(2013) 32, 5129–5143 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www..com/onc

REVIEW and aging: the critical roles of

A Rufini1, P Tucci1,2, I Celardo1 and G Melino1,3

p53 functions as a involved in -cycle control, DNA repair, and cellular stress responses. However, besides inducing cell growth arrest and apoptosis, p53 activation also modulates cellular senescence and organismal aging. Senescence is an irreversible cell-cycle arrest that has a crucial role both in aging and as a robust physiological antitumor response, which counteracts oncogenic insults. Therefore, via the regulation of senescence, p53 contributes to tumor growth suppression, in a manner strictly dependent by its expression and cellular context. In this review, we focus on the recent advances on the contribution of p53 to cellular senescence and its implication for therapy, and we will discuss p53’s impact on animal lifespan. Moreover, we describe p53-mediated regulation of several physiological pathways that could mediate its role in both senescence and aging.

Oncogene (2013) 32, 5129–5143; doi:10.1038/onc.2012.640; published online 18 February 2013 Keywords: senescence; aging; p53; mTOR; mitochondria; ROS

INTRODUCTION that both initiation and maintenance of senescence are p53 12–15 Senescence represents a stress response in which cells withdraw dependent, as discussed later in more detail. Regardless, from the and lose the capability to proliferate in senescence is functional to both tumor suppression and response to growth factors or mitogens.1,2 Senescent cells show organismal aging, which means that p53 ability to regulate both very distinctive changes in morphology, acquiring a typical flat processes may heavily rely on its fundamental role in eliciting and enlarged shape and increase expression of recognized cellular senescence. of senescence, including staining for b-galactosidase at pH of 6.0 (senescence-associated-b-gal or SA-b-gal), decreased replicative capacity, increased expression of p53, , and MOLECULAR MECHANISMS IN SENESCENCE AND AGING other cyclin-dependent kinase inhibitors, such as p27 and p15. The senescence pathway can be triggered by multiple mechan- Finally, accumulation of transcriptionally inactive heterochromatic isms. Originally, it was associated with replication exhaustion at structure (senescence-associated heterochromatic foci or SAHF) the end of the cellular lifespan, a process currently defined as has been reported, particularly in the promoters of E2F-target replicative senescence. Replicative senescence results from a genes.2–4 combination of events that include the progressive erosion of Initially thought to be a cell culture artifact, senescence has during . This phenomenon can lead to been more recently observed in vivo in cancer lesions and during critically short telomeres that are sensed by the cells as double- physiological aging.3,5–10 Hence, recently, increasing interest has strand breaks. Double-strand breaks trigger the DNA damage focused on senescence as a novel approach in cancer therapy, response (DDR), a signaling cascade centered around the ataxia because of its inherent property to suppress cell proliferation, teleangectasia-mutated (ATM) kinase that activates p53 to elicit senescence may protect against cancer onset.2 Intriguingly, cell-cycle arrest and to execute senescence.16–20 Ectopic senescence is also intimately related to aging as both shared expression of telomerase, the enzyme responsible for ability to limit lifespan. The constant regeneration of somatic stabilization, circumvents replicative senescence in human cells,21 tissues leads to accumulation of senescent cells, which limits and stabilization of telomeres is essential for tumor progression. tissue renewal, perturbs normal tissue homeostasis and ultimately Telomeres are also important during aging. In humans, a positive elicits aging. Recent findings have established a causal role correlation between telomere length and longevity has been between senescence and aging: selective killing of p16-positive suggested.20,22,23 In addition, a mouse model with depletion of senescent cells in vivo ameliorates aging-related features in a telomerase shows several signs of accelerated aging, including mouse model of progeroid syndrome.8,11 anemia, kyphosis, osteoporosis, glucose intolerance, alopecia and Senescence has been classically viewed as a state of permanent hair graying.24–26 Here, a crucial event points to a progressive loss growth arrest, during which cells are unable to re-enter the of the stem cell reservoir in these animals through induction cell cycle. Although this concept is still widely accepted, recent of apoptosis and senescence. These correlate studies have provided evidence that under certain conditions this with genomic instability and activation of p53,27 and genetic cellular status is reversible. In fact, stable suppression or even ablation of p53 ameliorates symptoms in mice with critically short more subtle changes in p53 expression in senescent fibroblasts telomeres.28 Thus telomere erosion links p53 to both senescence lead to rapid cell-cycle re-entry and immortalization, indicating and aging.

1Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK; 2Department of Pharmaco-Biology, University of Calabria, Rende (CS), Italy and 3University of Rome ‘Tor Vergata’, Department of Experimental Medicine and Biochemical Sciences, and Biochemistry Laboratory, Istituto Dermopatico dell’Immacolata, Rome, Italy. Correspondence: Dr G Melino, Medical Research Council, Toxicology Unit, Leicester University, Lancaster Road, Leicester LE1 9HN, UK. E-mail: [email protected] Received 9 October 2012; revised 30 November 2012; accepted 7 December 2012; published online 18 February 2013 Mechanisms of p53-mediated regulation A Rufini et al 5130 Relevant for tumorigenesis, persistent oncogenic signaling is anions, superoxide and hydroxyl radicals, and peroxides) formed another important trigger that activates a powerful senescence by partial reduction of oxygen, which, if not detoxified promptly response,29 known as oncogene-induced senescence (OIS). This by antioxidant agents, can oxidize macromolecules and damage process, indeed, prevents cellular transformation. Oncogenic organelles (Figure 1). Oxidation of DNA causes base modifications H-Ras and, more generally, activation of trigger (that is, ) leading to various pathologies in humans, such hyperproliferation. Enforced DNA replication results in DDR as cancer, whereas oxidized proteins tend to form aggregates followed by activation of senescence pathways, which must be resulting in diverse neurodegenerative pathologies. ROS are also overcome for transformation to occur. This process fails in cells involved in aging as oxidative damage to various constituents of that lack ATM activity or when cells cannot sense DNA damage or the cell may limit lifespan.35 In this regard, as mitochondria are the transduce DDR signals to p53.29 major source of ROS (Figure 1), a ‘mitochondrial free radical theory A major determinant of senescence, at the molecular level, is of aging’ has been postulated, arguing that mitochondrial- the intracellular accumulation of oxidative damage triggered by generated oxygen radicals cause widespread oxidative damage, (ROS).2,22,30–34 ROS are generally small, eventually resulting in aging.36,37 ROS enhance senescence and short-lived and highly reactive molecules (for example, oxygen aging, inducing toxicity, into a feed-forward cycle: ROS cause

