The cytoplasmic side of p53's oncosuppressive activities.

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Review

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The cytoplasmic side of p53’s oncosuppressive activities

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Anna Cornel 1, Giovanni Sorrentino 1, Valeria Capaci, Giannino Del Sal ⇑ Laboratorio Nazionale CIB (LNCIB), Area Science Park, 34149 Trieste, Italy Dipartimento di Scienze della Vita, Universita degli Studi di Trieste, 34127, Italy

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Article history: Received 23 March 2014 Revised 9 April 2014 Accepted 10 April 2014 Available online xxxx

Edited by Shairaz Baksh, Giovanni Blandino and Wilhelm Just

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Keywords: p53 Cytoplasm Mitochondria Apoptosis Metabolism Transcription-independent

a b s t r a c t The tumor suppressor p53 is a transcription factor that in response to a plethora of stress stimuli activates a complex and context-dependent cellular response ultimately protecting genome integrity. In the last two decades, the discovery of cytoplasmic p53 localization has driven an intense research on its extra-nuclear functions. The ability to induce apoptosis acting directly at mitochondria and the related mechanisms of p53 localization and translocation in the cytoplasm and mitochondria have been dissected. However, recent works indicate the involvement of cytoplasmic p53 also in biological processes such as autophagy, metabolism, oxidative stress and drug response. This review will focus on the mechanisms of cytoplasmic p53 activation and the pathophysiological role of p53’s transcription-independent functions, highlighting possible therapeutic implications. Ó 2014 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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1. Introduction

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The tumor suppressor TP53 has a central role in the biology of the cell and represents one of the most efficient barriers against cancer development and progression. Embedded within a complex and highly interconnected signaling pathway, p53 regulates key cellular process such as DNA repair, metabolism, stemness, development, inflammation, endocytosis and cell death. In addition to cancer, it becomes a critical player also in ischemia, neurodegeneration and ageing, in response to a variety of stress stimuli [1]. p53 exerts its activity primarily at the transcriptional level, directly binding to specific target sequences on DNA. Its timely activation/inactivation upon stress depends on a complex repertoire of post-translational modifications and protein–protein interactions to allow both direct and indirect transactivation of many coding and non-coding genes. However, as for several other transcription factors, it is now evident that the actions of p53, as well as its localization, are not restricted to the nucleus and over the years a clear transcriptional independent activity has emerged.

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⇑ Corresponding author at: Laboratorio Nazionale CIB (LNCIB), Area Science Park,

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34149 Trieste, Italy. E-mail address: [email protected] (G. Del Sal). 1 These authors contributed equally to this work.

One of the best characterized p53’s extra-nuclear activities is the induction of apoptosis in the cytoplasm. Among the tumor suppressive activities fostered by p53, apoptosis is undoubtedly the most studied mechanism in vitro and in vivo; in particular p53 is able to induce apoptosis through the intrinsic mitochondriamediated pathway, characterized by permeabilization of the outer membrane (MOMP) leading to programmed cell death. p53 can transcriptionally induce the pro-apoptotic members of this pathway and repress anti-apoptotic genes, but it is also able to induce apoptosis in a transcription-independent way. This alternative route has been tightly investigated in the recent years and the cytoplasmic and mitochondrial activities of p53 have been largely defined [2–4]. Out of the nucleus, p53 may also affect signal transduction, metabolism, autophagy, vesicular trafficking and cytoskeleton organization. Other findings also sustain a possible role of cytoplasmic p53 in regulating stem cell expansion, as for example the evidence that p53 is cytoplasmic in ES cells [5,6], where after ROS exposure it can induce mitochondrial dependent apoptosis [6]. However while several mechanisms of p53 cytoplasmic activation towards the apoptotic route have been discovered to date, most of the mechanisms regulating other arms of its cytoplasmic activities remain largely unknown, as well as the possible extension in this context of the gain-of-function concept related to some p53 missense mutants fostering oncogenesis.

http://dx.doi.org/10.1016/j.febslet.2014.04.015 0014-5793/Ó 2014 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Please cite this article in press as: Cornel, A., et al. The cytoplasmic side of p53’s oncosuppressive activities. FEBS Lett. (2014), http://dx.doi.org/10.1016/ j.febslet.2014.04.015

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In this review we will focus on these aspects of functions and dysfunctions of p53, on the mechanisms behind its action in the cytoplasm and its pathogenic implications pinpointing possible hubs for therapeutic intervention.

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2. Cytoplasmic localization: ‘‘should I stay or should I go’’

