Journal of Pathology J Pathol 2009; 219: 315 Published 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 apoptosis and cellular senescence

Valentina Zuckerman,1# Kamil Wolyniec,2# Ronit V Sionov,1 Sue Haupt2 and Ygal Haupt1,2 *
1 Lautenberg Centre for General and Tumour Immunology, The Hebrew University Hadassah Medical School, 2 Research Division, The Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne 3002, Victoria,

Jerusalem 91120, Israel Australia

*Correspondence to: Ygal Haupt, Lautenberg Centre for General and Tumour Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel. E-mail: ygal.haupt@petermac.org
# These authors contributed equally to this review.

Abstract
p53 is regarded as a central player in tumour suppression, as it controls programmed cell death (apoptosis) as well as cellular senescence. While apoptosis eliminates cells at high risk for oncogenic transformation, senescence acts as a barrier to tumourigenesis by imposing irreversible cell cycle arrest. p53 can act directly or indirectly at multiple levels of the tumour suppression network by invoking a myriad of mechanisms. p53 induces the extrinsic and intrinsic apoptotic pathways at multiple steps to ensure an efcient death response. This response involves transcriptional activation or repression of target genes, as well as the recently identied microRNAs, and transcription-independent functions. Importantly, p53 loss of function is required for tumour maintenance. Therefore, therapeutic strategies aimed at reactivation of p53 in tumours emerge as a promising approach for the treatment of cancer patients. Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: apoptosis; senescence; tumour suppression; p53; p73; cancer therapy; miRNA

No conicts of interest were declared.

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

Introduction
Cancer development results from the accumulation of genetic mutations, which lead to uncontrolled and unscheduled proliferation of cells that become immortalized and capable of invading other tissues. The p53 tumour suppressor is the major obstacle in this process. p53 is considered to be a key guardian of the genome. It senses DNA damage and in response induces a transient growth arrest, allowing DNA repair or, in the case of extensive damage, promoting irreversible growth arrest (senescence) or programmed cell death (apoptosis) [1,2]. This tumour-suppressive function of p53 prevents the propagation of abnormal cells at risk of becoming cancer cells. The critical role of p53 in the prevention of cancer development is demonstrated by p53 mutation in approximately 50% of human cancer cases. In the majority of the remaining cases p53 activities are compromised due to the deregulation of downstream or upstream signalling pathways [3]. In addition, LiFraumeni syndrome patients, carrying a mutant p53 allele, develop multiple tumour types at a high rate [4]. The contribution of mutant p53 gain of function to the development of cancer was demonstrated in a knockin mouse model expressing mutant p53 [58]. These mice develop a more metastatic cancer than mice lacking p53 [9]. In this review we will discuss the major mechanism by which p53 exerts its tumour suppression functions.

We will focus mainly on the apoptotic functions of p53 and the contribution of cellular senescence. We will also discuss evidence from recent in vivo studies shedding new light on the temporal restoration of tumour suppression by p53. It is also important to note that p53 has recently been implicated in autophagy and ageing as well as glycolysis [2,10]; however, due to space limitations, these will not be discussed here.

p53 and cellular senescence

Telomere shortening is a universal mechanism that limits the proliferative potential of normal cells (lacking endogenous telomerase) following extensive cell divisions. The process underlying this observation is known as replicative senescence. It is conjectured that telomere erosion beyond a certain limit triggers a DNA damage response and subsequent activation of the ATM/ATRp53 pathway, resulting in growth arrest [11,12]. Cells that fail to senesce and continue to proliferate despite dysfunctional telomeres develop chromosomal aberrations, which can result in malignant transformation [13]. The role of telomere exhaustion in the suppression of tumourigenesis in vivo, by initiating cellular senescence, has recently been demonstrated [1416]. A growing body of evidence suggests that cellular senescence is an important and evolutionarily conserved tumour-suppression mechanism, which

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V Zuckerman et al

acts as a natural barrier to cell immortalization and transformation [17,18]. The existence of non-telomere-induced senescence was anticipated by the behaviour of primary murine cells in culture. Normal mouse cells, like normal human cells, have a nite replicative potential. However, their replicative lifespan is substantially shorter than that of human cells (1015 population doublings compared to 5070), despite murine broblasts having very long telomeres (60 versus 12 kb) and in some cases expressing telomerase [19]. Thus, it is unlikely that the replicative senescence of primary mouse broblasts is due to telomere shortening. It has been proposed that the senescence phenomenon in primary mouse cells is due to a stress response to culture conditions, which can be overcome by decreasing the oxygen concentration [20]. Senescence mediated by non-telomeric signals is termed premature senescence, accelerated senescence or extrinsic senescence. Another term, stress- or aberrant signalling senescence (STASIS) has been suggested to describe the process of a senescence-like arrest mechanism in response to stress stimuli [21] (Figure 1). Abnormal activation of oncogenes, such as Ras, can promote cellular senescence in mouse and human cells. As long as the programme remains intact, the neoplastic growth process may remain benign for many years, possibly contributing to tumour dormancy [17]. A fundamental role in this fail-safe mechanism is attributed to the p53 and pRb pathways [22]. These pathways are critical for the initiation and maintenance of the senescent phenotype in human and mouse cells. Mutations in p53 or in the pRb pathway, commonly in p16INK4a , is sufcient to prevent cellular senescence, thereby

