loss of function is required for tumour maintenance. Therefore, therapeutic strategies aimedat reactivation of p53 in tumours emerge as a promising approach for the treatment of cancerpatients.Copyright 2009 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: apoptosis; senescence; tumour suppression; p53; p73; cancer therapy; miRNA

Introduction

Cancer development results from the accumulationof genetic mutations, which lead to uncontrolledand unscheduled proliferation of cells that becomeimmortalized and capable of invading other tissues.The p53 tumour suppressor is the major obstacle inthis process. p53 is considered to be a key guardian ofthe genome. It senses DNA damage and in responseinduces a transient growth arrest, allowing DNArepair or, in the case of extensive damage, promotingirreversible growth arrest (senescence) or programmedcell death (apoptosis) [1,2]. This tumour-suppressivefunction of p53 prevents the propagation of abnormalcells at risk of becoming cancer cells. The criticalrole of p53 in the prevention of cancer developmentis demonstrated by p53 mutation in approximately50% of human cancer cases. In the majority of theremaining cases p53 activities are compromised due tothe deregulation of downstream or upstream signallingpathways [3]. In addition, Li–Fraumeni syndromepatients, carrying a mutant p53 allele, develop multipletumour types at a high rate [4]. The contribution ofmutant p53 ‘gain of function’ to the development ofcancer was demonstrated in a knockin mouse modelexpressing mutant p53 [5–8]. These mice develop amore metastatic cancer than mice lacking p53 [9]. Inthis review we will discuss the major mechanism bywhich p53 exerts its tumour suppression functions.

We will focus mainly on the apoptotic functions ofp53 and the contribution of cellular senescence. Wewill also discuss evidence from recent in vivo studiesshedding new light on the temporal restoration oftumour suppression by p53. It is also important to notethat p53 has recently been implicated in autophagy andageing as well as glycolysis [2,10]; however, due tospace limitations, these will not be discussed here.

p53 and cellular senescence

Telomere shortening is a universal mechanism thatlimits the proliferative potential of normal cells (lack-ing endogenous telomerase) following extensive celldivisions. The process underlying this observation isknown as replicative senescence. It is conjectured thattelomere erosion beyond a certain limit triggers a DNAdamage response and subsequent activation of theATM/ATR–p53 pathway, resulting in growth arrest[11,12]. Cells that fail to senesce and continue to pro-liferate despite dysfunctional telomeres develop chro-mosomal aberrations, which can result in malignanttransformation [13]. The role of telomere exhaustion inthe suppression of tumourigenesis in vivo, by initiatingcellular senescence, has recently been demonstrated[14–16]. A growing body of evidence suggests thatcellular senescence is an important and evolutionar-ily conserved tumour-suppression mechanism, which

Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.www.pathsoc.org.uk

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acts as a natural barrier to cell immortalization andtransformation [17,18].

The existence of non-telomere-induced senescencewas anticipated by the behaviour of primary murinecells in culture. Normal mouse cells, like normalhuman cells, have a finite replicative potential. How-ever, their replicative lifespan is substantially shorterthan that of human cells (10–15 population doublingscompared to 50–70), despite murine fibroblasts hav-ing very long telomeres (∼60 versus ∼12 kb) andin some cases expressing telomerase [19]. Thus, itis unlikely that the replicative senescence of primarymouse fibroblasts is due to telomere shortening. Ithas been proposed that the senescence phenomenon inprimary mouse cells is due to a stress response to cul-ture conditions, which can be overcome by decreasingthe oxygen concentration [20]. Senescence mediatedby non-telomeric signals is termed ‘premature senes-cence’, ‘accelerated senescence’ or ‘extrinsic senes-cence’. Another term, ‘stress- or aberrant signallingsenescence’ (STASIS) has been suggested to describethe process of a senescence-like arrest mechanism inresponse to stress stimuli [21] (Figure 1).

Abnormal activation of oncogenes, such as Ras, canpromote cellular senescence in mouse and human cells.As long as the programme remains intact, the neoplas-tic growth process may remain benign for many years,possibly contributing to tumour dormancy [17]. A fun-damental role in this fail-safe mechanism is attributedto the p53 and pRb pathways [22]. These pathways arecritical for the initiation and maintenance of the senes-cent phenotype in human and mouse cells. Mutationsin p53 or in the pRb pathway, commonly in p16INK4a,is sufficient to prevent cellular senescence, thereby

removing the obstacle from tumour progression [17].In mouse embryo fibroblasts (MEFs) disruption ofp53 alone is sufficient to prevent senescence. Notably,p53-null MEFs acquire an immortal phenotype andp53-null mice are highly susceptible to spontaneoustumourigenesis [9,23]. While inactivation of pRb onits own is insufficient to overcome senescence, theconcomitant inactivation of its family members, p107and p130, allows MEFs to escape senescence [24,25].Thus, at least in MEFs, both p53 and the pRb familyoperate in a linear signalling pathway to induce senes-cence, whereby stress-activated p53 activates pRb toinduce senescence. The p21WAF1 protein, an inhibitorof cyclin E/Cdk2 complexes, which is a direct tran-scriptional target of p53, links these two pathways. Inhuman cells, however, inactivation of both p53 andpRb is essential to prevent the onset of replicativesenescence, whereas disruption of only one of thesepathways only delays the onset of senescence [26].

