Claude Universite

Bernard -Lyon

Secrétaire Général

M François-Noël

Gilly M Le Professeur

Hamda Ben

Hadid M Le Professeur

Philippe Lalle

M Le Professeur

Germain Gillet

M Alain Helleu

Composantes Sante

J Etienne

G Kirkorian

D Bourgeois

La Mme

C Professeure

Vinciguerra

Y Matillon

P Farge

Are the two N-terminal isoforms functionally equivalent or do they each have a specific functional and regulatory profile?

Given the potential of these isoforms to antagonise p53 function, can their overexpression provide an alternative mechanism to inactivate p53 in tumours without

TP53 mutation?

This manuscript describes our attempts to address and answer these questions. It opens with a bibliographic survey in which we review the role of p53 as a key element of the molecular hallmarks of cancer and we describe the current, state-of-the-art knowledge on p53 isoforms. After a brief section presenting the specific aims and objectives, the Result section includes 3 manuscripts either submitted, published or in preparation, which illustrate the progress we have made in resolving the questions outlined above. In the Discussion, we have taken a step ahead to propose hypotheses on the role of N-terminal isoforms in p53 regulation.

. They acquire the characteristic to evade immunological destruction by T and B lymphocytes, macrophages and natural killer cells. Acquisition of these multiple Hallmarks depends in large part on a succession of alterations in the genomes of neoplastic cells. Genomic instability and thus mutability endow cancer cells with genetic alterations that drive tumour progression.

Another dimension of complexity in tumours arises from their capacity to develop an intricate network of interactions between neoplastic and normal cells that contribute to the acquisition of hallmark characteristics by creating the "tumour microenvironment" (Hanahan and Weinberg, 2011). Such considerations suggest that drug development and the design of treatment protocols will benefit from incorporating the concept of hallmark capabilities, resulting in a more effective and durable therapies for human cancer.

COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE

To all my dear friends and colleagues that I saw leaving one by one from our group so friendly, "multi-cultural", "multi-language" and so pleasurable to work with! Amina, Chiara, Suna and Yayun, it was a big pleasure to share the office with you. I will never forget the wonderful time we spent together. You have always been here for me, in good and bad moments, and I am deeply thankful for that. Yayun, thank you very much for all these nice talks, your help, your advices, and your friendship during all these years; I hope to see you soon (and your baby!). Dominique, merci pour ces fous rires, ces coups de gueule, ta franchise et ton honnêteté. Les fins de journée, lorsque tout le monde partait, étaient moins monotones grâce à toi ! Promis, je viens te voir prochainement ! Nathalia, le rayon de soleil brésilien du labo ! Merci pour ta grande gentillesse, ces fous rires, ces super moments ensemble au labo et surtout le week-end génial que tu m'as fait passer à Rio !! Daniela, it was a great pleasure to collaborate with you. Thank you very much for your valuable help, the hundreds of experiments you performed and your enthusiasm doing them! You were the best trainee I have ever worked with and I am glad we achieved to finish the paper together.

Sandra, mi querida amiga! Fue un gran placer discutir contigo durante horas y horas!! Je te remercie pour avoir été là, pour toujours réussir à apporter ta touche d'humour dans n'importe quelle situation, bref pour être una verdadera colombiana !! Eve, merci énormément pour ton aide, ton écoute, ton soutien et tes encouragements. Tu as cru en moi jusqu'au bout et je t'en suis extrêmement reconnaissante. Je ne connais personne capable de voir toujours les choses du bon côté comme toi. C'est un vrai plaisir ! Ghyslaine, tu m'as beaucoup soutenue et encouragée dans les moments difficiles. Je te remercie pour tout et surtout pour ton excellente expertise technique ! Michelle, merci pour ton indispensable soutien administratif, ta persévérance à me placer dans l'agenda de Pierre et ta spéciale "English touch" inimitable ! Sid, un grand grand merci pour m'avoir proposé ton aide tant de fois, mais aussi pour ton sourire et ta véritable gentillesse. Je ne sais pas comment j'aurais fait sans toi ! Zdenko, I would like to thank you very much for all what you have done to help me to finish my thesis in the best conditions. I would like to express you my deep gratitude for your valuable support and your enthusiastic encouragements.

Agnès, comment te remercier pour l'aide précieuse que tu m'as apportée pendant ma thèse ? C'était un véritable plaisir de travailler avec toi, toujours avec efficacité, discrétion et dans la bonne humeur. Tu as laissé une grande place après ton départ qui n'a jamais été comblée.

Reetta, one of the last survivors! Je suis ravie d'avoir été entourée par quelqu'un d'intègre, authentique et juste comme toi pendant cette "aventure". On a su rester soudés et surtout bien rigoler malgré tout ! Merci pour avoir partagé tout ça avec moi.

Andre, a real friendship is born between us thanks to your kindness, sincerity and of course your absolute "locura" who makes you so unique! Thank you so much for all these great moments in and outside of the lab. The atmosphere is always more fun when you are around! Felipe, el mejor compañero de oficina del mundo! I will miss our nice talks about "life", your nice compliments every morning, your attitude as a real gentleman… Muchas gracias for your excellent company in the office, for being a real friend and for simply being you.

Mes années au CIRC n'auraient pas été les mêmes sans la rencontre de certaines personnes qui comptent aujourd'hui beaucoup pour moi.

Roland, mon paparazzi-conseiller-psychologue-thérapeute préféré ! Ta générosité et ta qualité d'écoute ont été sans faille depuis le jour où je t'ai rencontré. Merci pour avoir fait de moi tantôt égérie Chanel, tantôt modèle de couverture pour le volume des Monographies, tantôt top model,… Tu as inlassablement réussi à "capturer" mes moments de blues et les "développer" en moments de rigolade. Même si ce ne sera jamais suffisant, je te remercie profondément pour ta présence, ton écoute et les mots justes que tu as toujours trouvés pour me réconforter et me faire rire.

Gertrude, ta grande gentillesse, ton grand coeur et ta jovialité m'ont tout de suite touchée.

Merci énormément pour ton soutien, ton écoute et tes conseils précieux. Tu aimes donner sans espérer recevoir et c'est une qualité noble et rare de nos jours.

Anouar, merci pour ton soutien, ton écoute, tes conseils, mais aussi pour m'énerver comme personne d'autre n'y arrive ! Ta générosité et ta gentillesse sont toujours sans limite pour moi, c'est pourquoi je t'en suis infiniment reconnaissante.

A ma famille et mes amies, qui sont toujours là lorsque j'en ai besoin et qui me soutiennent et m'accompagnent dans les moments heureux mais aussi moins heureux de ma vie : Papa, Maman, c'est grâce à vous si je suis arrivée là aujourd'hui. Vous m'avez inlassablement poussée et encouragée pendant toutes mes études, sans jamais lésiner sur les moyens, jusqu'à la toute fin. J'espère vous rendre aujourd'hui, par ce titre de Docteur, une partie de tout ce que vous avez fait pour moi. Je vous aime fort.

Sarah, la plus belle et la plus intelligente de toutes les soeurs ! Tu as connu de très près la

"galère" de la thèse et c'est ce qui fait de toi la meilleure des soeurs. Pour toutes ces heures passées devant l'ordinateur mais surtout pour ce lien immuable qui nous unit, merci à toi et merci d'être là (même quand tu n'en as pas envie !).

Mounia, mon amie, ma complice, ma confidente. Tu as toujours été là pour moi depuis la toute première fois où je t'ai rencontrée (je me souviendrais toujours lorsque tu m'as apporté un gilet quand j'avais froid dans le bureau de Michelle). Cinq ans après, rien n'a changé… Les amis se comptent sur les doigts de la main et ce n'est pas nécessaire d'en avoir plus lorsqu'ils ont toutes tes qualités.

Maïlys, à mon tour de te dire que tu es une véritable amie sur qui je sais que je peux compter.

Merci pour m'avoir réconfortée à chaque fois que j'ai douté. Ton énergie, ta joie de vivre, ta sincérité, ta sensibilité, sans parler de ton extrême gentillesse et ta générosité expliquent très bien pourquoi nos chemins ne se sont jamais séparés depuis notre rencontre. Tu es une fille brillante, battante, toujours volontaire ; merci pour ce que tu es et ce que tu donnes.

Table of contents

List of figures and tables

Foreword

Cancer is a widespread disease present in the global population. Despite the clinical and molecular heterogeneity of this pathology, there is one molecular event that is commonly observed in many cancers: p53 inactivation. Mutated in more than half of cancers or repressed by other mechanisms, p53 seems to be one of the cornerstones of tumourigenesis.

The p53 protein (encoded by the TP53 gene) is an inducible transcription factor, which exerts widespread anti-proliferative effects through cell growth control and regulation of cellcycle arrest, apoptosis, senescence, differentiation and basal bioenergetics metabolism. Loss of p53 function therefore eliminates a critical emergency brake that normally prevents abnormal cell growth and transformation.

During the past 10 years, evidence has accumulated that p53 was not expressed as a single protein. The discovery of multiple isoforms of p53, differing from the full-length protein by truncations in the N-and C-terminal domains, has added another layer of complexity to the many complex transcriptional and post-translational mechanisms that regulate p53 functions.

So far, up to 12 p53 isoforms have been postulated and detected at mRNA level, although only a subset has been unambiguously identified at protein level. Some isoforms activate and others inhibit p53 functions, multiplying the possible biological effects. Do they play a role in basal conditions or after a stress? Are they constantly expressed in the cell or are they finely regulated? And by which mechanisms? Do they have a role in cancer?

These questions formed the basis of my PhD Thesis project. We have focused on one particular group of isoforms, the N-terminal isoforms lacking the main transactivation domain. This group includes two main members, 40p53 and 133p53, lacking respectively, the first 39 and the first 132 residues of the full-length protein. We have addressed three main

Introduction

INTRODUCTION

Cancer: from society to genes 1.The global burden of cancer

The global burden of cancer has more than doubled during the past thirty years. In 2008, it is estimated that there were 12.4 million new cases diagnosed with cancer, 7.6 million deaths from the disease and over 25 million persons alive with cancer (Boyle P and Levin B, World Cancer Report 2008). The most common cancers in the world in term of incidence were lung (1.52 million cases), breast (1.29 million) and colorectal (1.15 million). Because of its poor prognosis, lung cancer was also the most common cause of death (1.31 million), followed by stomach cancer (780,000 deaths) and liver cancer (699,000 deaths).

In developed countries, the overall cancer mortality is more than twice as high as in developing countries (Figure 1). The main reasons for the greater cancer burden of affluent societies are the earlier onset of the tobacco epidemic, the earlier exposure to occupational carcinogens and the Western diet and lifestyle. In developing countries, up to one quarter of malignancies are caused by infectious agents, including the hepatitis (HBV and HCV), and human papillomaviruses (HPV). Nevertheless, today the situation has dramatically changed, with the majority of the global cancer burden found in low-and medium-resource countries.

Overall, 53% of the total number of new cancer cases and 60% of the total number of deaths occur in the less developed countries. One third of cancers in high-resource countries are caused by tobacco smoking, which also causes a large proportion of deaths from other chronic disease including vascular disease and chronic obstructive pulmonary disease. Currently, the most common forms of cancer differ between high-income countries and the remainder. In high-income countries, cancers of the lung, breast, prostate and colorectum dominate, and one third of cancers are caused by tobacco use and 10% by chronic infection (Stewart BW and Kleihues P, World Cancer Report 2003). In low-resource and medium-resource countries, cancers of the stomach, liver, oral cavity, and cervix dominate [START_REF] Boyle | The globalisation of cancer[END_REF][START_REF] Parkin | The global health burden of infection-associated cancers in the year 2002[END_REF] (Figure 2). One quarter of the cancer burden in low resource countries appears to be attributable to chronic infection, but 12% is currently caused by tobacco, and this proportion is growing [START_REF] Parkin | The global health burden of infection-associated cancers in the year 2002[END_REF].

Figure 1. Global incidence of cancers

Estimated age-standardised incidence rate per 100,000 in male (left) and female (right). The highest rates are recorded in affluent countries. Non-melanoma skin cancer is not included. Globocan 2008, IARC. A better clinical diagnosis has led to more cases of cancer being diagnosed, a proportion of which would have previously been missed. There has been a remarkable improvement in imaging and other diagnostic techniques, which have contributed substantially to an increased chance of diagnosis and to a more accurate diagnosis of cancer [START_REF] Fass | Imaging and cancer: a review[END_REF]. While there are reasons for an artefactual increase in the cancer burden, there has undoubtedly been a real increase in the number of people who develop cancer due to an increased exposure to etiological agents.

Cancer is caused by both internal factors (such as inherited mutations, hormones, and immune conditions) and environmental factors (such as tobacco smoke, alcohol consumption, diet, radiation, and infectious organisms) (Table 1). The variation in incidence and progression of cancers among individuals can be attributed to interindividual variation in genetic makeup. Cancer genetic susceptibility is due to inheritance of specific sequence variants in cancer susceptibility genes that confer increased risk of cancer. The Li-Fraumeni susceptibility gene TP53 and the breast/ovarian cancer susceptibility gene BRCA1 are classic examples.

Table 1. Some examples of carcinogenic agents

IARC Monographs, http://monographs.iarc.fr.

Identification of risk factors for cancers as well as delivering effective prevention will reduce the burden of cancer.

The concept of the Hallmarks of Cancer

Cancer is a complex disease that is very variable in its presentation, development and outcome from one patient to the other. The same heterogeneity and variability exist at the cellular and molecular level. Cancer is a multi-step process during which cells undergo profound metabolic and behavioural changes, leading them to proliferate in an excessive and untimely way, to escape surveillance by the immune system, and to invade distant tissues to form metastases [START_REF] Merlo | Cancer as an evolutionary and ecological process[END_REF]. Metastasis is the main cause of death by cancer.

In 2000, Hanahan and Weinberg proposed the model of "Hallmarks of cancer" describing six biological capabilities that cells need to acquire to become tumourigenic and eventually malignant [START_REF] Hanahan | The hallmarks of cancer[END_REF] (Figure 3). These Hallmarks include sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Remarkable technical progress in cancer research in the last decade has led to a deeper understanding of these Hallmarks, as well as the emergence of two new Hallmarks: reprogramming of energy metabolism and evading immune destruction [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. Moreover, their acquisition is made possible by two "enabling characteristics", namely genomic instability and tumour-promoting inflammation, which occur in some, if not all, cancer cells (Figure 4). [START_REF] Hanahan | The hallmarks of cancer[END_REF]. [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF].

Normal tissues carefully control the balance between production of growth-promoting and anti-proliferating signals, thereby ensuring a homeostasis of cell numbers and maintenance of the tissue architecture and functions. Cancer cells can acquire the capability to sustain proliferative signalling through growth factors production or growth receptor activation [START_REF] Witsch | Roles for growth factors in cancer progression[END_REF]. In addition, cancer cells must circumvent powerful programs that negatively regulate cell proliferation, mainly by inactivating tumour suppressor genes like Retinoblastoma (Rb) and TP53. Natural "emergency brakes" exist in normal cell lineages in the body, which enable them to pass through only a limited number of successive cell growth and division cycles. This limitation has been associated with two distinct barriers to proliferation: senescence, a typically irreversible entrance into a non proliferative but viable state, and crisis, which involves apoptosis. When cells emerge from a population in crisis and exhibit unlimited replicative potential, they circumvent these barriers and become immortalized. A major reprogramming of cellular energy metabolism then occurs in order to support continuous cell growth and proliferation. An ''angiogenic switch'' is almost always activated and remains on, causing normally quiescent vasculature to continually sprout new vessels that help sustain expanding neoplastic growths [START_REF] Hanahan | Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis[END_REF]. The capability of invasion and metastasis enables cancer cells to escape the primary tumour mass

p53: the molecular gatekeeper 1.2.1 From oncogene to tumour suppressor gene

Around thirty years after its discovery in 1979, the p53 protein has achieved stardom status among tumour suppressors [START_REF] Hainaut | 30 years and a long way into p53 research[END_REF]. With 63,999 papers published in September 2012, and a steady flow of new ones every day, p53 is undoubtedly one of the most extensively studied genes and proteins. However, the idea that p53 is a pivotal tumour suppressor and a mainstay of our body's natural anticancer defence did not come easily [START_REF] Levine | The first 30 years of p53: growing ever more complex[END_REF]. The road leading to p53's eventual rise to prominence and its recognition as the most frequently altered gene in human cancer was long and winding, with concepts being repeatedly revised, extensively modified and sometimes even turned totally upside down (Figure 5).

The p53 protein owes its name to its apparent molecular weight of 53 kDa and was initially identified by two parallel approaches. First, p53 was shown to complex with the Large T antigen of the SV40 virus, thus representing critical cellular target for transformation induced by this virus [START_REF] Tegtmeyer | Modification of simian virus 40 protein A[END_REF][START_REF] Tegtmeyer | Regulation of tumor antigen synthesis by simain virus 40 gene A[END_REF]. Second, it was found that the serum of mice (and later, patients) carrying tumours often contained antibodies reacting with the same 53 kDa cellular phosphoprotein [START_REF] Deleo | Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse[END_REF]. In these early years, p53 was thought of as a type of oncogene. In 1984, Jenkins and colleagues cloned the full-length mouse p53 cDNA and further experiments demonstrated that when p53 expression plasmid was transfected into rodent primary fibroblasts, it resulted in cell immortalization and transformation in presence of an activated Ras oncogene [START_REF] Eliyahu | Participation of p53 cellular tumour antigen in transformation of normal embryonic cells[END_REF]Jenkins et al., 1984a;Jenkins et al., 1984b). The notion of p53 as an oncogene received additional support from studies showing that point mutations introduced in the p53 protein sequence increased its capacity to participate in cellular transformation [START_REF] Jenkins | The cellular oncogene p53 can be activated by mutagenesis[END_REF].

Several observations were indications of major advances to follow. First, in 1984, Milner showed that different forms of p53 could be distinguished in non-dividing and dividing cells, leading to the concept of conformational changes in p53 which would later explain the effect of mutations in cancer [START_REF] Milner | Different forms of p53 detected by monoclonal antibodies in non-dividing and dividing lymphocytes[END_REF]. In the same year, Maltzman and Czyzyk provided the first evidence that p53 was induced after UV irradiation [START_REF] Maltzman | UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells[END_REF]. One year later, Benchimol and colleagues identified TP53 alterations in leukemia cell lines transformed by the Friend leukaemia virus [START_REF] Mowat | Rearrangements of the cellular p53 gene in erythroleukaemic cells transformed by Friend virus[END_REF]. In the late 1980s, the emergence of the concept of "tumour suppressor gene", largely based on studies of the retinoblastoma gene, paved the way for a paradigm shift that totally changed the understanding of p53 and of the molecular mechanisms of carcinogenesis. This shift was triggered by in vitro experiments and studies of human tumour samples. Levine and Oren hbhk (1979)(1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988). Blue: definition of p53 as a tumour suppressor and transcription factor involved in growth suppression after DNA damage (1988DNA damage ( -1994)). Green: current developments of drugs targeting p53 and prognostic studies of TP53 mutations in cancer (1995-present). Purple: projected applications of p53 research to cancer diagnosis and treatment [START_REF] Hainaut | 30 years and a long way into p53 research[END_REF].

showed that wild-type p53 cDNA clones could suppress transformation of rodent cells in culture, whereas point mutant versions of p53 were transforming them [START_REF] Eliyahu | Wild-type p53 can inhibit oncogene-mediated focus formation[END_REF][START_REF] Finlay | The p53 proto-oncogene can act as a suppressor of transformation[END_REF]. Similarly, Vogelstein and Harris, followed by many others, found that point mutations in TP53 were common in colorectal, lung, breast, liver, and many other cancers [START_REF] Baker | Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas[END_REF][START_REF] Hollstein | p53 mutations in human cancers[END_REF]. In 1990, Malkin and Srivastava showed that inherited TP53 mutations were the underlying genetic defect of Li-Fraumeni syndrome, a familial syndrome of predisposition to multiple, early cancers [START_REF] Malkin | Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms[END_REF][START_REF] Srivastava | Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome[END_REF]. All these findings led to the recognition of TP53 as "the ultimate tumoursuppressor gene" [START_REF] Oren | p53: the ultimate tumor suppressor gene?[END_REF].

In a "News and Views" article in Nature, Lane qualified p53 as "the guardian of the genome", an expression that captured its main physiological function [START_REF] Lane | CANCER -P53, GUARDIAN OF THE GENOME[END_REF]. After all of these developments, p53 eventually received the award from the Science journal as the "Molecule of the Year 1993" [START_REF] Koshland | Molecule of the year[END_REF].

