Coping with Stress: Multiple Ways to Activate P53

HF Horn and KH Vousden

The Beatson Institute for Cancer Research, Glasgow, UK

Over the years, p53 has been shown to sit at the centre of Srivastava et al., 1990). Furthermore, mouse studies an increasingly complexweb of incoming stress signals have corroborated the importance of p53 in tumour and outgoing effector pathways. The number and diversity development; p53 null mice can develop normally but of stress signals that lead to p53 activation illustrates the nearly all develop cancer before 6 months of age breadth of p53’s remit – responding to a wide variety of (Donehower et al., 1992; Jacks et al., 1994; Purdie potentially oncogenic insults to prevent tumour develop- et al., 1994). Taken together, a role for p53 as a key ment. Interestingly, different stress signals can use player in the inhibition of tumour development is different and independent pathways to activate p53, and beyond doubt. However, there is growing evidence that there is some evidence that different stress signals can p53 also contributes to other pathologies beyond cancer mediate different responses. How each of the responses to – including a possible contribution in determining p53 contributes to inhibition of malignant progression is longevity – making it unlikely that the high degree of beginning to be clarified, with the hope that identification interest in p53 will wane. of responses that are key to tumour suppression will allow a more focused and effective search for new therapeutic targets. In this review, we will highlight some recently identified roles for p53 in tumour suppression, and discuss The downstream response to p53 activation some of the numerous mechanisms through which p53 can be regulated and activated. The p53 protein is a transcription factor that regulates Oncogene (2007) 26, 1306–1316. doi:10.1038/sj.onc.1210263 the expression of a large number of target genes (Vogelstein et al., 2000). Through this mechanism p53 Keywords: p53; tumour suppression; MDM2; ubiquiti- can induce a number of differentresponses, ranging nation; apoptosis from the induction of cell cycle arrest, cell death and senescence to protective antioxidant activities, DNA repair and the control of mitochondrial respiration. One of the first transcriptional targets of p53 to be identified was the cyclin dependent kinase (CDK) Waf1/Cip1 Introduction inhibitor p21 (El-Deiry et al., 1994). CDKs play an important role in regulating progression through the cell Waf1/Cip1 Since its discovery over a quarter of a century ago, work cycle, and the inhibition of CDK activity by p21 on the tumour suppressor protein p53 has produced results in a cell cycle arrest (reviewed in Sherr and nearly 40 000 publications. This vast amount of work on Roberts, 1999). Thus, p53 can effectively cause cells to one protein is arguably justified by the importance of stop proliferating, which – among other things – allows p53 in cancer biology, as illustrated by the frequency for the repair of any damaged DNA, preventing muta- with which p53 function is lost during tumour develop- tions from being passed on to daughter cells. Interest- ment. Approximately 50% of all malignancies carry a ingly, a directrole for p53 in DNA repair has also been p53 mutation, and of those tumours that do not have demonstrated (reviewed in Gatz and Wiesmuller (2006)). mutated p53, a large proportion have inactivated p53 Recently, other activities of p53 that can inhibit the function by another mechanism (Hainaut and Hollstein, acquisition of potentially oncogenic mutations have 2000; Bykov and Wiman, 2003). Taken together, p53 been described. Several studies have shown an anti- function is lost in almost all tumours. The importance oxidant role for p53 resulting from the expression of of p53 as a tumour suppressor is illustrated by target genes that reduce intracellular reactive oxygen the observation that many individuals affected by Li species (ROS) levels and so preventgenotoxic damage Fraumeni syndrome – and so displaying an abnormally (Budanov et al., 2004; Yoon et al., 2004; Sablina et al., high and early incidence of tumour development – suffer 2005; Bensaad et al., 2006). This function of p53 appears a germline mutation in p53 (Malkin et al., 1990; to be important even in the absence of acute, high levels of stress – and probably reflects a response to lower levels of constitutive stress that accompany everyday life Correspondence: Professor KH Vousden, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 of complex multicellular organisms (Sablina et al., 1BD, UK. 2005). These antioxidant functions of p53 are linked E-mail: [email protected] with enhanced survival of cells expressing p53, and so Activation of p53 HF Horn and KH Vousden 1307 seem to be at odds with another important activity of suggesting that both transcriptional and non-transcrip- p53, which is to induce cell death through apoptosis or tional activities of p53 are important for the induction of autophagy. Why would p53 induce repair of a cell that it cell death (reviewed in Yee and Vousden, 2005). then targets for destruction? While some of the answer clearly lies in cell-type dependent differences, an interesting model is emerging where p53 behaves Regulation of p53 function differently depending on the severity of the stress (Bensaad and Vousden, 2005). In response to low, The anti-proliferative activities of p53 play an important constitutive stress, p53 activates expression of genes that role in tumour suppression, but require strong negative drive a temporary cell cycle arrest, allowing for the regulation to allow for normal growth and development. removal of intracellular ROS and repair of any damage Not surprisingly, this regulation of p53 takes many that may have occurred. But when the stress-induced forms, including the regulation of expression, protein damage is too severe for the cell to recover, p53 initiates stability and protein activity. Many post-transcriptional programmed cell death – so eliminating cells that may modifications of p53 have been described including have acquired irreparable and potentially oncogenic phosphorylation, acetylation, methylation, glycosyla- alterations. The transcriptional targets of p53 include a tion, ribosylation and, most recently, O-GlcNAcylation number of pro-apoptotic proteins such as the BH3 only (Bode and Dong, 2004; Yang et al., 2006c). However, proteins Puma and Noxa (Oda et al., 2000; Nakano and in vivo models have suggested that these modifications Vousden, 2001; Yu et al., 2001). These proteins function play only very subtle, modulatory roles in regulating by inducing the loss of inner mitochondrial membrane p53 function (Toledo and Wahl, 2006). The interaction potential, leading to the release of cytochrome C and of p53 with proteins that control p53 function, either other apoptogenic factors and the activation of the directly or through the regulation of p53 stability, caspase cascade that results in apoptotic cell death appears to be key in turning on and off the p53 (Jeffers et al., 2003; Villunger et al., 2003; Yu et al., 2003 response. and reviewed in Danial and Korsmeyer, 2004). Activa- tion of autophagy by the p53-induced protein DRAM (damage-regulated autophagy modulator) has also Regulation of p53 protein stability been described to be an important contributor of the p53 is degraded via the proteasome by a predominately apoptotic response (Crighton et al., 2006). ubiquitin-dependent mechanism, and a number of Inkeeping with its function as a transcription factor, ubiquitin ligases that promote the ubiquitination and p53 is predominately a nuclear protein. However, a subsequentdegradationof p53 have been identified fraction of p53 can be found in the cytoplasm. Recent (Table 1). studies have demonstrated an unexpected transcription- By far the best studied of these is Mdm2 (sometimes ally independent function for this pool of p53 as a BH3- referred to as HDM2 in humans) – a RING finger only protein, inducing apoptosis by directly binding to protein that is essential to regulate p53 and allow the pro-apoptotic proteins Bax and Bak, as well as the survival during normal development(Jones et al., 1995; anti-apoptotic proteins like BclxL (Mihara et al., 2003; Montes de Oca Luna et al., 1995). Mdm2 functions as a Chipuk et al., 2004; Leu et al., 2004). Interestingly ubiquitin ligase, in which the RING domain is necessary PUMA, a transcriptional target of p53, can activate to promote the ubiquitination and degradation of its this cytoplasmic function of p53 (Chipuk et al., 2005), target proteins, including p53 (Fang et al., 2000). Mdm2

Table 1 E3s that target p53, including the class of E3 and other targets E3 Class Other targets Referencess