removing the obstacle from tumour progression [17]. In mouse embryo broblasts (MEFs) disruption of p53 alone is sufcient to prevent senescence. Notably, p53-null MEFs acquire an immortal phenotype and p53-null mice are highly susceptible to spontaneous tumourigenesis [9,23]. While inactivation of pRb on its own is insufcient to overcome senescence, the concomitant inactivation of its family members, p107 and p130, allows MEFs to escape senescence [24,25]. Thus, at least in MEFs, both p53 and the pRb family operate in a linear signalling pathway to induce senescence, whereby stress-activated p53 activates pRb to induce senescence. The p21WAF1 protein, an inhibitor of cyclin E/Cdk2 complexes, which is a direct transcriptional target of p53, links these two pathways. In human cells, however, inactivation of both p53 and pRb is essential to prevent the onset of replicative senescence, whereas disruption of only one of these pathways 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, chemotherapeutic drugs (such as etoposide or cyclophosphamide) or telomere dysfunction, appear to drive senescence primarily via the p53p21 pathway (Figure 1). Disruption of DNA repair genes, such as Brca1 and DNA ligase IV, induces premature ageing in mice and premature senescence of MEFs decient in these genes. Interestingly, many of these senescent phenotypes can be rescued by p53 inactivation, indicating a pivotal role for p53 in DNA damage-induced senescence [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-telomeric signals, such as DNA-damaging agents, oncogenic signalling, oxidative stress, -interferon, HDAC inhibitors and depletion of heat shock proteins, induce p53-dependent senescence
J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumour suppression by p53: importance of apoptosis and cellular senescence

intact p53 are much more susceptible to senescence in response to chemotherapy [2931]. The induction of p21 is important for DNA damage-induced senescence, as well as its well-established role in transient growth arrest. The effectors determining the decision between these outcomes remain largely elusive. It has been suggested that efcient DNA repair inhibits p53p21 signalling, allowing cell cycle progression, whereas irreparable DNA lesions sustain the ATM/ATRp53p21 DNA damage response, maintaining the senescent phenotype [32]. A number of oncogenes, such as RAS [33], E2F [34], RUNX1 [35,36] and RUNX1ETO [36], trigger p53-induced senescence. While activation of oncogenes such as RAS or MOS involve a DNA damage response [18], others, such as RUNX1 or RUNX1ETO induce p53-dependent senescence independently of replicative stress and without DNA damage [36]. In addition, hydrogen peroxide-mediated oxidative stress also induces p53-dependent senescence [37]. Intriguingly, chemical inhibition of histone deacetylase, resulting in chromatin decondensation and the formation of euchromatin, was also found to cause p53-mediated senescence in MEFs [38]. Recent ndings demonstrate that specic depletion of chaperones such as members of Hsp70 (heat shock proteins) causes activation of the p53 pathway and subsequently cellular senescence. This may explain why certain cancer cells over-express Hsp70 [39].

between p53 and PML [47]. Further, PRAK (p38regulated/activated protein kinase), a downstream regulator of p38 MAPK, is essential for RAS-induced transcriptional activity of p53. PRAK phosphorylates p53 on Ser37, and p38 phosphorylates p53 on Ser33 and Ser46 during RAS-induced senescence [48].

Effectors of p53-mediated growth arrest and senescence

p53 mediates cell growth arrest by inducing the expression of cell cycle regulatory target genes [49]. The p21Waf1 protein inhibits CDK2/Cyclin E activity, while Gadd45 and 1433 inhibit Cdc2 activity (Figure 2). 1433 binds the Cdc2Cyclin B complex and Cdc25C phosphatase, and sequesters these in the cytoplasm [50,51], whereas Gadd45 inhibits Cdc2Cyclin B complex formation [52,53]. p53 is also capable of repressing the expression of cell cycle progression 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 target genes have been identied. These include dualspecicity phosphatase 11 (DUSP11 ), response gene to complement 32 (RGC32 ) or protein tyrosine phosphatase (PTPRV ) [5759]. In contrast to the induction of cell cycle arrest, the mechanisms by which p53 induces senescence are partially understood. p53 regulates plasminogen activator inhibitor-1 (PAI-1) expression by stabilizing its mRNA through direct binding to its mRNA 3 -UTR [60]. PAI-1 has been considered a well-established marker of senescent cells [61,62]. Importantly, downregulation of PAI-1 results in escape from replicative senescence in murine and human primary broblasts, through sustained activation of the PI3KAkt survival pathway and nuclear retention of Cyclin D [63]. Another p53-induced gene implicated in senescence is MIC-1, a cytokine of the TGF family. MIC-1 secretion from the cell may propagate a senescence programme by autocrine and paracrine induction [39].