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Activation of p53 involves a very complex cascade of events that spans from protein oligomerization to a repertoire of posttranslational modifications and protein interactions to accumulation in specific cellular compartments. Although the key steps leading to the full activation of the transcriptional program of p53 have been intensively studied and defined [7], most of the regulatory mechanisms governing its cytoplasmic localization and activities remain largely unknown, with the majority of reports regarding the transcription-independent apoptotic context. That said, a few requisites can be traced. The increase of p53 abundance in the cytoplasm is the first required condition to trigger the majority of the extra-nuclear activities of p53 [2,3,8]. However, several reports indicate that cytoplasmic p53 is able to exert its action particularly after cell exposure to specific stimuli [2,9–12]. These observations suggest that besides protein accumulation and cytoplasmic shuttling, other events like specific posttranslational modifications and protein–protein interactions could be critical also for p53 activities outside of the nucleus. The oligomeric state of p53 is a fundamental determinant of its functions. While tetramerization has been shown to be essential for p53 nuclear retention by masking its C-terminal nuclear export signal (NES) [13], other forms of p53 could be important for its nuclear export. Indeed, it has been demonstrated that the transcription factor Foxo3a promotes p53 nuclear export and transcription-independent apoptosis by fostering its interaction with the nuclear-export protein CRM1 and p53 protein oligomerization has been proposed as a mechanism governing Foxo3a mediated nuclear p53 exclusion [14,15]. Among post-translational modifications, ubiquitylation has been clearly linked to p53 subcellular localization. Poly-ubiquitylation of p53 causes its proteasome-mediated degradation, while mono- and multi-mono-ubiquitylation promotes its cytoplasmic relocalization [14,16]. In particular, Marchenko et al. claimed the role of the ubiquitylation as a specific signal for p53 mitochondrial trafficking (p53 lacks a mitochondrial targeting sequence). They identified the MDM2-mediated p53 multi-mono-ubiquitylation as the main signal governing p53 delivery to mitochondria

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necessary for transcription-independent apoptosis induction after stress [10] (Fig. 1). In cells, the relative stoichiometry of p53 and MDM2, the main regulator of p53 protein abundance and also a p53 target gene, plays critical role in switching from poly- to mono-ubiquitylation, with low levels of MDM2 promoting monoversus poly-ubiquitylation of p53. Of note, upon arrival at the mitochondria, the authors demonstrated that p53 undergoes active de-ubiquitylation by resident HAUSP [10,17]. This has been the first demonstration that the cytoplasmic compartment is the source of p53 that translocates to mitochondria thus opening the debate about the role of nuclear exclusion process in this context. Despite early investigations failed to find causality between other specific p53 modifications (e.g. acetylation and phosphorylation) and its pro-apoptotic role at mitochondria [18,19], recent works unveiled a prominent role of these post-translational modification for p53-induced transcription-independent apoptosis. Sykes et al. found that the Lys-120-acetylated form of p53 is enriched at mitochondrial outer membrane, after DNA damage, and that this modification is not required for p53 mitochondrial translocation but rather to displace the anti-apoptotic Mcl-1 from the apoptosis effector Bak at the mitochondria [20]. Interestingly, p53 acetylation was found to be required also for transcriptionindependent apoptosis induced by HDAC inhibitors [21]. Recently, we and others identified DNA-damage induced phosphorylation of p53 on Ser46 as a key event for p53 mitochondrial translocation [9,22]. In this context a pivotal role is played by the prolylisomerase Pinl, a phospho-specific prolyl-isomerase that transduces post-translational modifications into conformational changes relevant for full activity of p53 and other proteins [23]. After DNA damage, Pinl promotes phospho-dependent cis-trans p53 isomerization that lowers its affinity for MDM2 with consequent activation of nuclear p53 pro-apoptotic program [24–26]. Similar mechanisms of p53 activation appear to govern its transcription-independent activity in the cytoplasm. Mechanistically, the kinase HIPK2, activated by DNA-damage [27,28], phosphorylates p53 at Ser46 residue recruiting Pinl that leads to a p53 conformational change that alters its affinity for MDM2. Besides activating the nuclear pro-apoptotic route of p53, this event results in an increase of the monoubiquitylated pool of p53 that is engaged for mitochondrial translocation [9,29]. Ser46-phosphorylation on p53 is maintained at the mitochondria and mediates the anchorage of p53 to resident MDM4 that, in turn, facilitates the interaction between p53 and the anti-apoptotic protein Bcl-2 and the consequent MOMP induction [22,30] (Fig. 1). Recent evidences suggest that this circuit

Fig. 1. Regulation of p53 transcriptional-independent activities by post-translational modifications and protein–protein interactions. Acetylation, phosphorylation and ubiquitylation control p53 mitochondrial translocation and activity after stress. After cellular stress (DNA damage, oxidative stress and others), p53 is actively acetylated and this modified p53 is found enriched at mitochondria, where it can displace the anti-apoptotic Mcl-1 from Bak causing consequent cytochrome c release. DNA damage induces also Pin 1-dependent HIPK2 activation that phosphorylates p53 on Ser46. By triggering Pin 1-dependent conformational change, this phosphorylation represents a key step in promoting MDM2-mediated p53 mono-ubiquitylation and mitochondrial relocalization. Here, after being deubiquitylated by HAUSP, phosphorylated p53 binds resident MDM4, which, in turn, facilitates p53 with anti-apoptotic Bcl-2 and cytochrome c release.