Structure of human p53 gene and protein

TP53 gene and its promoters

The human TP53 gene (OMIM 191170) spans about 20 kb of DNA and is localized on the short arm of the chromosome 17 on position 17p13.1. This gene is composed of 11 exons, the first of which is non coding and localized 10 kb away from exon 2 (reviewed in [START_REF] May | Twenty years of p53 research: structural and functional aspects of the p53 protein[END_REF]). Two main promoters P1 and P2 regulate the TP53 gene. Although TP53 gene lacks a TATA box, it has been demonstrated that a region of about 300 bp upstream of the initiation site of the non-coding exon 1 (P1 promoter) contains responsive elements (REs) for AP1, NF-B, PAX and members of the HOX family, among others [START_REF] Hollstein | Massively regulated genes: the example of TP53[END_REF][START_REF] Reisman | Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1[END_REF]. The precise role of each is not clear; while HoxA5 induces p53 expression, AP1 and NF-B have been described as promoter repressors [START_REF] Raman | Compromised HOXA5 function can limit p53 expression in human breast tumours[END_REF][START_REF] Webster | Transcriptional cross talk between NF-kappaB and p53[END_REF]. A second promoter (minimal P1' promoter) is located within intron 1 [START_REF] Reisman | Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1[END_REF]. This promoter does not appear to activate p53 transcription but to control a transcript of unknown function located within the non-coding p53 intron 1 (Hp53int1). A third promoter (P2 promoter), has been recently identified within TP53 gene in a region that spans the distal part of intron 1 to the proximal part of exon 5, covering about 1.5 kb (Bourdon et al., 2005). It has been described to contain several REs in intron 4 for TFIID, SP1, AP1, NF-B and p53 itself (Bourdon et al., 2005;[START_REF] Shamsher | Analysis of intron 4 of the p53 gene in human cutaneous melanoma[END_REF]. This promoter directs the expression of a short form of p53 mRNA (p53I4 mRNA). This transcript is initiated in the middle of intron 4 and generates 133p53 isoform by translation from an AUG at position 133 (see paragraph 1.5.1). The presence of up to four p53 REs suggests an auto-regulatory loop by which p53 controls the expression of 133p53 (Bourdon et al., 2005;Marcel and Hainaut, 2009). Interestingly, both P1 and P2 promoters are relatively conserved among species and also among the members of the TP53 family.

p53 protein: domain organization and functions

The nuclear phosphoprotein p53 contains 393 amino acids and is divided in four major functional domains (Figure 6). The N-terminal region consists of an acidic transactivation domain (TAD), which is often sub-divided in two sub-domains TAD1 (residues 1-42) and TAD2 (residues 43-73), and a proline-rich region (residues 61-94). It is followed by the DNA-binding core domain that is responsible for the sequence-specific DNA binding (residues 102-292). The C-terminal region (residues 300-393) contains a flexible linker region (residues 300-318 approximately) and a tetramerization domain (residues 326-355), which contains a so called regulatory domain (Joerger and Fersht, 2007a). Here, we give a brief description of the structure and function of each p53 domain. [START_REF] Cho | Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations[END_REF].

. . . . The amino-terminal transactivation region

This region consists of an acidic TAD subdivided into two sub-domains TAD1 (residues 1-42) and TAD2 (residues 43-73), a proline-rich region (residues 61-94) and a nuclear export signal (Figure 6). The transactivation domains are pivotal to p53 function in cells. The transcriptional activity of this domain resides in protein-protein interactions. For example, the TAD is a binding site for components of the transcription machinery, namely TFIID subunits (TATA box Binding Protein (TBP), TFII40 and TFII60 (TBP-associated factors)) [START_REF] Seto | Wild-type p53 binds to the TATA-binding protein and represses transcription[END_REF][START_REF] Truant | Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein[END_REF][START_REF] Xing | p53 Stimulates TFIID-TFIIA-promoter complex assembly, and p53-T antigen complex inhibits TATA binding protein-TATA interaction[END_REF]. p53 also interacts with transcriptional coactivators such as CBP (CREB Binding Protein)/p300, an histone deacetylase which is essential for the transcriptional function of p53 [START_REF] Dornan | The proline repeat domain of p53 binds directly to the transcriptional coactivator p300 and allosterically controls DNA-dependent acetylation of p53[END_REF][START_REF] Gu | Synergistic activation of transcription by CBP and p53[END_REF][START_REF] Liu | The activation domains, the proline-rich domain, and the Cterminal basic domain in p53 are necessary for acetylation of histones on the proximal p21 promoter and interaction with p300/CREB-binding protein[END_REF] or RPA (Replication protein A) [START_REF] Dutta | Inhibition of DNA replication factor RPA by p53[END_REF]. Through this domain p53 also binds viral proteins, such as E1B, which are inhibitors of its transcriptional activity [START_REF] Yew | Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein[END_REF].

The main transactivation domain corresponds to TAD1, whereas it is still debated whether TAD2 has an independent transactivation capacity [START_REF] Attardi | Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis[END_REF][START_REF] Haupt | Induction of apoptosis in HeLa cells by trans-activation-deficient p53[END_REF].

However, TAD2 appears to modulate the transactivation of specific subsets of target genes mediated by TAD1. Within TAD1, amino acids 22 and 23 have been shown to be required for transcriptional activation of p21WAF1, a gene encoding an inhibitor of cyclin kinase that regulate cell cycle progression in G1/S and G2/M phases [START_REF] El-Deiry | WAF1, a potential mediator of p53 tumor suppression[END_REF], but are dispensable for apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. However, mutation of amino acids 53 and 54 abrogates transactivation and apoptosis capacities of p53 [START_REF] Venot | Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains[END_REF].

The transactivation domain also contains the binding site of Human double-minute 2 and 4 (Hdm2/Hdm4) (residues 17-29) (Figure 6), E3-ubiquitin ligases which mediate p53 ubiquitination in the C-terminal region, its nuclear export and its cytoplasmic degradation by the proteasome [START_REF] Haupt | Mdm2 promotes the rapid degradation of p53[END_REF][START_REF] Kubbutat | Regulation of p53 stability by Mdm2[END_REF][START_REF] Marine | Mdmx as an essential regulator of p53 activity[END_REF][START_REF] Marine | Mdm2-mediated ubiquitylation: p53 and beyond[END_REF].

The N-terminus of p53 is highly post-translationally modified [START_REF] Appella | Post-translational modifications and activation of p53 by genotoxic stresses[END_REF] (see paragraph . Multiple phosphorylation events in this region have been implicated in both the stabilization of p53 and the specificity of target transactivation [START_REF] Chao | Cell type-and promoter-specific roles of Ser18 phosphorylation in regulating p53 responses[END_REF]. Specifically, phosphorylation of residues such as Serine 15 and Threonin 18 are critical for the post-translational control of p53 interactions with protein regulating its stability such as the Hdm2/Hdm4 complex, which prevents p53 interaction with Hdm2/Hdm4 that results in both p53 stabilization and nuclear localization by altering the accessibility of the nuclear export signal (NES, residues 11-27) [START_REF] Craig | Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers[END_REF][START_REF] Zhang | A p53 amino-terminal nuclear export signal inhibited by DNA damage-induced phosphorylation[END_REF]. Phosphorylation of p53 at Serine 15 has also been shown to inhibit binding of TFIID in vitro [START_REF] Pise-Masison | Phosphorylation of p53: a novel pathway for p53 inactivation in human T-cell lymphotropic virus type 1-transformed cells[END_REF], and Ser15/Ser37 phosphorylation to correlate inversely with Hdm2 binding [START_REF] Shieh | DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2[END_REF]. CBP/p300, through its interaction with the N-terminus of p53, acts as a coactivator for p53 and increases the sequence-specific DNA-binding activity of p53 by acetylating its C-terminus [START_REF] Gu | Synergistic activation of transcription by CBP and p53[END_REF]. Phosphorylation of p53 at Serine 15 increases its ability to recruit CBP/p300 and stimulates p53-dependent transactivation [START_REF] Dumaz | Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2[END_REF][START_REF] Lambert | Phosphorylation of p53 serine 15 increases interaction with CBP[END_REF].

. . . . Polyproline domain

The proline-rich domain (residues 61-94) contains five repeats of the sequence PXXP, where P represents Proline and X, any other amino acid [START_REF] Walker | Identification of a novel p53 functional domain that is necessary for efficient growth suppression[END_REF] (Figure 6). This region is structured as a SH3 (Src homology 3) binding domain, which provides a rigid link between the N-terminus and the DNA-binding domain. It has been shown to play a role in signal transduction via its SH3 domain binding activity. Indeed, it is required for p53-mediated apoptosis and transmission of anti-proliferative signals [START_REF] Sakamuro | The polyproline region of p53 is required to activate apoptosis but not growth arrest[END_REF][START_REF] Walker | Identification of a novel p53 functional domain that is necessary for efficient growth suppression[END_REF]. Of note, this domain contains a common polymorphic site at codon 72, which results in either a Proline (P) or an Arginine (R). Interestingly, the replacement of Proline with Arginine alters one of the five PXXP motifs [START_REF] Matlashewski | Alternative splicing of human p53 transcripts[END_REF].

. . . . DNA-binding domain

The DNA-binding domain (DBD) is localized between amino acids 102 and 292 (Figure 6). Sequencing of the p53 locus from over 25,000 human tumours provided striking evidence for the importance of the sequence-specific DNA binding domain for intact tumour suppressor function [START_REF] Olivier | The IARC TP53 database: new online mutation analysis and recommendations to users[END_REF]. This region lies in the centre of the protein and specifies a globular, folded domain made of a complex of two beta sheets interconnected by large loops and helixes. The loops are kept together by the binding of a Zn 2+ ion on a cluster of 3 cysteines and one histidine, conferring to the domain a dependence upon Zinc availability and oxidation-reduction conditions for correct folding (see Review in paragraph 1.2.4 Regulation of p53 in basal conditions). Consensus sequences for p53 binding contain two copies of the inverted pentameric sequence PuPuPuCA/TT/AGPyPy (Pu: purine, Py: Pyrimidine) separated by 0 to 13 base pairs with the 4th C and 7th G being the least variant.

Sequences generally conforming to this consensus have been found in many genes that are induced by p53. In most cases, repeats of imperfect consensus sequences are found in promoters or in introns, the invariant requirement being the conservation of the CA/TA/TG motif at the centre of the consensus. This motif is present at thousand of positions in the human genome, identifying an extremely wide potential spectrum of p53-regulated genes [START_REF] Jordan | Noncanonical DNA motifs as transactivation targets by wild type and mutant p53[END_REF][START_REF] Wei | A global map of p53 transcription-factor binding sites in the human genome[END_REF].

The isolated core domain is capable of binding target sites in a cooperative manner as a tetramer, even without the aid of the C-terminal tetramerization domain [START_REF] Ko | p53: puzzle and paradigm[END_REF] and there is some evidence that protein-protein interactions between core molecules partially mediate this phenomenon [START_REF] Rippin | Recognition of DNA by p53 core domain and location of intermolecular contacts of cooperative binding[END_REF]. It has been found that this domain is able to interact with pro-apoptotic proteins such as Bcl-XL [START_REF] Mihara | p53 has a direct apoptogenic role at the mitochondria[END_REF] and also with the main regulator of p53, Hdm2 [START_REF] Shimizu | The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo[END_REF]. Diverse post-translational modifications have been described in this domain such as acetylations, that regulate apoptosis induction [START_REF] Sykes | Acetylation of the p53 DNA-binding domain regulates apoptosis induction[END_REF]; conformation-dependent phosphorylations [START_REF] Adler | Conformation-dependent phosphorylation of p53[END_REF] and Sglutathionylations during oxidative stress [START_REF] Velu | Human p53 is inhibited by glutathionylation of cysteines present in the proximal DNA-binding domain during oxidative stress[END_REF].

Interestingly, it has been suggested that p53 binds with higher affinity to cell cycle control response elements than to those involved in apoptosis, suggesting a transcriptional regulation based on the nature of response elements [START_REF] Weinberg | Comparative binding of p53 to its promoter and DNA recognition elements[END_REF].

. . . . Oligomerization domain

The oligomerization domain is located between amino acids 326-355 (Figure 6). This domain is responsible for the tetrameric state of p53. The structure of the tetramerization domain has been determined and forms a "dimer of dimers". The domain is made of a betastrand followed by a short alpha-helix. While beta-strands of two monomers assemble in a beta-sheet to form a dimer, the alpha-helices of two dimers form hydrogen bonds that bring two dimers into a tetramer (Figure 6). The oligomerization domain is preceded by a nuclear localization signal (NLS, residues 316-325) [START_REF] Shaulsky | Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis[END_REF]. A nuclear export signal (NES, residues 340-351), which may require ubiquitination for efficient usage, also resides in the tetramerization domain (residues 340-351) (Stommel et al., 1999).

. . . . C-terminal basic domain

This highly basic domain is located between the C-terminal amino acids 363-393 and is itself capable of interacting with DNA in a non-specific sequence fashion [START_REF] Foord | A DNA binding domain is contained in the C-terminus of wild type p53 protein[END_REF][START_REF] Wang | p53 domains: identification and characterization of two autonomous DNAbinding regions[END_REF] (Figure 6). Two minor nuclear localization signals (NLS) reside in this region [START_REF] Shaulsky | Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis[END_REF]) that also contains multiple ubiquitination sites [START_REF] Michael | The p53-Mdm2 module and the ubiquitin system[END_REF], the major site of sumoylation at Lysine 386 [START_REF] Rodriguez | SUMO-1 modification activates the transcriptional response of p53[END_REF] and many other stress-inducible modification sites including phosphorylation, acetylation and glycosylation [START_REF] Appella | Post-translational modifications and activation of p53 by genotoxic stresses[END_REF]. In particular, phosphorylation at the Serine 392 stabilizes p53 as a tetramer [START_REF] Warnock | Influence of tetramerisation on site-specific post-translational modifications of p53: comparison of human and murine p53 tumor suppressor protein[END_REF]. This domain also interacts with proteins such as topoisomerase II [START_REF] Cowell | Human topoisomerase IIalpha and IIbeta interact with the C-terminal region of p53[END_REF], c-Abl [START_REF] Nie | Stimulation of p53 DNA binding by c-Abl requires the p53 C terminus and tetramerization[END_REF] and HBx [START_REF] Lin | The transactivation and p53-interacting functions of hepatitis B virus X protein are mutually interfering but distinct[END_REF].

It has been proposed that the C-terminus maintains p53 in a conformationally inert "latent" state which could be converted allosterically to an activated state by different posttranslational modifications induced by several treatments or stresses [START_REF] Hupp | Allosteric activation of latent p53 tetramers[END_REF].

As a result, binding of p53 to DNA in vitro requires the neutralisation of the extreme Cterminus, either by clipping out the last 30 residues, or by interaction with antibodies that bind specifically to C-terminal epitopes [START_REF] Hupp | Regulation of the specific DNA binding function of p53[END_REF].

Regulation of p53 activation and stability

p53 is a protein with a short half-life (around 20 minutes in most cells and tissues) being almost undetectable in unstressed cells. Several types of stresses induce post-translational modifications in p53 leading to its activation and accumulation. Activated p53 can, either by gene transactivation/transrepression or by protein interactions, trigger a response that either takes care of the damage by inducing cell cycle arrest and DNA repair and/or senescence, or eliminates the affected cells from the replicative pool through induction of apoptosis, thereby preventing its expansion into a large population of malignant progeny. However, despite its rapid turnover, there is evidence that p53 may regulate a number of processes even when expressed at basal levels. For example, p53 plays a role in the control of a number of genes involved in energy metabolism, downregulating glucose usage and increasing oxidative phosphorylation by the mitochondria (see paragraph 1.2.4 Regulation of p53 in basal conditions).

p53/Hdm2 feedback loop

The E3-ubiquitin ligase Hdm2 protein is integral to the process of maintaining p53 at low levels by targeting p53 for degradation by ubiquitin-mediated proteolysis [START_REF] Haupt | Mdm2 promotes the rapid degradation of p53[END_REF][START_REF] Kubbutat | Regulation of p53 stability by Mdm2[END_REF]. Endogenous levels of Hdm2 are sufficient to regulate p53 stability, and overexpression of Hdm2 can reduce the amount of endogenous p53. On the other hand, p53 activates the expression of the Hdm2 gene in an auto-regulatory feedback loop [START_REF] Wu | The p53-mdm-2 autoregulatory feedback loop[END_REF].

The other prominent regulator of Hdm2 activity is Hdm4 (also known as Hdmx). Like Hdm2, Hdm4 is a critical negative regulator of p53. Hdm4 does not have intrinsic E3-ligase activity for p53 but instead represses p53-mediated transcriptional activation [START_REF] Marine | Mdmx as an essential regulator of p53 activity[END_REF]. Hdm4 physically interacts with p53, forming a protein complex on target gene promoters that represses p53 function by preventing access to the general transcriptional machinery [START_REF] Finch | mdmx is a negative regulator of p53 activity in vivo[END_REF][START_REF] Migliorini | Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development[END_REF][START_REF] Parant | Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53[END_REF]. Thus, the major role of Hdm4 is to repress but not degrade p53 [START_REF] Kruse | Modes of p53 regulation[END_REF]. It has also been reported that Hdm4 stimulates Hdm2-mediated ubiquitination of p53. Thus, Hdm4 acts as a stimulator, rather than as an inhibitor, of the E3 activity of Hdm2 and, at least in certain conditions, Hdm4 is actively involved in the degradation of both p53 and Hdm2 [START_REF] Linares | HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53[END_REF].

So, although the oncoprotein Hdm2 is considered to be the primary E3 ubiquitin ligase for the p53 tumour suppressor, an increasing amount of data suggests that p53 ubiquitination and degradation are more complex than once thought. The discoveries of Hdm4, HAUSP, ARF, COP1, Pirh2, and ARF-BP1 continue to uncover the multiple facets of this pathway (Figure 7). There is no question that Hdm2 plays a pivotal role in down-regulating p53 activities in numerous cellular settings. Nevertheless, growing evidence challenges the conventional view that Hdm2 is essential for p53 turnover [START_REF] Brooks | p53 ubiquitination: Mdm2 and beyond[END_REF].

Post-translational modifications

Upon stress, p53 protein is stabilized due to a range of post-translational modifications.

Post-translational modifications have been described to occur in all domains with different effects on protein stability and function (Figure 8). The number of post-translational modifications on p53 that have been reported appears endless. Mono-and poly-ubiquitination, sumoylation, methylation, neddylation, acetylation, as well as phosphorylation on multiple sites were found to have physiological relevance [START_REF] Bode | Post-translational modification of p53 in tumorigenesis[END_REF]. [START_REF] Toledo | Regulating the p53 pathway: in vitro hypotheses, in vivo veritas[END_REF].

Several transducer proteins are involved in these post-translational modifications which are stress-specific: ionizing radiations induce DNA-PK [START_REF] Morozov | DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps[END_REF] as well as ATM (ataxia telangiectasia mutated) [START_REF] Canman | Activation of the ATM kinase by ionizing radiation and phosphorylation of p53[END_REF], kinases that both phosphorylate Ser15 in the N-terminus of p53. This serine is located in the Hdm2-binding region of the protein and blocks the interaction between p53 and Hdm2, thus preventing p53 degradation.

UV induce ATM and ATR, which will activate Chk2 [START_REF] Matsuoka | Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro[END_REF] and Chk1 [START_REF] Guo | Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UVdamaged DNA in Xenopus egg extracts[END_REF] kinases, respectively, and the result in both cases is the Ser20 phosphorylation of p53 that also blocks p53-Hdm2 interaction [START_REF] Chehab | Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage[END_REF]. Spindle poisons, such as nocodazole and Taxol, induce the spindle checkpoint kinase TTK/hMps1 which phosphorylates the N-terminal domain of p53 at Thr18, and this phosphorylation disrupts the interaction with Hdm2 and abrogates Hdm2-mediated p53 ubiquitination [START_REF] Huang | TTK/hMps1 mediates the p53-dependent postmitotic checkpoint by phosphorylating p53 at Thr18[END_REF]. p53 activity may also be regulated through interactions with proteins such as the ASPP1/2, p300, and the two other members of the p53 family p63 and p73. ASPPs and p300 are cofactors of p53, stimulating the p53-dependent apoptosis or potentiating by acetylation transcriptional activating function of p53, respectively [START_REF] Dornan | The proline repeat domain of p53 binds directly to the transcriptional coactivator p300 and allosterically controls DNA-dependent acetylation of p53[END_REF][START_REF] Samuels-Lev | ASPP proteins specifically stimulate the apoptotic function of p53[END_REF]. A recent study indicates that sumoylation of p53 at K386 blocks acetylation by p300 and impairs DNA binding [START_REF] Wu | Crosstalk between sumoylation and acetylation regulates p53dependent chromatin transcription and DNA binding[END_REF]. p53 C-terminal lysines are modified by ubiquitination, acetylation, sumoylation, neddylation and methylation. Neddylation seems to inhibit transactivation, whereas sumoylation can positively or negatively affect p53 function [START_REF] Bode | Post-translational modification of p53 in tumorigenesis[END_REF]. Unlike lysines 372, 373, 381 and 382, which are acetylated by p300 and ubiquitinated by Hdm2, lysine 320 is acetylated by the p300 and CBP associated factor (PCAF) [START_REF] Di Stefano | HIPK2 contributes to PCAF-mediated p53 acetylation and selective transactivation of p21Waf1 after nonapoptotic DNA damage[END_REF][START_REF] Knights | Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate[END_REF][START_REF] Sakaguchi | DNA damage activates p53 through a phosphorylation-acetylation cascade[END_REF].

It has been also found that p53 regulates and is regulated by microRNA. For example, it was shown that miR-125b was an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response [START_REF] Le | MicroRNA-125b is a novel negative regulator of p53[END_REF]. Another study showed that miR-34a was a direct proapoptotic transcriptional target of p53 that could mediate some of p53's biological effects. They suggested that perturbation of miR-34a expression, as occuring in some human cancers, may contribute to tumourigenesis by attenuating p53-dependent apoptosis [START_REF] Raver-Shapira | Transcriptional activation of miR-34a contributes to p53-mediated apoptosis[END_REF].

Finally, several studies have shown that the activity of p53 and its ability to bind DNA was influenced by its redox status. Indeed, oxidation of p53 leads to the loss of the wild-type conformation and consequently to the loss of DNA binding activity, while reduction promotes wild-type p53 conformation and binding to DNA [START_REF] Hainaut | Redox modulation of p53 conformation and sequence-specific DNA binding in vitro[END_REF]. Redox status of p53 influences its binding to the DNA consensus sequence, however binding to unspecific sequences remains unaffected [START_REF] Parks | Redox state regulates binding of p53 to sequencespecific DNA, but not to non-specific or mismatched DNA[END_REF].

Regulation of p53 in basal conditions

Beyond the indisputable importance of p53 as a tumour suppressor, an increasing number of new roles for p53 in basal conditions (i.e. in absence of stress) have recently been reported. These functions of p53 highlight an involvement in physiological processes that include the ability to regulate basal oxidative cell metabolism, stem cell maintenance, aging and senescence, and various aspects of differentiation and development.