Regulation of p53-induced senescence

Mechanisms responsible for activation of p53 in senescent cells are incompletely understood; however, some molecular details are emerging. One cause of p53 activation appears to be an increase in the expression of ARF (p14ARF in human; p19ARF in mouse), a tumour suppressor encoded by the INK4aARF locus. ARF stimulates p53 activity by sequestrating HDM2 (MDM2 in mice), which is an E3 ubiquitin ligase that targets p53 for proteosome-mediated degradation. Thus, ARF acts to prevent the MDM2-driven negativefeedback regulation of p53 by MDM2 [40,41]. ARF is up-regulated in cultured senescent murine cells as well as during premature senescence induced by HRASV12 in MEFs, and is required for telomeric and non-telomeric senescence in MEFs [42]. In human cells the role of ARF and p53 is more complicated. For example, ARF and p53 are critical regulators of E2F-induced senescence [34], but not RAS-induced senescence in human primary broblasts [43,44]. Another important activator of p53 is the promyelocytic leukaemia (PML) tumour suppressor. PML has been implicated in replicative senescence and in premature senescence in response to oncogenic RAS. PML interacts with CBP/p300 acetyltransferase and stabilizes p53 through acetylation [45,46]. PML has been also recently identied as a direct target of p53, hence revealing a regulatory positive feedback loop

p53 and cell death

Apoptosis is a well-studied and well-understood process that has been considered to play an important role in tumour suppression. Apoptosis is triggered in response to a variety of signals, which can activate the extrinsic and/or intrinsic death pathways; or when cells are deprived of pro-survival signals [1]. p53 acts at multiple levels of the intrinsic and extrinsic pathways [64] through the induction of multiple apoptotic target genes, as well as through transcription-independent mechanisms (Figure 3) [65,66].

Transcription-dependent apoptosis by p53

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

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

V Zuckerman et al

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) accumulate in response to mitogenic signals and initiate the phosphorylation of pRb, a process that is completed by cyclin ECdk2. Once cells enter S phase, cyclin E is degraded and cyclin A enters into complexes with Cdk2. Cyclin BCdc2 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) specically inhibit cyclin E-cdk2. Gadd45 and 1433 , which are transactivated by p53, interfere with cyclin B-Cdc2. Cdc25A and Cdc25C phosphatases, which are repressed by p53, activate cyclin ECDK2 or cyclin Bcdc2, respectively

Figure 3. Multiple mechanisms involved in p53-mediated apoptosis. p53 exerts its pro-apoptotic effect by transcription-dependent and transcription-independent modes of action. The targets of p53 transactivation represent a range of molecules, with various functions, such as the Bcl-2 family (Bax, Bid, Puma, Noxa), the apoptotic effector machinery (Apaf-1, Caspase-8, Caspase-6), cell death receptors (DR5, FAS), cell death ligands (TNFSF10, TNFS6) and other less dened 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 the mitochondria, as it can facilitate oligomerization of Bax and Bak as well as interact with anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 proteins

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumour suppression by p53: importance of apoptosis and cellular senescence

genes [64]. These target genes belong to the intrinsic and extrinsic apoptotic pathways. Within the intrinsic death pathway, p53 induces the expression of the proapoptotic Bcl-2 family members bax and BH3-only genes bid, puma and noxa, which can function as activators, which directly stimulate Bax/Bak oligomerization, and derepressors, which promote apoptosis through displacement of anti-apoptotic proteins from the BaxBak complex (Figure 3) [67]. Further, p53 represses the apoptosis repressor with caspase recruitment domain (ARC) protein, which counteracts the apoptotic functions of Puma and Bad [68]. p53 can also promote cytochrome c release by inducing the expression of the OKL38 tumour suppressor gene, which localizes to the mitochondria and augments cytochrome c release. Loss of OKL38 correlates with tumourigenesis, and its over-expression induces apoptosis in several carcinoma cell lines [69]. Further, p53 mediates HIPK2 kinase-dependent down-regulation of Galectin-3. Galectin-3 inhibits cytochrome c release from the mitochondria and is over-expressed in a large number of human cancers [70]. Within the extrinsic pathway (Figure 3), p53 induces the expression of the death receptor Fas (CD95) and DR5 (TRAIL receptor 2) [64]. In addition, p53 induces the expression of the TNFSF10 (TRAIL) death ligand itself and the Fas ligand, TNFSF6 (FasL) [71,72]. This induction of apoptotic genes at multiple levels augments the apoptotic signalling. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells and in natural killer cells in vivo after systemic treatment with 5-uorouracil. It has been proposed that TRAIL induction links p53 with the host immune response during cancer therapy [71]. A growing body of evidence implies that p53 is also capable of transactivating several other vital elements of the apoptotic machinery, including apaf-1, caspase 8 and caspase 6 [64], the apoptosis-enhancing nuclease (AEN), which is implicated in DNA fragmentation [73], PIG3, a gene involved in redox metabolism [74], and others [75].