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could also be fed by a positive feedback mechanism in which autophosphorylated HIPK2 interacts with Pinl after DNA-damage. Pinl mediates a conformational change of HIPK2 that releases it from the inhibitory E3 ubiquitin ligase Siah-1 thus triggering its stabilization, pro-apoptotic activation and consequent nuclear and cytoplasmic p53 activation [31] (Fig. 1). Pinl has been found aberrantly expressed in a variety of human tumors still expressing wt p53 [32,33]. It has been suggested that the higher levels of Pinl in these cells, by enhancing cytoplasmic apoptotic activity of p53, could sensitize cancer cells to drugs activating wt p53 minimizing undesired side effects in normal cells [9]. The HIPK2-p53Ser46-MDM4 recently emerged as an important oncosuppressive axis in glioblastoma. The ubiquitin specific protease USP2a and MDM4, in fact, are highly expressed in good prognosis glioma tissues. Here USP2a stabilizes mitochondrial MDM4-p53Ser46 interaction allowing efficient p53-mediated mitochondrial apoptosis [34]. p53 possesses also intrinsic genetic properties that define its ability to localize at mitochondria and to induce apoptosis. Dumont et al. unveiled a functional difference between p53 codon 72 polymorphic variant Pro72 and Arg72, being the last more prone to induce apoptosis [8]. This difference reflects the impaired ability of p53 Pro72 variant to localize at mitochondria due to its reduced binding to CRM1, essential for the nucleo-cytoplasmic shuttling [14]. In line, we previously demonstrated that Arg72 is more phosphorylated on Ser46 and binds Pinl more efficiently as compared to the Pro72 counterpart [25]. At mitochondria, Arg72 variant interacts with resident proteins GRP75 and Hsp60. Since these proteins are in intimate contact with pro-apoptotic factors it is possible that p53 engages these interactions in order to alter the function of these proteins and to consequently trigger apoptosis [35].

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One of the main outcomes of p53 activation by genotoxic stress is the induction of apoptosis through the intrinsic mitochondriamediated pathway. This route implies the permeabilization of the mitochondrial outer membrane (MOMP), which is regulated by the proteins belonging to the Bcl-2 family and particularly induced by the pro-apoptotic factors Bax or Bak [36], The Bcl-2 family proteins all share the characteristic Bcl-2 homology domain (BH). The family is divided in anti-apoptotic members, such as Bcl-2, Bcl-xL and Mcl-1, which contain BH1, BH2, BH3 and BH4 domains, and the pro-apoptotic members, which either contain BH1, BH2 and BH3 domains such as the MOMP effectors Bax and Bak, or only the BH3 domain class such as the activators tBid and Bim [37]. To efficiently induce apoptosis, the effector proteins are activated by a conformational change and an homo-oligomerization, which in turn leads to their translocation and insertion into the outer mitochondrial membrane to generate pores that release pro-apoptotic factors from the mitochondrial intramembranous space (e.g. cytochrome c). The BH3-only class of proteins covers the role of favoring such activation, either by stimulating the oligomerization of Bak and Bax (tBid and Bim proteins) or inhibiting the binding between anti-apoptotic proteins and the effectors of apoptosis (Puma, Noxa and Bad proteins) [38]. As said, MOMP is governed by p53. In the nucleus p53 induces the transcription of pro-apoptotic Bcl-2 members including Bax, Puma, Noxa and Bid, and represses the transcription of antiapoptotic genes including Bcl-2 and Bcl-xL [39–41]. In addition to these activities, about twenty years ago, a p53 transcriptionindependent route for MOMP has been discovered and starting from this in the last years a lot of effort has been made in elucidating p53 pro-apoptotic activities in the cytoplasm [42–44].

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The hallmark of transcription-independent apoptosis induction by p53 is the stress-dependent accumulation of the protein in the cytoplasm and mitochondria that directly activates the apoptosis effectors Bax and Bak. Starting from this first observation that stress-induced p53 translocation to the mitochondria precedes cytochrome c release and caspase activation [12], a great number of studies performed in the recent years provided definitive proof that this is a general mechanism of cell death responding to a wide spectrum of p53 activating stresses (DNA damaging agents, activated oncogenes, hypoxia and others) and in different cellular models, for example primary, immortal and malignant cells of human and mouse origin [45–47]. Also in vivo observations on p53 engineered proteins, either unable to translocate into the nucleus [2] or with enhanced capability to translocate to the mitochondria, strengthened this idea [12]. All these observations, together with the evidence that recombinant p53 can rapidly induce MOMP and cytochrome c release from isolated mitochondria in vitro, fully demonstrate that the p53 effect on MOMP is direct [2]. Experiments with recombinant p53 allowed to unveil the complex network of interactions that p53 entails at mitochondria. Purified p53 is able to disrupt the complex formed by tBid, Bax and Bcl-xL in vitro thus displacing the pro-apoptotic protein from their negative regulator [3,45–48]. This effect is indeed mediated by direct interaction with Bcl-xL, as shown by several structural studies that confirmed also the interaction with the other antiapoptotic protein Bcl-2 [3,48–50]. p53 also interacts with Bak [3,4], although the binding appears to be 10-fold weaker. It is not definitively proven whether these interactions happen in vivo, but it is clear that stress-induced p53 is able to disrupt the complex between Bak and its anti-apoptotic partner Mcl-1, by directly binding to Bak at the outer mitochondrial membrane, leading to oligomerization and activation of Bak and cytochrome c release [4,48,50,51]. In addition to Bak, p53 seems to work like a direct activator of Bax in absence of other proteins, by triggering its translocation to mitochondria and oligomerization. These events lead consequently to permeabilization of artificial membranes and isolated mitochondria in vitro [2,11,52]. Nevertheless a stable p53-Bax complex has never been detected [2]; for this reason, a ‘hit-and-run’ mechanism was proposed to explain the direct activation of Bax by p53, involving a direct activity of p53 to promote conformational change of Bax but without a stable interaction between the two proteins [2]. For p53-Bax interaction the proline-rich domain at N-terminal of p53 is required [2,52]. All the mentioned interactions can be declined in different ways in order to explain the sequence of events that lead to apoptosis. In particular, two different models have been proposed, the mitochondrial pathway and the cytosolic mechanism (Fig. 2). Starting from the strong evidence of p53 localization at mitochondria, the first model suggests that binding of p53 to Bcl-xL/ Bcl-2 neutralizes their anti-apoptotic effect leading to Bax or Bak-dependent apoptosis [3]. Among the experimental evidences in this direction there is the fact that all the p53 mutants unable to bind to Bcl-xL/Bcl-2 also lack apoptotic activity [3,48]. Indeed it was demonstrated that genotoxic stress is able to induce wild type p53 binding to Bcl-2 at mitochondria and this binding results in a reduced association of the anti-apoptotic protein with Bax, that can therefore oligomerize and induce MOMP [53]. Conversely the other model, integrating the previous one, sustains that the cytoplasmic pool of p53 is responsible for apoptosis induction and also takes into account the control of transcription exerted by nuclear p53. In unstressed cells, p53 is sequestered in the cytoplasm by the interaction with Bcl-xL, thus keeping under control the induction of apoptosis. Upon genotoxic stress, cytoplasmic Bcl-xL is bound by the BH3-only protein Puma rapidly tran-