Stimuli and regulation of p53-induced senescence

The signals that induce a DNA damage response,such as sublethal doses of radiation, chemotherapeu-tic drugs (such as etoposide or cyclophosphamide)or telomere dysfunction, appear to drive senescenceprimarily via the p53–p21 pathway (Figure 1). Dis-ruption of DNA repair genes, such as Brca1 andDNA ligase IV, induces premature ageing in miceand premature senescence of MEFs deficient in thesegenes. Interestingly, many of these senescent pheno-types can be rescued by p53 inactivation, indicat-ing a pivotal role for p53 in DNA damage-inducedsenescence [27,28]. Moreover, cancer cells that retain

Figure 1. p53-dependent senescence is triggered by a wide spectrum of stimuli. Telomere shortening as well as non-telomericsignals, such as DNA-damaging agents, oncogenic signalling, oxidative stress, β-interferon, HDAC inhibitors and depletion of heatshock proteins, induce p53-dependent senescence

J Pathol 2009; 219: 3–15 DOI: 10.1002/pathCopyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumour suppression by p53: importance of apoptosis and cellular senescence 5

intact p53 are much more susceptible to senescencein response to chemotherapy [29–31]. The induc-tion of p21 is important for DNA damage-inducedsenescence, as well as its well-established role intransient growth arrest. The effectors determining thedecision between these outcomes remain largely elu-sive. It has been suggested that efficient DNA repairinhibits p53–p21 signalling, allowing cell cycle pro-gression, whereas irreparable DNA lesions sustain theATM/ATR–p53–p21 DNA damage response, main-taining the senescent phenotype [32].

A number of oncogenes, such as RAS [33], E2F[34], RUNX1 [35,36] and RUNX1–ETO [36], trig-ger p53-induced senescence. While activation ofoncogenes such as RAS or MOS involve a DNAdamage response [18], others, such as RUNX1 orRUNX1–ETO induce p53-dependent senescence inde-pendently of replicative stress and without DNA dam-age [36]. In addition, hydrogen peroxide-mediatedoxidative stress also induces p53-dependent senes-cence [37]. Intriguingly, chemical inhibition of histonedeacetylase, resulting in chromatin decondensation andthe formation of euchromatin, was also found to causep53-mediated senescence in MEFs [38]. Recent find-ings demonstrate that specific depletion of chaper-ones such as members of Hsp70 (heat shock proteins)causes activation of the p53 pathway and subsequentlycellular senescence. This may explain why certain can-cer cells over-express Hsp70 [39].

Regulation of p53-induced senescence

Mechanisms responsible for activation of p53 insenescent cells are incompletely understood; however,some molecular details are emerging. One cause of p53activation appears to be an increase in the expressionof ARF (p14ARF in human; p19ARF in mouse), atumour suppressor encoded by the INK4a–ARF locus.ARF stimulates p53 activity by sequestrating HDM2(MDM2 in mice), which is an E3 ubiquitin ligasethat targets p53 for proteosome-mediated degradation.Thus, ARF acts to prevent the MDM2-driven negative-feedback regulation of p53 by MDM2 [40,41]. ARFis up-regulated in cultured senescent murine cells aswell as during premature senescence induced by H-RASV12 in MEFs, and is required for telomeric andnon-telomeric senescence in MEFs [42]. In humancells the role of ARF and p53 is more complicated.For example, ARF and p53 are critical regulators ofE2F-induced senescence [34], but not RAS-inducedsenescence in human primary fibroblasts [43,44].

Another important activator of p53 is the promyelo-cytic leukaemia (PML) tumour suppressor. PML hasbeen implicated in replicative senescence and in pre-mature senescence in response to oncogenic RAS.PML interacts with CBP/p300 acetyltransferase andstabilizes p53 through acetylation [45,46]. PML hasbeen also recently identified as a direct target of p53,hence revealing a regulatory positive feedback loop

between p53 and PML [47]. Further, PRAK (p38-regulated/activated protein kinase), a downstream reg-ulator of p38 MAPK, is essential for RAS-inducedtranscriptional activity of p53. PRAK phosphorylatesp53 on Ser37, and p38 phosphorylates p53 on Ser33and Ser46 during RAS-induced senescence [48].

Effectors of p53-mediated growth arrestand senescence

p53 mediates cell growth arrest by inducing theexpression of cell cycle regulatory target genes [49].The p21Waf1 protein inhibits CDK2/Cyclin E activ-ity, while Gadd45 and 14–3–3 inhibit Cdc2 activity(Figure 2). 14–3–3 binds the Cdc2–Cyclin B com-plex and Cdc25C phosphatase, and sequesters thesein the cytoplasm [50,51], whereas Gadd45 inhibitsCdc2–Cyclin B complex formation [52,53]. p53 isalso capable of repressing the expression of cell cycleprogression genes, such as CDK4 and Cyclin E2 [54],and the cell cycle phosphatases, Cdc25A and Cdc25C,[55,56] (Figure 2). In addition, a few more p53 tar-get genes have been identified. These include dual-specificity phosphatase 11 (DUSP11 ), response geneto complement 32 (RGC32 ) or protein tyrosine phos-phatase (PTPRV ) [57–59].