In the following review entitled "Redox control and interplay between p53 isoforms:

Roles in the regulation of basal p53 levels, cell fate, and senescence" and published in

Antioxidants & Redox Signaling, we propose that there are two main levels of regulation of basal p53 through: (1) p53 sensing redox changes and (2) the expression of p53 isoforms, which act as modulators of p53 functions. Indeed, there is growing evidence that p53 is a central player in the network of redox signalling mechanisms, acting in cellular antioxidant defense systems, and in metabolic pathways involved in energy production. On the other hand, p53 isoforms and in particular 40p53 and 133p53 have been demonstrated to regulate senescence and aging in cellular and animal models. An increased production of reactive oxygen species (ROS) is one of the characteristics of aging process, suggesting a role for p53 isoforms in controlling levels of p53 activation in response to endogenous oxidative stress, thus linking redox regulation, p53 isoforms and organismal senescence.

Altogether, the vision of p53 as a transcription factor that promotes a tumour suppressive response to acute stress is now enlarged by its capacities at the basal state to regulate redoxdependent physiological processes.

TP53 and the Hallmarks of Cancer

The tumour suppressor gene TP53, encoding the p53 phosphoprotein, plays a key role in the complex and powerful networks that counteract the effects of the Hallmarks of cancer.

The diverse characteristics and capacities of p53 allow it to play critical roles in each of the Hallmarks of cancer. As a result, the p53 signalling pathway can be seen as a molecular device integrating the Hallmarks processes into a coherent biological program. A direct consequence of this role is that p53 function is at least partially disabled in most of cancers [START_REF] Pfeifer | Next-generation sequencing: emerging lessons on the origins of human cancer[END_REF]. Taking into account the multiplicity of mechanisms other than mutation that may inactivate p53 functions (such as enhanced protein degradation through increased expression of cellular or viral proteins that target p53 for destruction by the proteasome), it may be considered that p53 function is inactivated, or somehow made deficient, in the vast majority of cancers. The fact that this deficiency may occur -or may show its effects-at any step in the carcinogenic process from pre-cancer condition to highly aggressive, metastatic lesions, makes it very complex to address the prognostic or predictive significance of measurable p53 alterations. Furthermore, such a sweeping role for a single molecule in such a mechanistically complex, multi-factorial disease raises the suspicion that p53 does not act as a frontline driver but as a kind of facilitator of carcinogenesis. In a recent review, P. Hainaut has developed the concept that the uniqueness of p53 as a target for inactivation in cancer may be due to the fact that this protein interferes in each of the ten Hallmarks of cancer processes, supporting the view that loss of p53 function not only make cells permissive to the acquisition of Hallmarks capabilities but also removes a critical brake that prevents the expression of Hallmarks capabilities. According to this model, p53 is not a specific component of any particular Hallmarks process but rather a molecular device that organizes the Hallmarks processes into a coherent biological program (Figure 9). In the section below, I am discussing this model based on the review by P. Hainaut [START_REF] Dichtel-Danjoy | Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation[END_REF]. This model represents p53 as a central and multifunctional molecular device controlling each of the Hallmarks proposed by [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. Adapted from [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF] and [START_REF] Dichtel-Danjoy | Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation[END_REF].

Repressing proliferative signalling

The most fundamental characteristic of cancer cells is their capability to sustain chronic proliferation. Unlike normal tissues, cancer cells can deregulate proliferative signalling through the production of growth-promoting signals, and through the pathways that regulate the progression through the cell cycle. The activation of the p53 protein and its network of genes sets in motion an elaborate process, which connect the p53 pathway to other signal transduction pathways to coordinate the cellular signals for growth and division. For instance, p53 regulates the transcription of PTEN (phosphatase and tensin homolog), the second most frequently mutated gene in human cancer after p53 [START_REF] Bostrom | Mutation of the PTEN (MMAC1) tumor suppressor gene in a subset of glioblastomas but not in meningiomas with loss of chromosome arm 10q[END_REF][START_REF] Li | PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer[END_REF][START_REF] Suzuki | Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues[END_REF]. PTEN protein phosphatase can dephosphorylate protein substrates on serine, threonine and tyrosine residues [START_REF] Myers | The lipid phosphatase activity of PTEN is critical for its tumor supressor function[END_REF]. Induction of PTEN suppresses cell survival and proliferation by antagonizing the phosphoinositol-3-kinase (PI3K)/AKT signalling pathway [START_REF] Maehama | The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate[END_REF].

In response to aberrant growth or oncogenic stress, p14 ARF protein is expressed. This protein is encoded by the alternative open reading frame of the INK4a/CDKN2a locus located on chromosome 9p21, which also expresses the cyclin kinase inhibitor p16 [START_REF] Kamijo | Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF[END_REF]. p14 ARF , an activator of p53, interacts with Hdm2 and blocks its shuttling between the nucleus and cytoplasm via the nucleolus [START_REF] Tao | P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2[END_REF]. Sequestration of Hdm2 in the nucleolus prevents it from binding and degrading p53, thus resulting in the accumulation and activation of p53.

The major effect of activated p53 is to block the cell cycle. Growth arrest is a reversible halt of the cell cycle that occurs at either the G1/S or G2/M barrier. Progress through cell cycle is thought to be mediated by sequential phosphorylation of the retinoblastoma (Rb) protein by G1 cyclin-dependent kinases, resulting in the release of active E2F transcription factor, which activates genes required for S phase transition [START_REF] Kato | Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4[END_REF][START_REF] Mudryj | Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A[END_REF]. The critical mediator of the p53-mediated G1 arrest response is p21WAF1 [START_REF] El-Deiry | WAF1, a potential mediator of p53 tumor suppression[END_REF]. Following activation by DNA damage, p53 induces the expression of CDKN1A (encoding p21WAF1), which is able to inhibit cdk2 and cyclin D/cdk4 complexes [START_REF] Labaer | New functional activities for the p21 family of CDK inhibitors[END_REF].

The second checkpoint corresponds to the G2/M phase. p21WAF1 also plays a role in mediating cell cycle arrest in the G2/M phase as it is induced after p53 over-expression [START_REF] Agarwal | p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts[END_REF]. Another p53 target gene involved in the G2 arrest is GADD45, which interacts with cdc2 and inhibits its kinase activity, causing the dissociation of the cyclin B1/cdc2 complex [START_REF] Wang | GADD45 induction of a G2/M cell cycle checkpoint[END_REF]. Moreover, through induction of 14-3-3 , p53 can also induce G2/M arrest in cells that have sustained DNA damage [START_REF] Hermeking | 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression[END_REF]. The 14-3-3 kinase phosphorylates cdc25, preventing the activation of the cyclin B1/cdc2 complex [START_REF] Peng | Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216[END_REF].

The S-phase checkpoint involves three different modes of control: the replication control, the intra-S-phase checkpoint and the S/M checkpoint. p53 has been proposed to be responsible of ensuring that cells do not enter mitosis with unreplicated DNA [START_REF] Taylor | p53 inhibits entry into mitosis when DNA synthesis is blocked[END_REF]. In addition, p53 has been shown to play a key role in the maintenance of the S-phase arrest induced by cdc7 down-regulation [START_REF] Montagnoli | Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells[END_REF], as well as in promoting chromatin condensation, a hallmark of mammalian cells that enter mitosis before completing DNA replication [START_REF] Nghiem | ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint[END_REF]. Finally, the spindle assembly checkpoint ensures that cells do not enter anaphase until all chromosomes are aligned and attached to the microtubules of the mitotic spindle. Initially it was proposed that p53 was an important component of a spindle checkpoint in mice, as it is required during normal cell division [START_REF] Cross | A p53-dependent mouse spindle checkpoint[END_REF]. However, more recent evidence revealed that expression of p53 occurred after cells had exited mitosis and progressed to a G1-like state and that p53 plays a critical role in preventing aneuploidy by blocking endoreduplication of tetraploid cells that result from mitotic cells [START_REF] Vogel | Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy[END_REF].

Enhancing the effects of growth suppressors

Besides the hallmark capacity of inducing and sustaining growth-promoting signals, cancer cells must also evade powerful programs that negatively regulate cell proliferation.

Many of these programs depend on tumour suppressor genes actions.

The growth suppressor CDKN1A, encoding the p21WAF1 cyclin kinase inhibitor, was the first one to be identified as a direct transcriptional target of p53 and is a central effector of the growth suppressive effect of p53 in response to intracellular stress signals such as DNA damage, levels of nucleotide pools or availability of oxygen [START_REF] El-Deiry | WAF1, a potential mediator of p53 tumor suppression[END_REF][START_REF] Roninson | Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts[END_REF]. However, the effects of p21WAF1 knockout in mice and its expression patterns in human cancer are consistent with a role for p21WAF1 as both a tumour suppressor and an oncogene [START_REF] Roninson | Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts[END_REF].

The cell-to-cell contacts formed by propagating cells in culture operate to suppress further cell proliferation. This "contact inhibition" is abrogated in a variety of cancer cells in culture, suggesting the existence of a comparable mechanism in vivo to maintain a normal tissue homeostasis. The mechanistic basis of contact inhibition is beginning to emerge. Merlin (also known as schwannomin), the product of the NF2 (neurofibromatosis type 2) tumour suppressor gene, coordinates the processes of adherens junction stabilization and negative regulation of epidermal growth factor receptor (EGFR) signalling by restraining the EGFR into a membrane compartment from which it can neither signal nor be internalized [START_REF] Curto | Contact-dependent inhibition of EGFR signaling by Nf2/Merlin[END_REF]. Merlin also mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane [START_REF] Okada | Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane[END_REF]. p53 stability and activity is increased by Merlin, through the inhibition of the Hdm2-mediated degradation of p53. Moreover, overexpression of Merlin leads to p53-dependent apoptosis in response to serum starvation or to a chemotherapeutic agent [START_REF] Kim | Merlin neutralizes the inhibitory effect of Mdm2 on p53[END_REF].

Another mechanism of contact inhibition involves p53 and the tumour suppressor LKB1, which organizes epithelial structure and polarity, and helps maintain tissue integrity. LKB1 is genetically inactivated in patients with Peutz-Jeghers syndrome and somatically mutated in a variety of cancers. Over-expression of LKB1 inhibits cell proliferation through the degradation of the c-myc oncogene protein [START_REF] Liang | Overexpression of the LKB1 gene inhibits lung carcinoma cell proliferation partly through degradation of c-myc protein[END_REF]. In contrast, knockdown of LKB1 accelerates cell cycle progression through G1/S checkpoint and enhanced cell proliferation mediated at least in part by a decline of p53 and p16 growth suppressor pathways [START_REF] Liang | Endogenous LKB1 knockdown accelerates G(1)/S transition through p53 and p16 pathways[END_REF].

The pleiotropic cytokine, transforming growth factor-(TGF-), is one of the main regulators of anti-proliferative pathways. TGF-has received much attention as a major inducer of Epithelium to Mesenchyme Transition (EMT) during embryogenesis, cancer progression and fibrosis [START_REF] Wendt | Mechanisms of the epithelialmesenchymal transition by TGF-beta[END_REF][START_REF] Xu | TGF-beta-induced epithelial to mesenchymal transition[END_REF]. TGF-ligands bind to cognate serine/threonine kinase receptors leading, intracellularly, to phosphorylation and activation of the Smad family of signal transducers [START_REF] Massague | How cells read TGF-beta signals[END_REF]. A recent study has identified p53 as an in vivo relevant partner of Smad2 in the activation of multiple TGF-target genes. Smad and p53 protein complexes bind on a target promoter and synergistically activate TGFinduced transcription [START_REF] Cordenonsi | Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads[END_REF]. Another p53-dependent mechanism involves the SnoN proto-oncogene product, which negatively regulates TGF-signalling through its interactions with Smad2 and Smad3 complexes. The tumour suppressor role of SnoN results from its interaction with the promyelocytic leukaemia (PML) protein and the accumulation of SnoN in PML nuclear bodies, thus allowing SnoN to stabilize p53 and induce premature senescence (Figure 10) [START_REF] Lamouille | Oncogene and tumour suppressor: the two faces of SnoN[END_REF][START_REF] Pan | SnoN functions as a tumour suppressor by inducing premature senescence[END_REF].

Figure 10. Oncogenic and anti-oncogenic roles of SnoN

In the nucleus, SnoN can bind the Smad complex with transcription factors (TF) and represses TGF-induced inhibition of proliferation, thus acting as an oncogene. Independently of the Smad interactions, SnoN can interact with the promyelocytic leukaemia (PML) protein in PML nuclear bodies (PML-NB), resulting in stabilization of p53. This leads to premature senescence and defines an anti-oncogenic role for SnoN [START_REF] Lamouille | Oncogene and tumour suppressor: the two faces of SnoN[END_REF].

Sensitizing cells to death

In response to exposure to radiation or various chemotherapeutic drugs that induce double strand DNA breaks, p53 initiates an apoptotic program in a number of cell types. p53 controls the transcription of several classes of genes involved at different levels in apoptosis signalling. The execution of apoptosis involves two main pathways: the death receptor pathway (extrinsic) triggered by so called death receptors (DR) such as CD95/Fas and Killer/DR5 [START_REF] Danial | Cell death: critical control points[END_REF]; and the mitochondrial pathway (intrinsic) where many components have been found to be p53 target genes as described below.

The intrinsic pathway is also termed the "mitochondrial pathway" as the mitochondria acts as the centrepiece (Figure 11A). APAF-1, the key component of the apoptosome, required for pro-caspase-9 recruitment and activation, has been reported to be a primary p53 response gene [START_REF] Moroni | Apaf-1 is a transcriptional target for E2F and p53[END_REF]. Also relevant for the pro-apoptotic p53 response appears to be its ability to regulate Bcl-2 family members, such as Bcl-2-associated protein X (Bax), and the BH3-only proteins: p53-upregulated modulator of apoptosis (Puma), Noxa and Bcl-2interacting domain death agonist (Bid); or directly by transrepressing Bcl2 [START_REF] Hoffman | Transcriptional repression of the anti-apoptotic survivin gene by wild type p53[END_REF][START_REF] Sugars | A minimal Bcl-x promoter is activated by Brn-3a and repressed by p53[END_REF]. BH3-only proteins most likely trigger apoptosis by engaging multiple Bcl-2 pro-survival proteins in a stimulus-and possibly also cell-type-dependent manner, leading to Bax and/or Bcl-2 antagonist/killer (Bak) oligomerization, mitochondrial outer membrane permeabilization accompanied by cytochrome c release and subsequent cell death [START_REF] Mikhailov | Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release[END_REF][START_REF] Willis | Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3only proteins[END_REF].

As for APAF-1, p53-mediated induction of Bax is not critical for apoptosis initiation but may rather serve to amplify apoptosis signalling. However, there is solid evidence that induction of BH3-only proteins is essential for p53-induced apoptosis. For example, the BH3only protein Bid, which is considered to amplify apoptosis signalling particularly by linking the DR pathway to the intrinsic pathway, was reported to be induced transcriptionally by p53 in response to -irradiation [START_REF] Sax | BID regulation by p53 contributes to chemosensitivity[END_REF]. Another example is Puma, whose mRNA and protein were both reported to be induced, in normal as well as in malignant cells, following DNA damage or in response to oncogenic stress; and this induction in response to DNA damage strictly depends on p53 [START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF]. Moreover, Noxa (whose promoter region contains a functional p53-binding site [START_REF] Oda | Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53induced apoptosis[END_REF]) and Puma appear to play an overlapping role in mediating cell death in response to DNA damage caused by cytotoxic drugs, such as etoposide or -irradiation [START_REF] Villunger | p53-and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa[END_REF].

Induction of the extrinsic pathway, also known as the death receptor pathway, occurs following p53 transactivation of genes encoding Fas (also known as Apo-1 or CD95), TNF receptor-1 (TNFR-1), DR5/Killer and TNF-related apoptosis-inducing ligand (TRAIL) in the cell plasma membrane, leading to activation of pro-caspase 8 and apoptosis [START_REF] Wu | KILLER/DR5, a novel DNA-damage inducible death receptor gene, links the p53-tumor suppressor to caspase activation and apoptotic death[END_REF] (Figure 11B). p53 also represses the pathway regulating the bioavailability of survival factors (Figure 11C). It induces the expression of insulin-like growth factor binding protein 3 (IGF-BP3), which encodes a protein that binds and neutralizes IGF-1 and IGF2, preventing Bcl-2 and Bcl-XL activation [START_REF] Buckbinder | Induction of the growth inhibitor IGF-binding protein 3 by p53[END_REF][START_REF] Butt | The IGF axis and programmed cell death[END_REF][START_REF] Kooijman | Regulation of apoptosis by insulin-like growth factor (IGF)-I[END_REF]. Autophagy is induced in response to various stress stimuli, including starvation, trophic factor deprivation, hypoxia, endoplasmic reticulum stress and oxidative stress [START_REF] Levine | p53: The Janus of autophagy?[END_REF]. Autophagy has diverse physiological functions, including stress adaptation, development, immunity and protection against neurodegeneration. It can also function as a tumour suppressor or cell-survival pathway as demonstrated by the fact that loss of autophagy genes leads to increased DNA damage, chromosomal instability and deregulated control of cell growth. This indicates a potential overlap in tumour suppressor-related autophagy effects and p53 activities. However, the p53 tumour suppressor, previously thought to positively regulate autophagy, may also inhibit it.

For example, genotoxic stresses caused by DNA damaging agents induce p53-dependent autophagy [START_REF] Feng | The coordinate regulation of the p53 and mTOR pathways in cells[END_REF][START_REF] Zeng | A novel role for DNA mismatch repair and the autophagic processing of chemotherapy drugs in human tumor cells[END_REF]. Similarly, forced expression of p14ARF or oncogenic activation of p53, induce autophagy in human cancer cells [START_REF] Abida | p53-Dependent and p53-independent activation of autophagy by ARF[END_REF]. The mechanisms of p53-dependent induction of autophagy are still incompletely understood, but are thought to involve both transcription-independent functions (AMPK activation), as well as transcription-dependent functions (upregulation of mTOR inhibitors, PTEN and TSC1, or the p53-regulated autophagy and cell death gene, DRAM) [START_REF] Crighton | DRAM links autophagy to p53 and programmed cell death[END_REF][START_REF] Feng | The coordinate regulation of the p53 and mTOR pathways in cells[END_REF].

Paradoxically, elevated autophagy, often associated with the tumour microenvironment and/or treatment with cytotoxic agents, can also increase tumour cell survival and in this sense, autophagy is considered to be pro-oncogenic. The mysteries underlying p53 regulation of autophagy extend beyond the question of whether p53-mediated autophagy is pro-death or pro-survival. Tasdemir et al. directly challenge the notion that p53 is a positive regulator of autophagy by showing that chemical inhibition of p53 increases autophagy in both normal and transformed cells in a transcription-independent way [START_REF] Tasdemir | A dual role of p53 in the control of autophagy[END_REF].

Suppressing replicative immortality through senescence

The senescence phenomenon was first described by Hayflick and Moorhead in human fibroblasts [START_REF] Hayflick | The serial cultivation of human diploid cell strains[END_REF]. When maintained in culture, normal cells undergo a phase of expansion through division until they enter senescence, which is characterized by large cell size, flat vacuolated morphology, inability to synthesize DNA, and the presence of the senescence-associated -galactosidase (SA--gal) marker [START_REF] Dimri | What has senescence got to do with cancer?[END_REF]. The telomeres, located at the end of chromosomes and composed of tandem repeats of 6 nucleotides (TTAGGG), determine how many rounds of division a cell can achieve before reaching replicative senescence [START_REF] Harley | Telomeres shorten during ageing of human fibroblasts[END_REF][START_REF] Podlevsky | It all comes together at the ends: telomerase structure, function, and biogenesis[END_REF]. Maintenance of telomere stability is required for cells to escape from replicative senescence and proliferate indefinitely. Telomerase is a cellular ribonucleoprotein reverse transcriptase which stabilizes telomere length by adding telomere repeats to the ends of the chromosomes, thus compensating for the continued erosion of telomeres. This enzymatic activity is almost absent in normal, post-mitotic cells but is expressed at high levels in 90% of immortalized and transformed human cells [START_REF] Shay | Telomeres and telomerase: implications for cancer and aging[END_REF].

Recently, it has been shown that even brief reactivation of endogenous p53 in p53deficient tumours can produce complete tumour regressions through induction of senescence [START_REF] Ventura | Restoration of p53 function leads to tumour regression in vivo[END_REF][START_REF] Xue | Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas[END_REF]. In these studies, the primary response to p53 reactivation was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. This indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.

p53-dependent senescence may also be induced in cells with dysfunctional telomeres. Indeed, it was shown that p53 abrogation rescued the small size phenotype and the functionality of epidermal stem cells of telomerase-deficient mice with dysfunctional telomeres. This suggested the existence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres that is actively limiting their contribution to tissue regeneration, thereby impinging on tissue fitness [START_REF] Flores | A p53-dependent response limits epidermal stem cell functionality and organismal size in mice with short telomeres[END_REF].

Exerting anti-angiogenic effects

During tumour formation, a disruption in the balance between productions of pro-and anti-angiogenic molecules occurs allowing continued proliferation and growth of tumour cells [START_REF] Bergers | Tumorigenesis and the angiogenic switch[END_REF].

p53 is able to block this process either by activating anti-angiogenic molecules or by repressing pro-angiogenic ones. For example, p53 transcriptionally activates the alpha (II) collagen prolyl-4-hydroxylase (alpha(II)PH) gene, resulting in the extracellular release of anti-angiogenic fragments of collagen type 4 and 18 [START_REF] Teodoro | p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase[END_REF]. In contrast, p53 significantly inhibited the transcription of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), which is considered to be the most important directly acting angiogenic protein, by regulating the transcriptional activity of Sp1 and also by downregulating the Src kinase activity [START_REF] Pal | Central role of p53 on regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) expression in mammary carcinoma[END_REF]. In addition, p53 inhibits angiogenesis by inducing the production of Arresten, a collagen-derived anti-angiogenic factor (CDAF) that is processed from α1 collagen IV (COL4A1). The p53 protein directly activates the transcription of COL4A1 and also increases the metalloproteinase-mediated release of Arresten, thus controlling the production of an important anti-angiogenic factor [START_REF] Assadian | p53 inhibits angiogenesis by inducing the production of Arresten[END_REF].