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Fig. 2. Schematic representation of the different routes of p53 induced transcription-independent cell-death. The apoptosisprogram (On the left). The figure resumes the pathways of cytoplasmic and mitochondrial apoptosis induced by p53. Cytoplasmic apoptosis (upper panel): under genotoxic stress activated p53 rapidly regulates the expression of several apoptosis-related genes; among them, increased levels of Puma are able to release p53 from a pre-existing complex with Bcl-xL. Free-activated p53 in the cytoplasm with a hit-and-run model binds Bax and promotes its mitochondrial translocation and homo-oligomerization, thus inducing mitochondrial membrane permeabilization and cytochrome c release. Mitochondrial apoptosis (lower panel): under cell death stimuli p53 is activated, translocates to the mitochondria where, through binding the anti-apoptotic factors Bcl-xL/Bcl-2 and Mcl-1, allows Bak release, prompting its homo-oligomerization that forms pores on mitochondrial outer membrane and induces cytochrome c release. The p53 necrosis program (On the right). ROS induce p53 activation and its mitochondrial translocation. In the mitochondrial matrix p53 interacts with cyclophilin D inducing VDAC oligomerization and PTP pore formation. These events cause H2 O intake, swelling of mitochondria and necrotic cell death. 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318

scribed by nuclear p53 and this relases cytoplasmic p53 that can activate monomeric Bax in the cytoplasm [51]. Notably however, it has been demonstrated that transcription-independent p53 apoptosis happens also in the absence of Puma [51,54,55]. The intrinsic pathway of apoptosis fostered by p53 causes the activation of the executioner caspases [56]. These effector caspases, including caspases-3, -6 and -7, cleave key substrates within the cell in order to carry out the apoptotic process [57]. In a recent elegant work, Murphy and colleagues performed a mass spectrometry analysis for p53 interactors at mitochondria and identified pro-caspase-3 as a novel binding partner of p53; interestingly it was demonstrated that this interaction was able to enhance caspase-3 activation. Also mutant p53 retains the capability to bind caspase-3, but in this case the effect is the inhibition of procaspase-3 activation, which is protected from cleavage mediated by upstream caspases with the final result of impairing the execution of apoptotic cell death [58].

p53 activities at mitochondria are not only restricted to the regulation of apoptosis. Recently a new role of p53 has been described in inducing programmed necrotic cell death [59]. In response to many insults and in direct relation to the strength of the stimulus, it has been demonstrated that p53 can induce necrosis instead of apoptosis. Necrosis is an irreversible route leading to cell death due to bioenergetic failure, mainly driven by excess cytosolic Ca2þ and ROS levels generated during oxidative damage and released by mitochondria during the mitochondrial permeability transition (mPT). Interestingly, upon oxidative stress p53 accumulates in the mitochondrial matrix and binds directly to cyclophilin D, the key regulator of the mitochondrial permeability transition pore (PTP) whose opening is able to induce necrosis via mPT. In accordance with this it has been demonstrated by several reports that p53 can change the oligomeric state of the inner membrane protein VDAC, leading to cytochrome c release even in the absence of Bax and Bak [59] (Fig. 2). The mechanism has been reported to


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be causative in brain ischemia/reperfusion injury but the results were reproducible also in HCT116 cancer cells, thus indicating that necrosis could also happen in cancer development. Although the ability of p53 to localize at the mitochondrial outer membrane thanks to interaction with the resident BH3-containing proteins is clear, the mechanism of p53 entry in the mitochondrial matrix is still unknown and not dependent from a mitochondrial entry sequence of which the protein is devoided [10].