In contrast to the induction of cell cycle arrest,the mechanisms by which p53 induces senescenceare partially understood. p53 regulates plasminogenactivator inhibitor-1 (PAI-1) expression by stabilizingits mRNA through direct binding to its mRNA 3′-UTR[60]. PAI-1 has been considered a well-establishedmarker of senescent cells [61,62]. Importantly, down-regulation of PAI-1 results in escape from replicativesenescence in murine and human primary fibroblasts,through sustained activation of the PI3K–Akt survivalpathway and nuclear retention of Cyclin D [63].Another p53-induced gene implicated in senescenceis MIC-1, a cytokine of the TGFβ family. MIC-1secretion from the cell may propagate a senescenceprogramme by autocrine and paracrine induction [39].

p53 and cell death

Apoptosis is a well-studied and well-understood pro-cess that has been considered to play an importantrole in tumour suppression. Apoptosis is triggered inresponse to a variety of signals, which can activate theextrinsic and/or intrinsic death pathways; or when cellsare deprived of pro-survival signals [1]. p53 acts atmultiple levels of the intrinsic and extrinsic pathways[64] through the induction of multiple apoptotic tar-get genes, as well as through transcription-independentmechanisms (Figure 3) [65,66].

Transcription-dependent apoptosis by p53

Stress-activated p53 can trigger apoptosis throughthe transcriptional activation of pro-apoptotic target


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Figure 2. Role of p53 in the regulation of checkpoints in the eukaryotic cell cycle. The cell cycle consists of alternating S phase(DNA synthesis) and M phase (mitosis), separated by two gap phases, G1 and G2. Cyclin D-dependent kinases (CDKs) accumulatein response to mitogenic signals and initiate the phosphorylation of pRb, a process that is completed by cyclin E–Cdk2. Oncecells enter S phase, cyclin E is degraded and cyclin A enters into complexes with Cdk2. Cyclin B–Cdc2 mediates G2 –M transition.INK4 proteins (eg p16INK4a, p15INK4b, p18INK4c, p19INK4d) oppose the activities of the various cyclin D-dependent kinases,whereas Cip/Kip proteins (eg p21) specifically inhibit cyclin E-cdk2. Gadd45 and 14–3–3σ , which are transactivated by p53,interfere with cyclin B-Cdc2. Cdc25A and Cdc25C phosphatases, which are repressed by p53, activate cyclin E–CDK2 or cyclinB–cdc2, respectively

Figure 3. Multiple mechanisms involved in p53-mediated apoptosis. p53 exerts its pro-apoptotic effect by transcription-dependentand transcription-independent modes of action. The targets of p53 transactivation represent a range of molecules, with variousfunctions, such as the Bcl-2 family (Bax, Bid, Puma, Noxa), the apoptotic effector machinery (Apaf-1, Caspase-8, Caspase-6), celldeath receptors (DR5, FAS), cell death ligands (TNFSF10, TNFS6) and other less defined factors (AEN, p53AIP, PERP, PIG3).Well-established transrepressed genes are: Bcl-2, Survivin, ARC and Gelactin-3. The p53 protein can also have direct effects in themitochondria, as it can facilitate oligomerization of Bax and Bak as well as interact with anti-apoptotic Bcl-2, Bcl-xl and Mcl-1proteins



genes [64]. These target genes belong to the intrinsicand extrinsic apoptotic pathways. Within the intrinsicdeath pathway, p53 induces the expression of the pro-apoptotic Bcl-2 family members bax and BH3-onlygenes bid, puma and noxa, which can function as ‘acti-vators’, which directly stimulate Bax/Bak oligomer-ization, and ‘derepressors’, which promote apoptosisthrough displacement of anti-apoptotic proteins fromthe Bax–Bak complex (Figure 3) [67]. Further, p53represses the apoptosis repressor with caspase recruit-ment domain (ARC) protein, which counteracts theapoptotic functions of Puma and Bad [68]. p53 canalso promote cytochrome c release by inducing theexpression of the OKL38 tumour suppressor gene,which localizes to the mitochondria and augmentscytochrome c release. Loss of OKL38 correlates withtumourigenesis, and its over-expression induces apop-tosis in several carcinoma cell lines [69]. Further, p53mediates HIPK2 kinase-dependent down-regulation ofGalectin-3. Galectin-3 inhibits cytochrome c releasefrom the mitochondria and is over-expressed in a largenumber of human cancers [70].