Mdm2 RING Mdm2, MdmX, Histone H2A and H2B, hnRNP Iwakuma and Lozano (2003), Brady et al. (2005), Zhang and K, RB, b-arrestin, MTBP, E2F/DP1, p21(Waf1) Zhang (2005), Cheng and Cohen (2007), Salcedo et al. (2006), Numb, PSD-95, androgen receptor, PCAF, Yang et al. (2006a) E-cadherin, G-protein coupled receptor kinase 2, TSG101, Tip60, Insulin-like growth factor 1 receptor, glucocorticoid receptor Cop1 RING Cop1, acetyl-coenzyme A carboxylase (ACC) Dornan et al. (2004, 2006), Qi et al. (2006) Pirh2 RING HDAC1 Leng et al. (2003), Logan et al. (2006) ARF BP1/ HECT MCL1, Myc Adhikary et al. (2005), Chen et al. (2005b), Zhong et al. (2005) HectH9/ MULE E6AP HECT AIB1 Reviewed in Longworth and Laimins (2004), Mani et al. (2006) CHIP U-box A range of chaperone-bound proteins, HSP70 McDonough and Patterson (2003), Esser et al. (2005) Cullin 7 RING Andrews et al. (2006) WWP1 HECT KLF2, KLF5, Smad4 Zhang et al. (2004), Moren et al. (2005), Chen et al. (2005a), Laine and Ronai (2006) Topors RING Hairy (Drosophila) Rajendra et al. (2004), Secombe and Parkhurst(2004) Carps RING Caspase 8 and 10 McDonald and El-Deiry (2004), Yang et al. (2006b) E4F1 Atypical Le Cam et al. (2006)

Oncogene Activation of p53 HF Horn and KH Vousden 1308 is itself a transcriptional target of p53 (Zauberman et al., being themselves transcriptional targets of p53 1993), establishing a negative feedback loop in which (Leng et al., 2003; Dornan et al., 2004). While each of Mdm2 can keep the physiological levels of p53 low, and these ligases have been shown to regulate p53 stability in contribute to the recovery phase at the end of a p53 cells, mouse knockoutstudieswill prove useful in response. determining their relative contribution to regulating While Mdm2 is sufficient to target p53 for degrada- p53 levels in vivo. tion, there is good evidence that it does not function Finally, ithas been shown thatp53can be degraded alone. Several proteins have recently been shown to through an ubiquitin-independent mechanism as well. cooperate with Mdm2 in the regulation of p53. The Yin This pathway involves the 20S proteasome and is Yang 1 (YY1) transcription factor, a protein that plays regulated by the NAD(P)H quinone oxidoreductase 1 a key role in development, can increase the interaction (NQO1) (Asher et al., 2001). The major pool of NQO1 between p53 and Mdm2, so enhancing Mdm2- can be found at the 20S proteasome, where it is dependent p53 poly-ubiquitination and degradation thought to act as a gatekeeper. NQO1 can bind directly (Gronroos et al., 2004; Sui et al., 2004). to p53, preventing it from being degraded in a 20S A similar function has also been attributed to proteasomal manner. Genotoxic insults such as ionizing gankyrin, with the gankyrin/Mdm2 interaction promot- radiation lead to increased association between p53 ing p53 binding and degradation (Higashitsuji et al., and NQO1, and so to p53 stabilization (Asher and 2005a, b). The mechanism underlying this activity of Shaul, 2005). gankyrin is suggested by the observation that gankyrin associates with the S6 proteasomal ATPase. This SUMOylation and NEDDylation interaction of gankyrin, Mdm2 and p53 also promotes While ubiquitination is the most studied protein increased association of the Mdm2-p53 complex to the modification of p53, the ubiquitin-like proteins 26S proteasome, and so promotes more efficient SUMO-1 and Nedd8 can also be conjugated to p53 degradation of p53 and Mdm2 (Dawson et al., 2002; and modify its function (recently reviewed in Watson Higashitsuji et al., 2005a). The frequentoverexpression and Irwin (2006)). As in ubiquitination, specific E3 of gankyrin in hepatocellular carcinomas may therefore ligases mediate the modification of p53 by SUMO-1 and be an important mechanism for inactivating the p53 Nedd8. Interestingly, Mdm2 also functions as an E3 for pathway in these tumours (Fu et al., 2002; Nagao et al., Nedd8 – promoting the conjugation of Nedd8 to three 2003; Higashitsuji et al., 2005b). C-terminal lysines in p53 that are also targeted by p300/CBP has also been shown to cooperate ubiquitination (Xirodimas et al., 2004). Neddylation of with Mdm2 to promote poly-ubiquitination of p53 p53 can also be mediated by FBX011, a component (Grossman et al., 2003). p300 has a well established role of the SCF complex (Abida et al., 2006). A number of in promoting acetylation of p53, thereby enhancing its proteins have been shown to function in the regulation transcriptional activity (Grossman, 2001). However, a of p53 SUMOylation, including the family of PIAS recent report has suggested that under most conditions ligases (PIAS1, PIASxb and PIASy), (Kahyo et al., Mdm2 can only promote the mono-ubiquitination of 2001; Nelson et al., 2001; Megidish et al., 2002; Schmidt p53, and that the binding of p300 to Mdm2 is necessary and Muller, 2002). While Neddylation appears to result for efficient poly-ubiquitination to occur (Grossman in the inhibition of p53 function, there have been et al., 2003). As poly-ubiquitination is required for conflicting reports of the effect of SUMOylation recognition by the proteasome and degradation (re- (reviewed in Melchior and Hengst(2002)). However, viewed in Weissman, 2001), proteins like p300 may be recent studies have demonstrated that SUMO-modifica- key in controlling p53 stability. tion can activate p53 and so contribute to the induction The recent identification of HAUSP (herpesvirus of p53-mediated senescence and apoptosis (Bischof associated ubiquitin-specific protease) and other de- et al., 2006; Li et al., 2006). Whereas the modification ubiquitinating (DUB) enzymes has added a further level by SUMO-1 and Nedd8 does notappear tomodulate of complexity to the regulation of p53 stability. p53 stability directly, SUMOylation can mediate an Although HAUSP can de-ubiquitinate p53 directly (Li indirect effect by regulating Mdm2’s stability and et al., 2002), its principal function in the p53 pathway function (Lee et al., 2006). seems to be in the de-ubiquitination and stabilization of Mdm2 (Cummins and Vogelstein, 2004; Li et al., 2004), resulting in enhanced degradation of p53. This modi- MdmX fication is influenced by yet another protein, Daxx Another key regulator of p53 is MdmX (Mdm4), a (death domain-associated protein), which forms a structural relative of Mdm2. Like Mdm2, MdmX complex with Mdm2 and HAUSP, preventing the contains an N-terminal p53-binding domain and a auto-ubiquitination of Mdm2 and thus promoting p53 RING domain at its C-terminus (Shvarts et al., 1996). degradation (Tang et al., 2006). However, MdmX does not show E3 activity (Jackson Of the other p53 ubiquitin ligases listed in Table 1, and Berberich, 2000; Stad et al., 2001) and functions Cop1, Pirh2, HectH9/MULE/ARF-BP1, E6AP and predominantly by directly inhibiting p53’s transcrip- CHIP have all been shown to promote the degradation tional activity (Shvarts et al., 1996; Francoz et al., 2006). of p53 under varying conditions. Like Mdm2, Cop1 Although this function of MdmX is independent of and Pirh2 also reside in negative feedback loops by Mdm2 (Francoz et al., 2006; Xiong et al., 2006), there