Repressing invasion and metastasis

Another tumour suppressive activity of p53, which is still poorly understood, is its ability to modulate cell migration. It has been shown that p53 inhibits cdc42-induced filipodia formation [START_REF] Gadea | Regulation of Cdc42-mediated morphological effects: a novel function for p53[END_REF]. In mouse embryonic fibroblast cells, absence of p53, or overexpression of its dominant negative isoform 133p53, induced rounded blebbing movements through over-activation of RhoA and ROCK (Rho-associated coil-containing protein kinase)-dependent translocation of RhoA to membrane blebbing structures. Thus, loss of p53 (as well as the expression of one p53 isoform, 133p53 ) increased cell motility and may thus contribute to tumour invasiveness [START_REF] Gadea | Loss of p53 promotes RhoA-ROCKdependent cell migration and invasion in 3D matrices[END_REF].

Another example is the transrepression of focal adhesion kinase (FAK) gene by p53 [START_REF] Golubovskaya | p53 regulates FAK expression in human tumor cells[END_REF]. FAK is a non-receptor tyrosine kinase localized at the contact points between cells and their substratum [START_REF] Burridge | Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly[END_REF]. FAK associates with integrin receptors and recruits other molecules to the site of this interaction thus forming a signalling complex that transmits signals from the extracellular matrix to the cell cytoskeleton [START_REF] Mitra | Integrin-regulated FAK-Src signaling in normal and cancer cells[END_REF]. FAK has been shown to play a role in proliferation and migration, but also in survival and angiogenesis, and has been proposed as a new potential therapeutic target in cancer [START_REF] Mclean | The role of focal-adhesion kinase in cancer -a new therapeutic opportunity[END_REF][START_REF] Van Nimwegen | Focal adhesion kinase: a potential target in cancer therapy[END_REF].

Analysis of human FAK gene promoter has identified p53 binding sites [START_REF] Golubovskaya | Cloning and characterization of the promoter region of human focal adhesion kinase gene: nuclear factor kappa B and p53 binding sites[END_REF] and more recently, it has been found that DNA-damaged-induced p53 showed an increased binding to FAK promoter resulting in the repression of FAK mRNA and protein expression [START_REF] Golubovskaya | p53 regulates FAK expression in human tumor cells[END_REF].

Another important connection between p53 and metastasis involves the Twist1 protein, a regulator of embryogenesis. Twist1 has been shown to induce EMT and is over-expressed in a large fraction of human cancers [START_REF] Ansieau | Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence[END_REF]. A common cancer-derived mutant p53 protein, p.R175H, up-regulates Twist1 expression in several cancer cell lines, suggesting that increased Twist1 might occur in cancer cells as the consequence of gain-of-function mutations in TP53 [START_REF] Kogan-Sakin | Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells[END_REF].

Promoting genetic and genomic stability

Among the main properties of p53 as a regulator of cell-cycle progression and apoptosis, it has been rapidly identified as a major candidate in maintaining genetic stability, a property that earned it the appellation of "guardian of the genome". Indeed, by promoting cell cycle arrest and DNA repair in cells with genetic damage, p53 protects cells from acquiring further DNA defects that may initiate cancer or drive its progression.

Exposure to UV light, reactive oxygen species or to some drugs can lead to DNA damage such as modifications of the bases, DNA strand breaks or adducts formation. These damages are repaired by specific mechanisms, including nucleotide excision repair (NER), base exclusion repair (BER) and homologous recombination (HR) after double strand breaks. p53 has been shown to play a role in each of these mechanisms.

The NER pathway is the primary mechanism in cells for the removal of helix-distorting, replication-blocking DNA adducts induced by exogenous agents such as UV radiation and a variety of genotoxic chemicals [START_REF] Sancar | Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints[END_REF]. NER can be regulated by transcriptional and post-transcriptional control of the XPA (Xeroderma pigmentosum group A) protein [START_REF] Fan | SIRT1 regulates UV-induced DNA repair through deacetylating XPA[END_REF][START_REF] Kang | Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase[END_REF][START_REF] Kang | Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein[END_REF]. Functionally, XPA is believed to play roles in verifying DNA damage, stabilizing repair intermediates, and recruiting other NER factors to the damaged site [START_REF] Batty | Damage recognition in nucleotide excision repair of DNA[END_REF][START_REF] Yang | Specific and efficient binding of xeroderma pigmentosum complementation group A to double-strand/single-strand DNA junctions with 3'-and/or 5'-ssDNA branches[END_REF]. Both ATM and ATR (ATM and RAD3-related) are protein kinases belonging to the phosphoinositide 3-kinase-like kinase (PIKK) family. These pathways are comprised of a series of DNA damage sensors, signal mediators and transducers, and downstream effectors [START_REF] Branzei | Regulation of DNA repair throughout the cell cycle[END_REF][START_REF] Sancar | Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints[END_REF]. p53, Checkpoint kinase-1 (Chk1), and MAPKAP Kinase-2 (MK2) are the three main downstream checkpoint proteins that can be directly or indirectly activated by ATR following UV irradiation [START_REF] Helt | Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target specificities in response to different forms of DNA damage[END_REF][START_REF] Reinhardt | p53-deficient cells rely on ATMand ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage[END_REF]. The DNA damageinduced response of XPA nuclear import is significantly reduced in p53-deficient cells, suggesting that the ATR/p53-dependent checkpoint is required for the UV-induced XPA nuclear import and the removal of UV-DNA damage (Li et al., 2011a;[START_REF] Li | Differential DNA damage responses in p53 proficient and deficient cells: cisplatin-induced nuclear import of XPA is independent of ATR checkpoint in p53deficient lung cancer cells[END_REF]. BER is the main pathway responsible for the repair of DNA damage caused by hydrolysis, oxygen free radicals, and simple alkylating agents. BER is initiated by the hydrolysis of the N-glycosylic bond linking a modified base to the deoxyribose, which excises the damaged base. As a result of glycosylase activity, apurinic or apyrimidinic (AP) sites are generated.

The removal of AP sites is initiated by a second class of BER enzymes, the apurinic/apyrimidinic endonuclease redox factor 1 (APE1/Ref1). The resulting AP sites are cleaved by the AP endonuclease APE1/Ref1 and the resulting single-strands are processed by either short-patch (where a single nucleotide is replaced) or long-patch BER [START_REF] Gatz | p53 in recombination and repair[END_REF][START_REF] Seeberg | The base excision repair pathway[END_REF]. Since 1999, Rotter's group has provided accumulating evidence suggesting a direct involvement of p53 in BER [START_REF] Offer | Structural and functional involvement of p53 in BER in vitro and in vivo[END_REF][START_REF] Offer | Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery[END_REF]. In these studies, an augmented BER activity was observed when p53 was over-expressed in cultured cells. Meanwhile, p53 was shown to interact with polymerase indicating a direct role of p53 in BER [START_REF] Zhou | A role for p53 in base excision repair[END_REF]. Furthermore, APE1/Ref-1 also binds to p53, contributes to redox-dependent p53 protein stabilization, and enhances both DNA binding and p53 transactivation [START_REF] Gaiddon | Ref-1 regulates the transactivation and proapoptotic functions of p53 in vivo[END_REF][START_REF] Jayaraman | Identification of redox/repair protein Ref-1 as a potent activator of p53[END_REF][START_REF] Seemann | Roles of thioredoxin reductase 1 and APE/Ref-1 in the control of basal p53 stability and activity[END_REF]. The p53 protein contributes to BER at several levels, both in a positive and in a negative fashion. Indeed, the initial step in BER is also subject to negative transcriptional regulation by p53. Following exposure to nitric oxide (NO) p53 downregulates 3-methyl adenine (3-MeAde) DNA glycosylase via transcriptional trans-repression [START_REF] Zurer | The role of p53 in base excision repair following genotoxic stress[END_REF]. Moreover, p53 induces PPM1D encoding protein phosphatase 1D (or Wip1), which interacts with Uracil DNA glycosylase 2 (Ung2) and suppresses BER. PPM1D, in turn, inhibits p53 activity through inactivating dephosphorylation of the p38 MAP kinase (Lu et al., 2004a;Lu et al., 2004b). In analogy to the Hdm2 feedback loop, these negative regulatory mechanisms executed by p53 may serve to return DNA repair to the deactivated state. The general message emerging from these observations is that p53 may enhance BER as part of its genome-stabilizing, tumour-suppressive activities. However, after activation of p53 in response to stress, high levels of p53 may repress BER to return DNA repair to the deactivated state and therefore participate to maintenance of basal DNA damage capacity. In conditions where p53 becomes activated to very high levels, such as for example in response p53 target genes has linked p53 to MMR [START_REF] Chen | Identification of the mismatch repair genes PMS2 and MLH1 as p53 target genes by using serial analysis of binding elements[END_REF][START_REF] Scherer | p53 and c-Jun functionally synergize in the regulation of the DNA repair gene hMSH2 in response to UV[END_REF][START_REF] Warnick | Identification of a p53 response element in the promoter region of the hMSH2 gene required for expression in A2780 ovarian cancer cells[END_REF]. Additionally, the p53 target genes MSH2, MLH1 and PMS2 are able to signal cell cycle arrest and apoptosis after certain types of DNA damage via p53 or its homologue p73 (Stojic et al., 2004).

p53 appears to be a major component of the DNA damage response pathway.

Nevertheless, there is only limited evidence that impaired p53 function leads to an increased mutator phenotype, which is predisposing to different types of cancer, both familial and sporadic. Spontaneous tumours in a mouse model of Li-Fraumeni Syndrome (LFS) (TRP53 +/- murine model) show only modest increase in mutation loads [START_REF] Hill | Most spontaneous tumors in a mouse model of Li-Fraumeni syndrome do not have a mutator phenotype[END_REF]. In humans, there is a highly significant increase in copy number variations among carriers of germline TP53 mutations with a familial cancer history [START_REF] Shlien | Excessive genomic DNA copy number variation in the Li-Fraumeni cancer predisposition syndrome[END_REF]. However, as discussed in Palmero et al., TP53 mutation carriers do not appear to be hyper-sensitive to mutagens although radio-sensitivity has been demonstrated in cultured cells and observed in some LFS patients who received radiation-based treatments [START_REF] Palmero | Tumor protein 53 mutations and inherited cancer: beyond Li-Fraumeni syndrome[END_REF]. ), and peroxynitrite (ONOO -) (de [START_REF] De Moraes | Cross-talks between cyclooxygenase-2 and tumor suppressor protein p53: Balancing life and death during inflammatory stress and carcinogenesis[END_REF]. It was in 1863 that Rudolf Virchow noted the presence of large amount of leucocytes in neoplastic tissues and made a connection between inflammation and cancer. He suggested that the "lymphoreticular infiltrate" reflected the origin of cancer at sites of chronic inflammation [START_REF] Balkwill | Inflammation and cancer: back to Virchow?[END_REF]. Indeed, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma, and DNA-damage-promoting agents, turns these protective responses into a cancer-promoting process [START_REF] Coussens | Inflammation and cancer[END_REF][START_REF] Kundu | Inflammation: gearing the journey to cancer[END_REF][START_REF] Wu | Inflammation: a driving force speeds cancer metastasis[END_REF]. Many aspects of chronic inflammation involve p53 in a direct or indirect way [START_REF] Kamp | Chronic inflammation and cancer: the role of the mitochondria[END_REF]. First, following DNA damage caused by generated ROS and RNS, p53 is activated and induces a growth suppressive response that contributes to preserve cell viability and genetic integrity [START_REF] Hafsi | Redox Control and Interplay Between p53 Isoforms: Roles in the Regulation of Basal p53 Levels, Cell Fate, and Senescence[END_REF]. Second, p53 regulates the expression of genes encoding enzymes involved in the detoxification and protection against ROS. Among these genes, p53 has been shown to up-regulate cyclooxygenase 2 (COX2) (de [START_REF] De Moraes | Cross-talks between cyclooxygenase-2 and tumor suppressor protein p53: Balancing life and death during inflammatory stress and carcinogenesis[END_REF], down-regulate mitochondrial superoxide dismutase 2 (SOD2) [START_REF] Hussain | p53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis[END_REF], and activate glutathione peroxidase (GPX1) [START_REF] Tan | Transcriptional activation of the human glutathione peroxidase promoter by p53[END_REF] and aldehyde dehydrogenase 4 A1 (ALDH4A1) [START_REF] Yoon | Identification of ALDH4 as a p53-inducible gene and its protective role in cellular stresses[END_REF]. Third, p53 represses the expression of inducible nitric oxide synthase 2 (NOS2), protecting cells against excess damage by NO and derivatives [START_REF] Ambs | Up-regulation of inducible nitric oxide synthase expression in cancer-prone p53 knockout mice[END_REF][START_REF] Forrester | Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53[END_REF]. It has also been demonstrated that TP53 mutation is correlated with increased expression of NOS2 and enhanced damage by nitrogen species in cancers developing in a context of chronic inflammation [START_REF] Ambs | Relationship between p53 mutations and inducible nitric oxide synthase expression in human colorectal cancer[END_REF][START_REF] Vaninetti | Inducible nitric oxide synthase, nitrotyrosine and p53 mutations in the molecular pathogenesis of Barrett's esophagus and esophageal adenocarcinoma[END_REF]. Fourth, p53 entertains a complex network of functional cross-talks with NF-B, the main transcription factor involved in the regulation of inflammatory responses. For example, p53 and NF-B cooperate in up-regulating COX2 expression, which provides a survival mechanism in chronically inflamed tissues [START_REF] Benoit | Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB[END_REF][START_REF] De Moraes | Cross-talks between cyclooxygenase-2 and tumor suppressor protein p53: Balancing life and death during inflammatory stress and carcinogenesis[END_REF]. Recently, Liu and colleagues demonstrated the involvement of p53 in the responses of inflammatory cells to lipopolysaccharide (LPS). Culture of mice LPS-stimulated neutrophils and macrophages with nutlin-3a, a specific inducer of p53 stabilization, attenuated NF-κB DNA-binding activity and production of proinflammatory cytokines [START_REF] Liu | p53 Attenuates lipopolysaccharide-induced NF-kappaB activation and acute lung injury[END_REF]. In addition, a novel mechanism of p53 in the repression of NF-B by the glucocorticoid receptor has been described where p53 loss severely impaired glucocorticoid receptor target genes downstream NF-B signalling in a mouse model of LPS shock [START_REF] Murphy | Tumor suppressor protein (p)53, is a regulator of NF-kappaB repression by the glucocorticoid receptor[END_REF]. The functional cross-talk between p53 and NF-B may play an important role in regulating organismal senescence and life span: it seems that the efficiency of p53 signalling declines during aging whereas that of NF-B is clearly enhanced.

Controlling tumour-promoting inflammation

Regulating cell bioenergetics

Cancer cells consume large quantities of glucose and primarily use glycolysis for ATP production, even in the presence of adequate oxygen. This metabolic signature (aerobic glycolysis or the Warburg effect) enables cancer cells to direct glucose to biosynthesis, supporting their rapid growth and proliferation (Warburg, 1956a, b;[START_REF] Warburg | THE METABOLISM OF TUMORS IN THE BODY[END_REF].

One of the most spectacular developments in the understanding of p53 functions is the discovery that it plays a fundamental role in cell bioenergetics. Loss of p53 activity may be pivotal for the Warburg effect [START_REF] Gottlieb | p53 regulation of metabolic pathways[END_REF][START_REF] Hafsi | Redox Control and Interplay Between p53 Isoforms: Roles in the Regulation of Basal p53 Levels, Cell Fate, and Senescence[END_REF]). p53 appears to favour oxidative metabolism against glycolysis [START_REF] Cheung | The role of p53 in glucose metabolism[END_REF]. This includes down-regulation of glucose transporters at the plasma membrane through direct inhibition of GLUT-1 and GLUT-4 (Schwartzenberg- [START_REF] Schwartzenberg-Bar-Yoseph | The tumor suppressor p53 down-regulates glucose transporters GLUT1 and GLUT4 gene expression[END_REF], and indirect inhibition of GLUT-3 by a mechanism involving NF-B [START_REF] Kawauchi | p53 regulates glucose metabolism through an IKK-NF-kappaB pathway and inhibits cell transformation[END_REF]. Further downstream, p53 regulates the synthesis of two rate limiting enzymes in the glycolytic pathway. It induces TIGAR (TP53-induced glycolysis and apoptosis regulator), an enzyme with fructose bi-phosphatase activities that counteracts the activity of 6-phosphofructo-1 kinase [START_REF] Bensaad | TIGAR, a p53-inducible regulator of glycolysis and apoptosis[END_REF][START_REF] Li | Structural and biochemical studies of TIGAR (TP53-induced glycolysis and apoptosis regulator)[END_REF]. In contrast, it down-regulates PGM (phosphoglycerate mutase), which converts 3-phosphoglycerate into 2-phosphoglycerate during the late ATP-generating steps of glycolysis [START_REF] Kondoh | Glycolytic enzymes can modulate cellular life span[END_REF]. This antiglycolytic activity is accompanied by the upregulation of enzymes that divert the metabolism of glucose to the pentose phosphate pathway (PPP), a metabolic shunt that uses glucose for biosynthesis rather than energetic purposes. The p53 protein binds to glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the PPP, and prevents the formation of the active dimer. Tumour-associated p53 mutants lack this G6PD-inhibitory activity [START_REF] Jiang | p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase[END_REF].

In parallel with its capacity to limit glycolysis, p53 maintains and promotes oxidative phosphorylation through at least two mechanisms. First, it activates AIF, encoding apoptosis inducing factor, a bifunctional protein with oxido-reductase function contributing to the assembly and function of complex I of the respiratory chain [START_REF] Stambolsky | Regulation of AIF expression by p53[END_REF][START_REF] Vahsen | AIF deficiency compromises oxidative phosphorylation[END_REF]. Second, p53 transactivates SCO2 (encoding synthesis of cytochrome c oxidase 2), a copper-dependent chaperone protein that is required for the assembly of complex IV of the respiratory chain [START_REF] Wanka | Synthesis of cytochrome C oxidase 2: a p53-dependent metabolic regulator that promotes respiratory function and protects glioma and colon cancer cells from hypoxia-induced cell death[END_REF]. Figure12 summarizes these recent observations on the role of p53 in energy metabolism.

Facilitating innate and adaptive immune response

The functions of p53 have been greatly extended beyond DNA repair and apoptosis.

Recent studies suggest a novel role for p53 in the control of human Toll-like receptor (TLR) gene expression [START_REF] Menendez | The Toll-like receptor gene family is integrated into human DNA damage and p53 networks[END_REF]. The TLR gene family mediates innate immunity to a wide variety of pathogenic agents through recognition of conserved pathogen-associated molecular motifs. The role of p53 in regulating TLR expression appears to be extremely diverse and complex. The promoters of most TLR family members contain both canonical and non-canonical p53 response elements, thus defining a potentially rich repertoire of dose-and context-dependent stress responses to p53 activation [START_REF] Menendez | The Toll-like receptor gene family is integrated into human DNA damage and p53 networks[END_REF]).

In addition, several studies suggest a potential role of p53 in antiviral immunity. Mice lacking p53 have impaired and delayed antiviral response to influenza A virus (IAV), caused by disruption of both innate and adaptive immunity [START_REF] Munoz-Fontela | p53 serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza A virus[END_REF]. In cells infected with hepatitis C virus (HCV), the HCV core protein induces the p53-dependent expression of TAP1 (encoding transporter associated with antigen processing 1) and consecutive major histocompatibility complex (MHC) class I up-regulation, leading to a significant down-regulation of the cytotoxic activity of natural killer (NK) cells against HCV infected cells and facilitating the establishment of a chronic infection [START_REF] Herzer | Upregulation of major histocompatibility complex class I on liver cells by hepatitis C virus core protein via p53 and TAP1 impairs natural killer cell cytotoxicity[END_REF].

Some other p53 activities might also directly contribute to help cancer cells to escape immune destruction. For instance, p53 represses ULBP2, a ligand for the natural killer cell immunoreceptor NKG2D, through miR-34a and miR-34c and loss of this function may contribute to eliminate an innate barrier against tumour development [START_REF] Heinemann | Tumor suppressive microRNAs miR-34a/c control cancer cell expression of ULBP2, a stress-induced ligand of the natural killer cell receptor NKG2D[END_REF].

This non-exhaustive list of the multiple roles of p53 in the Hallmarks of Cancer show that these processes are deeply interconnected, representing various pathways linked in a large functional network. The broad implication of p53 supports the idea that it represents a unifying factor between Hallmarks processes. While acquisition of one or several Hallmarks capabilities drives cells towards cancer, loss of p53 disintegrates the coherence between the Hallmarks processes and removes molecular obstacles to malignancy. Thus, in cancer cells having lost p53 function, DNA damage can accumulate without inducing cell growth arrest, accompanied by a metabolic switch that confers energetic autonomy, bypassing replicative

The TP53 family

For more than 20 years TP53 has appeared to stand alone among oncogenes and tumour suppressor genes by not being a member of a multi-gene family. Until the late nineties, it was even unclear whether a true equivalent to TP53 was present in organisms other than vertebrates. This view has considerably evolved over the past 15 years with the discovery of two homologues, TP63 and TP73, which share strong structural, biochemical and biological analogies with TP53. Recently, an additional layer of complexity has emerged when the p53 isoforms were identified, providing another similarity that p53 and its homologues have in common.