p53 rapidly translocated to the mitochondria prior to its transcriptional function in the nucleus [82]. This was proposed to trigger an early wave of apoptosis, followed by a second wave caused mostly by transcriptionally up-regulated Puma. Mitochondrial p53 was preferentially found in radiosensitive organs and in cultured cells that respond to p53 by undergoing apoptosis [82]. Interestingly, the human p53 Arg72 polymorphic variant, which has a stronger pro-apoptotic capability, localizes better to the mitochondria [83]. Altogether, these studies suggest a role for mitochondrial p53 in the induction of apoptosis [67].

microRNAs and p53-dependent apoptosis and senescence

It was recently shown by several groups that p53 regulates the expression of microRNAs (miRNAs), where a primary role has been attributed to the miR-34 family. Inactivation of miR-34a attenuated p53-mediated apoptosis in cells exposed to genotoxic stress, suggesting a role for this microRNA in regulating p53 responses. Ectopic miR-34 expression affected the transcription of multiple genes and induced apoptosis, cell cycle arrest as well as cellular senescence [84,85]. miR-34 represses genes involved in cell cycle control, such as Cdk4 and Cyclin E2, as well as down-regulating the hepatocyte growth factor receptor, c-Met. In addition, miR-34a down-regulates Notch1 and E2F1/3 transcription factors, which are important for cell cycle progression [84,85]. Notch1 inhibits p53-dependent apoptosis through activating the mTOR-dependent PI3KAkt/PKB survival pathway [86] and by direct interaction with p53, which leads to inhibition of p53 phosphorylation and transcriptional activation [87]. Additional p53-regulated miRs were reported, including miR-15/16, which target the anti-apoptotic Bcl-2 protein, let-7, that downregulates Ras and miR-221, which in turn downregulate the CDK inhibitor p27 [84,85]. It is important to note that miRNAs are implicated in cancer, as their expression is often lost in tumour cells, and forced reduction of global miRNA expression promotes cell transformation [88].

Transcription-independent regulation of apoptosis by p53

A number of early studies suggested that under certain conditions p53 can promote apoptosis without the transcriptional activation of target genes [76,77]. The current notion is that p53 can have direct apoptogenic effects at the mitochondria (Figure 3) [67]. Mitochondrially targeted wild-type p53 fusion proteins that bypass the nucleus were sufcient to trigger effective apoptosis in p53-decient cells [78,79] and to exert tumour-suppressive activities in vivo on p53-null or mutant backgrounds [80,81]. p53 directly activates the pro-apoptotic function of Bax, Bak and VDAC by inducing their oligomerizations. In addition, p53 has been shown to associate with anti-apoptotic Bcl-2, Bcl-xl and Mcl-1, leading to the release of pro-apoptotic BH3-only proteins [67]. In mice subjected to DNA damage treatment, a subpopulation of

Decision between apoptosis and growth arrest or senescence

The determinants of cell fate upon the activation of p53 are only patially understood. Notably, it seems that certain oncogenes, such as Myc, preferentially induce ARFp53-dependent apoptosis, whereas Ras is a prototypical mediator of senescence in primary broblasts [62,89]. Also, it appears that lymphocytes are intrinsically predisposed to apoptosis, whereas broblasts and epithelial cell undergo senescence. The crucial challenge is to understand what the molecular mechanisms are that determine whether cells undergo senescence