Oncogene Activation of p53 HF Horn and KH Vousden 1309 Acidic Zn A delicate balance p53 binding domain binding RING Finger MDM2 491 In both humans and mice, there is exquisite sensitivity to modulation of the p53 pathway, with relatively Acidic Zn p53 binding domain binding RING Finger modest changes in p53 function resulting in profound MDMX 490 effects. This has been highlighted by studies investigat- Figure 1 Mdm2 and MdmX. Comparison of the structural ing the consequences of modifying the naturally domains of Mdm2 and MdmX, showing the regions of similarity. occurring ratio between Mdm2 and p53. Increasing Although both proteins contain a RING domain, only Mdm2 is potential p53 activity, by adding an extra copy of p53 or functional as an E3 ubiquitin ligase (Marine and Jochemsen, 2005). reducing (butnoteliminating)expression of Mdm2, resulted in apparently normal mice that were resistant to are some interesting levels of cross talk between these tumour development (Garcia-Cao et al., 2002, 2006; two proteins. MdmX and Mdm2 hetero-oligomerize Mendrysa et al., 2006). However, mice in which over- through their RING domains (Sharp et al., 1999; expression of p53 was accompanied by an imbalance in Tanimura et al., 1999), which results in the stabilization the normal ratios of different p53 isoforms, showed an of Mdm2 (Stad et al., 2001; Gu et al., 2002) or the alarming premature aging phenotype (Tyner et al., 2002; Mdm2-mediated degradation of MdmX (de Graaf et al., Maier et al., 2004) that may be associated with p53 2003; Kawai et al., 2003; Pan and Chen, 2003), functions of driving senescence and modulating the depending on the relative expression levels of the two insulin pathway. The contribution of p53 to aging is still proteins. Recent studies have also suggested that a notclear, butitwould seem thatjustenough p53 – heteromeric complex between Mdm2 and MdmX can neither too much nor too little – is required for optimal retain ubiquitin ligase activity, suggesting that despite longevity (Mendrysa and Perry, 2006; Poyurovsky and the inactive RING, MdmX may play a direct role in E3 Prives, 2006). activity (Poyurovsky et al., 2006; Uldrijan et al., 2006) The consequences of small changes in p53 activity (Figure 1). have also been highlighted by the study of polymorphisms in the p53 pathway in humans. A SNP was recently discovered at position 309 of the first intron of Mdm2 p53 subcellular localization (Bond et al., 2004). This polymorphism alters the As might be expected for a protein with both nuclear affinity of an SP1 transcription factor-binding site, and and cytoplasmic functions, the subcellular localization results in a slightly higher expression of Mdm2 in these of p53 can also play an important role in regulating its individuals, dampening the activation of p53 in response activity. Mdm2 can promote the nuclear export of p53 to DNA damage. While this results in a relatively in a ubiquitin-ligase-dependent manner (Boyd et al., modest decrease in the activation of p53, there is a clear 2000; Lohrum et al., 2001; Li et al., 2003). Mdm2 and effect on tumour susceptibility in individuals carrying p53 do not leave the nucleus together; rather it seems this SNP. that mono-ubiquitination of p53 by Mdm2 leads to an A number of polymorphisms of p53 have also been unmasking of the nuclear export sequence within the described, including a SNP atamino acid position72 C-terminus of p53 (Gu et al., 2001; Li et al., 2003). that changes the amino acid from a proline (P) to an Two other ubiquitin ligases also promote the cytoplas- arginine (R). The P72 variant is somewhat better at mic localization of p53, Cullin 7 (CUL7) and WWP1 promoting a cell cycle arrest, while the R72 variant is (Andrews et al., 2006; Laine and Ronai, 2006). Neither better at inducing apoptosis (Pim and Banks, 2004) and of these E3s target p53 for degradation, but result in as such, tumours carrying the R72 variant are generally the accumulation of transcriptionally inactive p53 more responsive to treatment and have a better survival in the cytoplasm. Similarly, regulation of p53 ubiquiti- rate, when compared to tumours carrying the P72 nation by the E2 ubiquitin-conjugating enzyme Ubc13 variant(reviewed in Pietsch et al. (2006)). has been shown to drive nuclear export of p53 (Laine et al., 2006). In a further twist, ubiquitination of p53 by E4F1 results in neither degradation nor Stress induced stabilization and activation of p53 nuclear export, but enhances transcriptional activation resulting selectively in the cell cycle arrest response Efficient control of p53 is critical to allow normal cell (Le Cam et al., 2006). growth. But clearly these brakes to p53 function While cytoplasmic sequestration inhibits p53 – and is seen as a mechanisms to inactivate p53 in some breast must be released in order for p53 to act as an effective cancers and neuroblastomas (Moll et al., 1992, 1995) – tumour suppressor, and a number of mechanisms the relocalization of at least some p53 from the nucleus that lead to the stabilization and activation of p53 is important for the cytoplasmic functions of p53. The have been identified. A growing number of stress signals effect of Mdm2 on p53 function is therefore complex – that can lead to p53 activation are being identified, while degradation of p53 clearly inactivates all func- including DNA damage, oncogene activation and tions, some level of Mdm2-mediated ubiquitination may more. Interestingly, different stress signals seem to be required for export and full apoptotic activity (Moll utilize different pathways to allow for the activation et al., 2006). of p53.

Oncogene Activation of p53 HF Horn and KH Vousden 1310 DNA damage MdmX, thereby releasing p53 from the negative DNA damage efﬁciently induces a p53 response, in part regulation exerted by both proteins (Gilkes et al., through the activation of ATM/ATR kinases that result 2006). While somewhatsurprising, a clear role for in the phosphorylation of p53, Mdm2 and MdmX. While ribosomal proteins in tumour suppressor pathways has phosphorylation of p53 and Mdm2 can inhibit the also been established in zebraﬁsh (Amsterdam et al., interaction between these two proteins, allowing the 2004). The importance of these interactions in humans is stabilization of p53 (Shieh et al., 1997; Siliciano et al., also strongly suggested by the observation that cancer 1997; Banin et al., 1998; Khosravi et al., 1999; de Toledo associated mutations in Mdm2 can prevent the interac- et al., 2000; Sakaguchi et al., 2000; Maya et al., 2001), tion with L11 and L5 (Lindstrom et al., 2006). phosphorylation of Mdm2 and MdmX also enhances the degradation of these proteins by reducing their association Cell–cell contacts with HAUSP – further releasing p53 from this level of Another DNA-damage independent stimulus that leads negative regulation (Cummins and Vogelstein, 2004; Li to the activation of p53 is the loss of cell–matrix et al., 2004; Meulmeester et al., 2005a, b; Tang et al., adhesion (reviewed in Grossmann (2002)). Loss of cell– 2006). The COP1 ubiquitin ligase is also phosphorylated cell or cell–matrix contact occurs in migrating cells, in an ATM-dependentfashion, leading toa dissociation especially when they enter the blood or lymph systems. of COP1 from p53 and so promoting p53 stabilization In non-transformed cells, the loss of adhesion can (Dornan et al., 2006). In addition to the stabilization of trigger a form of apoptosis known as anoikis, which in p53, DNA damage-induced kinases may also play a role some cell types has been shown to be p53 dependent. in activating p53 as a transcription factor, in part by The ability to overcome anoikis may be an important promoting acetylation within the C-terminus of p53 step in tumour progression towards metastasis, and the (Lambert et al., 1998; Dumaz and Meek, 1999). loss of p53 is critical for this step in some cell types. The PTEN phosphatase, a target of p53, appears to play Oncogene activation an important role, as the expression of PTEN in a p53 One of the key mediators of oncogene-induced activation of null background can restore the sensitivity to anoikis p53 is p14ARF(ARF) (recently reviewed in Sherr, 2006). (Lu et al., 1999). While the pathways leading to the ARF, the alternate reading frame productof thep16/ activation of p53 in response to loss of adhesion are not INK4A locus, has been shown to have a variety of clear, signalling through integrins can provide adhesion- functions, with one of its key roles being the binding and associated survival signals to prevent the activation of inhibition of Mdm2. Loss of ARF can, to a large degree, p53 and cell death (Wang et al., 2002). substitute for loss of p53 in tumour development, suggesting that disabling this pathway of p53 activation is critical Hypoxia for cancer progression (Sherr, 2006). Interestingly, loss of Cells at the centre of expanding tumours often ARF does notpreventthe activationof p53 in response to experience hypoxic conditions, due to the lack of DNA damage, indicating that these are independent – angiogenesis to provide the ever increasing mass of cells although clearly complementary and cooperative – signals with oxygen. Hypoxia activates p53 and stimulates to p53 (Stott et al., 1998; Kamijo et al., 1999). p53-dependent apoptosis through mechanisms that – although not clear – appear to involve ATR, HIF 1a Ribosomal stress (hypoxia inducible factor) and VHL (von Hippel- A number of ribosomal proteins, in particular L5, L11 Lindau) (reviewed in Hammond and Giaccia, 2005). and L23, have been shown to bind and inhibit Mdm2, Under normoxic conditions, HIF 1a is degraded et al thereby stabilizing and activating p53 (Lohrum et al., through its interaction with VHL (Maxwell ., 2003; Zhang et al., 2003; Dai and Lu, 2004; Dai et al., 1999; Semenza, 2001). Hypoxic or anoxic conditions 2004). Indeed, treating cells with siRNA for L5 or L11 promote the stabilization of HIF 1a by disrupting its abrogates the induction of p53 in response to ribosomal interaction with VHL, and HIF 1a is able to bind p53 stress, such as treatment with the RNA PolI inhibitor and promote its stabilization. Furthermore, VHL can Actinomycin D (Bhat et al., 2004; Dai and Lu, 2004). also interact with p53 directly and promote p53 Physiological stimuli that have been shown to signal phosphorylation and acetylation, leading to the activa- et al through the ribosomal stress pathway include serum tion (Roe ., 2006). Interestingly, p53 induced by starvation and cell–cell contact growth inhibition (Bhat hypoxia shows a different pattern of target gene et al., 2004). Furthermore, inactivation of the transcrip- activation than seen following DNA damage (reviewed et al tion initiation factor 1A (TIF-IA), which is responsible in Hammond and Giaccia (2005), Krieg . (2006)), for regulating the transcription of ribosomal RNA, suggesting that p53 may mediate a quite different induced binding of ribosomal proteins, including L11, to response to hypoxia compared to other stress signals. Mdm2, and the activation of p53 (Yuan et al., 2005). Interestingly, the DNA damage signal to p53 also depends on nucleolar disruption (Rubbi and Milner, All signals are not equal 2003), suggesting a cooperation between kinases such as ATM/ATR and the ribosomal proteins. Ribosomal The realization that different stress signals can activate stress also enhances Mdm2-mediated degradation of p53 through different pathways raises the question – are