TP53 homologues

1.4.1.1 p63 and p73 protein organization . . . . Homologies with p TP63 (OMIM 602273) and TP73 (OMIM 601990) genes are respectively located in positions 3q27.29 and 1p36 [START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF][START_REF] Yang | p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[END_REF]. They present the same intron/exon organization as p53, with a non-coding exon 1 and a long intron 1 (around 10kb in TP53). All family members have 3 structural domains that are essential for their function: an N-terminal transactivation domain (TAD), a DNA-binding domain (DBD) and an oligomerization domain (OD) (Figure 13). In addition, p63 and p73 have another protein/protein interaction domain at their C-terminus known as sterile alpha motif (SAM). The three main domains are well conserved and the highest level of homology is reached in the DBD (65% identity between p53 and p73, and 60% identity between p53 and p63), which suggests that the three proteins can bind to the same DNA sequences and transactivate the same promoters [START_REF] Levrero | The p53/p63/p73 family of transcription factors: overlapping and distinct functions[END_REF].

This high structural homology reveals that p63 and p73 both acts as transcription factors like p53. They bind specifically to DNA onto conserved p53 response elements (p53REs) by using their DNA-binding domain [START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF][START_REF] Shimada | The transcriptional activities of p53 and its homologue p51/p63: similarities and differences[END_REF][START_REF] Yang | p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[END_REF]. The E3 ubiquitin ligase Hdm2 binds also to p73 but instead of promoting its degradation through poly-ubiquitination, it determines its re-localization to cytoplasm and changes its transcriptional activity through attachment of the ubiquitin-like protein NEDD8 [START_REF] Watson | Mdm2-mediated NEDD8 modification of TAp73 regulates its transactivation function[END_REF]. Binding of Hdm2 to p63 has also been demonstrated but the functional outcome is still a matter of debate [START_REF] Wang | MDM2 and MDMX can interact differently with ARF and members of the p53 family[END_REF][START_REF] Zdzalik | Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73[END_REF]. These proteins can undergo post-translational modifications. For example, PIAS-1 (the protein inhibitor of STAT-1) sumoylates p73, which inhibits its transcriptional activity and promotes exit from the G1 phase of the cell cycle [START_REF] Munarriz | PIAS-1 is a checkpoint regulator which affects exit from G1 and G2 by sumoylation of p73[END_REF]. p300 and pCAF acetyl-transferases promote p63 and p73 acetylation and their transcriptional activity [START_REF] Chae | Cell density-dependent acetylation of DeltaNp63alpha is associated with p53-dependent cell cycle arrest[END_REF][START_REF] Macpartlin | p300 regulates p63 transcriptional activity[END_REF] while deacetylases such as SIRT1 inhibit this function [START_REF] Dai | SIRT1 interacts with p73 and suppresses p73-dependent transcriptional activity[END_REF]. Phosphorylation of p73 by the tyrosine kinase c-Abl or by p38 MAP kinase in response to DNA damage stabilizes and activates p73, while threonine phosphorylation by Cyclin/CDK complexes inhibits p73 transcriptional activity promoting progression through the cell cycle [START_REF] Rufini | p73 in Cancer[END_REF][START_REF] Sanchez-Prieto | Regulation of p73 by c-Abl through the p38 MAP kinase pathway[END_REF].

. . . . p and p isoforms

In the late nineties, the cloning of TP63 and TP73 revealed an elaborate pattern of mRNA expression resulting in several protein isoforms [START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF][START_REF] Yang | p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[END_REF] (Figure 13). Both TP63 and TP73 genes can be transcribed from the proximal promoter P1 in exon 1, producing the TA forms which contain the entire TAD. An alternative promoter P2 located in the intron 3 will lead to the expression of N-terminally truncated isoforms ( Np63 and Np73) [START_REF] Ishimoto | Possible oncogenic potential of DeltaNp73: a newly identified isoform of human p73[END_REF][START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF]. Additional p73 isoforms truncated in their N-terminal domain have been described, resulting from alternative splicing of the mRNA produced from the P1 promoter: Ex2p73 (missing exon 2), Ex3p73 (missing exon 3), and Ex2/3p73 (missing exons 2 and 3) [START_REF] Ishimoto | Possible oncogenic potential of DeltaNp73: a newly identified isoform of human p73[END_REF][START_REF] Yang | p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[END_REF].

These isoforms were found combined with several C-terminal forms generated by alternative splicing in exons 10 to 15 for TP63 (producing five p63 isoforms: to ), and exons 10 to 14

for TP73 (producing seven p73 isoforms: to ) [START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF][START_REF] Yang | p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[END_REF].

p53 family members are able to modulate the activity of each others. p53 and p73 can regulate the same response element in the TP73 P2 promoter to induce Np73 isoforms expression [START_REF] Grob | Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53[END_REF]. Moreover, p53 positively regulates Np63 promoter activity, while Np63 mediates the silencing of its own promoter thereby altering the pattern of p53target gene expression [START_REF] Harmes | Positive and negative regulation of deltaN-p63 promoter activity by p53 and deltaN-p63-alpha contributes to differential regulation of p53 target genes[END_REF]. Overall, p53 family members seem to be able to interplay with each other and to modulate their expression and activity in a finely-tuned manner.

p63 and p73 functions . . . . Intrinsic functions

Although p53, p63 and p73 share structural and sequence similarities, p63 and p73 are rarely mutated in tumours, in contrast with p53. Instead, the TP63 locus is amplified in 20-30% of squamous cell carcinomas of the head and neck, oesophagus or lung [START_REF] Crook | High level expression of deltaN-p63: a mechanism for the inactivation of p53 in undifferentiated nasopharyngeal carcinoma (NPC)?[END_REF][START_REF] Hibi | AIS is an oncogene amplified in squamous cell carcinoma[END_REF][START_REF] Taniere | TP53 mutations, amplification of P63 and expression of cell cycle proteins in squamous cell carcinoma of the oesophagus from a low incidence area in Western Europe[END_REF], and p73 is over-expressed in diverse types of cancers such as, bladder, prostate, liver and colorectal cancer [START_REF] Rufini | p73 in Cancer[END_REF]. While mice deficient for p53 function develop early, multiple cancers, knockdown of the entire TP63 and TP73 loci (targeting all isoforms) revealed the roles of p63 and p73 in epithelial differentiation and neuronal development, respectively [START_REF] Mills | p63 is a p53 homologue required for limb and epidermal morphogenesis[END_REF][START_REF] Yang | p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours[END_REF].

p73 -/-mice showed increased apoptosis of sympathetic neurons in the superior cervical ganglion and reduced cortical thickness [START_REF] Killick | p73: a multifunctional protein in neurobiology[END_REF][START_REF] Yang | p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours[END_REF]. Interestingly, when aged, p73 +/-mice develop an Alzheimer-like phenotype, with reduced motor and cognitive functions. The brains of these animals show atrophy and neuronal degeneration [START_REF] Wetzel | p73 regulates neurodegeneration and phospho-tau accumulation during aging and Alzheimer's disease[END_REF][START_REF] Yang | p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours[END_REF]. The fact that no spontaneous tumours were observed could be due to the reduced lifespan of these p73 -/-mice. It later emerged that heterozygosity of p73 substantially shortened the animal's life span, and half of the p73 +/-cohort developed malignant tumours by 15 months of age. Importantly, these tumours show loss of heterozygosity of the remaining wild-type p73 allele, which is a typical hallmark of tumour suppressor inactivation in cancer. Moreover, survival of double heterozygote p53 +/-/p73 +/- mice is further reduced (from 10 to 6 months), due to an increased tumour burden and more aggressive metastatic disease. In many of the tumour specimens analyzed, the loss of heterozygosity of p73 was more frequent than the loss of the remaining p53 allele, revealing a potent tumour suppressor function for p73 [START_REF] Flores | Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family[END_REF]. Specific TAp73 isoform KO mice showed an increased susceptibility to spontaneous and induced carcinogenesis, with more than 70% of the KO animals developing tumours, mainly lung adenocarcinomas [START_REF] Tomasini | TAp73 knockout shows genomic instability with infertility and tumor suppressor functions[END_REF]. In contrast, there is no evidence of tumour development in ΔNp73 -/- mice, they are viable and fertile but display signs of neurodegeneration. Cells derived from Np73 -/-mice were sensitized to DNA-damaging agents and showed an increase in p53dependent apoptosis, suggesting a new role for Np73 in inhibiting the molecular signal emanating from a DNA break to the DNA-damage response pathway [START_REF] Wilhelm | Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway[END_REF].

Moreover, overexpression of oncogenic Ras influenced the TAp73/ Np73 ratio in favour of Np73, and the resulting downregulation of TAp73 and upregulation of Np73 were important for Ras transforming activity [START_REF] Beitzinger | p73 poses a barrier to malignant transformation by limiting anchorage-independent growth[END_REF]. Taken together, these findings support a tumour suppressive function for TAp73 and an oncogenic role for Nterminal truncated isoforms.

Differently from p73, p63 deletion does not seem to affect the central nervous system development [START_REF] Holembowski | While p73 is essential, p63 is completely dispensable for the development of the central nervous system[END_REF]. Total p63 KO mice show profound alterations of skin and other stratified epithelia as well as epithelial appendages including mammary and salivary glands, teeth and hair follicles. These animals also show truncated limbs and craniofacial abnormalities as a consequence of an alteration of the apical ectodermic ridge and die soon after birth [START_REF] Mills | p63 is a p53 homologue required for limb and epidermal morphogenesis[END_REF][START_REF] Yang | p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development[END_REF]. Similarly heterozygous mutations of p63 in humans are responsible for a number of syndromes known as ectodermal dysplasias, characterized by defects of ectodermal structures such as hair, teeth, digits, sweat glands and nails [START_REF] Celli | Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome[END_REF]. Two independent groups have generated these mice and have given a different interpretation of the phenotype. One group suggests that p63 is required for commitment and differentiation of epithelial precursors while the other proposes that it is essential for maintenance of the progenitor population [START_REF] Mills | p63 is a p53 homologue required for limb and epidermal morphogenesis[END_REF][START_REF] Yang | p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development[END_REF].

However, Np63 facilitates the formation of the basal layer and TAp63 is able to drive the expression of proteins of the upper layers of the epidermis, suggesting that both models are not incompatible [START_REF] Candi | Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice[END_REF]. The involvement of Np63 in proliferation is supported by the observations in squamous cell carcinoma cell lines of head and neck cancer where TP63 is amplified, inducing an overexpression of Np63 [START_REF] Hibi | AIS is an oncogene amplified in squamous cell carcinoma[END_REF]. In addition,

Np63 isoforms are commonly expressed as the major, if not the only, type of p63 isoform in epidermal stem cells (ESC) and in squamous cell progenitors [START_REF] Medawar | DeltaNp63 is essential for epidermal commitment of embryonic stem cells[END_REF]. In contrast, in squamous epithelia, TAp63 expression is restricted to differentiated cells [START_REF] Nylander | Differential expression of p63 isoforms in normal tissues and neoplastic cells[END_REF]. Thus, this isoform may have an oncogenic potential. Moreover, after treatment with etoposide and doxorubicin of hepatocellular carcinoma cell lines, the expression of TAp63 isoforms was clearly induced and was accompanied by an upregulation of cell cycle arrest genes [START_REF] Petitjean | The expression of TA and DeltaNp63 are regulated by different mechanisms in liver cells[END_REF]. TAp63 has recently been identified to play a role in NER [START_REF] Liu | TAp63gamma enhances nucleotide excision repair through transcriptional regulation of DNA repair genes[END_REF]. Overexpression of TAp63 significantly enhances NER of UVinduced DNA damage in H1299 and Saos-2 cells. TAp63 , but not TAp63α/ and TAp73α/ / isoforms, transcriptionally activates the expression of DDB2, XPC and GADD45

through the regulation of the p53 binding elements in these genes.

. . . . Roles in the modulation of p activity p63/p73 isoforms have opposite effects: N forms are involved in cell proliferation and survival, while TA forms have a role in cell cycle arrest and apoptosis. In addition, N isoforms act as dominant negatives on the TA isoforms [START_REF] Nakagawa | Autoinhibitory regulation of p73 by Delta Np73 to modulate cell survival and death through a p73-specific target element within the Delta Np73 promoter[END_REF][START_REF] Yang | p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[END_REF]. The analogy between the TP53 family members led to studies on the effects of Np63 and Np73 on p53. These two isoforms inhibit p53 transactivation activity and its ability to induce apoptosis [START_REF] Vossio | DN-p73 is activated after DNA damage in a p53-dependent manner to regulate p53-induced cell cycle arrest[END_REF]. The dominant negative property of ΔNp63/ Np73 isoforms could be exerted via their OD [START_REF] Nakagawa | Autoinhibitory regulation of p73 by Delta Np73 to modulate cell survival and death through a p73-specific target element within the Delta Np73 promoter[END_REF], but this hypothesis is controversial [START_REF] Ishimoto | Possible oncogenic potential of DeltaNp73: a newly identified isoform of human p73[END_REF].

Interestingly, both p53 and TAp73 induce Np73 creating an auto-regulatory feedback loop [START_REF] Grob | Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53[END_REF]. By interfering with p53 and TAp73 the N isoforms inhibit TAp63 to induce cell cycle arrest or apoptosis [START_REF] Liefer | Downregulation of p63 is required for epidermal UV-B-induced apoptosis[END_REF][START_REF] Marchbank | The CUSP DeltaNp63alpha isoform of human p63 is downregulated by solarsimulated ultraviolet radiation[END_REF].

The same decrease of expression after DNA damage was described for Np73 isoform [START_REF] Maisse | DNA damage induces the rapid and selective degradation of the DeltaNp73 isoform, allowing apoptosis to occur[END_REF].

p63 and p73 proteins not only modulate p53 suppressive functions, but also the oncogenic functions of p53 mutants. Studies show that mutant p53 (like p53R175H and p53R248W) can physically and functionally interact with p63 and p73 isoforms [START_REF] Gaiddon | A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain[END_REF][START_REF] Strano | Physical and functional interaction between p53 mutants and different isoforms of p73[END_REF]. These findings define a network involving mutant p53 and the various spliced isoforms of p63 and p73 that may confer upon tumour cells a selective survival advantage.

The p53 isoforms

The p53 isoforms were first identified in early studies investigating p53 expression patterns. In 1984, Matlashewski et al. cloned an N-terminal variant of the human p53 mRNA, whereas in 1985, Rotter and co-workers detected an alternatively spliced C-terminal variant of mouse p53, latter isolated in human cells [START_REF] Matlashewski | Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene[END_REF][START_REF] Wolf | Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule[END_REF].

However, the "p53 isoform" field has only really emerged in the past 10 years, when it became clear that TP53 retained the elaborate patterns of isoform expression that characterizes its homologues, TP63 and TP73 (Marcel et al., 2011a).

Mechanisms of production of p53 isoforms

Like TP63 and TP73, the human TP53 gene encodes several p53 protein isoforms through conserved mechanisms [START_REF] Khoury | The Isoforms of the p53 Protein[END_REF]Marcel et al., 2011a;Marcel and Hainaut, 2009). The main and most abundant p53 isoform is the canonical p53 protein, also termed TAp53 , as it contains an entire TAD and the longest C-terminal domain (Figure 14).

In addition to the TA forms, three N forms have been identified that differ by their translation initiation site. This is used to designate them as 40p53, 133p53 and 160p53.

The four N-terminal p53 forms can be combined with three different C-terminal domains ( , , ). Recently, some cis-and trans-regulators have been identified as specific modulators of p53 isoform expression (Figure 14).

40p53 expression: two mechanisms for one protein

Compared to TAp53, the human N-terminal 40p53 forms lack the first 39 amino acids corresponding to the main TAD (Figure 14). Matlashewski et al. described for the first time an altered human p53 transcript arising by alternative splicing and retaining the entire intron 2 [START_REF] Matlashewski | Alternative splicing of human p53 transcripts[END_REF]. They later observed that the p53I2 mRNA contained three stop codons in-frame with the first start codon of exon 2, leading to a transcript starting to the next methionine in codon 40 [START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF]. In addition to alternative splicing, 40p53 forms can be encoded from the fully-spliced p53 (FSp53) mRNA, which encodes the full-length p53 protein, through an internal initiation of translation at codon 40 (Courtois et al., 2002;Yin et al., 2002). Indeed, two internal ribosome entry sites (IRES) have been identified to regulate the translation of either p53 or 40p53 [START_REF] Candeias | Expression of p53 and p53/47 are controlled by alternative mechanisms of messenger RNA translation initiation[END_REF][START_REF] Ray | Two internal ribosome entry sites mediate the translation of p53 isoforms[END_REF]. The relative contribution in vivo of these two mechanisms of expression of 40p53 still remains to be fully established.

Alternative splicing of intron 2 can be regulated through G-quadruplex structures located in intron 3 of p53 pre-mRNA (Marcel et al., 2011c). G-quadruplexes are three-dimensional structures, which result of the propensity of Guanine-rich sequences to fold into fourstranded, monovalent cation-dependent structures [START_REF] Simonsson | G-quadruplex DNA structures--variations on a theme[END_REF]. Using reporter assays and RNA-G-quadruplex ligands, G-quadruplex structures appeared to promote the correct splice-out of intron 2, leading to the FSp53 mRNA. Disruption of the G-quadruplex structures however favoured the retention of intron 2 and thus p53I2 mRNA expression. This is the first evidence that the TP53 sequence itself can modulate its own isoforms' expression through regulation of alternative splicing.

Production of 133p53 and 160p53

Like TP63 and TP73, TP53 contains an internal promoter that control the expression of two N-terminal isoforms (Bourdon et al., 2005). In addition to the proximal P1 promoter regulating p53 and 40p53 expression, an internal P2 promoter located between intron 1 and exon 5 regulates the transcription of p53 mRNAs initiated in intron 4 (p53I4) (Figure 14A andB).

Experimental studies showed that the translation of p53I4 mRNAs can be initiated at two different codons, AUG133 and AUG160, leading to the expression of 133p53 and 160p53, respectively, which lack the TAD and part of the DBD (Figure 14C) (Marcel et al., 2010a).

Indeed, in the open reading frame of the human 133p53 mRNA, a second methionine is present at codon 160, which is contained within a Kozak's consensus motif conserved through evolution [START_REF] Kozak | At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells[END_REF]Marcel et al., 2010a). These observations evoke those of Courtois et al., describing the production of 40p53 isoform using AUG40 from p53 transcript (as described above) (Courtois et al., 2002).

Several studies have shown the capacity of p53 to regulate 133p53 expression (Aoubala et al., 2011;[START_REF] Marcel | p53 regulates the transcription of its Delta 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]. The internal P2 promoter contains p53REs located at the junction of exon 4/intron 4. p53 is able to bind these REs, resulting in an increased expression of 133p53 at both mRNA and protein levels. The expression of 133p53 can also be increased through the knockdown of p68, an RNA-helicase co-activator of p53-dependent transcription in response to DNA-damage (Moore et al., 2010).

C-terminal p53 isoforms

The alternative splicing of intron 9 of human TP53 produces three different C-terminal p53 forms ( , and ) (Figure 14) (Bourdon et al., 2005;[START_REF] Flaman | The human tumour suppressor gene p53 is alternatively spliced in normal cells[END_REF]. Complete excision of intron 9 results in the expression of the -forms corresponding to the classical p53 C-terminal domain. On the other hand, partial retention of intron 9 generates the -orforms, in which the OD is replaced by 10 or 15 new amino acids, respectively. However, the mechanisms that control the alternative splicing of intron 9 are unknown. The classical p53 Cterminal domain contains the main post-translational modification sites regulating p53 stability, such as the lysines residues ubiquitinated by Hdm2 [START_REF] Meek | Posttranslational modification of p53: cooperative integrators of function[END_REF].

Their absence in -and -forms led to the investigation of the stability of the p53 and p53 proteins by the Hdm2 ubiquitin proteasome pathway. To date, results remain controversial. For instance, whereas constitutively overexpressed FLAG-tagged p53 or p53

do not appear to interact with Hdm2 or to be degraded in a proteasome-dependent manner [START_REF] Graupner | Functional characterization of p53beta and p53gamma, two isoforms of the tumor suppressor p53[END_REF], Bourdon and co-workers observed that the endogenous p53 protein can be degraded by the proteasome in an Hdm2-dependent manner [START_REF] Camus | The p53 isoforms are differentially modified by Mdm2[END_REF].

Biological functions of p53 isoforms

40p53 isoform

Based on their homologies with the p63 and p73 family members, the N-terminal p53 isoforms lacking the TAD (i.e., 40p53, 133p53 and 160p53) were expected to only act as dominant-negative regulators of p53 activity. Indeed, 40p53 expression down-regulated the p53-induced transactivation on reporter genes and counteracted p53-dependent growth suppression in colony formation assays (Courtois et al., 2002;[START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF]. In addition, in human diploid fibroblast WI38 cells, expression of endogenous 40p53 increased during the G1/S transition, in parallel with decreased expression of p21WAF1 (Courtois et al., 2002).

Furthermore, as 40p53 lacks the Hdm2-binding site, it escapes Hdm2-mediated degradation and does not accumulate in response to DNA damage, its expression persisting at low but stable amounts in many cell types (Courtois et al., 2002;Yin et al., 2002). These data support the notion that 40p53 inhibits basal p53 activities during cell cycle progression.

However, whether 40p53 can exert p53-independent effects is still unknown. 40p53 lacks the first TAD, which has been shown to be dispensable for p53 transcriptional activity, and retains the second TAD, which can regulate gene expression [START_REF] Brady | Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression[END_REF][START_REF] Ohki | Dissecting functional roles of p53 N-terminal transactivation domains by microarray expression analysis[END_REF]. Therefore, it is premature to consider 40p53 as a simple dominant negative inhibitor of p53.

A recent study by [START_REF] Ungewitter | Delta40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs[END_REF] has shed a new and original light on the possible physiological role of 40p53 [START_REF] Ungewitter | Delta40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs[END_REF]. These authors reported that 40p53, in addition to being highly expressed in embryonic stem cells (ESCs), was the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of 40p53 in ESCs, they identified a critical role for this isoform in maintaining the ESC state.