À Figure 1. Mitochondrial ROS. (a) A schematic model of ROS generation in the mitochondria. Superoxide (O2 ) generated by the respiratory À chain is mostly released to the matrix at complex I and the IMS at complex III. O2 can naturally dismute to hydrogen peroxide (H2O2)oris enzymatically dismuted by matrix MnSOD or Cu/ZnSOD in the IMS or cytosol. H2O2 is detoxified in the matrix by catalase, the thioredoxin/ thioredoxin peroxidase system (TPx), or the glutathione/glutathione peroxidase system (GPx). Alternately, H2O2 can react with metal ions to À generate the highly reactive hydroxyl radical ( Á OH) via Fenton chemistry. O2 is not membrane permeable but can pass through ion channels (solid lines), whereas H2O2 can pass freely through membranes. (b) ROS can have both endogenous or exogenous sources. The overall balance in ROS cellular content is the result of ROS production and removal from specialized scavenging enzymes.

Oncogene (2013) 5129 – 5143 & 2013 Macmillan Publishers Limited Mechanisms of p53-mediated regulation A Rufini et al 5131 damage to mitochondrial constituents, and, subsequently, aging , possibility in agreement with studies damaged mitochondria produce more ROS.35,38–40 The highlighting an ‘aging’ process affecting stem cells.70 Although senescence–ROS correlation has attracted great interest, the mechanisms underlining these phenotypes are still unclear, prompted by studies in several organisms, in which a negative these results led to the notion that excessive p53 activity relationship between mitochondrial ROS production and lifespan compromises healthy aging. On the other hand, whether lack or has been found.41,42 Moreover, senescence and aging are reduced p53 activity affects lifespan has been difficult to assess, associated with an increase in the levels of oxidative-damaged owing to the severe tumor phenotype that accompanies loss of proteins, lipids and DNA,43–45 consequently to ROS-mediated p53.71 Nonetheless, recently developed in vivo models have shed damage to macromolecules such as proteins, nucleic acids and light on the issue. A fundamental residue in p53 is Serine 15 (Ser-15) lipids. Mitochondrial-generated ROS have also been involved in (ser-18 in mouse): phosphorylation of Ser-15 by ATM activates p53 OIS: Ras-driven senescence is associated with the accumulation of in response to DNA damage. In 2006, Armata and colleagues dysfunctional mitochondria, a sharp rise in ROS and a drop in ATP analyzed the phenotype of KI mice where Ser-18 of p53 was levels.46 Accordingly, chemical or genetic inhibition of the electron replaced with non-phosphorylable alanine. These mice developed transport chain suffices in inducing senescence in human signs of accelerated aging, indicating that physiological p53 fibroblasts.46 activity may preserve tissues from aging-related damage.72,73 The Mechanistically, senescence relies on two main molecular super-arf/p53 mouse model, developed by Serrano’s group, pathways: p53–p21 (discussed later) and p16INK4A-Rb. p16, a provided an additional striking support to the anti-aging activity cyclin/cdk inhibitor, prevents phosphorylation of Rb by cyclin/cdk of p53. These transgenic mice bear long genomic sequence of p53 complexes. Hypophosphorylated Rb halts cell proliferation by and p19arf, allowing their increased expression (owing to inhibitory binding to E2Fs transcription factors, thus preventing increased copy number, up to 4n), but maintaining endogenous them from stimulating transcription of genes involved in cellular regulation (as the regulatory region of the loci are preserved). In proliferation and DNA replication.17 In this context, p16-Rb these circumstances, the authors noted an increase in lifespan and axis is pivotal to the establishment of cell-cycle arrest. During an overall improvement of the aging-related health decline.74 OIS, suppression of Rb abolishes the establishment of a Although p19arf may act independently of p53, it is worth proper senescent phenotype, but it is not sufficient to overcome remembering that it does increase p53 activity preventing MDM2- cell-cycle arrest; this depends on the concomitant p53-dependent mediated p53 proteasomal degradation.75 Overall, these findings cell-cycle arrest.47 suggest that loss of p53 is detrimental to aging. In summary, the model that is emerging is an intensity- based model: physiological p53 activity prevents from cancer p53 IN SENESCENCE AND AGING and protects from aging, whereas unrestrained and excessive p53 is a tetrameric transcription factor heavily regulated by p53 activation still protects from cancer, but is detrimental to posttranscriptional modifications.48–52 It is regarded as one of the healthy aging. most powerful tumor suppressor genes owing to its ability to halt Induction of p53 is pivotal for the establishment of senescence, cell proliferation and induce apoptosis and its activity is pivotal to mainly following its activation by the DDR.76,77 Indeed, depletion successful traditional chemotherapy, as many DNA-damage- of p53 or abrogation of the upstream DDR signaling is sufficient to inducing drugs target tumors via p53-mediated apoptosis.49,53–55 impair OIS.29 Several p53-targets and regulators have been linked Consequently, p53 is mutated or lost in the vast majority of to induction of senescence, including microRNAs,22,78,79 but the human and considerable effort is focused on recovering molecular mechanisms are still elusive. One of the most well- its function in anticancer therapy.56–61 established p53-target genes, CDKN1A/p21, has been proved to p53 is clearly involved in cancer, but the existence of p53 in be upregulated during replicative senescence.80–83 p21 has been short-living organisms that do not develop cancers, such as among the first identified downstream targets of p53, and it is an flies and worms, suggests that tumor suppression is not its only essential mediator of p53-dependent cell-cycle arrest. p21- and, probably, original function. Indeed, recent studies have depleted mouse embryonic fibroblasts are unable to undergo shown that p53 influences development,62,63 reproduction,64 p53-dependent G1 arrest after DNA damage.84 The obvious metabolism65 and longevity. dependency of p53 on p21 for the induction of cell-cycle arrest The first evidence linking p53 to aging arose from the analysis and the established role of p21 as inhibitor of proliferation of a mutant mouse model: in the attempt to develop a knock-in suggest a crucial role for this gene in the induction of p53- (KI) of p53, Tyson and colleagues obtained an aberrant dependent senescence (Figure 2). Indeed, lack of p21 abrogates serendipitous truncation of the N-terminal portion of the gene. senescence in several settings.85–87 The truncated mutant proteins showed a robust constitutive p53 Nonetheless, although p21 contributes to the growth arrest of activity and the mutant mice presented an array of aging-related senescent cells, it is unlikely to be solely responsible for the features and severely reduced lifespan. In 2004, Scrable’s group complex and paramount changes underpinning senescence and, produced a transgenic mouse model overexpressing the trun- even more, aging. Moreover, p53 regulates a plethora of target cated DNp53 or p44 isoform of p53.66,67 This mouse showed a genes affecting several physiological and metabolic pathways, all striking defect in growth with associated reduced lifespan and heavily involved in regulation of aging and establishment of accelerated aging. Interestingly, p44 overexpression resulted in senescence.88 Here, we review several of these pathways and hyperactive p53 and increased IGF signaling, a master regulator of discuss their potential implication in p53-induced senescence and aging.68 Recently, a KI mouse model of p53 was developed in p53-regulated aging (Figure 2). order to mimic constitutive phosphorylation (that is, activation) of p53. This mouse model showed striking aging features, which seemed to result from widespread apoptosis affecting the stem p53 and E2F7 cell compartments of several organs, hence compromising tissue In agreement with the idea that p21 is not sufficient to explain the self-renewal.69 PUMA is a proapoptotic protein and a well- essential need for p53 in the establishment of senescence, two characterized p53 target that exerts a fundamental role in recent papers have described E2F7 as a new p53 target involved induction of apoptosis and survival of stem cells: notably in cell-cycle arrest and senescence.47,89 In particular, Aksoi and depletion of PUMA in the context of p53 mutations rescued the colleagues, in Scott Lowe’s laboratory, showed that E2F7 is stem cell loss and ameliorated the aging phenotype.69 Thus, upregulated in a p53-dependent fashion during proliferative as widespread apoptosis of stem cells may underline p53-mediated well as OIS. This gene is an atypical member of the E2F-family of