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4. Regulation of Autophagy by p53

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Macroautophagy (generally referred to as autophagy) is a conserved catabolic pathway evolved to deliver superfluous proteins and damaged organelles to lysosomes for degradation and represents an essential process for survival under unfavorable metabolic conditions (e.g. nutritional stress) [60]. Reported to be involved in numerous physiological processes and in multiple pathological conditions from neurodegeneration to cancer, autophagy is able to drive the choice between cell survival and cell death, thus playing an important role in cancer development. In this context, autophagy may have a pro-survival effect in conditions of mild nutritional stress, while it can lead to cell death when taken to extremes. Several genetic/epigenetic alterations associated with cancer development negatively regulate autophagy, suggesting that basal autophagy may function as a tumor suppressive mechanism. Conversely, autophagy may represent an important way to escape cell death for tumors exposed to a challenging microenvironment or subjected to chemotherapy [61,62]. In the recent years it has been demonstrated that p53 can control the process of autophagy both positively and negatively. When exposed to stress, nuclear p53 can induce autophagy by inhibiting

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the master negative regulator of autophagy, the mTOR protein kinase, acting at multiple levels of the AMPK-mTOR axis. First of all, p53 can upregulate the transcription of mTOR negative modulator TSC2 (tuberous sclerosis 2). Nuclear p53 also induces the transcription of the beta 1 and beta 2 subunits of AMP-activated protein kinase (AMPK), an evolutionarily conserved sensor of cellular energy levels that phosphorylates and thus activates TSC1 and TSC2. Moreover p53 enhances sestrins 1 and 2 transcription, two main activators of AMPK [63–65] (Fig. 3). Another important transcriptional target of p53 involved in autophagy modulation is DRAM (damage-regulated autophagy modulator), a lysosomal protein that induces macroautophagy and is also able to regulate cell death [66,67]. Interestingly, the role of DRAM unveils the tight connection existing between autophagy and apoptosis, which can partially explain the complexity of their mutual regulation and the outcome of nuclear p53 activation. Indeed, regarding this crosstalk, another pro-autophagic role of p53 is played through the members of the Bcl-2 protein family, whose transcription is repressed by p53 after stress and whose reduction induces the release the autophagic protein beclin-1 from sequestration by Bcl-2, Bcl-xL and Mcl-1. The same mechanism can work via transcriptional induction of the proteins Bax, Bad and Puma [68,69]. In contrast to these autophagy-promoting functions of p53 in the nucleus, the cytoplasmic pool of p53 suppresses autophagy in several model organisms as demonstrated by the fact that pharmacological inhibition of p53 can trigger autophagy in cytoplasts [70] (Fig. 3). Interestingly, in human, mouse and nematode cells depletion or inactivation of p53 by different means induced autophagy relying on mTOR inhibition. Another demonstration of this p53 cytoplasmic activity is the fact that canonical inducers

Fig. 3. Cytoplasmic p53 regulates key metabolic pathways and the oxidative stress response. (A) By interacting with clathrin heavy chain wild type p53 fosters EGFR internalization in stimulated cells, thus promoting its lysosomal degradation. On the other hand mutant p53 promotes the recycling of internalized EGFR by sustaining RCPdependent vesicular trafficking, thus prolonging EGF pathway activation. (B) Cytoplasmic p53 interacts with rate-limiting enzymes of glycolysis and pentose phosphate pathway (PPP) and inhibits their activity. Via inhibition of the PPP, cytoplasmic p53 fosters ROS production, an effect augmented by the inhibition of MnSOD activity at mitochondria. (B) Cytoplasmic p53 is able to inhibit autophagy, in contrast with what is reported for the transcriptional activity; mechanistic insights for this inhibition are still lacking.


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with SREBP family of transcription factors, leading to their aberrant activation [80]. This event has the consequence of fostering the flux of mevalonate pathway with increase in lipid biosynthesis and protein geranyl-geranylation that, in turn, impinges on several oncogenes such as YAP and TAZ [81]. Since RAB proteins require prenylation for proper membrane localization, the positive effect of mutant p53 on the mevalonate pathway could contribute to the observed effect on receptor recycling. Moreover, beyond receptor internalization, mutant p53 has also been reported to control the glucose influx stimulating glucose transporters (GLUT1) translocation to the plasma membrane by promoting the RhoA/ROCK/Factin axis [82].

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of autophagy, as for example starvation, enhance p53 degradation and this degradation can be counteracted by the inhibition of either MDM2 or of the proteasome. In line with this, restoration of cytoplasmic p53 in p53-deficient HCT116 cells can inhibit autophagy induction. Notably, this inhibition is cell cycle dependent and causes autophagy mostly in the Go/Gi phase [70]. The meaning of the divergence of nuclear and cytoplasmic p53 effects on autophagy is still controversial. By inducing the activation of the protein DRAM, p53 could favor cell death (DRAM is required for p53-dependent cell-death) or counteract it (autophagy induction can substitute apoptosis in the response to stress). In this context it has been demonstrated that in many cell types wild type p53 confers increased survival upon chronic starvation by posttranscriptionally downregulating microtubule-associated protein 1 A/lB-light chain 3 (LC3), a key component of the autophagic machinery [71].