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

V Zuckerman et al

or apoptosis, and how this knowledge could be utilized to modulate p53 responses in cancer cells. The stress-induced cellular response depends on the nature, intensity (the threshold level of stress signal) and duration of the stress signal, as well as the cell type [90]. As p53 is an integral and central part of a network of proteins responding to genotoxic stimuli, the decision between growth arrest, senescence or apoptosis is believed to be determined by the appropriate qualitative status of p53, which we term p53 conformation, localization, activity and stability status (CLASS). As suggested, the p53 CLASS can be modied by certain post-translational modications (PTMs) or by direct proteinprotein interactions [9092]. Signicantly, there are multitudes of other gene-specic regulatory components within the p53 network that are independent of and do not affect p53 status, but can shift the balance towards specic cellular outcome in response to stress [90]. p53 undergoes multiple modications in the N- and C-terminal regions [92] that mostly contribute to generic activation of p53 CLASS. This p53 activation is achieved by PTMs through a variety of mechanisms; eg by inhibiting p53 interaction with its negative regulators Mdm2 and Mdmx (Mdm4), by recruiting essential co-activators, by affecting its localization or by promoting subsequent p53 conformational changes. The dissociation from Mdm2, for example, leads to p53 stabilization and an increase in its nuclear and mitochondrial concentration. As p53 has different afnities for the promoters of distinct p53-target genes, its nuclear concentration per se can modulate the transcriptional programme [90]. Thus, the level of generic activation of p53 may be decisive for the cellular response: low levels of p53 may favour growth arrest, whereas higher levels would trigger apoptosis. Alternatively, some of p53 PTMs were proposed to enhance p53 transcriptional activity towards selective target promoters. For example, one factor that may inuence the decision to preferentially undergo apoptosis after severe DNA damage is the acetylation of p53 at Lys120, within its DNA-binding domain mediated by the MYST histone acetyltransferases (HAT) Tip60 and hMOF [93,94]. Prevention of this acetylation by mutation selectively reduced p53-induction of the pro-apoptotic bax and puma genes, without affecting p21 or mdm2. The stress-induced phosphorylation of p53 at Ser46 by either p38, DYRK2 or HIPK2 kinases also selectively enhanced direct p53 apoptotic promoter activity [9598]. One possible mechanism for this enhancement is that phosphorylation of Ser46 enables prolyl isomerase Pin1-mediated conformational change and dissociation of p53 from the inhibitor of apoptosis, iASPP, thereby promoting cell death [99]. A recent study suggested that modications of K320 and K373 modulate p53 N-terminus phosphorylations and affect the repertoire of genes induced by p53 [100]. Acetylation of K320 by PCAF causes hypophosphorylation of N-terminal residues and allows activation of p21, whereas acetylation of K373 enhances N-terminal

phosphorylations and the induction of pro-apoptotic genes. Intriguingly, mono-ubiquitylation of K320 by the zinc-nger protein E4F1 enhances the specicity of p53 towards the induction of cell cycle arrestpromoting genes [101]. A recent study using chromatin immunoprecipitation (ChIP) on Chip revealed that the binding of p53 to its target promotes is irrespective of the cellular outcome [102], suggesting that the selection for promoter specicity may occur, if at all, at the transcriptional level rather than promoter recognition. Gaining insight into the critical determinants that inuence the cellular outcome to p53 activation is particularly important for effective therapeutics by favouring apoptosis over growth arrest.

Role of p53 in tumour maintenance

The important question related to p53-based cancer therapies and tumour suppression is whether p53 loss is essential for the maintenance of established tumours. This issue has been recently addressed by several groups, using elegant mouse models to control the temporal activation of p53. Martins et al [103] demonstrated that restoring p53 function in p53-decient Emyc-driven lymphomas triggered a rapid and extensive cell death [103]. Seven days of p53 restoration conferred a 50% increase in mean survival. These mice developed secondary lymphomas by escaping from p53-mediated tumour suppression, which involved either deletion of the p53ER (oestrogen receptor fusion) allele or loss of p19ARF. Importantly, a p53 response could still be activated in E-myc secondary lymphomas with inactivated p19ARF. The observation that p53 restoration in lymphomas causes cancer regression was supported by Dickins et al [104], who used the RNAi strategy to reset active p53 in tumours. Interestingly, restoration of p53 in two types of solid tumour, radiation-induced sarcomas and hepatocellular carcinoma, led to cellular senescence and tumour clearance [105,106]. In the liver model the senescent cells were effectively eliminated by an innate immune-mediated mechanism, stimulated by pro-inammatory cytokines, which may explain the shrinkage of tumours without apoptosis being involved. Intriguingly, despite the restoration of p53 in normal and neoplastic tissues, only in the latter was p53 activated [105]. These experiments support the notion that p53 activating signals exist and persist in tumour cells, making the strategy of p53 restoration very attractive for cancer therapy. It is important to note that in these experimental models p53 was restored in the context of a p53-null background. p53 is infrequently absent from human cancers but instead is directly mutated, or its function is compromised indirectly. In both cases, the activity of restored p53 is anticipated to be at least partially compromised. Another important question to address was to determine the relative importance of DNA damage and oncogenic stress in p53-mediated