Oncogene Activation of p53 HF Horn and KH Vousden 1311 all these pathways equally important for tumour Constitutive/mild damage suppression? The DNA damage pathway has been shown to be constitutively active in tumours (DiTullio Severe DNA damage Oncogene activation et al., 2002), with evidence that the DNA damage response pathways are activated even before cells become tumorigenic. The resultant activation of p53 in early hyperplastic and pre-cancerous lesions appears to prevent the formation of tumours (Bartkova et al., 2005; p53 Gorgoulis et al., 2005), highlighting the importance of DNA damage pathways. However, recent studies have also suggested the signalling through the ARF pathway – which is required Toxicity Tumor suppression to respond to oncogene activation but not DNA damage Tumour supression Antioxidant Toxicity Repair – is the response that is critical for tumour suppression. Survival These studies suggest that while the DNA damage- induced activation of p53 certainly produces a strong Figure 2 Activation and response to p53. p53 is activated by many stress signals that can differ in type and severity, which can lead to response, this is not necessary for tumour suppression, the activation of different responses, not all of which are clearly which requires ARF and is therefore more likely to beneficial to the organism. Induction of cell death or senescence in reflect the induction of p53 in response to oncogene response to severe DNA damage or oncogene activation are activation (Christophorou et al., 2006; Efeyan et al., thought to be amongst the most important mechanisms of p53- mediated tumour suppression. While there is evidence that DNA 2006) (Figure 2). Is it possible that activation of p53 by damage signalling in premalignancies activates p53 and contributes acute genotoxic stress is only deleterious to health, to tumour suppression, other work has suggested that the response leading to radiation sickness that might be avoided in to acute genotoxic stress results predominantly in toxicity, and that the absence of functional p53? p53 may become the response to oncogene activation plays the most important role beneficial only once mostof thedamage has been in tumour suppression. In addition to these two acute insults, p53 responds to a variety of milder, constitutive stresses – such as resolved, to drive elimination of the rare cells that have the generation of ROS by normal metabolism – and under acquired an oncogenic mutation. Further studies of the these conditions p53 functions to allow survival and repair of the relative contribution of each of the p53 activating damage, and so may even contribute to the longevity of the signals will be very revealing. organism.

Regulation of p53 stability and cancer therapy 2006). However, other studies have suggested that in some cells, Nutlin-3 treatment can result in the degradation of A large number of human tumours retain wild-type p53, MdmX (Wade et al., 2006) – possibly through the action of and many of these show evidence of perturbations in the Mdm2 released from p53 – and so the utility of Nutlin-3 pathways that allow stabilization and activation of p53 in as a therapy is likely to be strongly context dependent. response to stress. These pathways present attractive Other approaches to reactivating p53 have included targets for developing treatments that could restore p53 directly targeting the Mdm2 E3 ligase activity (Yang function in these tumours. Examples of these include et al., 2005); and the direct targeting of MdmX or drugs that might inhibit the interaction between p53 and HAUSP may also prove fruitful. It is clear that as the Mdm2, such as Nutlin-3 (Vassilev et al., 2004) or RITA secrets of p53 regulation are uncovered, more potential (Issaeva et al., 2004). While there is some contro- targets for the design of novel therapies will be revealed. versy concerning the mechanisms of function of RITA It seems likely that the next 40 000 reports on p53 will be (Krajewski et al., 2005), both compounds allow for the as exciting as the first. stabilization of active p53 and show promising results in animal tumour models. Interestingly, Nutlin-3 does not Acknowledgements efficiently block the interaction between p53 and MdmX, resulting in inefficient activation of p53 in tumours We are grateful to Cancer Research UK for support and overexpressing MdmX (Hu et al., 2006; Patton et al., funding.

References

Abida WM, Nikolaev A, Zhao W, Zhang W, Gu W. (2006). Amsterdam A, Sadler KC, Lai K, Farrington S, Bronson RT, FBXO11 promotes the neddylation of p53 and inhibits Lees JA et al. (2004). Many ribosomal protein genes are its transcriptional activity. J Biol Chem [Epub ahead cancer genes in zebraﬁsh. PLoS Biol 2: E139. of print]. Andrews P, He YJ, Xiong Y. (2006). Cytoplasmic localized Adhikary S, Marinoni F, Hock A, Hulleman E, Popov N, ubiquitin ligase cullin 7 binds to p53 and promotes cell growth Beier R et al. (2005). The ubiquitin ligase HectH9 regulates by antagonizing p53 function. Oncogene 25: 4534–4548. transcriptional activation by Myc and is essential for tumor Asher G, Lotem J, Cohen B, Sachs L, Shaul Y. (2001). cell proliferation. Cell 123: 409–421. Regulation of p53 stability and p53-dependent apoptosis by