Haploinsufficiency for 40p53 causes a loss of pluripotency in ESCs and acquisition of a cell cycle progression pattern, which is characteristic of somatic, differentiated cells, while increased dosage of 40p53 prolonged pluripotency and inhibited progression to a more differentiated state. At the molecular level, 40p53 appeared to control the switch from pluripotent ESCs to differentiated somatic cells by modulating the activity of full-length p53

at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). These results are consistent with results in a mouse model expressing a 40p53 transgene, which suggested that 40p53 may operate as a regulator of IGF-I receptor signalling [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF]. These observations led Ungewitter and Maier to propose that the primary function of 40p53 in cells of the early embryo and stem cells, is to control their pluripotent status. However, it is unclear how 40p53 exert this effect through full-length p53, and how this particular activity is regulated during differentiation.

133p53 isoforms

Compared with 40p53 , the available data have clearly revealed that 133p53 controls p53 activity. In reporter assays, 133p53 acts as a dominant-negative inhibitor of p53 (Bourdon et al., 2005;Murray-Zmijewski et al., 2006). 133p53 does not bind to consensus p53REs in vitro, consistent with its partial lack of the DBD, and thus can also behave as a dominant mutant p53 protein [START_REF] Marcel | p53 regulates the transcription of its Delta 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]. Nevertheless, instead of being a strict dominant inhibitor, 133p53 is instead a fine modulator of p53's suppressive activity as its expression determines cell fate in response to stress. Indeed, the induction of 133p53 expression inhibits p53-mediated apoptosis and G1 cell cycle arrest in response to doxorubicin treatment, without altering the p53-dependent G2 cell cycle arrest (Aoubala et al., 2011). These effects may be due to the ability of 133p53 to modulate gene expression in a promoter-dependent manner, as observed for p21WAF1, Hdm2 and Bcl-2 (Aoubala et al., 2011). Interestingly, 133p53 silencing has also been associated with replicative, but not oncogene-induced, senescence in normal human fibroblasts through transcriptional regulation of p53-target genes, including p21WAF1 and miR-34a [START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF]. Overall, these results are consistent with an oncogenic capacity of 133p53.

In H1299 cells, co-expression of Δ133p53 or Δ133p53 with p53 does not alter p53 transcriptional activity on the p21WAF1 and Bax promoters or p53-mediated apoptosis (Bourdon et al., 2005;Murray-Zmijewski et al., 2006). Δ133p53 and Δ133p53 are expressed in normal human tissues. However, several clinical studies have shown that Δ133p53 and Δ133p53 are abnormally expressed in tumours, suggesting that they play a role in carcinogenesis (Bourdon et al., 2005;[START_REF] Song | Expression of p53 isoforms in renal cell carcinoma[END_REF].

160p53 isoforms

Endogenous expression of Δ160p53 protein was detected in U2OS, T47D, and K562 cells (Marcel et al., 2010a). Interestingly, the human erythroleukaemic K562 cells were described as devoid of p53 expression because of a base insertion at codon 135, leading to a frameshift and a premature stop codon, which prevents p53 protein expression [START_REF] Neubauer | Genetic alterations in the p53 gene in the blast crisis of chronic myelogenous leukemia: analysis by polymerase chain reaction based techniques[END_REF].

However, K562 cells express Δ160p53α and Δ160p53 both at the mRNA and protein levels.

Moreover, upon hemin-induced erythroid differentiation of K562 cells, only Δ160p53

expression was reduced, while Δ160p53α expression was stable, suggesting that Δ160p53 could play a role in erythroid differentiation.

C-terminal p53 isoforms

The biological functions of the C-terminal p53 isoforms (i.e., p53 and p53 ) remain poorly described and controversial. Bourdon et al. showed that, in the absence of stress, endogenous p53 bound to the Bax and p21WAF1 promoters, but only weakly to that of Hdm2 (Bourdon et al., 2005). Moreover, in luciferase reporter assays in the absence of stress, the co-expression of p53 and p53 enhanced the p53 transcriptional activity on the p21WAF1 promoter but not on the Bax promoter, suggesting a promoter-dependent effect. These observations are consistent with the demonstration that p53 cooperates with p53 to accelerate senescence in human fibroblasts [START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF]. By contrast, experimental studies failed to observe binding of FLAG-tagged p53 or p53 onto p53RE consensus and to show a role of FLAG-tagged p53 or p53 in p53-dependent apoptosis or senescence in cells constitutively overexpressing FLAG-tagged p53 or p53 , and selected to grow in presence of neomycin [START_REF] Graupner | Functional characterization of p53beta and p53gamma, two isoforms of the tumor suppressor p53[END_REF]. Thus, there is still debate on whether p53 or p53 exert their activities in an autonomous manner or through an interaction with p53. Furthermore, there is no evidence of distinct biological activities for p53 or p53 . Overall, current experimental data on the biological roles of p53 isoforms are fragmented. Given that p53 isoforms differ from each other in the three functional domains (TAD, DBD and OD), their potential to modulate p53-dependent responses is expected to be diverse and cell typedependent.

Lessons from animal models

Sequences homologous to p53/p63/p73 have been identified in vertebrates and even invertebrates. Surprisingly, the observation of the same pattern of expression of the human p53 family members into two classes of isoforms, TA and N, is reported in animal models studies. The main three models studied to date are the drosophila, the zebrafish and the mouse, giving further insight into which of the several functions of p53 isoforms are of physiological or pathological relevance.

The Drosophila model

The diversification of the p53 gene family into three members occurred in vertebrates.

Hence, invertebrates such as Drosophila contain a single p53-related gene, which contains two alternative promoters and encodes three protein isoforms: Dp53, containing 495 aminoacids and corresponding to the human full-length p53; D Np53, containing 385 amino-acids and considered as a general counterpart of the human N-terminal p53 forms as it is encoded by an mRNA transcribed from an internal promoter (i.e., human 133) and contains a truncated TAD followed by a complete DBD and OD (i.e., human 40); and Dp53 C, encoded by a short transcript leading to a putative isoform bearing only the TAD (Bourdon et al., 2005;[START_REF] Brodsky | Drosophila p53 binds a damage response element at the reaper locus[END_REF][START_REF] Jin | Identification and characterization of a p53 homologue in Drosophila melanogaster[END_REF][START_REF] Ollmann | Drosophila p53 is a structural and functional homolog of the tumor suppressor p53[END_REF] (Figure 15). The Drosophila p53 gene is activated by irradiation and exerts broad suppressive effects recapitulating those of the p53-family members, including regulation of apoptosis, aging, autophagy, differentiation and growth [START_REF] Rutkowski | Phylogeny and function of the invertebrate p53 superfamily[END_REF]. Historically, D Np53 was the first form identified and previously termed Dp53; hence most of the functional studies to date have focused on the role of this particular isoform. The subsequent identification of a form of Drosophila p53 matching the mammalian full-length (TA) p53 protein has led to a reassessment of this nomenclature, with the name Dp53 to designate the full-length protein isoform and D Np53 for the N-terminal truncated form.

Studies on the morphogenesis of imaginal discs have highlighted the role of D Np53 isoforms in the control of cell death [START_REF] Brodsky | Drosophila p53 binds a damage response element at the reaper locus[END_REF][START_REF] Brodsky | Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage[END_REF]Fan et al., 2010;[START_REF] Ollmann | Drosophila p53 is a structural and functional homolog of the tumor suppressor p53[END_REF]. In this system, D Np53 controls apoptosis through the Reaper-Hid-Grim (RHG) cascade. Indeed, irradiated imaginal discs from flies with a mutant Dp53 gene show reduced apoptosis but normal cell-cycle arrest, suggesting a specific regulatory role of the Dp53 gene product in apoptosis [START_REF] Brodsky | Drosophila p53 binds a damage response element at the reaper locus[END_REF][START_REF] Brodsky | Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage[END_REF].

Apoptosis-induced proliferation requires Drosophila p53 and the caspase Dronc, and involves the release of mitogens such as Wingless (Wg) and Decapentaplegic (Dpp) that induce the growth of the surrounding tissues [START_REF] Fan | Apoptosis-induced compensatory proliferation. The Cell is dead[END_REF][START_REF] Kondo | DRONC coordinates cell death and compensatory proliferation[END_REF][START_REF] Wells | Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity[END_REF]. So far, the main isoform implicated in apoptosis appears to be D Np53, which directly regulates the reaper (rpr) gene expression, whose protein product activates caspases by inhibiting Drosophila Inhibitor of Apoptosis Protein (DIAP1) [START_REF] Brodsky | Drosophila p53 binds a damage response element at the reaper locus[END_REF][START_REF] Jin | Identification and characterization of a p53 homologue in Drosophila melanogaster[END_REF]. Recently, Mollereau and co-workers found that both isoforms were capable of (Fan et al., 2010;[START_REF] Peterson | reaper is required for neuroblast apoptosis during Drosophila development[END_REF]. Moreover, D Np53 appears to inhibit cellular differentiation in the retina independently of its apoptotic function (Fan et al., 2010).

The Dp53 gene product also exerts important roles in controlling lifespan in a sex-and stage-dependent manner. When overexpressed in adult flies, D Np53 limited lifespan in females and extended it in males. By contrast, when overexpressed during development, D Np53 exerted a similar dose-dependent effect on longevity in both sexes [START_REF] Waskar | Drosophila melanogaster p53 has developmental stage-specific and sex-specific effects on adult life span indicative of sexual antagonistic pleiotropy[END_REF]. Conversely, inactivation of the Dp53 locus increased lifespan in females but had only minor effects in males. Similar phenotypes were observed in Drosophila expressing dominant-negative Dp53 mutant transgenes or overexpressing D Np53, suggesting that D Np53 interferes with Dp53 activity [START_REF] Bauer | Neuronal expression of p53 dominant-negative proteins in adult Drosophila melanogaster extends life span[END_REF]. The role of Drosophila p53 has also been investigated in detail in other cellular functions such as DNA repair, cell cycle or aging [START_REF] Biteau | It's all about balance: p53 and aging[END_REF][START_REF] Brodsky | Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage[END_REF][START_REF] Jassim | Dmp53 protects the Drosophila retina during a developmentally regulated DNA damage response[END_REF][START_REF] Mandal | Metabolic control of G1-S transition: cyclin E degradation by p53-induced activation of the ubiquitin-proteasome system[END_REF].

The study of the specific roles of each Drosophila p53 isoform, Dp53 and D Np53, will allow shedding new light on the roles of p53 isoforms in humans.

Figure 15. Structural organization of p53 isoforms in animal models

The three main domains (TAD, DBD and OD) present in human p53 are conserved in the mouse, drosophila and zebrafish. Only the mouse Mp53 contains the PXXP (purple) and NLS (green). AS: alternative splicing (green boxes); grey box: different residues compared with the full-length p53 protein (Marcel et al., 2011a).

The Zebrafish model

Zebrafish models have significantly contributed to our understanding of vertebrate development and, more recently, human disease. The growing number of genetic tools available in zebrafish research has resulted in the identification of many genes involved in developmental and disease processes. In particular, studies in the zebrafish have clarified roles of the p53 tumour suppressor in the formation of specific tumour types, as well as roles of p53 family members during embryonic development. The zebrafish p53 protein, Zp53, recapitulates the suppressive and pro-apoptotic functions of human p53 upon genotoxic stress [START_REF] Storer | Zebrafish models of p53 functions[END_REF]. So far, only N-terminal Zp53 isoforms have been identified

(Figure 15): Zp53, corresponding to the human p53 protein [START_REF] Cheng | Zebrafish (Danio rerio) p53 tumor suppressor gene: cDNA sequence and expression during embryogenesis[END_REF]; Z Np53, produced through an alternative splicing of intron 2 and thus similar to the human 40p53 forms [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]; and Z 113p53, produced by an internal promoter located within the zebrafish Zp53 gene that is regulated by Zp53 itself, and thus equivalent to the human 133p53 isoform (Chen et al., 2005). In contrast to the human 40p53, AUG1 of Z Np53 is located within the partial intronic sequence retained by alternative splicing [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]. Thus in Z Np53, the 38 N-terminal residues containing the TAD are replaced by 33 residues derived from the intron 2 sequence. There is evidence that the Z Np53 transcript accumulates in response to -ray irradiation [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]. In addition, ectopic expression of Z Np53 resulted in a strong developmental phenotype with hypoplasia and malformation of the head, eyes and somites. This phenotype is dependent on the presence of Zp53, the two isoforms forming a protein complex through their ODs.

Overexpression of an OD-mutant Z Np53 or wild-type Z Np53 in a mutant Zp53 background is phenotypically ineffective. These observations suggest that Z Np53 exerts its effects by modulating the activity of Zp53 during zebrafish development.

Z 113p53 was discovered in a different context. Chen and co-workers found that in zebrafish embryos, loss of the Digestive organ Expansion Factor gene (Def) led to defects in the morphogenesis of digestive organs. In a genome-wide screen, they identified a shorter form of Zp53 whose expression was upregulated in Def -/-embryos (Chen et al., 2005).

Upregulation of Z 113p53 correlated with increased expression of p53-target genes involved in cell-cycle progression such as cyclin-G1 and p21WAF1, whereas pro-apoptotic genes such as Bax and Reprimo were not activated. Furthermore, Z 113p53 was found to selectively upregulate the Bcl-2L anti-apoptotic gene [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF]. This pattern of effects is consistent with the notion that Z 113p53, like 133p53, operates as a modulator of p53 in selectively activating defined target genes.

The Mouse model

Six p53 isoforms have been described in mice, resulting from combination of three Nterminal p53 isoforms with two different C-terminal isoforms (Figure 15). The first report revealing that the mouse TP53 gene codes for more than one functional protein was published by Rotter and colleagues in 1985, describing the presence of another p53 variant in transformed mouse fibroblasts [START_REF] Wolf | Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule[END_REF]. This p53 variant was reported to be generated by alternative splicing using a cryptic 3′ splicing site in intron 10, located 96 bp upstream of the regular 3′ splicing site of exon 11, thereby naming it Mp53AS (alternatively spliced) [START_REF] Arai | Immunologically distinct p53 molecules generated by alternative splicing[END_REF]. A stop codon in the 96-bp insert from intron 10 leads to the production of a truncated p53 protein, in which the last 26 amino acids of mouse p53 are missing and are replaced by 17 new amino acids homologous to the human p53 [START_REF] Arai | Immunologically distinct p53 molecules generated by alternative splicing[END_REF][START_REF] Wolf | Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule[END_REF]. Later, M 41p53 (also named p44) was isolated, the mouse counterpart of human 40p53 forms [START_REF] Mowat | Rearrangements of the cellular p53 gene in erythroleukaemic cells transformed by Friend virus[END_REF]. In addition, Khoury et when compared to that induced by p53, accompanied with a delay in Bax induction and Bcl-2 repression [START_REF] Almog | The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells[END_REF]. Indeed, Mp53AS is a less potent transactivator of Bax and p21WAF1 promoters, although Mp53AS has a stronger affinity to p53REs [START_REF] Almog | p53-dependent apoptosis is regulated by a Cterminally alternatively spliced form of murine p53[END_REF]. Interestingly, coexpression of Mp53 and Mp53AS in stably transfected myeloid cells or in transiently transfected p53-null H1299 cells results in an inhibition of p53 transcriptional activity and p53-mediated apoptosis [START_REF] Almog | p53-dependent apoptosis is regulated by a Cterminally alternatively spliced form of murine p53[END_REF]. The p53AS inhibitory effect on p53-mediated apoptosis is Hdm2 dependent, as p53AS is able to induce higher levels of the Hdm2 protein than p53 [START_REF] Almog | The role of the C' terminus of murine p53 in the p53/mdm-2 regulatory loop[END_REF].

In 2004, Maier et al. described a transgenic mouse overexpressing M 41p53 [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF]. When expressed in a p53-null background, this isoform did not induce any particular phenotype. However, when expressed in a p53-competent background, an increased dosage of M 41p53 led to reduced size, accelerated aging and a shorter lifespan associated with hypoinsulinemia and glucose insufficiency [START_REF] Hinault | Delta40 Isoform of p53 controls beta-cell proliferation and glucose homeostasis in mice[END_REF][START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF][START_REF] Pehar | Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death[END_REF]. These effects were attributed to the hyper-activation of the insulin-like growth factor (IGF)-signalling axis by M 41p53, setting in motion a cascade that clamps unimpeded growth through p21WAF1 [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF]. Furthermore, these M 41p53-overexpressing mice show cognitive decline and synaptic impairment early in life, also attributable to the hyper-activation of the IGF-1-signalling pathway [START_REF] Pehar | Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death[END_REF]. These observations are consistent with studies performed in vitro and in the zebrafish model, indicating that 40p53 isoforms may regulate growth suppression through modulation of p53 activity.

A premature aging phenotype was also reported in a non-physiological knock-in p53 mouse model expressing an M 122p53 form, truncated for the first 122 residues [START_REF] Slatter | Hyperproliferation, cancer, and inflammation in mice expressing a Delta133p53-like isoform[END_REF]. Although M 122p53 has no physiological equivalent in mouse, it can be considered as an "intermediate" between the M 41p53 and M 157p53 isoforms, as it lacks the TAD and part of the DNA-binding domain. During adulthood, transgenic p53 122p53/ 122p53 mice showed premature aging symptoms, such as balding and arthritis, similar to that observed in the p53 +/m mice (deletion exons 1-6) [START_REF] Slatter | Hyperproliferation, cancer, and inflammation in mice expressing a Delta133p53-like isoform[END_REF][START_REF] Tyner | p53 mutant mice that display early ageing-associated phenotypes[END_REF]. In addition, earlier tumour onset and shortened lifespan were observed in p53 +/ 122p53 mice as compared with p53 +/-mice.

Overall, these results support the hypothesis that mouse N-terminal p53 forms may operate as dominant oncogenes to promote cell proliferation and inflammation. The studies summarized above highlight common themes in the p53 isoform field. First, overall patterns of isoform expression are well-conserved throughout evolution. Second, N-terminal isoforms have a major role as regulators of physiological processes related to development, aging, lifespan and, possibly, carcinogenesis. In this respect, two key mechanisms are emerging. The 40p53 form exerts regulatory effects on signalling cascades controlled by p53, perhaps through direct interaction between the two isoforms. Conversely, isoforms corresponding to 133p53 modulate cell response by regulating gene expression in a p53-dependent andindependent manner. It should, however, be remembered that the current view of isoform activities remains fragmentary and that further studies are needed to better understand their roles and underlying mechanisms.

Functional inactivation of p53 in cancer

The regulation of p53 function is highly complex and occurs at many levels, thus involving a spectrum of determinants that activate specific components of the p53 network appropriate for the circumstances. Each of the stress responses tends to prevent cellular transformation and tumourigenesis by removing damaged cells from the proliferative cycle. It is thus obvious that inactivation of p53 by either mutation or deregulation of its pathway is commonly observed in human cancer. This part will summarise the principal mechanisms by which p53 function is altered in cancer, highlighting recent discoveries. Within this section p53-based anti-cancer therapeutic strategies developed to reactivate or restore p53 functions will be briefly presented.

Viruses

The small DNA tumour viruses, Polyoma virus, Simian Vacuolating Virus 40 (SV40), the Papilloma viruses and the human Adenoviruses, were first described during a period of intense virus discovery (1930-1960s) and shown to produce tumours in animals (reviewed in [START_REF] Levine | The common mechanisms of transformation by the small DNA tumor viruses: The inactivation of tumor suppressor gene products: p53[END_REF]). In each of these cases the viral DNA was shown to persist (commonly integrated into a host chromosome) and only a selected portion of this DNA was expressed as mRNA and proteins in these cancers [START_REF] Botchan | Viral DNA in transformed cells. III. The amounts of different regions of the SV40 genome present in a line of transformed mouse cells[END_REF][START_REF] Gallimore | Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA IN NINE LINES OF TRANSFORMED RAT CELLS USING SPECIFIC FRAGMENTS OF THE VIRAL GENOME[END_REF]. For SV40, this region corresponded to the portion of the viral chromosome expressed early after infection and encoded two proteins, the SV40 large T-antigen and the small t-antigen [START_REF] Huebner | SPECIFIC ADENOVIRUS COMPLEMENT-FIXING ANTIGENS IN VIRUS-FREE HAMSTER AND RAT TUMORS[END_REF]. For the Adenoviruses, this region corresponds to a subset of the early genes termed the E1A proteins and the E1B proteins composed of the E1B-55Kd and E1B-21Kd proteins (Ross et al., 1980a;[START_REF] Ross | Early viral proteins in HeLa cells infected with adenovirus type 5 host range mutants[END_REF]. Subsequent experiments with the human papilloma viruses (HPV) types 16 and 18 uncovered a similar set of early proteins called the E6 and E7 proteins [START_REF] Scheffner | Functions of human papillomavirus proteins[END_REF]. The E6 and E7 proteins, encoded by the oncogenic HPV types 16 and 18, are viral products expressed in HPV-associated cancers. The viral encoded tumour antigens were identified and shown to be required to both establish the tumour and maintain the transformed cell phenotype [START_REF] Howie | Papillomavirus E6 proteins[END_REF][START_REF] Pipas | Role of T antigen interactions with p53 in tumorigenesis[END_REF]Ross et al., 1980a).

The functions of these viral tumour antigens were explored and shown to have common features and mechanisms even though they appear to have evolved from diverse genes. The SV40 large T-antigen, the HPV E7 protein and the Adenovirus E1A protein were shown to bind to and inactivate the functions of the Retinoblastoma proteins in transformed cells [START_REF] Decaprio | SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene[END_REF][START_REF] Dyson | The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product[END_REF][START_REF] Whyte | Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product[END_REF]. This resulted in the activation of the E2F and DP transcription factors and the entry of cells into the S-phase of DNA synthesis which was required for viral DNA replication. These events triggered the activation of p53, which promotes apoptosis of these virus-infected cells limiting virus replication and tumour formation [START_REF] Wu | p53 and E2F-1 cooperate to mediate apoptosis[END_REF]. These viruses responded by evolving and producing the SV40 large T-antigen, the HPV E6 protein and the Adenovirus E1B-55Kd protein, which bind to and inactivate the p53 functions in both the infected and transformed cells.