Within the extrinsic pathway (Figure 3), p53 inducesthe expression of the death receptor Fas (CD95) andDR5 (TRAIL receptor 2) [64]. In addition, p53 inducesthe expression of the TNFSF10 (TRAIL) death liganditself and the Fas ligand, TNFSF6 (FasL) [71,72]. Thisinduction of apoptotic genes at multiple levels aug-ments the apoptotic signalling. TRAIL protein expres-sion was elevated in adriamycin-treated breast cancercells and in natural killer cells in vivo after systemictreatment with 5-fluorouracil. It has been proposedthat TRAIL induction links p53 with the host immuneresponse during cancer therapy [71]. A growing bodyof evidence implies that p53 is also capable of trans-activating several other vital elements of the apoptoticmachinery, including apaf-1, caspase 8 and caspase 6[64], the apoptosis-enhancing nuclease (AEN), whichis implicated in DNA fragmentation [73], PIG3, a geneinvolved in redox metabolism [74], and others [75].

Transcription-independent regulation of apoptosisby p53

A number of early studies suggested that under cer-tain conditions p53 can promote apoptosis withoutthe transcriptional activation of target genes [76,77].The current notion is that p53 can have direct apop-togenic effects at the mitochondria (Figure 3) [67].Mitochondrially targeted wild-type p53 fusion pro-teins that bypass the nucleus were sufficient to trig-ger effective apoptosis in p53-deficient cells [78,79]and to exert tumour-suppressive activities in vivo onp53-null or mutant backgrounds [80,81]. p53 directlyactivates the pro-apoptotic function of Bax, Bak andVDAC by inducing their oligomerizations. In addition,p53 has been shown to associate with anti-apoptoticBcl-2, Bcl-xl and Mcl-1, leading to the release ofpro-apoptotic BH3-only proteins [67]. In mice sub-jected to DNA damage treatment, a subpopulation of

p53 rapidly translocated to the mitochondria prior toits transcriptional function in the nucleus [82]. Thiswas proposed to trigger an early wave of apoptosis,followed by a second wave caused mostly by transcrip-tionally up-regulated Puma. Mitochondrial p53 waspreferentially found in radiosensitive organs and incultured cells that respond to p53 by undergoing apop-tosis [82]. Interestingly, the human p53 Arg72 poly-morphic variant, which has a stronger pro-apoptoticcapability, localizes better to the mitochondria [83].Altogether, these studies suggest a role for mitochon-drial p53 in the induction of apoptosis [67].

microRNAs and p53-dependent apoptosisand senescence

It was recently shown by several groups that p53 reg-ulates the expression of microRNAs (miRNAs), wherea primary role has been attributed to the miR-34 fam-ily. Inactivation of miR-34a attenuated p53-mediatedapoptosis in cells exposed to genotoxic stress, sug-gesting a role for this microRNA in regulating p53responses. Ectopic miR-34 expression affected thetranscription of multiple genes and induced apop-tosis, cell cycle arrest as well as cellular senes-cence [84,85]. miR-34 represses genes involved incell cycle control, such as Cdk4 and Cyclin E2, aswell as down-regulating the hepatocyte growth factorreceptor, c-Met. In addition, miR-34a down-regulatesNotch1 and E2F1/3 transcription factors, which areimportant for cell cycle progression [84,85]. Notch1inhibits p53-dependent apoptosis through activatingthe mTOR-dependent PI3K–Akt/PKB survival path-way [86] and by direct interaction with p53, whichleads to inhibition of p53 phosphorylation and tran-scriptional activation [87]. Additional p53-regulatedmiRs were reported, including miR-15/16, which tar-get the anti-apoptotic Bcl-2 protein, let-7, that down-regulates Ras and miR-221, which in turn down-regulate the CDK inhibitor p27 [84,85]. It is importantto note that miRNAs are implicated in cancer, as theirexpression is often lost in tumour cells, and forcedreduction of global miRNA expression promotes celltransformation [88].

Decision between apoptosis and growtharrest or senescence

The determinants of cell fate upon the activation ofp53 are only patially understood. Notably, it seems thatcertain oncogenes, such as Myc, preferentially induceARF–p53-dependent apoptosis, whereas Ras is a pro-totypical mediator of senescence in primary fibroblasts[62,89]. Also, it appears that lymphocytes are intrinsi-cally predisposed to apoptosis, whereas fibroblasts andepithelial cell undergo senescence. The crucial chal-lenge is to understand what the molecular mechanismsare that determine whether cells undergo senescence


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or apoptosis, and how this knowledge could be utilizedto modulate p53 responses in cancer cells.