& 2013 Macmillan Publishers Limited Oncogene (2013) 5129 – 5143 Mechanisms of p53-mediated regulation A Rufini et al 5132

Figure 2. Senescence regulation by p53. Several posttranslational modifications regulate p53 activity. Stimuli that activate the DDR, lead to active p53 via ATM-mediated phosphorylation. Active p53 triggers expression of pro-senescence targets such as p21, responsible for G1 cell- cycle arrest and E2F7, pivotal in repression of mitotic genes. In addition, p53 controls other pathways linked to aging, including ROS generation and mTOR. In this regard, while steady-state levels of p53 are able to dampen ROS and limit oxidative damage, active p53 elevates intracellular ROS, which participate in its proapoptotic and pro-senescent activities. Overall, the physiological role of p53-mediated regulation of ROS in senescence and aging is still unclear.

transcription factors, as, unlike canonical E2Fs, it does not longevity. From yeast and Caenorhabditis elegans and up to mice heterodimerize with DP1 proteins,90,91 but binds DNA as a and primates, one of the most effective methods in prolonging monomer and promotes repression of several E2F target genes, lifespan is caloric restriction (CR),103 achieved decreasing caloric including . Moreover, many genes essential for , such intake, without malnutrition. Importantly, mTOR is necessary for as cyclin A, cyclin B and cdc2/cdk1, are repressed in senescent the CR beneficial effect and CR fails to extend lifespan in cells in a E2F7-dependent way. Hence, functionally, E2F7 arrests organisms where mTOR signaling has been reduced.104 cell-cycle progression at the mitotic phase. This may have some Moreover, in Drosophila melanogaster, inhibition of mTOR during important implications for tumorigenesis, explaining an apparent CR results in selective increased translation of components of the conundrum. In fact, both p53 and Rb are necessary for a full mitochondrial electron transport chain mediated by increased establishment of senescence. But, whereas p53-depleted cells are activation of eIF4E-binding protein 1. This selective upregulation immortalized and readily transformed by exogenous oncogenic leads to improved mitochondrial respiration, decreased ROS Ras alone, Rb-depleted cells, despite failing to undergo production and results in reduced ROS-dependent senescence proper senescent arrest, are not immortalized and expression of and prolonged lifespan.105 Strikingly, the drug rapamycin, a Ras is not sufficient for their transformation and does not endow chemical inhibitor of mTOR, has been recently shown to prolong them with tumorigenic capability.92 Aksoi and colleagues lifespan in mammals.101 demonstrate that p53-mediated upregulation of E2F7 is Active mTOR signaling promotes tumor growth and malignancy potentially responsible for this difference. In fact, concomitant and, to some extent, mTOR partners behave like oncogenes.106–110 inactivation of both Rb and E2F7 immortalizes cells and allows Thus, even though it is normally related to cellular growth, mTOR Ras-mediated cellular transformation.47 activity reinforces certain types of senescence,.111–113 In this regard, it is of great interest that overexpression of the GTPase protein mTOR-activator Ras homolog enriched in brain (Rheb) p53 and mTOR triggers senescence in vivo and in vitro in an mTOR-dependent The kinase mechanistic target of rapamycin (mTOR), previously fashion.111 Similar results were described upon in vivo known as mammalian TOR, is at the interface between growth and overexpression of the mTOR downstream target eIF4E.114 starvation. When nutrients are available, mTOR is active and Moreover, the ability of mTOR to promote cell growth seems to promotes organism growth and anabolism. Conversely, in the case be pivotal to the establishment of senescence in cell-cycle- of nutrient depletion, mTOR is promptly inactivated to favor arrested cells. Indeed, expression of p21 induces senescence when catabolism and growth arrest. Mechanistically, mTOR phosphor- mTOR is active, but it promotes quiescence when cells are serum ylates its substrates S6 kinase 1 and eIF4E-binding protein 1 to starved (that is, mTOR is inactive) or upon pharmacological regulate mRNA translation initiation and progression, thus inhibition of mTOR by rapamycin.102,115 This may have important controlling the rate of protein synthesis.93 Hence, mTOR is implications in p53-induced senescence as detailed below. implicated in diseases showing growth deregulation and Nonetheless, this pro-senescence function of mTOR does not metabolic compromise, such as cancer, diabetes and obesity94 apply universally: DNA-damage-induced senescence seems and is a master regulator of senescence and aging in several refractory to mTOR inhibition,102 whereas in Ras-driven OIS animal models, such as yeast,95–98 worms,99 flies100 and mice,101 dampening of mTOR signaling has been reported116 (see where mTOR inhibition has been proved to prevent the chapter p53 and ). Moreover, mTOR promotes expression of some senescent markers.102 Overall, many findings senescence via autophagy inhibition, by decreasing lysosomal suggest that sustained mTOR signaling promotes cell and tissue degradation of intracellular components. Activated by nutrients, aging, fostering the idea that inhibition of mTOR may increase mTOR inhibits autophagy, a process that may contribute