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The process of endocytic membrane trafficking which involves cellular internalization and sorting of molecules, plasma membrane proteins and lipids, is essential for a variety of biological functions (e.g. nutrient uptake, cell adhesion and migration, receptor signaling and recycling, pathogen entry, cell polarity and fate) and is subverted in cancer cells [72]. p53 may affect the endocytic compartment in different ways. Indeed, p53 physically interacts with proteins like NUMB, an endocytic factor asymmetrically partitioned at stem cell mitosis and determining daughter cells fate [73]. In somatic stem cells (SC), p53 was shown to counteract stem cell expansion by several mechanisms, including block of reprogramming of somatic/progenitor cells into SC [74] and by imposing an asymmetric mode of selfrenewing divisions in mammary and neural SC [75,76], Even if p53 localization was not investigated in these studies, a possible involvement of p53 in the initial process of asymmetric cell division, likely by cytoplasmic positioning, can be envisaged. Undoubtedly, the roles of nuclear and cytoplasmic p53 in regulating normal and cancer stem cell niche homeostasis are worth to be investigated in depth in the future. A key step in the process of endocytosis is the establishment of clathrin-coated vesicles to select receptors for internalization. Notably, in unstressed conditions, cytoplasmic p53 has been shown to interact with clathrin heavy chain (CHC) to regulate the endocytosis of epidermal growth factor receptor (EGFR). In response to EGF stimulation, wt p53, but not mutant p53, has been found to localize at plasma membrane interacting with CHC. This event promotes EGFR internalization and alters its related signal transduction [77]. Intriguingly the association of clathrin with wt p53 has also been found in the nucleus, where it increases the transactivation functions of p53 [78]. This role of p53 on endocytosis may represent another important layer of its oncosuppressive activities in the cytoplasm and it is not excluded that this regulation may happen also for other receptors. Further investigations on these aspects is however required. Interestingly pro-oncogenic mutant forms of p53 have been found to possess endocytosisrelated gain-of-function activity contributing to aberrant migration. Indeed, Muller et al. demonstrated that mutant p53 governs the recycling of EGFR and integrins by enhancing the binding of the RAB-coupling protein (RCP) to recycling endosomes thus promoting the plasma-membrane localization of EGFR and integrins [79]. Different studies are beginning to unveil a role of p53 in regulating vesicle trafficking that is more complex than this, at least from a nuclear perspective. Vesicle trafficking is profoundly affected by lipid metabolism. In an elegant work, Prives and colleagues found that some mutant p53 gain the ability to interact

In addition to being fundamental for normal cell survival, the regulation of cell metabolism has recently emerged to play an important role in cancer. Tumor cells indeed display particular metabolic needs connected to their high proliferative rate, and consume large quantities of glucose. In fact it has been demonstrated that cancer cells primarily use glycolysis for ATP production, even in the presence of adequate oxygen and this metabolic signature, named ‘‘Warburg effect’’, enables cancer cells to direct glucose to macromolecules biosynthesis, supporting their rapid growth [83]. In the recent years, the concept that p53 can directly regulate glucose metabolism and oxidative phosphorylation has emerged [84–86]. The activity of p53 in metabolism involves several aspects spanning from oxidative stress regulation to the orchestration of glucose metabolism [85]. These p53 actions result from a coordination of transcriptional and cytoplasmic activities, whose net balance is not always easy to understand. Regarding the mitochondrial energetic regulation, p53 is able to promote both tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS). For the TCA cycle, p53 reduces the expression of the pyruvate dehydrogenase kinase isoenzyme-2 (Pdk2), which favors the flux of pyruvate to acetyl-CoA [87]. Moreover, p53 also regulates glutaminolysis by activating the expression of mitochondrial glutaminase (GLS2), which promotes glutamine to glutamate conversion leading to the formation of a-ketoglutarate, a TCA cycle intermediate [88,89]. As for mitochondrial respiratory chain, p53 is able to foster the pathway by transcriptionally activating synthesis of cytochrome c oxidase 2 (SCO2) expression, a regulator of complex IV [90], cytochrome c oxidase (COX) I subunit, and AIF, essential for mitochondrial respiratory complex I functional [91,92]. The reactive oxygen species produced by the enhancement of OXPHOS could be detrimental for cell survival, thus p53 upregulates antioxidant activities inducing the transcription of antioxidant targets such as GPX1, MnSOD, ALDH4, TPP53INP1, GLS2, the sestrins [65,88,89,93,94] and the TP53-induced glycolysis and apoptosis regulator, TIGAR. By limiting the activity of Phosphofructokinase 1 (PFK1), TIGAR reduces the rate of glycolysis while promoting pentose phosphate pathway (PPP) [95] (Fig. 3). The PPP is a central metabolic pathway that produces NADPH required for regeneration of reduced glutathione (GSH). Interestingly, it has been recently demonstrated that a pool of cytoplasmic p53 can inhibit glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of PPP, through transient interactions [96] (Fig. 3). In line with this, it was demonstrated that p53 is able to bind the antioxidant enzyme MnSOD in the mitochondrial matrix and inhibit its activity [97], thus fostering the propagation of oxidative stress [97]. The net effect of nuclear and cytoplasmic activity is not clear and may be cell context and stress-dependent. In HCT116 colon

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cancer cells p53 depletion is able to foster G6PDH flux and therefore raise NADPH levels. It is conceivable that cancer cells inactivation of p53 by different means accelerates glucose consumption and directs glucose for rapid production of macromolecules by an increase in PPP flux thus enhancing biosynthesis. In turn p53 loss could also contribute to the Warburg effect by fostering glycolysis; indeed p53 can negatively regulate, through the control of translation or protein stability, the phosphoglycerate mutase (PGM) thus reducing the glycolytic rate [98].