Oncogene Activation of p53 HF Horn and KH Vousden 1312 NADH quinone oxidoreductase 1. Proc Natl Acad Sci USA damage does not contribute to p53-mediated tumour 98: 1188–1193. suppression. Nature 14: 214–217. Asher G, Shaul Y. (2005). p53 proteasomal degradation: Crighton D, Wilkinson S, O’Prey J, Syed N, Harrison PR, poly-ubiquitination is not the whole story. Cell Cycle 4: Gasco M et al. (2006). DRAM, a p53-induced modulator of 1015–1018. autophagy, is critical for apoptosis. Cell 14: 121–134. Banin S, Moyal L, Shieh S-Y, Taya Y, Anderson CW, Cummins JM, Vogelstein B. (2004). HAUSP is required for Chessa L et al. (1998). Enhanced phosphorylation of p53 p53 destabilization. Cell Cycle 3: 689–692. by ATM in response to DNA damage. Science 281: Dai MS, Lu H. (2004). Inhibition of MDM2-mediated p53 1674–1677. ubiquitination and degradation by ribosomal protein L5. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K J Biol Chem 279: 44475–44482. et al. (2005). DNA damage response as a candidate anti- Dai MS, Zeng SX, Jin Y, Sun XX, David L, Lu H. (2004). cancer barrier in early human tumorigenesis. Nature 434: Ribosomal protein L23 activates p53 by inhibiting MDM2 864–870. function in response to ribosomal perturbation but not to Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, translation inhibition. Mol Biol Cell 24: 7654–7668. Bartrons R et al. (2006). TIGAR, a p53-inducible regulator Danial NN, Korsmeyer SJ. (2004). Cell death: critical control of glycolysis and apoptosis. Cell 126: 107–120. points. Cell 116: 205–219. Bensaad K, Vousden KH. (2005). Savior and slayer: the two Dawson S, Apcher S, Mee M, Higashitsuji H, Baker R, Uhle S faces of p53. Nat Med 11: 1278–1279. et al. (2002). Gankyrin is an ankyrin-repeat oncoprotein that Bhat KP, Itahana K, Jin A, Zhang Y. (2004). Essential role of interacts with CDK4 kinase and the S6 ATPase of the 26 S ribosomal protein L11 in mediating growth inhibition- proteasome. J Biol Chem 277: 10893–10902. induced p53 activation. EMBO J 23: 2402–2412. de Graaf P, Little NA, Ramos YF, Meulmeester E, Bischof O, Schwamborn K, Martin N, Werner A, Sustmann C, Letteboer SJ, Jochemsen AG. (2003). Hdmx protein stability Grosschedl R et al. (2006). The E3 SUMO ligase PIASy is a is regulated by the ubiquitin ligase activity of Mdm2. J Biol regulator of cellular senescence and apoptosis. Mol Cell 22: Chem 278: 38315–38324. 783–794. de Toledo SM, Azzam EI, Dahlberg WK, Gooding TB, Bode AM, Dong Z. (2004). Post-translational modification of Little JB. (2000). ATM complexes with HDM2 and p53 in tumorigenesis. Nat Rev Cancer 4: 793–805. promotes its rapid phosphorylation in a p53-independent Bond GL, Hu W, Bond EE, Robins H, Lutzker SG, Arva NC manner in normal and tumor human cells exposed to et al. (2004). A single nucleotide polymorphism in the ionizing radiation. Oncogene 19: 6185–6193. MDM2 promoter attenuates the p53 tumor suppressor DiTullio Jr RA, Mochan TA, Venere M, Bartkova J, Sehested M, pathway and accelerates tumor formation in humans. Cell Bartek J et al. (2002). 53BP1 functions in an ATM- 119: 591–602. dependent checkpoint pathway that is constitutively Boyd SD, Tsai KY, Jacks T. (2000). An intact HDM2 RING- activated in human cancer. Nat Cell Biol 4: 998–1002. finger domain is required for nuclear exclusion of p53. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Nature Cell Biol 2: 563–568. Montgomery Jr CA, Butel JS et al. (1992). Mice deficient Brady M, Vlatkovic N, Boyd MT. (2005). Regulation of p53 for p53 are developmentally normal but susceptible to and MDM2 activity by MTBP. Mol Cell Biol 25: 545–553. spontaneous tumours. Nature 356: 215–221. Budanov AV, Sablina AA, Feinstein E, Koonin EV, Dornan D, Shimizu H, Mah A, Dudhela T, Eby M, Chumakov PM. (2004). Regeneration of peroxiredoxins by O’Rourke K et al. (2006). ATM engages autodegradation p53-regulated sestrins, homologs of bacterial AhpD. Science of the E3 ubiquitin ligase COP1 after DNA damage. Science 304: 596–600. 313: 1122–1126. Bykov VJ, Wiman KG. (2003). Novel cancer therapy by Dornan D, Wertz I, Shimizu H, Arnott D, Frantz GD, reactivation of the p53 apoptosis pathway. Ann Med 35: Dowd P et al. (2004). The ubiquitin ligase COP1 is a critical 458–465. negative regulator of p53. Nature 429: 86–92. Chen C, Sun X, Guo P, Dong XY, Sethi P, Cheng X et al. Dumaz N, Meek DW. (1999). Serine15 phosphorylation (2005a). Human Kruppel-like factor 5 is a target of the E3 stimulates p53 transactivation but does not directly influence ubiquitin ligase WWP1 for proteolysis in epithelial cells. interaction with HDM2. EMBO J 18: 7002–7010. J Biol Chem 280: 41553–41561. Efeyan A, Garcia-Cao I, Herranz D, Velasco-Miguel S, Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. (2005b). Serrano M. (2006). Tumour biology: policing of oncogene ARF-BP1/Mule is a critical mediator of the ARF tumor activity by p53. Nature 443: 159. suppressor. Cell 121: 1071–1083. El-Deiry WS, Harper JW, Oconnor PM, Velculescu VE, Cheng TH, Cohen SN. (2007). Human MDM2 isoforms Canman CE, Jackman J et al. (1994). WAF1/CIP1 is translated differentially on constitutive vs p53-regulated induced in p53-mediated G1 arrest and apoptosis. Cancer transcripts have distinct functions in the p53/MDM2 and Research 54: 1169–1174. TSG101/MDM2 feedback control loops. Mol Cell Biol 27: Esser C, Scheffner M, Hohfeld J. (2005). The chaperone- 111–119. associated ubiquitin ligase CHIP is able to target Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, p53 for proteasomal degradation. J Biol Chem 280: Green DR. (2005). PUMA couples the nuclear and 27443–27448. cytoplasmic proapoptotic function of p53. Science 309: Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM. 1732–1735. (2000). Mdm2 is a RING finger-dependentubiquitin Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, protein ligase for itself and p53. J Biol Chem 275: Newmeyer DD, Schuler M et al. (2004). Direct activation 8945–8951. of Bax by p53 mediates mitochondrial membrane permea- Francoz S, Froment P, Bogaerts S, De Clercq S, Maetens M, bilization and apoptosis. Science 303: 1010–1014. DoumontG et al. (2006). Mdm4 and Mdm2 cooperate to Christophorou MA, Ringhausen I, Finch AJ, Brown Swigart L, inhibit p53 activity in proliferating and quiescent cells Evan GI. (2006). The pathological response to DNA in vivo. Proc Natl Acad Sci USA 103: 3232–3237.