Oncogene (2013) 5129 – 5143 & 2013 Macmillan Publishers Limited Mechanisms of p53-mediated regulation A Rufini et al 5133 to mitochondrial dysfunction ER stress and senescence. protein. In response to p53 activation, damage-regulated Indeed, autophagy seems to be required for the senescence autophagy modulator is upregulated and elicits autophagy that response116–118 (Figure 2). is necessary to mediate p53-dependent cell death. On the other Recent evidence indicates that p53 can also prevent cell hand, cytoplasmic p53 represses autophagic flux through a growth, interacting with the mTOR pathway. Interestingly, p53 substantially unknown mechanism. Kroemer’s group showed inhibits mTOR signaling through different ways.119 In fact, p53- that loss of p53 activity can enhance autophagy and that regulated sestrins repress mTOR activity directly.120 In addition, cytoplasmic, not nuclear, p53 is responsible for autophagy p53 triggers expression of the AMP-activated protein kinase inhibition. Importantly, in this context, inducers of autophagy, (AMPK), which, in turn, inactivates mTOR.121,122 Finally, p53 such as starvation or rapamycin, induce degradation of p53 that is upregulates PTEN, an inhibitor of the PI3K pathway, which is an necessary for autophagy induction.151 Although the physiological upstream-positive regulator of TOR. implications of p53-regulated autophagy are unknown with Lately, mTOR regulation by p53 has been implicated in a regard to senescence induction, there is evidence of their paradoxical antisenescence role of p53. Induction of p21 allows involvement in the regulation of lifespan. Indeed, knockdown of the establishment of an irreversible senescent arrest. Nonetheless, the C. elegans p53 ortholog Cep-1 increases lifespan, a phenotype further accumulation of transcriptional-competent (but even abrogated by inhibition of autophagy.154 unphosphorylated) p53 triggers inhibition of mTOR and switches 14,123 cell status to a reversible cell-cycle arrest. Unfortunately, the p53 and ROS p53-target(s) responsible for this phenotype is yet unidentified, As aforementioned, ROS or, more accurately, ROS-mediated but the ability of p53 to induce cell-cycle arrest and inhibiting damage have been extensively implicated in the induction of mTOR simultaneously could help explaining why moderate cellular senescence and in the onset of aging disorders. p53 shows increases of p53 activity protects from cancer and a Janus role, dictated by its dual capacity to inhibit or promote simultaneously prolongs lifespan. In addition, these data support senescence, by regulating ROS levels.14,42,155 Indeed, increasing our view that p53-mediated senescence is not simply an on–off evidence suggests that transcriptional regulation of antioxidant switch mediated by p21 induction, but it is a complex cellular genes (including mitochondrial superoxide dismutase 2, phenotype that can be fine tuned by regulation of several glutathione peroxidase 1 and mammalian sestrin homologs 1 additional targets and pathways. and 2) accounts for p53’s ability to repress senescence by dampening intracellular ROS levels.156–160 On the other hand, in p53 and autophagy cells sensitive to p53-mediated apoptosis, DNA-damage-activated p53 elicits a spike in intracellular ROS content, resulting in cell Autophagy is an evolutionary conserved self-eating mechanism by death or senescence.42,155,161,162 The p53-dependent ROS which cellular cytoplasmic portions and organelles are delivered generation may well represent a crucial event for senescence to the lysosome for degradation. Degraded products are then regulation, but its dual regulation of oxidative metabolism may recycled for energy production or other metabolic processes, confer to p53 a double-edged role in the senescence process. As which explains why autophagy is engaged in conditions of far as aging is concerned, the free radical theory of aging states nutrient deprivation.124 An additional role for the autophagic that ROS-mediated damage has a direct detrimental effect on process is the removal of damaged macromolecules and animal well-being.38 Hence, p53 may counteract aging by dysfunctional mitochondria, avoiding the build-up of damage. mitigating the oxidative burden, as suggested by reduced As such, autophagy is cytoprotective and can modulate aging and oxidative damage in long-lived super-arf/p53 mice.74 However, it influence cancer survival.125–127 is unclear whether induction of ROS by p53 in response to The longevity pathways interact with the autophagic process to stressors is involved in aging. regulate diverse cellular functions, including growth, differentia- tion, response to nutrient deprivation, , cell death, as well as macromolecule and organelle turnover. Indeed, p53 and mitochondria mutations in genes that promote autophagy reduce lifespan in Mitochondria have been linked to aging, neurodegeneration163–165 C. elegans, D. melanogaster and yeast.125,128–131 Moreover, CR and cancer.166 As stated, according to the free radical theory of induces autophagy via repression of the mTOR signaling, and aging, ROS-mediated damage to cellular components is the autophagy induction is essential for the anti-aging outcome of driving force behind aging.35 As mitochondria are the prime reduced caloric intake.132,133 source of ROS, they are as well the main targets of ROS-mediated On the other hand, the role of autophagy in cancer is more damage, a hypothesis known as ‘mitochondrial theory of aging’. debatable.127,134–136 Robust engagement of autophagy in tumor Impaired mitochondrial activity and the resulting imbalance in areas deprived of blood and nutrient supplies promotes survival of oxidative and energetic metabolism can indeed severely affect cancer cells, suggesting an ‘oncogenic’ role for autophagy, and lifespan and negatively impact on aging.167 Study of telomerase- several studies have proved that engagement of autophagy deficient animals has unveiled a link between p53, mitochondria protects cancer cell from chemotherapy.137–145 Conversely, allelic and aging. Telomerase maintains the stability of telomeres, the disruption of some autophagic genes predisposes to tumor nucleoprotein complexes responsible for the genomic integrity of development, indicating that autophagy may be required to chromosomal ends. Eroded telomeric ends trigger widespread repress tumor onset.146–150 As far as senescence is concerned, DNA damage, which activates p53 and results in age-related autophagy acts as an effector mechanism during OIS.116 In fact, disorders.24,25 Importantly, depletion of p53 ameliorates the age- autophagy is engaged during OIS in a PI3k-mTOR-dependent related degeneration in telomerase-deficient animals, partially fashion and its inhibition delays the onset of the senescent abolishing p53-mediated cell death.28 Intriguingly, upon telomere phenotype.116 dysfunction, active p53 represses expression of peroxisome p53 has a dual function in the control of autophagy: it can proliferator-activated receptor gamma, coactivator 1 alpha and either activate or repress autophagy.49,151–153 On the one hand, beta (PGC-1a/b). PGC proteins regulate mitochondrial physiology nuclear p53 can induce autophagy through transcriptional and energetic metabolism (Figure 3). Their repression decreases upregulation of targets such as AMPK, PTEN and sestrins that mitochondrial biogenesis, reduces oxygen consumption and activate autophagy mainly through inhibition of mTOR. Another increases ROS levels.168 These findings bridge DNA damage, pivotal p53-target and positive regulator of autophagy is damage- mitochondria and aging, and prove that p53 regulation of regulated autophagy modulator, which codes for a lysosomal mitochondrial respiration is likely to affect animal longevity.