The stress-induced cellular response depends on thenature, intensity (the threshold level of stress signal)and duration of the stress signal, as well as the celltype [90]. As p53 is an integral and central part ofa network of proteins responding to genotoxic stim-uli, the decision between growth arrest, senescence orapoptosis is believed to be determined by the appro-priate qualitative status of p53, which we term p53conformation, localization, activity and stability status(CLASS). As suggested, the p53 CLASS can be modi-fied by certain post-translational modifications (PTMs)or by direct protein–protein interactions [90–92]. Sig-nificantly, there are multitudes of other gene-specificregulatory components within the p53 network thatare independent of and do not affect p53 status, butcan shift the balance towards specific cellular outcomein response to stress [90]. p53 undergoes multiplemodifications in the N- and C-terminal regions [92]that mostly contribute to generic activation of p53CLASS. This p53 activation is achieved by PTMsthrough a variety of mechanisms; eg by inhibitingp53 interaction with its negative regulators Mdm2 andMdmx (Mdm4), by recruiting essential co-activators,by affecting its localization or by promoting subse-quent p53 conformational changes. The dissociationfrom Mdm2, for example, leads to p53 stabilizationand an increase in its nuclear and mitochondrial con-centration. As p53 has different affinities for the pro-moters of distinct p53-target genes, its nuclear con-centration per se can modulate the transcriptional pro-gramme [90]. Thus, the level of generic activation ofp53 may be decisive for the cellular response: low lev-els of p53 may favour growth arrest, whereas higherlevels would trigger apoptosis. Alternatively, some ofp53 PTMs were proposed to enhance p53 transcrip-tional activity towards selective target promoters. Forexample, one factor that may influence the decisionto preferentially undergo apoptosis after severe DNAdamage is the acetylation of p53 at Lys120, within itsDNA-binding domain mediated by the MYST histoneacetyltransferases (HAT) Tip60 and hMOF [93,94].Prevention of this acetylation by mutation selectivelyreduced p53-induction of the pro-apoptotic bax andpuma genes, without affecting p21 or mdm2.

The stress-induced phosphorylation of p53 at Ser46by either p38, DYRK2 or HIPK2 kinases also selec-tively enhanced direct p53 apoptotic promoter activity[95–98]. One possible mechanism for this enhance-ment is that phosphorylation of Ser46 enables prolylisomerase Pin1-mediated conformational change anddissociation of p53 from the inhibitor of apoptosis,iASPP, thereby promoting cell death [99]. A recentstudy suggested that modifications of K320 and K373modulate p53 N-terminus phosphorylations and affectthe repertoire of genes induced by p53 [100]. Acety-lation of K320 by PCAF causes hypophosphoryla-tion of N-terminal residues and allows activation ofp21, whereas acetylation of K373 enhances N-terminal

phosphorylations and the induction of pro-apoptoticgenes. Intriguingly, mono-ubiquitylation of K320 bythe zinc-finger protein E4F1 enhances the specificityof p53 towards the induction of cell cycle arrest-promoting genes [101]. A recent study using chro-matin immunoprecipitation (ChIP) on Chip revealedthat the binding of p53 to its target promotes is irre-spective of the cellular outcome [102], suggesting thatthe selection for promoter specificity may occur, if atall, at the transcriptional level rather than promoterrecognition. Gaining insight into the critical determi-nants that influence the cellular outcome to p53 activa-tion is particularly important for effective therapeuticsby favouring apoptosis over growth arrest.

Role of p53 in tumour maintenance

The important question related to p53-based cancertherapies and tumour suppression is whether p53 lossis essential for the maintenance of established tumours.This issue has been recently addressed by severalgroups, using elegant mouse models to control thetemporal activation of p53. Martins et al [103] demon-strated that restoring p53 function in p53-deficient Eµ-myc-driven lymphomas triggered a rapid and extensivecell death [103]. Seven days of p53 restoration con-ferred a 50% increase in mean survival. These micedeveloped secondary lymphomas by escaping fromp53-mediated tumour suppression, which involvedeither deletion of the p53ER (oestrogen receptorfusion) allele or loss of p19ARF. Importantly, a p53response could still be activated in Eµ-myc secondarylymphomas with inactivated p19ARF. The observa-tion that p53 restoration in lymphomas causes cancerregression was supported by Dickins et al [104], whoused the RNAi strategy to reset active p53 in tumours.

Interestingly, restoration of p53 in two types ofsolid tumour, radiation-induced sarcomas and hep-atocellular carcinoma, led to cellular senescenceand tumour clearance [105,106]. In the liver modelthe senescent cells were effectively eliminated byan innate immune-mediated mechanism, stimulatedby pro-inflammatory cytokines, which may explainthe shrinkage of tumours without apoptosis beinginvolved. Intriguingly, despite the restoration of p53in normal and neoplastic tissues, only in the lat-ter was p53 activated [105]. These experiments sup-port the notion that p53 activating signals exist andpersist in tumour cells, making the strategy of p53restoration very attractive for cancer therapy. It isimportant to note that in these experimental mod-els p53 was restored in the context of a p53-nullbackground. p53 is infrequently absent from humancancers but instead is directly mutated, or its func-tion is compromised indirectly. In both cases, theactivity of restored p53 is anticipated to be at leastpartially compromised. Another important question toaddress was to determine the relative importance ofDNA damage and oncogenic stress in p53-mediated



tumour suppression. Using switchable p53 ER knock-in mice, Christophorou et al [107] showed that restora-tion of p53 immediately prior to whole-body irra-diation resulted in an expected massive apoptosisresponse in all radiosensitive tissues. However, itdid not protect the mice from radiation-induced lym-phomagenesis. Thus, an immediate apoptotic responsedid not eliminate existing tumour cells. By contrast,delayed restoration of p53 after acute DNA dam-age responses resulted in suppression of tumourige-nesis and increased survival, despite the absence ofan immediate apoptotic response to irradiation. Theseresults imply that signals activating p53 persist in lym-phoma cells long after the DNA damage signals haveceased.