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The pathophysiological relevance of the p53 transcriptionindependent activities in vivo has been unveiled by some pivotal studies in animals. The classic model of p53-induced cell death is the apoptosis following c-irradiation. Irradiation treatments on mouse fibroblast revealed that transcription-independent apoptosis is the final determinant of cell death [11]. The group of Ute Moll demonstrated that in normal mouse exposed to whole body c-irradiation or a single injection of a dose of etoposide there is mitochondrial p53 accumulation in radiosensitive organs like thymus, spleen and testis but not in radioresistant organs like liver and kidney. p53 that accumulates in mitochondria in radiosensitive organs is able to trigger a p53-dependent first wave of caspase-3 activation within the first hours, followed by an early wave of apoptosis [99]. Importantly in this system the induction of p53 target genes occurs later and gives rise to a second wave of caspase-3 activation. Conversely, a lack of mitochondrial p53 accumulation in radioresistant organs correlates with cell cycle arrest and absence of apoptosis [54]. The functional importance of p53 extra-nuclear activities has been clarified by the use of the two molecules pifithrin-a and -l. Pifithrin-l (PFTl) selectively inhibits p53 mitochondrial translocation and reduces its affinity to Bcl-xL and Bcl-2, without interfering with its transcription function [100]. Pifithrin-a inhibits the transcriptonal activity of p53 [101,102]. PFT-l treatment of mice, before c-irradiation, was able to protect them from bone marrow failure reducing thymocyte death [100], although transcriptional activity was not affected. A key hallmark of cancer is the escape from oncosuppressive pathways such as apoptosis, autophagy and cell cycle arrest, mainly regulated by wild type p53. In order to escape this regulation cancer cells frequently bear p53 mutations (in nearly 50% of the cases), and these mutations occur mainly in the DNA binding domain (‘‘hotspot mutations’’) and abolish its transactivation functions. Whether the presence of mutations in p53 affects its translocation to the cytoplasm and its cytoplasmic and mitochondrial functions has not been thoroughly investigated. From several studies, however, the cytoplasmic and mitochondrial localization of mutant p53 [3,46,48,103] has emerged, even if the pattern of interactions entailed by mutant p53 at the mitochondria has still not been understood. Some transcriptionally impaired p53 mutants (e.g. R175H, R273H, C277F) have been shown to bind Bak in vitro and induce cytochrome c release from isolated mitochondria [103]. This is in contrast with the proto-oncogenic role of mutant p53, and indeed it was demonstrated that mutant p53 overexpressed in human cancer cell lines, even if able to bind Bak, did not display apoptotic activity [3]. In line with this, it was also shown that p53 mutants are unable to interact with Bcl-2 family proteins and thus to induce M0MP [2,3,46,48]. In general it is assumed that such ‘‘hot spot’’ p53 mutations also affect the mitochondrial functions of the protein, reducing its capacity to induce MOMP; intriguingly, however, it has been demonstrated that upon transfection in HCT116/ cells p53 mutants that are able to localize to the cytoplasm have an intact inhibitory effect on autophagy [104].

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Reactivation of wild type p53 in tumors in order to trigger apoptosis is a promising approach for cancer therapy [105–107]. So far, the majority of screens searching for p53-reactivating compounds have focused on molecules that would activate the wild type p53-dependent transcriptional program, although the p53 transcriptional response does not necessarily lead to apoptosis and can sometime favor the persistence of tumor cells in the body [108]. The search for molecules that specifically activate cytoplasmic p53 function can be therefore an attractive goal for drug development. Several drugs that can restore transcription-dependent apoptosis have been developed recently, starting from small molecules such as PRTMA-1 that can rescue oncogenic mutants to wild type [109,110]. Interestingly pharmacological modulation of p53 by PRIMA-1 can activate a transcription-independent apoptotic program [52], by inducing mitochondrial localization of Bax and consequent cytochrome c release. Conversely, in p53 wild type tumors, compounds such as Nutlin-3a and RITA, lead to p53 detachment from its negative regulator MDM2 thus unleashing its pro-apoptotic activity [105,106]. Although the apoptotic effects of Nutlin-3a and RITA has been initially linked exclusively to the transcriptional activities of p53 in the nucleus, it has recently emerged how the transcription-independent activity of p53 could be a major contributor to apoptosis induced by these drugs, at least in some cell lines [9,111]. Indeed, Vaseva and others found that p53 rapidly translocates to the mitochondria after treatment of cells with Nutlin-3a, and activates a pro-apoptotic signal that is strongly independent from its transcriptional activities [111–114]. Yet, we found that RITA, due to its ability in promoting HIPK2 activation and consequent p53 Ser46 phosphorylation, efficiently triggers apoptosis even without transcriptional cell activity [9,115]. A high number of new molecules able to unleash or restore p53 transcriptional activity have been discovered over the last years [116]; it is important to consider that also for these molecules, as happened for Nutlin-3a and RITA, p53-dependent transcription-independent oncosuppressive activities could be unveiled, thus providing new tools for cancer therapy. Another possible therapeutic approach, which may exploit the gene therapy technology, could be the specific delivery to the cells of p53 engineered to localize at mitochondria. This is the case of Lp53wt, generated by fusion of the import leader sequence of ornithine transcarbamylase to the N-terminus of p53 [117–119]. Lp53wt is able to interact with Bcl-xL and is imported in the mitochondrial matrix where the leader is cleaved off and the protein is redistributed in the mitochondria. Interestingly although unable to induce transcription, Lp53wt is capable of promoting transcriptionindependent apoptosis in p53-null human cancer cell lines [12] .