& 2013 Macmillan Publishers Limited Oncogene (2013) 5129 – 5143 Mechanisms of p53-mediated regulation A Rufini et al 5134

Figure 3. p53 regulates mitochondrial function. Mitochondria are pivotal regulators of aging and accumulating evidences link them to cellular senescence. In unstressed cells, p53 sustains mitochondrial respiration promoting the transcription of the nuclear-encoded mitochondrial protein synthesis of cytochrome c oxidase 2 (a). Conversely, stress-activated p53 (for example, consequently to telomere erosion) represses PGC1a, a positive regulator of mitochondrial function and biogenesis, resulting in impaired mitochondrial respiration and bioenergetic, which has been directly implicated in p53-mediated regulation of animal lifespan (b). In addition, potentially, p53 could affect mitochondria indirectly, through regulation of autophagy. Autophagy of mitochondria, or mitophagy, is essential for the removal of damaged organelles and, hence, the preservation of an efficient pool of healthy mitochondria. Nuclear p53 activates autophagy, whereas cytoplasmic p53 inhibits it. Whether this regulations affect mitochondria and, generally, their physiological implication in aging and senescence still awaits investigation (c,d).

Intriguingly, basal p53 activity is necessary for maintenance of DNA damage, SIRT1 relocalizes from its constitutive loci to sites of mitochondrial function. Indeed, p53 promotes the expression of DNA damage where it promotes DNA repair and hence genomic synthesis of cytochrome c oxidase 2, a component of the complex stability. Thus, both SIRT1 and p53 are /DNA responders IV of the electron transport chain. p53 null tissues and cells have that help maintain genomic stability and are coordinated so that reduced complex IV activity resulting in impaired oxygen SIRT1 favors repair and survival, while p53 elicits programmed consumption. However, whether this has a role in aging or removal of overly damaged cells via apoptosis. The presence of a senescence has not been investigated yet. chronic DDR (as may be seen in cancer cells), which is linked to the Intriguingly, mTOR signaling has been reported to sustain induction of senescence, can directly increase p53 acetylation respiration in human cells,169 whereas autophagy of mitochondria, by promoting the interaction with the acetyl transferases or mitophagy, removes damage organelles and helps maintain a CBP/p300.184 Acetylation of p53 is also seen to be important healthy pool of mitochondria.170,171 Hence, the ability of p53 to during Ras-induced or replicative senescence, where it is inhibit both mTOR and its dual regulation of autophagy may well antagonized by SIRT1.180,181,185,186 In keeping with this, cells be implicated in regulation of mitochondrial function during harboring p53 with acetyl-mimicking mutations of the last seven senescence or aging, a possibility that needs further investigation. lysine residues have an accelerated entry into senescence and are very resistant to senescence bypass,187 although the cell-cycle arrest response in these cells remains normal. Conversely, p53 and sirtuins mutations that abolish acetylation of the lysine residues located The crosstalk between p53 and Sirt1 represents a crucial point of in the DNA-binding domain fails to establish replicative as well as regulation of p53 signaling, implicated in many biological OIS.188 Thus, these data strongly suggest that deacetylation of p53 processes such as senescence. Sirt1 belongs to a family of by Sirt1 impedes the induction of senescence. Whether this is evolutionary conserved NAD þ -dependent protein deacetylase, relevant in tumor formation is unclear. Indeed, SIRT1-mediated classified as class III histone deacetylase, able to deacetylate target repression of p53 activity supports the idea that SIRT1 could be histone and non-histone proteins, and thus participates in the oncogenic. However, several mouse models proved that SIRT1 acts regulation of chromatin structure and of DNA accessibility for as a tumor suppressor reducing cancer incidence even in p53 processing and repair, as well as in transcriptional control heterozygous mice.189–191 Thus, the physiological meaning of networks via deacetylation of transcription factors and cofac- SIRT1-mediated deacetylation of p53 remains to be elucidated. tors.58,172–179 SIRT1 is necessary for the establishment of SIRT1 was initially identified as a ‘longevity’ gene in senescence,180,181 and SIRT1 is strongly downregulated in C. elegans,192 yeast193 and Drosophila,194 findings that spurred senescent cells. A major substrate for SIRT1 is p53, and the research in mammalian models. The idea that SIRT1 is necessary to deacetylation of p53 regulates cell cycle, cellular senescence and prolong animal lifespan has been heavily questioned.195 stress resistance in various cell types. Deacetylation inhibits p53’s Nonetheless, mice-overexpressing SIRT1 have a reduced ability to transcriptionally activate some, but not all, target incidence of age-related metabolic disorders, including diabetes, genes—including those involved in apoptosis, proliferation, ROS liver steatosis and196 cancer. In other words, it is now evident that production and presumably also senescence.58,182,183 Following SIRT1 is not necessary to live longer, but to live healthier.