Oncogene Activation of p53 HF Horn and KH Vousden 1313 Fu XY, Wang HY, Tan L, Liu SQ, Cao HF, Wu MC. (2002). Jeffers JR, Parganas E, Lee Y, Yang C, Wang J, Brennan J Overexpression of p28/gankyrin in human hepatocellular et al. (2003). Puma is an essential mediator of p53-dependent carcinoma and its clinical significance. World J Gastroenterol and -independent apoptotic pathways. Cancer Cell 4: 8: 638–643. 321–328. Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Criado Jones SN, Roe AE, Donehower LA, Bradley A. (1995). Rescue LM, Klatt P, Flores JM et al. (2002). ‘Super p53’ mice of embryonic lethality in Mdm2-deficient mice by absence of exhibitenhanced DNA damage response, are tumor p53. Nature 378: 206–208. resistant and age normally. EMBO J 21: 6225–6235. Kahyo T, Nishida T, Yasuda H. (2001). Involvementof PIAS1 Garcia-Cao I, Garcia-Cao M, Tomas-Loba A, Martin- in the sumoylation of tumor suppressor p53. Mol Cell 8: Caballero J, Flores JM, Klatt P et al. (2006). Increased 713–718. p53 activity does not accelerate telomere-driven ageing. Kamijo T, van de Kamp E, Chong MJ, Zindy F, Diehl JA, EMBO Rep 7: 546–552. Sherr CJ et al. (1999). Loss of the ARF tumor suppressor Gatz SA, Wiesmuller L. (2006). p53 in recombination and reverses premature replicative arrest but not radiation repair. Cell Death Diff 13: 1003–1016. hypersensitivity arising from disabled atm function. Cancer Gilkes DM, Chen L, Chen J. (2006). MDMX regulation of p53 Res 59: 2464–2469. response to ribosomal stress. EMBO J 25: 5614–5625. Kawai H, Wiederschain D, Kitao H, Stuart J, Tsai KK, Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Yuan ZM. (2003). DNA damage-induced MDMX degrada- Kotsinas A, Liloglou T et al. (2005). Activation of the DNA tion is mediated by MDM2. J Biol Chem 278: 45946–45953. damage checkpoint and genomic instability in human Khosravi R, Maya R, Gottlieb T, Oren M, Shiloh Y, Shkedy precancerous lesions. Nature 434: 907–913. D. (1999). Rapid ATM-dependentphosphorylationof Gronroos E, Terentiev AA, Punga T, Ericsson J. (2004). YY1 MDM2 precedes p53 accumulation in response to DNA inhibits the activation of the p53 tumor suppressor in damage. Proc Natl Acad Sci USA 96: 14973–14977. response to genotoxic stress. Proc Natl Acad Sci USA 101: Krajewski M, Ozdowy P, D’Silva L, Rothweiler U, Holak TA. 12165–12170. (2005). NMR indicates that the small molecule RITA Grossman SR. (2001). p300/CBP/p53 interaction and regula- does notblock p53-MDM2 binding in vitro. Nat Med 11: tion of the p53 response. Eur J Biochem 268: 2773–2778. 1135–1136; author reply 1136–1137. Grossman SR, Deato ME, Brignone C, Chan HM, Kung AL, Krieg AJ, Hammond EM, Giaccia AJ. (2006). Functional Tagami H et al. (2003). Polyubiquitination of p53 by a analysis of p53 binding under differential stresses. Mol Cell ubiquitin ligase activity of p300. Science 300: 342–344. Biol 26: 7030–7045. Grossmann J. (2002). Molecular mechanisms of ‘detachment- Laine A, Ronai Z. (2006). Regulation of p53 localization and induced apoptosis – Anoikis’. Apoptosis 7: 247–260. transcription by the HECT domain E3 ligase WWP1. Gu J, Kawai H, Nie L, Kitao H, Wiederschain D, Oncogene [Epub ahead of print]. Jochemsen AG et al. (2002). Mutual dependence of MDM2 Laine A, Topisirovic I, Zhai D, Reed JC, Borden KL, and MDMX in their functional inactivation of p53. Ronai Z. (2006). Regulation of p53 localization and activity J Biol Chem 277: 19251–19254. by Ubc13. Mol Cell Biol 26: 8901–8913. Gu J, Nie N, Wiederschain D, Yuan Z-M. (2001). Identification Lambert PF, Kashanchi F, Radonovich MF, Shiekhattar R, of p53 sequence elements that are required for MDM2- Brady JN. (1998). Phosphorylation of p53 serine 15 mediated nuclear export. Mol Cell Biol 21: 8533–8546. increases interaction with CBP. J Biol Chem 273: 33048– Hainaut P, Hollstein M. (2000). p53 and human cancer: the 33053. first ten thousand mutations. Adv Cancer Res 77: 81–137. Le Cam L, Linares LK, Paul C, Julien E, Lacroix M, Hatchi E Hammond EM, Giaccia AJ. (2005). The role of p53 in et al. (2006). E4F1 is an atypical ubiquitin ligase that hypoxia-induced apoptosis. Biochem Biophys Res Commun modulates p53 effector functions independently of degrada- 331: 718–725. tion. Cell 127: 775–788. Higashitsuji H, Higashitsuji H, Itoh K, Sakurai T, Nagao T, Lee MH, Lee SW, Lee EJ, Choi SJ, Chung SS, Lee JI et al. Sumitomo Y et al. (2005a). The oncoprotein gankyrin binds (2006). SUMO-specific protease SUSP4 positively regulates to MDM2/HDM2, enhancing ubiquitylation and degrada- p53 by promoting Mdm2 self-ubiquitination. Nat Cell Biol tion of p53. Cancer Cell 8: 75–87. 8: 1424–1431. Higashitsuji H, Liu Y, Mayer RJ, Fujita J. (2005b). The Leng RP, Lin Y, Ma W, Wu H, Lemmers B, Chung S et al. oncoprotein gankyrin negatively regulates both p53 and (2003). Pirh2, a p53-induced ubiquitin-protein ligase, RB by enhancing proteasomal degradation. Cell Cycle 4: promotes p53 degradation. Cell 112: 779–791. 1335–1337. Leu JI, DumontP, Hafey M, Murphy MP, George DL. Hu B, Gilkes DM, Farooqi B, Sebti SM, Chen J. (2006). (2004). Mitochondrial p53 activates Bak and causes disrup- MDMX overexpression prevents p53 activation by the tion of a Bak-Mcl1 complex. Nature Cell Biol 6: 443–450. MDM2 inhibitor Nutlin. J Biol Chem 281: 33030–33035. Li M, Brooks CL, Kon N, Gu W. (2004). A dynamic Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, role of HAUSP in the p53-Mdm2 pathway. Mol Cell 13: Masucci M et al. (2004). Small molecule RITA binds to p53, 879–886. blocks p53-HDM-2 interaction and activates p53 function in Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. (2003). tumors. Nat Med 10: 1321–1328. Mono- versus polyubiquitination: differential control of p53 Iwakuma T, Lozano G. (2003). MDM2, an introduction. Mol fate by Mdm2. Science 302: 1972–1975. Cancer Res 1: 933–1000. Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J et al. Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, (2002). Deubiquitination of p53 by HAUSP is an important Bronson RT et al. (1994). Tumor spectrum analysis in pathway for p53 stabilization. Nature 416: 648–652. p53-mutant mice. Current Biol 4: 1–7. Li T, Santockyte R, Shen RF, Tekle E, Wang G, Yang DC Jackson MW, Berberich SJ. (2000). MdmX protects p53 et al. (2006). Expression of SUMO-2/3 induced senescence from Mdm2-mediated degradation. Mol Cell Biol 20: through p53- and pRB-mediated pathways. J Biol Chem 1001–1007. 281: 36221–36227.