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Notwithstanding the growing number of papers reporting evidences of the oncosuppressive role of p53 in the cytoplasm, several aspects have not been clarified and some data remain elusive. One of the main points that need to be elucidated regards the co-regulation of the nuclear and the cytoplasmic routes of p53 activation. Indeed these two arms are tightly coordinated as demonstrated by the fact that both pathways can be triggered by the same stimuli in vitro and in vivo leading to their simultaneous activation. A major drawback of the approaches inspecting p53 roles in the cytoplasm is that the mechanisms have been primarily elucidated by in vitro studies, in artificial settings that have often led to controversial results. Dissecting these mechanistic aspects in vivo could help to clarify the real sequence of biochemical events

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by which p53 exerts its transcriptional independent pro-apoptotic activity. An intriguing point of investigation is whether in vivo differences in the type and strength of the biological and pharmacological stimuli could imbalance the p53 activity towards one of these specific routes. This could be of particular relevance in terms of exploiting the p53 mitochondrial route for therapeutic intervention. Indeed transcriptional activation of p53 in response to oncogenic stress and chemotherapy could lead to cell cycle arrest and senescence instead of leading to more desired outcomes such as apoptosis and necrosis. Several reports, in fact, have underlined the possible negative impact of p53-induced senescence on chemotherapy response [120], while it has been found that inactivation of p53 transcriptional activity by mutations [121] or mitochondrial sequestration [12] favors apoptotic response and tumor cell clearance. Moreover, the field needs to move forwards clarifying the real capability of missense p53 mutants to regulate mitochondrial apoptosis. The same should be done for the several isoforms of p53 found in mammalian cells, many of which are transcriptionally inactivated or cytoplasm-localized [122]. Despite, from a mechanistic point of view, a lot of effort has been done in defining the critical steps of p53 extra-nuclear activities, the localization and timing of the post-translational modifications (ubiquitylation, phosphorylation, acetylation) affecting p53 translocation and their real role in vivo still remain an open issue. Apart from apoptosis, there is a relevant lack of knowledge on how p53 orchestrates autophagy and metabolism in the cytoplasm and in particular on how extra nuclear activities are coordinated with the transcriptional response. Indeed the real final outcome derived from this interplay and how this could impact on tumorigenesis and cancer therapy is still controversial. To date, all the evidences point towards a role of p53 in tumor suppression, that goes well beyond its role as a nuclear resident transcription factor. For this reason a fine dissection of p53 interactions, modifications and subcellular localization in the cytoplasm may still reserve new unexpected aspects of this extensively studied protein and provide a rationale for therapeutic intervention.

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Acknowledgements

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We thank A. Testa for discussion and proofreading the manuscript. We also thank A. Rustighi and F. Demarchi for reading and providing inputs. We apologize to colleagues whose work has not been described due to space limitations. Work in GDS lab is supported by Grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ to G.D.S. and the Italian Ministry of Education, University and Research (COFIN, FIRBaccordi di programma 2010 cod.RBAP10XKNC_003) to G.D.S. A.C. and G.S. are fellows of the Fondazione Italiana per la Ricerca sul Cancro (FIRC).

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Oncosuppressive functions of decorin.

Binding of markers of either side of plasma membranes to the cytoplasmic side of lysosomes.

Mercury inhibits Na-K-ATPase primarily at the cytoplasmic side.

Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles.

Acetylcholinesterase is orientated facing the cytoplasmic side in membranes derived from sarcoplasmic reticulum.

Unexpected oncosuppressive role for STAT3 in KRAS-induced lung tumorigenesis.

Novel localization of formin mDia2: importin β-mediated delivery to and retention at the cytoplasmic side of the nuclear envelope.

Mutations in glioblastoma oncosuppressive pathways pave the way for oncomodulatory activity of cytomegalovirus.

Activities of the Cytoplasmic Domains of Patched-1 Modulate but Are Not Essential for the Regulation of Canonical Hedgehog Signaling.

Synthesis and biological activities of vitamin D3 derivatives with cyanoalkyl side chain at C-2 position.

Side by side: The work of Elizabeth and James Miller.

Side-to-side choledochoduodenostomy in the management of choledocholithiasis.

Oncogenic and oncosuppressive signal transduction at mitochondria-associated endoplasmic reticulum membranes.

The far side of auxin signaling: fundamental cellular activities and their contribution to a defined growth response in plants.

The side effects of imatinib.

Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope.

A Babesia bovis 225-kilodalton protein located on the cytoplasmic side of the erythrocyte membrane has sequence similarity with a region of glycogen phosphorylase.

The traumatic side of fibrinolysis.

The business side of telemedicine.

The Cognitive Side of M1.

The hypoglycemic side of hypothyroidism.

The dark side of medicine.

The human side of prayer.

The wrong side of history.