Oncogene (2013) 5129 – 5143 & 2013 Macmillan Publishers Limited Mechanisms of p53-mediated regulation A Rufini et al 5135 Currently, it is unclear whether p53 is required for the metabolic Table 1. Table reports anticancer drugs currently undergoing clinical function of SIRT1. trials (source www.clinicaltrial.gov), which induce cellular senescence in cancer cell lines and tumors p53-INDUCED SENESCENCE IN CANCER THERAPY Agent Mechanism p53 The cancer cell phenotype is characterized by sustained pro- Status liferative signaling, alteration of cellular homeostasis and meta- bolism.197,198 Senescence is a robust physiological antitumor Resveratrol ROS þ response that is engaged by tissues to counteract oncogenic Hydroxyurea ROS þ insults. Accumulating evidence of its involvement in the onset and Mitoxantrone DNA damage þ / À therapeutic response in humans has spurred considerable efforts Cyclophosphamide þ doxorubicin DNA damage ND towards its therapeutic exploitation.176,199 Indeed, therapy- þ 5-fluorouracil Carboplatin þ docetaxel DNA damage þ / À induced senescence is an emerging appealing approach to halt Diaziquone/AZQ DNA damage þ / À tumor growth; several agents are reported to induce senescence VO-OHpic PTEN þ by acting on different pathways, as demonstrated in vitro and in MLN4924 Cul1 SCF subunit þ / À human tumors and in tumor models (Table 1). The DDR entails inhibitor senescence as an anticancer mechanism; indeed, many drugs MLN8054 Aurora kinase A þ induce senescence following DNA damage. In OIS, the genetic inhibitor lesions that initiate tumorigenesis (for example, RAS overactiva- K858 KIF11 þ / À tion) promote senescence at the early stage of cellular transfor- Irotecan DNA damage þ mation. For transformation to occur, additional genetic Etoposide DNA damage þ / À 17,77,82,200,201 MS-275 HDAC inhibitor þ / À modifications are necessary to overcome senescence. Cetuximab þ radiation þ EGFR DNA damage þ / À Therefore, designing innovative therapeutic approaches to trigger inhibitor tumor regression re-activating senescence programs and effectors Pemetrexed Folate þ / À is appealing. In this regard, modulation of p53 activity may be antimetabolite appropriate.56,57,202–207 Recent findings have demonstrated that GSK690693 AKT inhibitor þ / À reactivation of p53 in tumors elicits a robust tumor regression Everolimus mTOR inhibitor þ 203,204 mediated by induction of senescence. These studies Abbreviations: mTOR, mechanistic target of rapamycin; ND, not deter- boosted the long-standing efforts to develop drugs able to mined; ROS, reactive oxygen species; WT, wild type. ‘p53 status’ indicates reactivate p53 in tumors-bearing null or mutant the p53 form expressed in cells in which the studies have been conducted: 59,73,186,205,208–211 p53. Nonetheless, the efficiency of p53- ‘ þ ’ denotes p53 WT-expressing cells, ‘ À‘’ denotes p53 null- or mutant- mediated tumor clearance is also stage-specific and dependent expressing cells, ‘ þ / À ’ denotes that studies have been conducted in both on the overall ‘oncogenic’ burden. Indeed, using a mouse model p53 WT- and null-/mutant-expressing cells. of K-Ras-driven lung cancer, two different research groups demonstrated that p53 reactivation is efficient only in advanced 218 cancers characterized by sustain Ras-Raf-Mef-Erk signaling. In R172P expression. Furthermore, the DDR activated by telomere dysfunction induces the ATM/ATR and Chk1/Chk2 activation, addition, in the same system, p53 tumor suppressor activity does 29 rely on co-expression of p19ARF. Although these results cast doubt which consequently phosphorylates and stabilizes p53. on the potential therapeutic benefit of p53 restoration in cancer Evidence that deletion of key regulators of senescence, for therapy, they also unveil an interesting parallelism between p53 example, p53, p27, PRAK or Arf, induces tumor progression and senescence block, connect the loss of senescence to tumor tumor suppressive function and regulation of aging. Indeed, in the 82,219–222 super-arf/p53 mouse model, increased expression of p19 is transformation. Eighty to ninety percentage of human cancers seem to be associated with unlimited proliferation due to essential to prolong lifespan, whereas increased dosage of p53 223,224 alone does not suffice.212,213 These findings highlight the activation of telomerase. Therefore, the inhibition of telomerase could be a promising therapeutic target for cancer, existence of a p19–p53 axis, where ARF expression seems to be 21 necessary to fully engage p53 activity. because telomere shortening induces senescence in cancer cells The phytoalexin resveratrol is known to possess a variety of and this kind of approach could offer the additional advantage to cancer-preventive, therapeutic and chemosensitizing properties. It specifically target cancer cells, characterized by telomerase has been reported that chronic treatment with resveratrol in a expression, unlike normal cells. subapoptotic concentration induces senescence-like growth arrest in tumor cells (Table 1). Resveratrol has proved to act by increasing the level of ROS and induce a p21–p53-dependent senescence.214 A FAMILY MATTER: p63 AND p73 IN SENESCENCE AND AGING This anticancer property of resveratrol is particularly intriguing on Two others p53 homologs of p53 have been characterized over the light of its debated role in regulating aging and sirtuin the past two decades: p63 and p73.225 Like p53, both proteins function. Because of the debate on the issue, we refer to other contain three domains: a N-terminal transactivation domain, a reviews for details,125,215 but in summary, there is evidence that DNA-binding domain and an oligomerization domain responsible resveratrol may improve healthy aging, especially by for tetramerization. In addition, the use of different promoters and counteracting obesity and diabetes, and that this could be alternative splicing results in the expression of multiple isoforms. mediated, at least partially, by activation of Sirt1 and induction Briefly, alternative splicing at the 30-end of the primary transcript of autophagy. Hence, resveratrol is able to suppress tumor growth, originates three isoforms in p63 and at least seven in p73, some of while improving organismal metabolism. which contain an additional C-terminal protein/protein interaction A promising target for senescence induction in cancer cells is domain, the sterile alpha motif, absent in p53.225 Although several the enzyme telomerase. Findings have demonstrated that short studies have attempted to dissect specific functions of these telomeres induce senescence, limiting tumor suppression.216,217 proteins, at present clear-cut roles for these different variants have The evidence that senescence induced by telomere shortening is not been attributed. As mentioned, the use of alternative an in vivo tumor suppression process comes from studies in promoters generates two additional N-terminal variants. An mTERC À / À mice, in which shortened telomeres decrease upstream promoter transcribed longer, transcriptionally tumorigenesis when block of apoptosis is due to p53-mutant competent isoforms containing a transactivation domain (TAp63