Oncogene Activation of p53 HF Horn and KH Vousden 1314 Lindstrom MS, Jin A, Deisenroth C, Wolf GW, Zhang Y. Meulmeester E, Maurice MM, Boutell C, Teunisse AFAS, (2006). Cancer-associated mutations in MDM2 zinc finger Ovaa H, Abraham TE et al. (2005a). Loss of HAUSP- domain disrupt ribosomal protein interaction and attenuate mediated deubiquitination contributes to DNA damage- MDM2-Induced p53 degradation. Mol Cell Biol [Epub induced destabilization of Hdmx and Hdm2. Mol Cell 18: ahead of print]. 565–576. Logan IR, Gaughan L, McCracken SR, Sapountzi V, Leung Meulmeester E, Pereg Y, Shiloh Y, Jochemsen AG. (2005b). HY, Robson CN. (2006). Human PIRH2 enhances andro- ATM-mediated phosphorylations inhibit Mdmx/Mdm2 gen receptor signaling through inhibition of histone stabilization by HAUSP in favor of p53 activation. Cell deacetylase 1 and is overexpressed in prostate cancer. Mol Cycle 4: 1166–1170. Cell Biol 26: 6502–6510. Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Lohrum MA, Ludwig RL, Kubbutat MHG, Hanlon M, Pancoska P et al. (2003). p53 has a directapoptogenicrole at Vousden KH. (2003). Regulation of HDM2 activity by the the mitochondria. Mol Cell 11: 577–590. ribosomal protein L11. Cancer Cell 3: 577–587. Moll UM, LaQuaglia M, Benard J, Riou G. (1995). Wild-type Lohrum MAE, Woods DB, Ludwig RL, Ba´ lintE, Vousden KH. p53 protein undergoes cytoplasmic sequestration in undif- (2001). C-terminal ubiquitination of p53 contributes to ferentiated neuroblastomas but not in differentiated tumors. nuclear export. Mol Cell Biol 21: 8521–8532. Proc Natl Acad Sci USA 92: 4407–4411. Longworth MS, Laimins LA. (2004). Pathogenesis of human Moll UM, Marchenko N, Zhang XK. (2006). p53 and papillomaviruses in differentiating epithelia. Microbiol Mol Nur77/TR3 – transcription factors that directly target Biol Rev 68: 362–372. mitochondria for cell death induction. Oncogene 25: Lu Y, Lin YZ, LaPushin R, Cuevas B, Fang X, Yu SX et al. 4725–4743. (1999). The PTEN/MMAC1/TEP tumor suppressor gene Moll UM, Riou G, Levine AJ. (1992). Two distinct mechan- decreases cell growth and induces apoptosis and anoikis in isms alter p53 in breast cancer: mutation and nuclear breastcancer cells. Oncogene 18: 7034–7045. exclusion. Proc Natl Acad Sci USA 89: 7262–7266. Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Montes de Oca Luna R, Wagner DS, Lozano G. (1995). Guise T et al. (2004). Modulation of mammalian life span by Rescue of early embryonic lethality in mdm2-deficient mice the short isoform of p53. Genes Dev 18: 306–319. by deletion of p53. Nature 378: 203–206. Malkin D, Li FP, Strong LC, Fraumini Jr JF, Nelson CE, Moren A, Imamura T, Miyazono K, Heldin CH, Moustakas A. Kim DH et al. (1990). Germ line p53 mutations in a familial (2005). Degradation of the tumor suppressor Smad4 by WW syndrome of breastcancer, sarcomas, and otherneoplasms. and HECT domain ubiquitin ligases. J Biol Chem 280: Science 250: 1233–1238. 22115–22123. Mani A, Oh AS, Bowden ET, Lahusen T, Lorick KL, Nagao T, Higashitsuji H, Nonoguchi K, Sakurai T, Dawson S, Weissman AM et al. (2006). E6AP mediates regulated Mayer RJ et al. (2003). MAGE-A4 interacts with the proteasomal degradation of the nuclear receptor coactivator liver oncoprotein gankyrin and suppresses its tumorigenic amplified in breastcancer 1 in immortalizedcells. Cancer activity. J Biol Chem 278: 10668–10674. Res 66: 8680–8686. Nakano K, Vousden KH. (2001). PUMA, a novel proapopto- Marine JC, Jochemsen AG. (2005). Mdmx as an essential tic gene, is induced by p53. Mol Cell 7: 683–694. regulator of p53 activity. Biochem Biophys Res Commun 331: Nelson V, Davis GE, Maxwell SA. (2001). A putative protein 750–760. inhibitor of activated STAT (PIASy) interacts with p53 and Maxwell PH, Wiesener MS, Chang GW, Clifford SC, inhibits p53-mediated transactivation but not apoptosis. Vaux EC, Cockman ME et al. (1999). The tumour Apoptosis 6: 221–234. suppressor protein VHL targets hypoxia-inducible OdaE,OhkiR,MurasawaH,NemotoJ,ShibueT,YamashitaT factors for oxygen-dependent proteolysis. Nature 399: et al. (2000). Noxa, a BH3-only member of the Bcl-2 family 271–275. and candidate mediator of p53-induced apoptosis. Science Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O 288: 1053–1058. et al. (2001). ATM-dependentphosphorylationof Mdm2 on Pan Y, Chen J. (2003). MDM2 promotes ubiquitination and serine 395: role in p53 activation by DNA damage. Genes degradation of MDMX. Mol Cell Biol 23: 5113–5121. Dev 15: 1067–1077. Patton JT, Mayo LD, Singhi AD, Gudkov AV, Stark GR, McDonald III ER, El-Deiry WS. (2004). Suppression of Jackson MW. (2006). Levels of HdmX expression dictate the caspase-8- and -10-associated RING proteins results in sensitivity of normal and transformed cells to Nutlin-3. sensitization to death ligands and inhibition of tumor cell Cancer Res 66: 3169–3176. growth. Proc Natl Acad Sci USA 101: 6170–6175. Pietsch EC, Humbey O, Murphey ME. (2006). Polymorphisms McDonough H, Patterson C. (2003). CHIP: a link between the in the p53 pathway. Oncogene 25: 1602–1611. chaperone and proteasome systems. Cell Stress Chaperones Pim D, Banks L. (2004). p53 polymorphic variants at codon 72 8: 303–308. exertdifferenteffectson cell cycle progression. Int J Cancer Megidish T, Xu JH, Xu CW. (2002). Activation of p53 by 108: 196–199. protein inhibitor of activated Stat1 (PIAS1). J Biol Chem Poyurovsky MV, PriestC, KentsisA, Borden KL, Pan ZQ, 277: 8255–8259. Pavletich N et al. (2006). EMBO J [Epub ahead of print]. Melchior F, HengstL. (2002). SUMO-1 and p53. Cell Cycle 1: Poyurovsky MV, Prives C. (2006). Unleashing the power of 245–249. p53: lessons from mice and men. Genes Dev 20: 125–131. Mendrysa SM, O’Leary KA, McElwee MK, Michalowski J, Purdie CA, Harrison DJ, Peter A, Dobbie L, White S, Eisenman RN, Powell DA et al. (2006). Tumor suppression Howie SE et al. (1994). Tumour incidence, spectrum and and normal aging in mice with constitutively high p53 ploidy in mice with a large deletion in the p53 gene. activity. Genes Dev 20: 16–21. Oncogene 9: 603–609. Mendrysa SM, Perry ME. (2006). Tumor suppression by p53 Qi L, Heredia JE, Altarejos JY, Screaton R, Goebel N, without accelerated aging: just enough of a good thing? Cell Niessen S et al. (2006). TRB3 links the E3 ubiquitin ligase Cycle 5: 714–717. COP1 to lipid metabolism. Science 312: 1763–1766.