One potential signal that may be generated as a con-sequence of the original genotoxic stress is oncogenicactivation, capable of stimulating p19ARF induction[62,89]. Critically, delayed p53 restoration did not pro-tect mice from radiation-induced lymphomas whenARF was absent [107]. This is consistent with theessential role of ARF in tumour suppression by p53[108]. This does not exclude the contribution of otherp53-activating signals in human tumours [109]. Over-all, these important studies demonstrated that inactiva-tion of the p53 tumour suppressor pathway is essentialfor tumour maintenance and that restoration of p53can promote tumour regression. The particular tumoursuppression response of p53 is tumour type- andcontext-dependent.

p53 and cancer therapy

Instilling malignancies with p53 that is competent fortumour suppression continues to be a primary ambi-tion of extensive research initiatives (refer to earlierreviews for the broader historical context [110,111]).The potential for such approaches will depend uponthe nature of the endogenous p53 gene. Furthermore,it is now clear that p73, the p53 family member whichfunctions as a critical determinate of chemosensitiv-ity, is also frequently activated by drugs designed toactivate p53. This observation has led to the design ofp73 activators.

When p53 is wild-type

The most extensively studied small molecule ther-apy applied to cancer cells bearing wt p53 is nutlin-3(used in the following to also refer to its active enan-tiomer, Nutlin-3a) (Figure 4). Nutlin-3 binds to thep53-binding pocket of Mdm2, inhibits p53 attachmentand consequently promotes p53 accumulation [112].Nutlin-3-mediated elevation of p53 levels, activationof transcriptional targets and induction of apoptosisoccurs independently of p53 N-terminal phosphoryla-tion associated with DNA damage-induced p53 stabi-lization [113]. Efficacy of nutlin-3 has been demon-strated in a xenograft mouse model [114]. A non-genotoxic therapy that is able to selectively targetcancer cells without detriment to the healthy cells ofthe body is the ambition of Nutlin therapy.

Figure 4. The tumour-suppressive functions of p53 can be imposed on cancer cells that have lost these capabilities, using genetherapy and pharmacological intervention. Nutlin-3 and RITA promote wild-type p53 activation by inhibiting interaction with itsnegative regulator, Mdm2. Mutant p53 can be converted to perform wild-type functions in the presence of CP-31398, PRIMA. p73chemosensitivity can be restored by exposure to RETRA or 37AA, if its activities are inhibited through sequestration by mutantp53 or iASPP, respectively


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Importantly, Nutlin-3 was also found to inhibitthe Mdmx–p53 interaction (despite its lower bind-ing affinity compared to that for Mdm2), due toconservation of the p53-binding domain between thetwo molecules. Retinoblastomas (RBs), initiated bythe inactivation of the retinoblastoma protein, com-monly advance due to the selective inactivation of thep53 pathway, mediated through the up-regulation ofMdmx. Local application of Nutlin-3 and the p53 acti-vator topotecan were found to synergize to induce adramatic (82-fold) reduction in RB tumour burden ina mouse model, in the absence of systemic or ocu-lar side-effects (compared with the maximum five-foldreduction achieved through systemic application, withsignificant detrimental side-effects [115]). The advan-tage of local application of Nutlin-3 to target sites, ascompared with systemic delivery, is further suggestedby the ability of Nutlin-3 to induce cellular senescencein mouse [116] and human primary fibroblasts [117].

An important identification of Nutlin-3 enhancementhas been demonstrated using subgenotoxic levels ofcompounds known to induce DNA damage at higherconcentrations. The cyclin-dependent kinase (CDK)inhibitors roscovitine and DRB (which inhibits CDK9and hence RNA polymerase II-dependent transcrip-tion) synergize with Nutlin-3 to induce apoptosis andcontribute to the reduced cell viability. Importantly,the combined effect of these inhibitors is greater thaneither alone and, while active in promoting p53 tran-scription, does not stimulate a DNA damage responseinvolving Ser15 phosphorylation [118].

Significantly, as Nutlin-3 targets Mdm2 and Mdmxin a region that is not exclusive to p53 association,it was suspected that it may affect p53-independentfunctions, related to other partners that associate in thesame binding domain. Additional partners that havebeen identified to bind Mdm2 in this region includep73, p63, E2F-1, numb and the transcription fac-tors TFIIE [119] and HIF1a [120]. Nutlin-3 was alsodemonstrated to inhibit the binding between Mdm2and E2F-1 and subsequently induce the transcriptionalactivation of E2F-1 in the context of DNA damage,and requires wt p53 context. Thus, the actions of Nut-lins are still being delineated, with recent additionalstudies suggesting that at least in some leukaemic andcolonic lines, Nutlin can trigger rapid apoptosis onsetthrough the induction of p53 activity in the mitochon-dria, without the requirement for transcriptional targetactivation [121].

Another small molecular activator of wt p53 isreferred to as ‘RITA’, which is the acronym for‘reactivation of p53 and induction of tumour-cellapoptosis’ (Figure 4). In contrast to Nutlin-3, RITAwas characterized for its ability to bind to the Mdm2-binding pocket of p53 and consequently promotethe accumulation of ‘Mdm2-free’ p53 molecules andinduce apoptosis [122]. Subsequent studies have iden-tified that RITA can also interact with the p53-bindingdomain of Mdm2, although at lower affinity, thus

suggesting that RITA has at least two target molecules[123].