& 2013 Macmillan Publishers Limited Oncogene (2013) 5129 – 5143 Mechanisms of p53-mediated regulation A Rufini et al 5136 and TAp73), whereas an internal downstream promoter originates underwent senescence at higher rate than wild-type counterparts. shorter isoforms that lack the transactivation domain (DNp63 and In the quest for a mechanism underlining the observed DNp73) and are thought to act as dominant negatives.226 phenotypes, we showed that TAp73 regulates expression of a Both TAp63 and TAp73 are activated in response to DNA cytochrome c oxidase 4 subunit 1 (Cox4i1), a protein essential for damage by the non-receptor tyrosine kinase c-Abl227–232 and act assembly of fully functional mitochondrial complex 4. as proapoptotic molecules.233,234 Although they are involved in Consequently, lack of TAp73 impaired activity of the complex 4 cancer and chemotherapy response,226,235 p73 has a major role in of the electron transport chain and decreased oxygen regulation of inflammation236,237 and brain development through consumption both in vitro and in vivo, similarly to depletion of several mechanisms,238,239 including preservation of neural stem p53. This resulted in reduced ATP cellular content and increased cells240–242 and, importantly, its depletion predisposes to age- cellular ROS and oxygen sensitivity. Hence, we proposed that the related in mouse models.243–245 In addition, mitochondrial and metabolic dysfunction triggered by depletion TAp73 has been involved in the preservation of genomic stability of TAp73 underlined the accelerated aging in KO animals.34 and fertility, and is important for accurate mitotic and meiotic In summary, regulation of senescence and aging are shared division.64,246–248 p63’s role in cancer onset and metastatic spread functions of the p53-family members. has been widely investigated,249–251 but it also has a fundamental role in epithelial development and maintenance of the epithelial stem cell reservoir252–258 and protects the female germ line CONCLUDING REMARKS against DNA damage.259 Nonetheless, like their sibling p53, both The molecular mechanisms underlying the senescence pathway genes have a role in senescence and aging. Indeed, the recent are becoming increasingly topical owing to its role in tumor development of several N-terminal selective-knockout (KO) mouse suppression, giving great relevance for its potential exploitation in models has helped understand the involvement of these isoforms cancer therapy. Although these molecular mechanisms are only in complex biological processes, such as senescence and aging. partially elucidated and are currently under intense investigation, p63 null mice (that is, lacking all isoforms) show a very severe it is evident that p53 has a key role in its regulation. Indeed, p53 phenotype and die shortly after birth,260,261 although long-term can modulate senescence at different levels. Surprisingly, p53 analysis of heterozygous mice allowed detection of premature seems to show a dual effect, promoting or in same case inhibiting aging.262 These findings were strengthened by the development the senescence program. This dual effect of p53 is still unclear; a of inducible TAp63-KO mice.263 In this setting, depletion of p63 in possible explanation can be the dependence on the degree and the epithelial compartment was sufficient to accelerate aging, type of stress or the cellular milieu where p53 is active. Indeed, which correlated with accumulation of senescence markers in vivo mild stress can induce p53 to repair the cell and activate and in isolated keratinocytes. In particular, the establishment of antioxidant mechanisms, while more severe stress leads p53 to the senescent status relies on PML, a known mediator of induce apoptosis and senescence, via ROS generation. In the senescence.263–265 Further insights into p63 regulation of the context of cancer therapy, the ability of p53 to regulate aging process were provided by development of TAp63 isoform- senescence is emerging as a promising and alternative way to specific KO. Flores and colleagues showed that absence of TAp63 eliminate cancerous cells, because p53-signaling pathways can be has severe effects and results in ulceration, premature aging manipulated at several steps to stimulate senescence. Beyond its and reduced lifespan. Interestingly, this correlates with strong antitumor ability, senescence is emerging as a casual factor in cellular senescence triggered by genomic instability, which is aging.11 Presently, while it is clear that modulation of p53 activity responsible for the loss of the epithelial stem cell repertoire.266,267 affects lifespan, the contribution of senescence to this function Although these findings suggest that lack of p63 induces needs further investigation. In a recent mouse model of senescence, other reports have shown that TAp63 mediates the hyperactive p53 and consequent aggravation of aging disorders, induction of OIS in keratinocytes, similarly and independently of the concomitant removal of the proapoptotic p53 target PUMA p53.268 Hence, it appears that p63, especially its transcriptionally suffices to rescue the stem cell repertoire and increase the lifespan competent isoforms, share a Janus role with p53: their activation of mutant mice. This suggests that apoptosis may be critical to in response to oncogenic stress is necessary to halt transformation p53-induced aging. But, on the other hand, it does not exclude an via senescence, but their absence compromises the stem cell additional role for senescence. In particular, apoptosis can explain reservoir and promotes aging. Nonetheless, to fully address the the defect in tissues subject to extended self-renewal (for role of p63 in senescence and aging, it is necessary to take into example, bone marrow or gut), but it is unclear whether it could account the activity of the N-terminal truncated proteins. Indeed explain the aging-related degeneration that accompanies DNp63 isoforms are by far the most abundant isoforms expressed quiescent tissues (such as liver or brain).272 In this context, in epithelial cells. They have been consistently reported to support senescence may have a more critical role, which would be worth the maintenance of the stem cell compartment of the skin and to investigating. antagonize the induction of replicative senescence.255,269 Mechanistically, we have proposed several mechanisms that are Moreover, DNp63 downregulation by oncogenic K-Ras is at the crossroads between senescence and aging: ROS scavenging necessary for establishment of OIS and tumor prevention in and generation, mitochondrial function, mTOR signaling and keratinocytes.270 Unfortunately, it is still unclear to what extent autophagy. Importantly, these biological processes are intimately these isoforms affect aging, and data on isoform-specific KO are linked: mTOR positively regulates respiration in mammalian eagerly awaited. cells169,273 and renders cancer cells addicted to mitochondrial p73 has been linked to OIS and in fact expression of DNp73 has activity.274 Moreover, it inhibits autophagy, and autophagy of been reported to bypass Ras-induced senescence allowing cellular mitochondria (a process known as mitophagy) is essential to transformation to occur.271 Moreover, oncogenic Ras promotes a remove dysfunctional mitochondria, whose accumulation would switch from TAp73 to DNp73 expression to sustain transformation. result in increased ROS production and reduced energy efficiency. Indeed, transformed mouse fibroblasts, lacking DNp73, fails to Activation of autophagy depends on ROS, while autophagy form tumors in nude mice because of ensuing senescence.244 dampens intracellular ROS build-up.38,275–277 Intriguingly, SIRT1- Recently, by mean of isoform-specific KO models,247 we have mediated deacetylation of PGC-1a is required for its activation of demonstrated that mice lacking TAp73 show an aggravation of mitochondrial biogenesis and gluconeogenesis,278 thus several aging-related parameters (hunchback, cataract, alopecia antagonizing p53-negative regulation of PGC-1a. In other words, and skin thinning among others).34 In addition, fibroblasts isolated p53 is controlling a network of tightly connected biological from null embryos were more susceptible to oxidative stress and processes that impinge on senescence and aging. Future efforts