Oncogene Activation of p53 HF Horn and KH Vousden 1315 Rajendra R, Malegaonkar D, Pungaliya P, Marshall H, Tang J, Qu LK, Zhang J, Wang W, Michaelson JS, Rasheed Z, Brownell J et al. (2004). Topors functions as DegenhardtYY et al. (2006). Critical role for Daxx in an E3 ubiquitin ligase with specific E2 enzymes and regulating Mdm2. Nat Cell Biol 8: 855–862. ubiquitinates p53. J Biol Chem 279: 36440–36444. Tanimura S, Ohtsuka S, Mitsui K, Shirouzu K, Yoshimura A, Roe JS, Kim H, Lee SM, Kim ST, Cho EJ, Youn HD. (2006). Ohtsubo M. (1999). MDM2 interacts with MDMX through p53 stabilization and transactivation by a von Hippel- their RING finger domains. FEBS Lett 447: 5–9. Lindau protein. Mol Cell 22: 395–405. Toledo F, Wahl GM. (2006). Regulating the p53 pathway: Rubbi CP, Milner J. (2003). Disruption of the nucleolus in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6: mediates stabilization of p53 in response to DNA damage 909–923. and other stresses. EMBO J 22: 6068–6077. Tyner SD, Venkatachalam S, Choi J, Ghebranious N, Sablina AA, Budanov AV, Ilyinskaya GV, Agapova LS, Igelmann H, Lu X et al. (2002). p53 mutant mice Kravchenko JE, Chumakov PM. (2005). The antioxidant that display early ageing-associated phenotypes. Nature function of the p53 tumor suppressor. Nat Med 11: 415: 45–53. 1306–1313. Uldrijan S, Pannekoek WJ, Vousden KH. (2006). EMBO J Sakaguchi K, Saito S, Higashimoto Y, Roy S, Anderson CW, [Epub ahead of print]. Appella E. (2000). Damage-mediated phosphorylation of Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, human p53 threonine 18 through a cascade mediated by a Filipovic Z et al. (2004). In vivo activation of the p53 casein 1-like kinase. Effecton Mdm2 binding. J Biol Chem pathway by small-molecule antagonists of MDM2. Science 275: 9278–9283. 303: 844–848. Salcedo A, Mayor Jr F, Penela P. (2006). Mdm2 is involved in Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, the ubiquitination and degradation of G-protein-coupled Ausserlechner MJ et al. (2003). p53- and drug-induced receptor kinase 2. EMBO J 25: 4752–4762. apoptotic responses mediated by BH3-only proteins puma SchmidtD, Muller S. (2002). Members of thePIAS family act and noxa. Science 302: 1036–1038. as SUMO ligases for c-Jun and p53 and repress p53 activity. Vogelstein B, Lane D, Levine AJ. (2000). Surfing the p53 Proc Natl Acad Sci USA 99: 2872–2877. network. Nature 408: 307–310. Secombe J, ParkhurstSM. (2004). Drosophila Topors is Wade M, Wong ET, Tang M, Stommel JM, Wahl GM. (2006). a RING finger-containing protein that functions as a Hdmx modulates the outcome of p53 activation in human ubiquitin-protein isopeptide ligase for the hairy basic tumor cells. J Biol Chem 281: 33036–33044. helix-loop-helix repressor protein. J Biol Chem 279: Wang WJ, Kuo JC, Yao CC, Chen RH. (2002). DAP-kinase 17126–17133. induces apoptosis by suppressing integrin activity and Semenza GL. (2001). HIF-1, O(2), and the 3 PHDs: how disrupting matrix survival signals. J Cell Biol 159: 169–179. animal cells signal hypoxia to the nucleus. Cell 107: 1–3. Watson IR, Irwin MS. (2006). Ubiquitin and ubiquitin-like Sharp DA, Kratowicz SA, Sank MJ, George DL. (1999). modifications of the p53 family. Neoplasia 8: 655–666. Stabilization of the MDM2 oncoprotein by interaction with Weissman AM. (2001). Themes and variations on ubiquityla- the structurally related MDMX protein. J Biol Chem 274: tion. Nat Rev Mol Cell Biol 2: 169–178. 38189–38196. Xiong S, Van PeltCS, Elizondo-Fraire AC, Liu G, Lozano G. Sherr CJ. (2006). Divorcing ARF and p53: an unsettled case. (2006). Synergistic roles of Mdm2 and Mdm4 for p53 Nat Rev Cancer 6: 663–673. inhibition in central nervous system development. Proc Natl Sherr CJ, Roberts JM. (1999). CDK inhibitors: positive and Acad Sci USA 103: 3226–3231. negative regulators of G1-phase progression. Genes Dev 13: Xirodimas DP, Saville MK, Bourdon JC, Hay RT, Lane DP. 1501–1512. (2004). Mdm2-mediated NEDD8 conjugation of p53 Shieh S-Y, Ikeda M, Taya Y, Prives C. (1997). DNA damage- inhibits its transcriptional activity. Cell 118: 83–97. induced phosphorylation of p53 alleviates inhibition by Yang JY, Zong CS, Xia W, Wei Y, Ali-Seyed M, Li Z et al. MDM2. Cell 91: 325–334. (2006a). MDM2 promotes cell motility and invasiveness Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, by regulating E-cadherin degradation. Mol Cell Biol 26: Bazuine M et al. (1996). MDMX: a novel p53-binding 7269–7282. protein with some functional properties of MDM2. EMBO Yang W, Rozan LM, McDonald III ER, Navaraj A, Liu JJ, J 15: 5349–5357. Matthew EM et al. (2006b). Carps are ubiquitin ligases that Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, promote MDM2-independent P53 and phospho-P53ser20 Kastan MB. (1997). DNA damage induces phosphorylation degradation. J Biol Chem [Epub ahead of print]. of the amino terminus of p53. Genes Dev 11: 3471–3481. Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim YS et al. Srivastava S, Zou Z, Pirollo K, Blattner W, Chang EH. (1990). (2006c). Modification of p53 with O-linked N-acetylgluco- Germ-line transmission of a mutated p53 gene in a samine regulates p53 activity and stability. Nat Cell Biol 8: cancer-prone family with Li-Fraumeni syndrome. Nature 1074–1083. 348: 747–749. Yang Y, Ludwig RL, Jensens JP, Pierre S, Medaglia MV, Stad R, Little NA, Xirodimas DP, Frenk R, van der Eb AJ, Davydov I et al. (2005). Small molecule inhibitors of HDM2 Lane DP et al. (2001). Mdmx stabilizes p53 and Mdm2 via ubiquitin ligase activity stabilize and activate p53 in cells. two distinct mechanisms. EMBO Rep 2: 1029–1034. Cancer Cell 7: 547–559. Stott F, Bates SA, James M, McConnell BB, Starborg M, Yee KS, Vousden KH. (2005). Complicating the complexity of Brookes S et al. (1998). The alternative product from the p53. Carcinogenesis 26: 1317–1322. human CDKN2A locus, p14(ARF), participates in a Yoon KA, Nakamura Y, Arakawa H. (2004). Identification of regulatory feedback loop with p53 and MDM2. EMBO J ALDH4 as a p53-inducible gene and its protective role in 17: 5001–5014. cellular stresses. J Hum Genet 49: 134–140. Sui G, Affar el B, Shi Y, Brignone C, Wall NR, Yin P et al. Yu J, Kinzler KW, Vogelstein B, Zhang L. (2003). PUMA (2004). Yin Yang 1 is a negative regulator of p53. Cell 117: mediates the apoptotic response to p53 in colorectal cancer 859–872. cells. Proc Natl Acad Sci USA 100: 1931–1936.

Oncogene Activation of p53 HF Horn and KH Vousden 1316 Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. (2001). like factor, KLF2. Biochem Biophys Res Commun 316: PUMA induces the rapid apoptosis of colorectal cancer 139–148. cells. Mol Cell 7: 673–682. Zhang Y, Wolf GW, BhatK, Jin A, Allio T, BurkhartWA et al. Yuan X, Zhou Y, Casanova E, Chai M, Kiss E, Grone HJ (2003). Ribosomal protein L11 negatively regulates oncopro- et al. (2005). Genetic inactivation of the transcription factor tein MDM2 and mediates a p53-dependent ribosomal-stress TIF-IA leads to nucleolar disruption, cell cycle arrest, and checkpointpathway. Mol Cell Biol 23: 8902–8912. p53-mediated apoptosis. Mol Cell 19: 77–87. Zhang Z, Zhang R. (2005). p53-independent activities of Zauberman A, Barak Y, Ragimov N, Levy N, Oren M. (1993). MDM2 and their relevance to cancer therapy. Curr Cancer Sequence-speciﬁc DNA binding by p53: identiﬁcation of Drug Targets 5: 9–20. target sites and lack of binding to p53 – MDM2 complexes. Zhong Q, Gao W, Du F, Wang X. (2005). Mule/ARF-BP1, EMBO J 12: 2799–2808. a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquiti- Zhang X, Srinivasan SV, Lingrel JB. (2004). WWP1-dependent nation of Mcl-1 and regulates apoptosis. Cell 121: ubiquitination and degradation of the lung Kruppel- 1085–1095.

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