When p53 is mutant

Restoring wt p53 conformation and consequentlyDNA binding capacity to mutant p53 through chem-ical ‘correction’ is the ambition of the p53 ‘reac-tivation’ approach. Intriguingly, while p53 mutationalone is insufficient for inducing cancer onset, con-verting a mutant structure to a wild-type form can begrowth-constrictive, presumably because of the abnor-mally high levels and the activation signals perme-ating cancer cells [105]. A small molecule selectedfor restoring a wt p53 transcriptional transactivationfunction to mutant p53 is referred to as ‘PRIMA-1’ (Figure 4), the acronym for ‘p53 reactivation andinitiation of massive apoptosis’ [124]. PRIMA hasbeen demonstrated to induce p53-dependent growtharrest and apoptosis (although this capacity appears tobe mutation- and cellular context-dependent [125]. Astructural derivative PRIMA-1 (MET) reduced tumourgrowth in a mouse xenograft model [126].

CP-31 398 is a small molecule, styrylquinazoline,selected for its capacity to restore a wt p53 DNA-binding epitope to mutant p53 (Figure 4). CP-31 398has been demonstrated to not only ‘reactivate’ the wtp53 functions of cell cycle arrest and apoptosis in amutant p53 cellular context [127], but also to induceelevated wt p53 levels. Intriguingly, this effect appearsto be Mdm2-independent and p53–Mdm2 bindingdoes not appear to be disrupted during CP-31 398activation of p53. A novel mode of action has beenproposed for CP-31 398, in which it protects p53 fromubiquitination and thus high levels of transcriptionallyactive p53 accumulate [128]. The in vivo efficacy ofCP-31 398 has been demonstrated in both an athymicnude mouse model [129] and in a UVB-induced skincancer mouse model [130].

p73 activation

The ability of mutant p53 to sequester its fam-ily member p73 has been identified to enhancechemoresistance [131]. Specific activation of p73 hasrecently gained attention as a potential tumour ther-apy. Reactivation of transcriptional reporter activity(RETRA; Figure 4) is a synthetic small molecule thatreleases p73 from inactivating mutant p53 seques-tration. A p73-dependent up-regulation of p21 andPUMA transcription was induced in response. Theefficacy has been demonstrated in vivo in a xenograftmouse model [132].

A p53-derived peptide, ‘37AA’, binds iASPP, aninhibitor of p53 family members p53, p63 and p73. Inthe absence of p53, 37AA binding was noted to dis-rupt iASPP interaction with p73 37AA, activate p73transcriptional activity and induce cell death. Further,in vivo administration, involving transgene expression



of this peptide, led to a p73-dependent tumour repres-sion. In tumours where iASPP inactivates p53, p63 orp73, a potential therapeutic value of iASPP inhibitionhas been predicted [133].

In the absence of p53, it has been demonstratedthat Nutlin-3 promotes p73–Mdm2 dissociation, sta-bilizes p73 and enhances p73 transcriptional activity,resulting in the up-regulation of the p73 target genesnoxa, puma and p21 (common targets of p53) andenhances apoptosis [119]. In both a mutant p53 and ap53-null context, apoptosis was provoked through thesynergistic action of Nutlin-3 and DNA damage (drug-induced). Thus, a potential application for Nutlin-3 topromote chemosensitivity has been suggested also ina mutant p53 context [134], apparently by harnessingp73. Another avenue to be explored is the potentialdisruption of the interaction between p73 and Mdmx.Additional screening for small molecular activators ofp53 transcription have also identified compounds thatactivate p53 transcriptional targets even in the absenceof either wt or mutant p53 and this is also believed tobe mediated through p73 activation [129].

Gene therapy for wt and mutantp53-bearing tumours

Adenoviral-mediated p53 gene therapy (Ad-p53),in which replication-incompetent recombinant aden-ovirus carries normal p53 directly into tumours, hasbeen clinically trialled in a confined study in Amer-ica (Advexin) [135] and more extensively in China(Gendicine) [136]. Advexin is currently in Phase IIItrials [137] after demonstrating some effect in lim-ited earlier studies. Gendicine (injectable), launchedfor the treatment of squamous cell carcinoma of thehead and neck, was more effective when combinedwith chemotherapy or radiotherapy and its therapeuticpotential is currently being tested for other types ofcancer, such as ovarian, non-small cell lung and manyother solid tumours [136].