Oncogene (2013) 5129 – 5143 & 2013 Macmillan Publishers Limited Mechanisms of p53-mediated regulation A Rufini et al 5137

Figure 4. The unified theory of aging and the p53 family. Recent findings suggest that p53 regulates mitochondrial function in both stress and unstressed conditions. In unstressed cells, it regulates expression of synthesis of cytochrome c oxidase 2 (SCO2) and help sustaining and oxidative energy production. In response to stress, such as telomere erosion, p53 is activated in response to DNA damage and directly repress the expression of PGC1a/b protein, causing reduction in mitochondrial mass and function and increasing ROS content. Similar results were described for p73: deletion of the TA isoforms worsen aging in mice and results in increased cellular ROS levels, oxidative damage and senescence triggered by mitochondrial dysfunction. In this case, the target responsible, at least partially, for the described phenotype is cytochrome c oxidase 4 subunit 1 Cox4i1. Unfortunately, data are still missing with regards to p63 and it is not clear whether, upon stress, activated p63 and p73 are able to regulate mitochondrial function similarly to p53. Further studies aim to investigate these possibilities are eagerly awaited. are necessary to fully address how p53 regulates these processes, not investigate mitochondrial function in detail, they demonstrate how they interrelate in the context of p53 regulation and their that TAp63 null mice have reduced activity of SIRT1 and AMPK overall relevance to cancer and aging. in vivo, which renders these animals extremely susceptible to Finally, a growing body of evidence points to mitochondria as obesity and insulin resistance following high-fat diet regimen.287 crucial factors in aging and senescence. Indeed, dysfunctional Although further studies are necessary, it is becoming increasingly mitochondrial function accompanies OIS, and several mouse evident that regulation of energetic metabolism is a main function model of dysfunctional mitochondria show worsening of the of the p53 family of genes with essential consequences on animal aging phenotype.168,279–285 Strikingly, overexpression of the lifespan and well-being. scavenging enzyme catalase exerts negligible effects on longevity if targeted to the peroxisomes or the nuclei, but ABBREVIATIONS extends median lifespan by 20% when targeted to the DDR, DNA damage response; ROS, reactive oxygen species. mitochondria,286 and a recent study demonstrated that this genetic manipulation prevents the age-associated decline in 281 mitochondrial activity. Our recent data on TAp73-selective KO CONFLICT OF INTEREST and the findings of Ronald DePinho’ s Laboratory on the p53- The authors declare no conflict of interest. PGC1a axis both support a model whereby depletion or hyperactivation of the p53 family members leads to aging through impaired mitochondrial function and metabolic REFERENCES 272 compromise (Figure 4). These findings also explain the aging 1 Sherwood SW, Rush D, Ellsworth JL, Schimke RT. Defining cellular senescence decline of quiescent organs (such as liver) that do not depend on in IMR-90 cells: a flow cytometric analysis. Proc Natl Acad Sci USA 1988; 85: a strong stem cell pool (such as the haematopoietic system or 9086–9090. skin) and thereby are less susceptible to apoptosis mediated by 2 Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence. DNA damage and p53 activation. Future studies to thoroughly Genes Dev 2010; 24: 2463–2479. address the mitochondrial function and metabolic profile of long- 3 Romagosa C, Simonetti S, Lopez-Vicente L, Mazo A, Lleonart ME, Castellvi J et al. lived mouse models such as super-arf/p53 or short-lived p53- p16(Ink4a) overexpression in cancer: a tumor suppressor gene associated with knock-in mice are desirable. Similarly, these recent studies urge senescence and high-grade tumors. Oncogene 2011; 30: 2087–2097. immediate work to investigate whether p63 shares with its 4 Alessio N, Squillaro T, Cipollaro M, Bagella L, Giordano A, Galderisi U. The BRG1 ATPase of complexes is involved in modulation of siblings the ability to regulate oxidative metabolism, making it mesenchymal stem cell senescence through RB-P53 pathways. Oncogene 2010; definitively a family matter. Intriguingly, a recent paper 29: 5452–5463. demonstrated that TAp63 upregulates expression of SIRT1 and 5 Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M et al. Tumour AMPKa2 (one of the subunit of AMPK). Although the authors do biology: senescence in premalignant tumours. Nature 2005; 436: 642.

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