After initial observations that the SV40 large T-antigen was forming a complex with p53, it became later apparent that SV40 was in fact inactivating the functions of the p53 protein in tumours and transformed cells [START_REF] May | Twenty years of p53 research: structural and functional aspects of the p53 protein[END_REF][START_REF] Tegtmeyer | Modification of simian virus 40 protein A[END_REF][START_REF] Tegtmeyer | Regulation of tumor antigen synthesis by simain virus 40 gene A[END_REF]. Then, it was observed that an Adenovirus oncogene product, the E1B-55Kd protein, was able to form a protein complex with the p53 protein in virus infected and transformed cells [START_REF] Sarnow | Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells[END_REF]. This viral protein also inactivates the functions of the p53 protein and adds to the ability of this virus to transform cells and produce tumours in animals. Several years later, it was demonstrated that the E6 oncogene products of HPV-16 and 18 (the tumourigenic human Papilloma viruses) were bound to the p53 protein in HPV transformed cells [START_REF] Werness | Association of human papillomavirus types 16 and 18 E6 proteins with p53[END_REF]. The E6 protein recruited the E6-associated protein ligase (E6-AP) and this resulted in the poly-ubiquitination of the p53 protein and its rapid degradation in the proteasome of a transformed cell [START_REF] Scheffner | The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53[END_REF]. This observation explains the rarity of TP53 mutations in cervical cancers [START_REF] Crook | Clonal p53 mutation in primary cervical cancer: association with human-papillomavirus-negative tumours[END_REF].Thus SV40, the Adenoviruses and the tumourigenic Papilloma viruses all targeted the same cellular protein for inactivation with their oncogene products and in so doing eliminated a tumour suppressor activity from these cells. Because the SV40 T-antigen, the Adenovirus E1B-55Kd and the HPV E6 proteins are completely unrelated in their amino acid sequences, this is an example of convergent evolution to satisfy a need that must be required for these viruses to replicate in cells. Somatic TP53 mutations occur in almost every type of cancer at rates from 38%-50% in ovarian, oesophageal, colorectal, head and neck, larynx and lung cancers to about 5% in primary leukemia, sarcoma, testicular cancer, malignant melanoma and cervical cancer [START_REF] Olivier | TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use[END_REF]. The main modes of TP53 inactivation are single-base substitution and loss of alleles. In contrast to other tumour suppressor genes that are mainly altered by truncating mutations, most TP53 mutations are missense substitutions (75%) and cause single amino-acid changes at many different positions. Other alterations include frameshift insertions and deletions (9%), nonsense mutations (7%), silent mutations (5%) and other infrequent alterations [START_REF] Olivier | The IARC TP53 database: new online mutation analysis and recommendations to users[END_REF]. Of note, 74% of TP53 mutations fall within the DBD, more than 30% of which are at codons encoding residues that play essential structural and chemical roles in the contact with p53RE [START_REF] Hainaut | p53 and human cancer: the first ten thousand mutations[END_REF]Petitjean et al., 2007). These hotspot mutants have been classified as contact mutants, that retain the overall architecture of the DBD with loss of a critical DNA contact (i.e. R248 and R273); and structural mutants, which induce conformational changes in the DNA binding surface (i.e.

nonsense or frameshift [START_REF] Olivier | TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use[END_REF]. Mutants located in distant sites are presumed to inactivate p53 function by virtue of defects in protein conformation [START_REF] Goh | The role of mutant p53 in human cancer[END_REF].

Experimental assays have been performed in yeast and human cells to measure various It has been hypothesized that reactivation of mutant p53 in tumours should trigger massive apoptosis and eliminate tumour cells. PRIMA-1 is a molecule found to be capable of restoring sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells [START_REF] Bykov | Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound[END_REF]. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumour effect with no apparent toxicity (Bykov et al., 2002a). Another way to reactivate p53 is by impairing the interaction Hdm2-p53. Overexpression of Hdm2, found in many human tumours, effectively impairs p53 function. Small-molecule antagonists of Hdm2, such as Nutlins, have been found to bind Hdm2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumour xenografts in nude mice [START_REF] Vassilev | In vivo activation of the p53 pathway by small-molecule antagonists of MDM2[END_REF].

The p53/Hdm2/Hdm4 /p14ARF connexion

Abnormal accumulation of the Hdm2 protein is observed in many tumours, especially sarcomas [START_REF] Onel | MDM2 and prognosis[END_REF]. This accumulation can be due to amplification of the Hdm2 gene, enhanced transcription of the gene or enhanced translation of its messenger RNA [START_REF] Michael | The p53 and Mdm2 families in cancer[END_REF]. A database listing the Hdm2 gene amplifications showed that the overall frequency of Hdm2 amplification is 7% [START_REF] Momand | The MDM2 gene amplification database[END_REF]. The highest frequency is observed in soft tissue tumours (20%) and osteosarcomas have the second highest frequency of Hdm2 gene amplification (16%), followed by oesophageal carcinomas (13%). In melanomas, where TP53 gene is rarely mutated (95% wild-type), amplification of Hdm2 is detected in 3-5% of the cases [START_REF] Chin | The genetics of malignant melanoma: lessons from mouse and man[END_REF]Muthusamy et al., 2006).

In early studies, Hdm2 was shown to be amplified and overexpressed in soft tissue sarcomas, and its increased level was found to be mutually exclusive with mutation of TP53 [START_REF] Oliner | Amplification of a gene encoding a p53-associated protein in human sarcomas[END_REF][START_REF] Reifenberger | Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations[END_REF]. Although these tumours would be expected to no longer express p53, the opposite situation is generally observed, with a large number of tumours overexpressing both p53 and Hdm2. The reasons for this apparent paradox have not been elucidated. No formal exclusion between TP53 gene mutation and Hdm2 accumulation has been clearly demonstrated.

Interestingly, a recent study has demonstrated an overexpression of Hdm4 as an alternative way to inactivate p53 in human melanomas (Gembarska et al., 2012). In these tumours, Hdm4 is overexpressed in 65% of the cases, suggesting that Hdm4 has an important oncogenic role to alter p53 functions. Several efforts have focused on blocking Hdm2 as a strategy to reactivate p53 [START_REF] Canner | MI-63: a novel small-molecule inhibitor targets MDM2 and induces apoptosis in embryonal and alveolar rhabdomyosarcoma cells with wild-type p53[END_REF][START_REF] Hedstrom | Tumor-specific induction of apoptosis by a p53-reactivating compound[END_REF][START_REF] Issaeva | Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors[END_REF][START_REF] Shangary | Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer[END_REF][START_REF] Vassilev | In vivo activation of the p53 pathway by small-molecule antagonists of MDM2[END_REF]. One major limitation of Hdm2-specific therapy could be explained in tumour cells where Hdm2 expression is low and Hdm4 is high.

Alteration of upstream signals corresponds to inactivation of the CDKN2 locus, which expresses both p14ARF protein, an activator of p53 in response to an oncogenic stress and the cyclin kinase inhibitor p16. The interaction of p14ARF with Hdm2 blocks Hdm2 shuttling between the nucleus and cytoplasm via the nucleolus [START_REF] Iwakuma | MDM2, an introduction[END_REF].

Sequestration of Hdm2 in the nucleolus thus results in activation of p53. Germline alterations (point mutations) and somatic alterations (point mutations and hypermethylation of the promoter) of this gene are frequent in malignant melanoma and impair the induction of p53 after an oncogenic stress [START_REF] Chin | The INK4a/ARF tumor suppressor: one gene--two products--two pathways[END_REF].

Alternative pathways

Twist1 and Twist2 are closely related members of a family of basic helix-loophelix (bHLH) transcription factors involved in gastrulation and mesoderm specification.

Typically, Twist proteins regulate the expression of target genes by binding, as homo-or heterodimers, to E-box-containing promoters.

By investigating the role of Twist1 in the context of sarcomas, this study highlights an alternative mechanism of p53 inactivation and provides evidence that Twist1 promotes Hdm2-mediated degradation of p53 by directly interacting with its C-terminal regulatory domain and by interfering with key phosphorylations [START_REF] Piccinin | A "Twist box" Code of p53 Inactivation: Twist box:p53 Interaction Promotes p53 Degradation[END_REF].

In inflammatory breast cancers or neuroblastomas, molecular and immunohistochemical analyses demonstrate accumulation of wild-type p53 in the cytoplasm of tumour cells, leading to functional inactivation of p53 [START_REF] Moll | Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage[END_REF]. A new protein has been isolated, Parc (a Parkin-like ubiquitin ligase), that has been found to sequester p53 in the cytoplasm of cells in the absence of any lesions [START_REF] Nikolaev | Parc: a cytoplasmic anchor for p53[END_REF]. Abnormal accumulation of this protein is observed in neuroblastomas cells and could therefore account for functional inactivation of p53 in this cancer.

TP53 polymorphisms

The TP53 gene is highly polymorphic, with over 80% of known single-nucleotide polymorphisms (SNPs) located within introns or non-coding 5' and 3' sequences (Petitjean et al., 2007). Marcel et al. showed that G-quadruplex structures, formed in intron 3 and regulating intron 2 splicing (and thus 40 form expression), overlap a common polymorphism, TP53 PIN3, which consists of a 16-bp duplication (A1, non-duplicated allele;

A2, duplicated allele) [START_REF] Lazar | Simple sequence repeat polymorphism within the p53 gene[END_REF]Marcel et al., 2011c). Therefore, this polymorphism may modulate the structure and/or the stability of the G-quadruplexes, and affect 40p53 expression.

Consistent with a functional effect, TP53 PIN3 has recently been identified as a strong genetic modifier of germline TP53 mutations. Indeed, carriers bearing A1A1 genotypes developed their first cancer on average 20 years earlier than carriers with an A1A2 genotype (Marcel et al., 2009). This effect was detected in a Brazilian cohort, in which the p.R337H mutation of partial penetrance is very common owing to a widespread founder effect [START_REF] Garritano | Detailed haplotype analysis at the TP53 locus in p.R337H mutation carriers in the population of Southern Brazil: evidence for a founder effect[END_REF]. It remains to be demonstrated whether a similar effect is observed in carriers of other TP53 mutation types. Landi and co-workers have identified 11 different haplotypes defined by eight SNPs in a region from exon 3 to intron 4, overlapping part of the internal P2 promoter regulating 133p53 and 160p53 expression [START_REF] Bellini | DeltaN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region[END_REF]. Using these different haplotypes as promoters to drive luciferase expression, they found significant differences in basal promoter activity that were confirmed by analysing endogenous 133p53 expression in samples of normal colonic mucosa. Furthermore, in silico and in vitro DNA binding analyses suggested that a SNP in intron 4 (rs179287; C>T) affects proteins binding within the P2 promoter [START_REF] Bellini | DeltaN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region[END_REF]. These results suggest that genetic polymorphisms may modulate basal 133p53 expression. Whether these effects influence cancer risk remains to be demonstrated.

A role for p53 isoforms?

Given the fact that N-terminal p53 isoforms act as inhibitors of p53 transactivation, it can be expected that overexpression of certain isoforms may represent an alternative to a mutation in TP53 for inactivating p53 in cancers, as observed in some small clinical studies (Avery-Kiejda et al., 2008;Bourdon et al., 2005;[START_REF] Machado-Silva | p53 family members in cancer diagnosis and treatment[END_REF].

There is emerging evidence that p53 isoform expression is deregulated in human cancers.

In a study of 245 primary ovarian cancers, Hofstetter et al. observed that expression of p53 was associated with serous and poorly differentiated cancers, and, when expressed together with the functional p53 protein, it was correlated with poor recurrence-free and overall survival [START_REF] Hofstetter | Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer[END_REF]. By contrast, tumours expressing functional p53 and 40p53

showed improved recurrence-free survival of patients compared with tumours expressing no 40p53. Studies of patients with acute myeloid leukemia have shown that elevated expression of p53 and/or p53 in blood cells was correlated with improved responses to chemotherapy [START_REF] Anensen | A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia[END_REF], which may predict decreased chemoresistance and improved overall

survival. An effect of isoform expression has also been observed on breast cancer prognosis.

In a cohort of 127 breast cancer patients, Thompson and co-workers reported that patients whose tumours expressed both mutant p53 and p53 mRNAs had a prognosis as good as patients whose tumours expressed a wild-type p53, suggesting that the expression of p53 may abrogate the poor prognosis commonly associated with TP53 mutations [START_REF] Bourdon | p53 mutant breast cancer patients expressing p53gamma have as good a prognosis as wild-type p53 breast cancer patients[END_REF].

CONTEXT, OBJECTIVES AND APPROACHES 2.1 Context

For a long time after its discovery in 1979, p53 has been considered as an "orphan"

protein, the product of a gene having no homolog in the genome of vertebrates. The discovery in 1997-98 of two distinct p53-related factors, p63 and p73, has led to re-think p53 as one member of a multi-gene family [START_REF] Courtois | p53 protein variants: structural and functional similarities with p63 and p73 isoforms[END_REF]. Significantly, p63 and p73 were shown to retain a number of conserved features in their structure and expression patterns. In particular, these two proteins were found to be expressed as multiple isoforms due to alternative splicing, alternative promoter usage and alternative initiation of translation [START_REF] Petitjean | The expression of TA and DeltaNp63 are regulated by different mechanisms in liver cells[END_REF]. These isoforms consisted in versions of p63 with defined truncations in the N-terminal domain (encoding the main transactivation domain) or the C-terminal domain (encoding a number of regulatory signals). The Np63 and Np73 isoforms were found to represent major isoforms of the protein, responsible for the bulk of protein expression in many cells. In contrast, TAp63 and TAp73 forms were found to be expressed at relatively low levels.

This observation attracted attention on the possibility that p53 may also retain a standard pattern of isoform expression. Studies published before the discovery of p63 and p73 had reported alternative forms of p53 in mouse and humans, although these forms appeared to have a restricted, and perhaps tissue-specific, expression patterns [START_REF] Matlashewski | Alternative splicing of human p53 transcripts[END_REF][START_REF] Wolf | Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule[END_REF]. The field of p53 isoforms started to emerge in 2002-2004 when it became apparent that many p53 isoforms could fit into the same N-or C-terminal truncation pattern as p63/p73 isoforms. Following the initial discovery of 40p53 (initially called Np53) (Courtois et al., 2002;Yin et al., 2002), a wide range of N-and C-terminal isoforms has been identified, raising the current isoform tally to at least 12 potential isoforms (Bourdon et al., 2005;Marcel and Hainaut, 2009). However, so far, the biochemical functions of each isoform, and its functional interaction with TAp53, have remained elusive.

Currently, two distinct N-terminal isoforms of p53 have been widely recognized, 40p53 and 133p53. Early studies have shown that each of them is impaired in its transcriptional activity due to lack of the main transactivation domain (located in residues 1-42 of wild-type p53) (Aoubala et al., 2011;Courtois et al., 2002;[START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF][START_REF] Marcel | p53 regulates the transcription of its Delta 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]Yin et al., 2002). Therefore, they have the potential to operate as dominant-negative inhibitors of p53 functions. However, there are so far very limited clues on how, when and where p53 isoforms exert their physiological functions. Moreover, whether expression of isoforms is deregulated in cancer has not been systematically addressed. Indeed, given their potential to inhibit p53 transactivation capacity, 40p53 and 133 p53 may represent a plausible alternative mechanism to mutation for inactivation of p53 function.

Animal models of overexpression of p53 isoforms in vertebrates are available in the mouse ( 40p53) [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF] and in the Zebrafish ( 40p53, 133p53) [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]. These models have shown surprising results. Whereas the expression of N-terminal isoforms in a p53-null background has no clear phenotypic effect, expression in a background of wild-type TAp53 induced a phenotype of premature aging in the mouse and of perturbed development in Zebrafish. These observations suggested that N-terminal isoforms could exert strong effects by modulating the activity of wild-type TAp53.

Approaches

To address the first two of these points, we have used several well-defined cultured cancer cell lines that lack functional p53 protein and we have transfected p53 isoforms together with TAp53 in defined concentrations and conditions. We have then assessed the effects on p53 transactivation capacity and on DNA-binding activity. For 133p53, we have further analysed the nature of the specific promoter regulating this isoform and we have shown that it contains p53-dependent Response Elements, suggesting that the two p53 isoforms, although having overlapping molecular and biochemical effects, might be regulated in response to very different signals.

To address the third point, we have turned to cutaneous melanoma, a highly metastatic tumour in which TP53 mutation is rare and evidence for functional inactivation of wild-type p53 is building-up. We have analysed 40p53 and 133p53 expression levels in wellcharacterized lesions grouped in categories according to the extent of metastatic spread. Our results show enhanced isoform expression in most highly metastatic lesions, consistent with the hypothesis that isoform expression may play an active role in neutralizing p53 in melanoma.

Overall, our results contribute to the view that p53 isoform expression is not a side-effect of p53 regulation but is a potentially important mechanism for the fine-tuning of p53 functions. Furthermore, they indicate that deregulation of isoform expression may selectively contribute to cancer progression in tumours which retain wild-type p53 alleles.

Results

Results |1. Effects of ∆40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumour suppressor protein p53 108

RESULTS

Effects of 40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumour suppressor protein p53

Article No.1

Submitted to: BMC Cancer

Context

The p53 protein is expressed as multiple isoforms that differ in their N-and C-terminus due to alternative splicing, promoter or codon initiation usage (Marcel and Hainaut, 2009).

40p53 can be produced from two different mechanisms, initiation of translation at AUG 40 in fully spliced p53 mRNA or by alternative splicing retaining intron 2 (Courtois et al., 2002;[START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF]Yin et al., 2002). Consequently, 40p53 lacks the first 39 residues containing the main transcriptional activation domain. It has been previously shown that 40p53 was unable to transactivate p53 target genes using luciferase reporter constructs, and that overexpression of 40p53 antagonized wild-type p53 transcriptional activity (Courtois et al., 2002). However, animal models overexpressing 40p53 in mouse or zebrafish have shown complex phenotypes suggestive of p53-dependent growth suppressive effects [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF][START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF][START_REF] Pehar | Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death[END_REF]. These observations are not compatible with a simple role of 40p53 as an antagonist of wild-type p53, suggesting that when expressed in conditions where proteins are at roughly equivalent effect, 40p53 may modulate TAp53 activity in a subtle way.

The aim of this study is to analyse in vitro the effects of 40p53 on TAp53 transcriptional activity in conditions where both proteins are expressed at roughly equivalent levels.

Strategy

We performed co-transfections of constitutive (CMV-driven) expression vectors for TAp53 (full-length p53, corresponding to the C-terminal isoform) and 40p53 (also as C-terminal isoform) in p53-null cell lines to identify the formation of hetero-tetramers between both isoforms. We have analysed the capacity of these hetero-oligomers to bind specifically to DNA, using an Electro-Mobility-Shift Assay (EMSA) with a 32 P-labeled p53 Response Element (p53RE) consensus oligonucleotide. We then measured in Saos-2 and H1299 cells the transcriptional activity of p53, using a -galactosidase reporter gene assay, in presence of different amounts of 40p53. To precisely assess the inhibitory power of low levels of 40p53 on TAp53 activity, we have used a strategy previously developed by [START_REF] Chan | How many mutant p53 molecules are needed to inactivate a tetramer?[END_REF] to investigate how many mutant p53 molecules were needed to inactivate a p53 tetramer [START_REF] Chan | How many mutant p53 molecules are needed to inactivate a tetramer?[END_REF]. Finally, to determine how 40p53 may modulate the transcriptional activity of TAp53, we hypothesized that 40p53 could interfere with Hdm2mediated degradation of mixed p53/ 40p53 oligomers. We thus co-transfected p53, 40p53

and Hdm2 expression vectors in Saos-2 cells and analysed p53 stability.

Results

We first analysed the intrinsic turnover of 40p53 in two cell lines that constitutively express detectable levels of this isoform, either together with TAp53 (HCT116) or alone (21PT). The breast cancer cell line 21PT contains an insertion of 1 base at codon 33 in TP53, leading to frameshift and to a premature stop downstream of the initiation codon at AUG 1.

However, the sequence downstream of the internal initiation codon at AUG 40 is intact and usage of this codon results in the expression of 40p53. We show that, in both cell contexts, 40p53 has a slow turnover (several hours) as compared to the rapid turnover of TAp53, consistent with the notion that this isoform lacks the binding domain from Hdm2 and therefore escapes Hdm2-mediated degradation. Next, we show that 40p53 can form heterooligomers with p53. By EMSA and super-shifting experiments using an antibody to the Nterminus of TAp53 (DO-7), we showed that these hetero-oligomers could bind DNA in configurations compatible with the presence of 1, 2 or 3 monomers of 40p53 within a p53 tetramer. By co-transfection into two different p53-null cell lines, H1299 and Saos-2, we showed that 40p53 could modulate the transcriptional activity of TAp53 towards a generic p53-dependent -galactosidase reporter gene. However, the amplitude and dose-dependence upon 40p53 of these effects differed between the two cell lines. In H1299 cells, co-Results |1. Effects of ∆40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumour suppressor protein p53 110 expression of the two proteins induced a decrease in transcription with an amplitude that depended upon the predicted composition of the hetero-tetramer and a dose-dependent variation that suggested that the inhibitory potential of 40p53 was stronger at low levels 40p53/TAp53 ratios than at high ratios (Figure 19). In Saos-2, a paradoxical effect was observed, with first a small but reproducible increase in activity for hetero-tetramers predicted to contain 1 or 2 monomers of 40p53, followed by a sharp decrease at higher 40p53/TAp53 ratios. These results could not be interpreted solely on the basis of a dose-dependent titration of TAp53 transcriptional activity by 40p53. To determine whether 40p53 may also modulate transcriptional activity by interfering with Hdm2 binding, we co-transfected TAp53, 40p53

and Hdm2 in Saos-2 cells. Results show that expression of Hdm2 prevented the degradation of p53, suggesting that it interferes with the binding of Hdm2 to the hetero-tetramer. This effect may account for an increased stability of hetero-oligomers that have incorporated 1 or 2 monomers of 40p53.