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumour suppression by p53: importance of apoptosis and cellular senescence

tumour suppression. Using switchable p53 ER knockin mice, Christophorou et al [107] showed that restoration of p53 immediately prior to whole-body irradiation resulted in an expected massive apoptosis response in all radiosensitive tissues. However, it did not protect the mice from radiation-induced lymphomagenesis. Thus, an immediate apoptotic response did not eliminate existing tumour cells. By contrast, delayed restoration of p53 after acute DNA damage responses resulted in suppression of tumourigenesis and increased survival, despite the absence of an immediate apoptotic response to irradiation. These results imply that signals activating p53 persist in lymphoma cells long after the DNA damage signals have ceased. One potential signal that may be generated as a consequence of the original genotoxic stress is oncogenic activation, capable of stimulating p19ARF induction [62,89]. Critically, delayed p53 restoration did not protect mice from radiation-induced lymphomas when ARF was absent [107]. This is consistent with the essential role of ARF in tumour suppression by p53 [108]. This does not exclude the contribution of other p53-activating signals in human tumours [109]. Overall, these important studies demonstrated that inactivation of the p53 tumour suppressor pathway is essential for tumour maintenance and that restoration of p53 can promote tumour regression. The particular tumour suppression response of p53 is tumour type- and context-dependent.

p53 and cancer therapy

Instilling malignancies with p53 that is competent for tumour suppression continues to be a primary ambition of extensive research initiatives (refer to earlier reviews for the broader historical context [110,111]). The potential for such approaches will depend upon the nature of the endogenous p53 gene. Furthermore, it is now clear that p73, the p53 family member which functions as a critical determinate of chemosensitivity, is also frequently activated by drugs designed to activate p53. This observation has led to the design of p73 activators.

When p53 is wild-type

The most extensively studied small molecule therapy applied to cancer cells bearing wt p53 is nutlin-3 (used in the following to also refer to its active enantiomer, Nutlin-3a) (Figure 4). Nutlin-3 binds to the p53-binding pocket of Mdm2, inhibits p53 attachment and consequently promotes p53 accumulation [112]. Nutlin-3-mediated elevation of p53 levels, activation of transcriptional targets and induction of apoptosis occurs independently of p53 N-terminal phosphorylation associated with DNA damage-induced p53 stabilization [113]. Efcacy of nutlin-3 has been demonstrated in a xenograft mouse model [114]. A nongenotoxic therapy that is able to selectively target cancer cells without detriment to the healthy cells of the 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 gene therapy and pharmacological intervention. Nutlin-3 and RITA promote wild-type p53 activation by inhibiting interaction with its negative regulator, Mdm2. Mutant p53 can be converted to perform wild-type functions in the presence of CP-31398, PRIMA. p73 chemosensitivity can be restored by exposure to RETRA or 37AA, if its activities are inhibited through sequestration by mutant p53 or iASPP, respectively
J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Importantly, Nutlin-3 was also found to inhibit the Mdmxp53 interaction (despite its lower binding afnity compared to that for Mdm2), due to conservation of the p53-binding domain between the two molecules. Retinoblastomas (RBs), initiated by the inactivation of the retinoblastoma protein, commonly advance due to the selective inactivation of the p53 pathway, mediated through the up-regulation of Mdmx. Local application of Nutlin-3 and the p53 activator topotecan were found to synergize to induce a dramatic (82-fold) reduction in RB tumour burden in a mouse model, in the absence of systemic or ocular side-effects (compared with the maximum ve-fold reduction achieved through systemic application, with signicant detrimental side-effects [115]). The advantage of local application of Nutlin-3 to target sites, as compared with systemic delivery, is further suggested by the ability of Nutlin-3 to induce cellular senescence in mouse [116] and human primary broblasts [117]. An important identication of Nutlin-3 enhancement has been demonstrated using subgenotoxic levels of compounds known to induce DNA damage at higher concentrations. The cyclin-dependent kinase (CDK) inhibitors roscovitine and DRB (which inhibits CDK9 and hence RNA polymerase II-dependent transcription) synergize with Nutlin-3 to induce apoptosis and contribute to the reduced cell viability. Importantly, the combined effect of these inhibitors is greater than either alone and, while active in promoting p53 transcription, does not stimulate a DNA damage response involving Ser15 phosphorylation [118]. Signicantly, as Nutlin-3 targets Mdm2 and Mdmx in a region that is not exclusive to p53 association, it was suspected that it may affect p53-independent functions, related to other partners that associate in the same binding domain. Additional partners that have been identied to bind Mdm2 in this region include p73, p63, E2F-1, numb and the transcription factors TFIIE [119] and HIF1a [120]. Nutlin-3 was also demonstrated to inhibit the binding between Mdm2 and E2F-1 and subsequently induce the transcriptional activation of E2F-1 in the context of DNA damage, and requires wt p53 context. Thus, the actions of Nutlins are still being delineated, with recent additional studies suggesting that at least in some leukaemic and colonic lines, Nutlin can trigger rapid apoptosis onset through the induction of p53 activity in the mitochondria, without the requirement for transcriptional target activation [121]. Another small molecular activator of wt p53 is referred to as RITA, which is the acronym for reactivation of p53 and induction of tumour-cell apoptosis (Figure 4). In contrast to Nutlin-3, RITA was characterized for its ability to bind to the Mdm2binding pocket of p53 and consequently promote the accumulation of Mdm2-free p53 molecules and induce apoptosis [122]. Subsequent studies have identied that RITA can also interact with the p53-binding domain of Mdm2, although at lower afnity, thus