The endogenous p53 status, whether wt or mutant,also appears to be critically decisive to the responseof Ad-p53 therapy, where endogenous wt p53 is asso-ciated with reversible cell growth arrest and mutantp53 with apoptosis. Thus, ironically, a poorer responseand greater resistance to Ad-p53 therapy is observedin the wt p53 context, due to DNA repair and recov-ery from cell cycle arrest. Further, in cells undergoingapoptosis, phosphorylation of p53 at Thr18 and Ser20was identified, but not in those arresting. Strikingly,enhanced levels of apoptosis were identified follow-ing the introduction of Thr18/Ser20 p53 phosphory-lation mimic into wt p53 cells, and this was asso-ciated with enhanced expression of apoptosis-relatedgenes. While the risk to normal, healthy cells har-bouring wt p53 is obvious, the restricted distributionof the virus would be anticipated to act as a natu-ral barrier to broader body damage. In fact, in braintumour therapy, poor tumour penetration appears at

least partially responsible for the reported limited effi-cacy of this approach. However, in the case of gliomas,in which 70% bear wt p53, this innovative approachdoes appear to offer therapeutic promise [138]. Inter-estingly, a complementary study identified that acety-lation of p53, which also promotes p53 transcrip-tion and apoptosis, induced through histone deacety-lase inhibition (FK228), in conjunction with Ad-p53,enhanced the therapeutic efficacy in human cancer(expressing wt p53) xenograft mouse model [139].Another interesting possibility for Ad-p53 therapy wasdemonstrated by the ability of retrovirally transferred,mitochondrially-targeted p53 (which is excluded fromthe nucleus and thus unable to act as a transcrip-tion factor), to induce tumour cell death in a mousexenograft model [140].

An additional approach to adenoviral gene therapy isthe introduction of AD-p73 into the HPV-wtp53 con-text, where p53 levels are low through the combinedeffect of viral E6 proteins and host E6-associated pro-tein. As p73 is not targeted by E6, it is stable andable to induce apoptosis. Selectivity for cancer cellsover normal was proposed through the application of atumour-specific promoter (ESM6), suggested as a ther-apy for life-threatening uterine cervical cancers [141].

The significance and biology of p53 isoforms

Bourdon and colleagues have identified nine noveltranscripts of p53: TAp53α, TAp53β, TAp53γ ,�Np53 (�Np53α), �Np53β, �Np53γ , �133p53(�133p53α), �133p53β and �133p53γ , which arisefrom internal promoter and alternative splicing [142].An additional transcript, �p53, which lacks the C-terminal 65 amino acids, has been also identified [143].Much of the information to date on the function ofthese isoforms is based on their ectopic expression inculture. For instance, �Np53 inhibits p53 transcrip-tional activity [142] and suppression of colony growth.The �Np53 transgenic mice expressed higher levels ofp53 and presented decreased body mass and prema-ture ageing associated with slower proliferation andenhanced senescence [144]. On the other hand, theTAp53β isoform elevates p53-mediated bax induction,but not p21, and increases the induction of apopto-sis [142]. By contrast, �p53 elevates p21 levels andinduces an intra-S-phase checkpoint arrest through ap53-independent transcriptional activity [143]. Thus,it appears that different isoforms of p53 exert dif-ferent effects on p53 activities and signalling. Impor-tantly, aberrant expression of some p53 isoforms wasobserved in certain types of cancer. For example, ele-vated expression of �133p53 and reduced TAp53β

expression was observed in breast cancer patients[142]; however, a different expression pattern wasobserved in head and neck cancer patients [145]. Whileit appears that p53 isoforms can affect tumour sup-pression by p53, additional studies are required to



determine their precise role and involvement in humancancer.

Conclusion

It is well established that p53 is a key tumour suppres-sor, integrating multiple stress conditions into appro-priate cellular responses. The molecular basis for howp53 induces a transient growth arrest is well under-stood. The explanation for the induction of apoptosishas been extensively studied and a wealth of informa-tion on the induction of apoptosis by p53 is available.The relative contribution of each downstream effectoris still to be explored. Likewise, the relative con-tribution of the transcriptional-independent roles ofp53 requires further study. A major challenge thathas been tackled by many laboratories concerns thecritical determinants that influence the particular cel-lular response to p53 activation. Recent studies haveshed some light on post-translational modificationsand factors that can influence this decision; however,additional studies are required to further explore thisimportant issue. The recent identifications of microR-NAs as vital effectors of p53 growth-inhibitory func-tions are likely to shed light on these issues. Whilemuch of this review has focused on the role of p53in tumour suppression in a cell autonomous manner,it is important to note that initial studies suggest thatp53 may act also in a non-cell autonomous manner.Future studies will explore the contribution of p53 totumour suppression by acting at the microinvironmentin healthy cells. While the answers to many questionsstill need to be completed, it is now apparent thattumours are addicted to a loss of p53 function, pro-viding a rationale for therapeutic reactivation of p53in cancer patients.

Acknowledgements

Due to space limitations, many original important studies havenot been cited directly but rather through recent reviews. Workin the authors’ laboratory was supported by the Israel ScienceFoundation (Grant No. 1341/05), by NHMRC project grants(Grant Nos 509196 and 509197), by the VESKI award, andby the EC FP6 funding of the European Commission (ContractNo. 503576). This publication reflects only the authors’ views.The European Commission is not liable for any use that maybe made of the information herein. We thank Gerard Tarulli forkindly helping with formatting the figures.

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Power Point slides of the figures from this Review may be found in the supporting information.


Tumour suppression by p53: the importance of apoptosis and cellular senescence

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