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

When p53 is mutant

Restoring wt p53 conformation and consequently DNA binding capacity to mutant p53 through chemical correction is the ambition of the p53 reactivation approach. Intriguingly, while p53 mutation alone is insufcient for inducing cancer onset, converting a mutant structure to a wild-type form can be growth-constrictive, presumably because of the abnormally high levels and the activation signals permeating cancer cells [105]. A small molecule selected for restoring a wt p53 transcriptional transactivation function to mutant p53 is referred to as PRIMA1 (Figure 4), the acronym for p53 reactivation and initiation of massive apoptosis [124]. PRIMA has been demonstrated to induce p53-dependent growth arrest and apoptosis (although this capacity appears to be mutation- and cellular context-dependent [125]. A structural derivative PRIMA-1 (MET) reduced tumour growth in a mouse xenograft model [126]. CP-31 398 is a small molecule, styrylquinazoline, selected for its capacity to restore a wt p53 DNAbinding epitope to mutant p53 (Figure 4). CP-31 398 has been demonstrated to not only reactivate the wt p53 functions of cell cycle arrest and apoptosis in a mutant p53 cellular context [127], but also to induce elevated wt p53 levels. Intriguingly, this effect appears to be Mdm2-independent and p53Mdm2 binding does not appear to be disrupted during CP-31 398 activation of p53. A novel mode of action has been proposed for CP-31 398, in which it protects p53 from ubiquitination and thus high levels of transcriptionally active p53 accumulate [128]. The in vivo efcacy of CP-31 398 has been demonstrated in both an athymic nude mouse model [129] and in a UVB-induced skin cancer mouse model [130].

p73 activation
The ability of mutant p53 to sequester its family member p73 has been identied to enhance chemoresistance [131]. Specic activation of p73 has recently gained attention as a potential tumour therapy. Reactivation of transcriptional reporter activity (RETRA; Figure 4) is a synthetic small molecule that releases p73 from inactivating mutant p53 sequestration. A p73-dependent up-regulation of p21 and PUMA transcription was induced in response. The efcacy has been demonstrated in vivo in a xenograft mouse model [132]. A p53-derived peptide, 37AA, binds iASPP, an inhibitor of p53 family members p53, p63 and p73. In the absence of p53, 37AA binding was noted to disrupt iASPP interaction with p73 37AA, activate p73 transcriptional activity and induce cell death. Further, in vivo administration, involving transgene expression

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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of this peptide, led to a p73-dependent tumour repression. In tumours where iASPP inactivates p53, p63 or p73, a potential therapeutic value of iASPP inhibition has been predicted [133]. In the absence of p53, it has been demonstrated that Nutlin-3 promotes p73Mdm2 dissociation, stabilizes p73 and enhances p73 transcriptional activity, resulting in the up-regulation of the p73 target genes noxa, puma and p21 (common targets of p53) and enhances apoptosis [119]. In both a mutant p53 and a p53-null context, apoptosis was provoked through the synergistic action of Nutlin-3 and DNA damage (druginduced). Thus, a potential application for Nutlin-3 to promote chemosensitivity has been suggested also in a mutant p53 context [134], apparently by harnessing p73. Another avenue to be explored is the potential disruption of the interaction between p73 and Mdmx. Additional screening for small molecular activators of p53 transcription have also identied compounds that activate p53 transcriptional targets even in the absence of either wt or mutant p53 and this is also believed to be mediated through p73 activation [129].

Gene therapy for wt and mutant p53-bearing tumours

Adenoviral-mediated p53 gene therapy (Ad-p53), in which replication-incompetent recombinant adenovirus carries normal p53 directly into tumours, has been clinically trialled in a conned study in America (Advexin) [135] and more extensively in China (Gendicine) [136]. Advexin is currently in Phase III trials [137] after demonstrating some effect in limited earlier studies. Gendicine (injectable), launched for the treatment of squamous cell carcinoma of the head and neck, was more effective when combined with chemotherapy or radiotherapy and its therapeutic potential is currently being tested for other types of cancer, such as ovarian, non-small cell lung and many other solid tumours [136]. The endogenous p53 status, whether wt or mutant, also appears to be critically decisive to the response of Ad-p53 therapy, where endogenous wt p53 is associated with reversible cell growth arrest and mutant p53 with apoptosis. Thus, ironically, a poorer response and greater resistance to Ad-p53 therapy is observed in the wt p53 context, due to DNA repair and recovery from cell cycle arrest. Further, in cells undergoing apoptosis, phosphorylation of p53 at Thr18 and Ser20 was identied, but not in those arresting. Strikingly, enhanced levels of apoptosis were identied following the introduction of Thr18/Ser20 p53 phosphorylation mimic into wt p53 cells, and this was associated with enhanced expression of apoptosis-related genes. While the risk to normal, healthy cells harbouring wt p53 is obvious, the restricted distribution of the virus would be anticipated to act as a natural barrier to broader body damage. In fact, in brain tumour therapy, poor tumour penetration appears at

least partially responsible for the reported limited efcacy of this approach. However, in the case of gliomas, in which 70% bear wt p53, this innovative approach does appear to offer therapeutic promise [138]. Interestingly, a complementary study identied that acetylation of p53, which also promotes p53 transcription and apoptosis, induced through histone deacetylase inhibition (FK228), in conjunction with Ad-p53, enhanced the therapeutic efcacy in human cancer (expressing wt p53) xenograft mouse model [139]. Another interesting possibility for Ad-p53 therapy was demonstrated by the ability of retrovirally transferred, mitochondrially-targeted p53 (which is excluded from the nucleus and thus unable to act as a transcription factor), to induce tumour cell death in a mouse xenograft model [140]. An additional approach to adenoviral gene therapy is the introduction of AD-p73 into the HPV-wtp53 context, where p53 levels are low through the combined effect of viral E6 proteins and host E6-associated protein. As p73 is not targeted by E6, it is stable and able to induce apoptosis. Selectivity for cancer cells over normal was proposed through the application of a tumour-specic promoter (ESM6), suggested as a therapy for life-threatening uterine cervical cancers [141].

The signicance and biology of p53 isoforms

Bourdon and colleagues have identied nine novel transcripts of p53: TAp53, TAp53, TAp53 , Np53 ( Np53), Np53, Np53 , 133p53 ( 133p53), 133p53 and 133p53 , which arise from internal promoter and alternative splicing [142]. An additional transcript, p53, which lacks the Cterminal 65 amino acids, has been also identied [143]. Much of the information to date on the function of these isoforms is based on their ectopic expression in culture. For instance, Np53 inhibits p53 transcriptional activity [142] and suppression of colony growth. The Np53 transgenic mice expressed higher levels of p53 and presented decreased body mass and premature ageing associated with slower proliferation and enhanced senescence [144]. On the other hand, the TAp53 isoform elevates p53-mediated bax induction, but not p21, and increases the induction of apoptosis [142]. By contrast, p53 elevates p21 levels and induces an intra-S-phase checkpoint arrest through a p53-independent transcriptional activity [143]. Thus, it appears that different isoforms of p53 exert different effects on p53 activities and signalling. Importantly, aberrant expression of some p53 isoforms was observed in certain types of cancer. For example, elevated expression of 133p53 and reduced TAp53 expression was observed in breast cancer patients [142]; however, a different expression pattern was observed in head and neck cancer patients [145]. While it appears that p53 isoforms can affect tumour suppression by p53, additional studies are required to

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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determine their precise role and involvement in human cancer.

Conclusion
It is well established that p53 is a key tumour suppressor, integrating multiple stress conditions into appropriate cellular responses. The molecular basis for how p53 induces a transient growth arrest is well understood. The explanation for the induction of apoptosis has been extensively studied and a wealth of information on the induction of apoptosis by p53 is available. The relative contribution of each downstream effector is still to be explored. Likewise, the relative contribution of the transcriptional-independent roles of p53 requires further study. A major challenge that has been tackled by many laboratories concerns the critical determinants that inuence the particular cellular response to p53 activation. Recent studies have shed some light on post-translational modications and factors that can inuence this decision; however, additional studies are required to further explore this important issue. The recent identications of microRNAs as vital effectors of p53 growth-inhibitory functions are likely to shed light on these issues. While much of this review has focused on the role of p53 in tumour suppression in a cell autonomous manner, it is important to note that initial studies suggest that p53 may act also in a non-cell autonomous manner. Future studies will explore the contribution of p53 to tumour suppression by acting at the microinvironment in healthy cells. While the answers to many questions still need to be completed, it is now apparent that tumours are addicted to a loss of p53 function, providing a rationale for therapeutic reactivation of p53 in cancer patients.

Acknowledgements
Due to space limitations, many original important studies have not been cited directly but rather through recent reviews. Work in the authors laboratory was supported by the Israel Science Foundation (Grant No. 1341/05), by NHMRC project grants (Grant Nos 509196 and 509197), by the VESKI award, and by the EC FP6 funding of the European Commission (Contract No. 503576). This publication reects only the authors views. The European Commission is not liable for any use that may be made of the information herein. We thank Gerard Tarulli for kindly helping with formatting the gures.

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Teaching Materials
Power Point slides of the gures from this Review may be found in the supporting information.

J Pathol 2009; 219: 315 DOI: 10.1002/path Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.