Conclusion

Our results confirm that 40p53 has an extended life-span and stability in cells as compared to TAp53. They also confirm previous findings that 40p53 could form mixed hetero-oligomers with TAp53, and further show that different configurations of these oligomers may bind to DNA. Thus, the effect of 40p53 on TAp53 cannot be interpreted as a simple competition for DNA-binding between two independent proteins. This effect consist into a more subtle impact of 40p53 on the activity of p53 tetramers, the strength of which may vary depending on how many 40p53 subunits are incorporated into the tetramers. This hypothesis was confirmed by analysing the effects of 40p53 on transactivation of a generic reported gene by TAp53 over a range of 40p53/TAp53 ratios. In two different cell lines, the effects of 40p53on TAp53 were not linear and were different from the predicted inhibition curves, suggesting that other factors than simple inhibition of transactivation by 40p53 were involved. Our results further show that, among such factors, interference with the binding of Hdm2 may have a critical role. Indeed, co-transfection of 40p53 in Saos-2 at least partially protected TAp53 against rapid degradation in the presence of ectopically expressed Hdm2.

Overall, these different effects suggest that 40p53 exerts subtle modulation on p53 activity, in particular in the range of expression levels where 40p53 is expressed at lower or equal levels to p53, which are generally observed in non-transfected cells. These modulation results from the combination of at least two effects resulting from the fact that 40p53 lacks residues 1-42 of TAp53, namely, escape from Hdm2-mediated degradation, which extends the lifespan of p53 oligomers and therefore transactivation capacity, and decreased transactivation due to decreased capacity of the hetero-oligomer to bind components of the transcription machinery. Altogether, our observations shed a new light on a possible role of 40p53 in the regulation of p53 activity and provide a model to explain the effects of 40p53 on TAp53 observed in animal models. Thus, 40p53 may either enhance or inhibit p53 function in a manner which is exquisitely regulated by the relative levels of each isoform and their interrelations with p53 regulatory systems such as Hdm2/Hdm4.

Results |2. p53 regulates the transcription of its ∆133p53 isoform through specific response elements contained within the TP53 P2 internal promoter 134

p53 regulates the transcription of its 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter

Article No.2

Oncogene, 29: 2691-2700, 2010

Context

The 133p53 isoform was discovered by JC Bourdon and collaborators in 2005, who first reported the existence of a p53 transcript initiated in the distal part of intron 4, independent from the main p53 transcript initiated upstream of exon 1 (Bourdon et al., 2005). These authors postulated that this transcript could drive the expression of an N-terminal truncated p53 by using as initiation codon an in-frame AUG located at position 133 in TAp53. Because of its regulation through an alternative internal promoter (P2 promoter), 133p53 shows a functional similarity with Np63, which is mainly expressed from an internal, intronic promoter regulated by both TAp63 and p53 [START_REF] Grob | Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53[END_REF][START_REF] Harmes | Positive and negative regulation of deltaN-p63 promoter activity by p53 and deltaN-p63-alpha contributes to differential regulation of p53 target genes[END_REF]. The P2 promoter spans the region between intron 1 and exon 5 of TP53 gene, covering around 1.5 kb (Bourdon et al., 2005). The resulting transcript, p53I4, contains the 3'end of intron 4 followed by all exons from 5 to 11. This p53I4 mRNA has been observed to be overexpressed in squamous cell carcinoma of the head and neck (Boldrup et al., 2007) and in breast cancers (Moore et al., 2010), suggesting a role for 133p53 isoform in tumourigenesis. In this study, we have used siRNA to the p53I4 mRNA to provide conclusive evidence for the existence of the 133p53 isoform and we have determined whether, similarly to Np53, this promoter could be regulated by TAp53. This work was carried out within collaboration with V. Marcel.

Strategy

To determine the role of p53 in 133p53 expression, we first over-expressed p53 in A549 cell lines. To examine whether p53 regulates p53I4 mRNA level and to demonstrate that this mRNA indeed supports the synthesis of 133p53, we silenced p53 expression in A549 cells

Results |2. p53 regulates the transcription of its ∆133p53 isoform through specific response elements contained within the TP53 P2 internal promoter 135 using a siRNA targeting exon 4 of TP53 gene. Then, we assessed p53I4 mRNA expression in response to DNA damage induced by doxorubicin to investigate the possible regulation of 133p53 by p53 in response to stress. To investigate whether 133p53 isoform may modulate p53 DNA-binding activity, we performed an EMSA using a 32 P-labeled p53RE consensus oligonucleotide. We then performed a colony formation assay using p53-null H1299 cells transfected with constitutive expression vectors for p53 or 133p53 transfected vectors to determine whether 133p53 may inhibit p53 suppressive function. Next, we generated a luciferase reporter driven by the P2 promoter sequence identified by Bourdon et al. (2005) consisting in a 1.5 kb fragment from intron 1 to exon 5 of TP53 gene, and by sequential deletions of this sequence, to determine whether p53 could transactivate the internal P2

promoter (Bourdon et al., 2005). This luciferase reporter was co-transfected in p53-null H1299 cells with various amounts of pcDNA3-p53 expression vector. Finally, chromatin immunoprecipitation (ChIP) and luciferase assays using P2 promoter deletion constructs were used to analyze the binding of p53 on different segments of the P2 promoter in A549 cells.

Results

Regulation of P2 promoter by TAp53

Induction of TAp53 in A549 cells (expressing wild-type p53) resulted in an increase in the levels of p53I4 mRNA and increased levels of a small-molecular weight variant of p53 protein, with an apparent molecular weight of 34 kDa. Usage of a siRNA to p53I4 decreased the levels of this 34 kDa species, identifying it as the 133p53 protein. Next, we identified putative p53 binding sequences in the P2 promoter. Using stepwise deletion mutants, we found that two of these sequences had a critical role in the activation of the promoter by p53.

By ChIP experiments, TAp53 was found to bind to promoter fragment containing these sequences. We also showed that 133p53 did not bind specifically to a p53 consensus DNA sequence in vitro, but competed with wild-type p53 in specific DNA-binding assays. Finally, we found that 133p53 counteracted p53-dependent growth suppression in a clonogenic assay (Figure 20). (B) Colony formation assays were performed using p53-null H1299 cells transfected with 0.5 μg/ml of wild-type pcDNA3-p53 vector and/or -133p53 vector during 14 days under 0.5 mg/ml of neomycin selection. As compared to wild-type p53, 133p53 did not suppress cell growth and partially reverted the suppression induced by wild-type p53.

Effect of 40p53 on P2 promoter activity

Since 40p53 had been shown to exert complex effects on the transcriptional activity of p53-dependent promoter, we investigated whether 40p53 could modulate the activation of the P2 promoter by TAp53. Both proteins were co-transfected into the p53-null cell line H1299 together with the P2-driven luciferase reporter. Results showed that 40p53 inhibited the activation of P2 promoter by TAp53, suggesting the existence of complex regulatory loops interconnecting TAp53, 40p53 and 133p53 (Figure 21).

Figure 21. P2 promoter activity is down-regulated by 40p53

In p53-null H1299 cells, luciferase assays (upper panel) were performed using a luciferase reporter under the control of the 1.5 kb segment of TP53 from intron 1 to exon 5, in the presence of both p53 and 40p53 in different ratio. The expression of both isoforms was verified by western blot with the p53 polyclonal antibody CM1 (lower panel). 40p53 decreases luciferase activity of P2 promoter. Basic: promoterless luciferase reporter; P2: luciferase reporter driven by TP53 segment; Ku80: loading control. Black arrowhead: p53 protein; grey arrowhead: 40p53 protein; Ku80: loading control.

Conclusion

These results identify 133p53 protein as an independent isoform of p53, expressed from the internal promoter P2. By demonstrating that this promoter was regulated by TAp53 in response to genotoxic stress, we identify a novel auto-regulatory loop by which p53 may control the expression of an isoform potentially acting as an inhibitor of p53 suppressor activities. From a biological viewpoint, 133p53 does not appear to bind DNA and prevents TAp53 to form stable p53:DNA complexes. These observations are in agreement with the predicted structure of 133p53 that lacks a part of the DNA-binding domain and therefore has an unfolded DNA-binding domain. Its inhibitory effect on TAp53 may thus be due to a transconformational effect of 133p53 on TAp53 DNA-binding domain, similar to the one exerted by mutant p53 on wild-type p53 conformation and binding activity [START_REF] Milner | Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation[END_REF]. Furthermore, 40p53 inhibited the induction of the P2 promoter by TAp53, suggesting that the three isoforms may have coordinated effects and may regulate each other. Overall, 133p53 is a novel target of p53 that may participate in a negative feedback loop controlling p53 function.

Results |3. Expression of ∆40p53 and ∆133p53, two isoforms of p53 lacking the N-terminus, in cutaneous melanoma 148

Expression of 40p53 and 133p53, two isoforms of p53 lacking the N-terminus, in cutaneous melanoma

Article No.3

To be submitted to Virchow Archiv

Context

Due to their capacity to antagonize p53 when expressed in excess to TAp53, N-terminal isoforms 40p53 and 133p53 may provide a functional mechanism to inactivate p53 in tumours that do not contain mutant TP53. Indeed, the N-terminal isoforms, 40p53 and 133p53, which lack the first 39 ( 40p53) or 132 ( 133p53) residues, both inhibit growth suppression in a dominant-negative manner when expressed in significant excess as compared to TAp53.

Cutaneous melanomas are highly malignant tumours that retain wild-type TP53 in over 90% of the cases, suggesting that p53 suppressive activity may be abrogated through alternative mechanisms (Chin et al., 2006;Tsao et al., 2012). Such alternative mechanisms have been demonstrated in cancers of the cervix, head and neck and in sarcomas, in which wild-type p53 is suppressed by targeting the protein for proteasome-mediated degradation through viral or cellular proteins acting as E3 ubiquitin ligases (Petitjean et al., 2007). In sarcomas, the main mechanism is amplification and over-expression of Hdm2, the main regulator of p53 protein stability [START_REF] Onel | MDM2 and prognosis[END_REF]. A recent study has shown that Hdm4, a RING finger protein which forms complexes with Hdm2, was up-regulated in a substantial proportion (65%) of stage I-IV metastatic melanomas (Gembarska et al., 2012).

Hdm4 (also known as Hdmx) cooperates with Hdm2 to induce poly-ubiquitination and proteosomal degradation of p53 (Wang, 2011). In this study, we have analysed the expression of p53I2 mRNA (encoding 40p53) and p53I4 mRNA (encoding 133p53) isoforms to determine their potential involvement in inactivating p53 in cutaneous melanomas.

Strategy

Through collaboration with JC Marine, University of Ghent, Belgium, we have used mRNA extracted from short-term cultures of melanoma cells, and from stage I-IV melanomas which had previously been used to analyse Hdm4 expression levels by Gembarska et al.

(2012) (Gembarska et al., 2012). A series of 40 cases was assembled, sub-grouped in 4 categories of 10 cases according to the extent of metastatic dissemination, with group 1: noninvasive melanomas (stage I-II), group 2: Regional Dermal (RD) melanomas; group 3: Nodal Metastatic (NM) melanomas and group 4: Distal Metastatic (DM) melanomas. The levels of expression of Fully-Spliced (FSp53), p53I2 and p53I4 mRNA were analysed by RT-PCR using GAPDH as internal standard and the Ct method to express the results. In cell lines, expression levels were further compared to the mean of levels in two primary cultures of normal melanocytes. In tumours, the mean Ct values in group 1 (non-invasive melanomas)

were used as reference.

Results

RT-qPCR provides for a clear and unambiguous, quantitative assessment of steady-state levels of FSp53, p53I2 and p52I4 mRNA in melanoma cell lines and tissues. In cultured melanoma cell lines, enhanced expression of FSp53, p53I2 or p53I4 compared to primary melanocytes was detected in 53% of the cases (9/17). Expression of FSp53 and p53I2 was often enhanced in a coordinated way. In contrast, p53I4 was substantially enhanced in only one cell line and was not correlated with FSp53 or p53I2. Compared to primary stage I-II melanoma, enhanced expression of either p53I2 or p53I4 was detected in a substantial proportion of metastatic cases (70%; 21/30). When sub-grouped according to metastatic status, enhanced expression of either p53I2 or p53I4 was more frequent in nodal (NM) (9/10) and distant (DM) (8/10) melanomas than in regional dermal (RD) (4/10) lesions (p=0.044, Fisher-Exact) (Figure 22). Whereas enhanced expression of FSp53 mRNA was detected only in cases with enhanced expression of p53I2, p53I4 alone was increased in 6/30 lesions (20%).

Figure 22. Representation of expression levels of FSp53, p53I2 and p53I4 mRNAs in melanoma tissues

qRT-PCR analysis were performed to determine the expression levels of FS-p53 (Fully-spliced p53, grey dots), p53I2 (retaining intron 2, white dots) and p53I4 (retaining intron 4, black dots). Bars indicate the average.

Conclusion

In this study, we have taken advantage of a pre-existing, well characterized series of melanoma cases sub-grouped according to metastatic status to analyse the levels of expression of mRNA encoding TAp53 (FSp53 mRNA), 40p53 (p53I2 mRNA) or 133p53 (p53I4 mRNA) using a qRT-PCR strategy. Our results show that levels of expression of these mRNA isoforms are increased in the majority of metastatic melanoma cases and was significantly higher and more frequent among melanoma with nodal (NM) or distant (DM) metastasis than among regional dermal (RD) or non-metastatic melanoma. Among the 3 NM and DM cases without enhanced p53I2 or p53I4 mRNA expression, one contained mutant TP53 (p.R342X) whereas the mutation status of the two other cases was not available. FSp53 and p53I2 mRNA showed an overlapping pattern of enhanced expression, whereas p53I4 mRNA expression is enhanced in cases that show neither FSp53 nor p53I2 mRNA expression. These results are compatible with the fact that FSp53 and p53I2 mRNA are alternative splicing variants expressed from the same promoter, whereas p53I4 mRNA is controlled by a distinct promoter located in intron 4. Thus, more than one mechanism may contribute to enhanced expression of p53 mRNA isoforms in metastatic melanoma. Overall, these results show that deregulation of expression of p53 mRNA encoding N-terminal truncated isoforms is common in nodal or distant metastatic melanoma lesions, suggesting that this phenomenon may contribute to functional inactivation of p53 during the spread of melanoma. Our study has strong limitations in the fact that it does not provide a direct assessment of p53 protein status.

Indeed, thus far, there is no specific antibody that specifically recognizes 40p53 or 133p53 isoforms. Since their amino-acid sequence is a part of TAp53 sequence, isoforms are recognized by antibodies that also react with TAp53, making it cumbersome to analyse p53 isoform expression using histochemistry. Further studies will be needed to assess the levels of p53 protein isoforms and to understand the dynamics of interactions between TAp53 and p53 isoforms in melanoma cells that overexpress Mdm4 or not.

Introduction

Cutaneous melanoma is the most lethal form of skin cancer and shows increasing incidence rates in most Western countries. This highly metastatic cancer is resistant to conventional therapy (Chin et al., 2006;Ibrahim and Haluska, 2009). Whereas the TP53 gene is wild-type in about 95% of melanomas, activating mutations in the catalytic domain of the BRAF serine-threonine kinase are detectable in over 50% of the cases, with the most common mutation leading to substitution of valine by glutamic acid at position 600 (V600E).

Other mutations occur in NRAS (in about 15% of melanoma), MEK1 or MEK2 (Chin et al., 2006;Tsao et al., 2012). Targeting the BRAF oncogene using the selective inhibitor verumafenib (PLX4032) has shown promising results in Phase III trials (Bollag et al., 2012).

However, most patients rapidly develop resistance, highlighting the need for combined therapy to improve the long-term effectiveness of such treatments.

In cancers with wild-type TP53 alleles, the suppressive activity of the p53 protein is frequently switched-off by alternative mechanisms such as targeting p53 for proteasomemediated degradation through viral or cellular proteins acting as E3 ubiquitin ligases (Petitjean et al., 2007). In melanoma, amplification of MDM2, encoding the main E3-ubiquitin ligase of p53, is detected in 3-5% of the cases (Muthusamy et al., 2006). A recent study has shown that another negative regulator of p53, Mdm4, was up-regulated in a substantial proportion (65%) of stage I-IV metastatic melanomas (Gembarska et al., 2012). Mdm4 (also known as Mdmx) cooperates with Mdm2 to induce polyubiquitination and proteosomal degradation of p53 (Wang, 2011). Inhibition of p53-Mdm4 interaction restored the proapoptotic functions of p53 in melanoma cells, resulting in increased sensitivity to cytotoxic treatments and to inhibitors of BRAF (V600E) oncogene, suggesting that enhanced Mdm4 expression antagonizes p53 suppressor functions (Gembarska et al., 2012).

Expression of p53 isoforms may represent another alternative mechanism for functional inactivation of p53 suppressor functions. These isoforms differ from full-length p53 (TAp53) by truncation of a variable portion of the N-terminus (DeltaN isoforms) and by alternative Cterminal portions (C-terminal isoforms) (Courtois et al., 2004a). The main N-terminal isoforms, Delta40p53 and Delta133p53, lack the first 39 and 132 residues, respectively, and are unable to transactivate p53 target genes (Bourdon et al., 2005;[START_REF] Marcel | p53 regulates the transcription of its Delta 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF].

Delta40p53 is generated by two mechanisms, internal initiation at AUG 40 using fully-spliced p53 mRNA (Courtois et al., 2002;Yin et al., 2002), or alternative splicing of p53 pre-mRNA that retains intron 2 to generate p53I2 mRNA, which introduces in-frame stop codons downstream of AUG 1 and leads to usage of AUG 40 as initiation codon [START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF].

Delta133p53 is expressed from an alternative promoter within intron 4, leading to the expression of p53I4 mRNA that carries AUG 132 as initiation codon (Bourdon et al., 2005;Murray-Zmijewski et al., 2006). Both isoforms inhibit growth suppression in a dominantnegative manner when expressed in significant excess as compared to TAp53 (Aoubala et al., 2011;Courtois et al., 2002;[START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF][START_REF] Marcel | p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]Yin et al., 2002).

Increased levels of mRNA encoding p53 isoforms have been reported in several cancers including breast and head and neck (Boldrup et al., 2007;Moore et al., 2010). In melanoma cell lines, Avery-Kiejda et al. have reported that small molecular weight variants of p53 were frequently, but not systematically overexpressed at both mRNA and protein levels (Avery-Kiejda et al., 2008). In contrast, these variants isoform were barely detectable in primary melanocytes. Furthermore, they show that ectopic expression of Delta40p53 in a melanoma cell line constitutively expressing low levels of this isoform inhibited the expression of two p53-dependent reporter genes (driven by the p53-response elements from p21/WAF1 or PUMA promoters, respectively). Together, these observations suggest a role for enhanced expression of N-terminal isoforms in the functional inactivation of wild-type p53 in cancer cells [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF].

In this study, we have used mRNA extracted short-term cultures of melanoma cell lines and from stage I-IV human cutaneous melanoma with well defined metastatic status to analyse the expression of p53, p53I2 (encoding Delta40p53 mRNA) and p53I4 (encoding Delta133p53 mRNA). These tumours have been previously analysed for their BRAF and NRAS mutation status and for enhanced expression of Mdm4 (Gembarska et al., 2012).

Patients, materials and methods

Patients and samples

All samples (melanoma tissues and cell lines) were kindly provided by Dr JC Marine, University of Ghent, Belgium. The panel of 17 short-term culture of melanoma cell lines has been described in Gembarska et al. (Gembarska et al., 2012) Gembarska et al., (2012), in which their characteristics are described in details (Gembarska et al., 2012). Briefly, melanoma patients were aged 19-82 years, with 46% of the cases over 60 years old. Pathological subtypes of primary tumours included mainly superficial spreading melanoma (46%), nodular melanoma (30%) and was unknown in 20% of the cases. BRAF V600E mutations were observed in 14/40 (35%) and NRAS mutations at Q61 in 6/40 cases (15%). There were no significant differences in the distribution of these mutations across the 4 sub-groups.

RNA extraction and quantitative Real-Time PCR (qRT-PCR)

RNA was extracted using the Rneasy minikit (Qiagen) according to the manufacturer's protocol and was quantified using a NanoDrop 1000 (Thermo Scientific). A total of 2 ug of RNA was reverse transcribed using the High-Capacity cDNA reverse transcription kit (Applied Biosystems). The same cDNA as for prevously reported Mdm4 qRT-PCR were used for analysis of FSp53, p53I2 and p53I4 mRNA using Brilliant SYBR Green master mix (Qiagen) as fluorescent readout. Primers for each mRNA isoform are listed in Table 1. For each isoform, expression levels were normalized to GAPDH using the DeltaCT method. To evaluate expression in cell lines, the mean of DeltaCT values in two cultures of primary melanocytes was used as reference. In metastatic lesions, expression was assessed using as reference the mean of DeltaCT values for Primary Melanomas. Results for each metastatic melanoma sample were expressed using the DeltaDeltaCT method.

Statistical analysis

For each data point, results were expressed as means of DeltaDeltaCT values ± standard deviation using data from at least 3 independent qRT-PCR replicates. These values were categorized in three groups defining low (mean DeltaDeltaCT value <0.3), normal (mean DeltaDeltaCT value between 0.3 and 3) or high expression (mean DeltaDeltaCT value >3).

The distribution of samples accross each expression group was analysed using the Freeman-Halton extension of the Fisher-Exact probability test for a two-rows by three-columns contingency table using the tools available at http://vassarstats/net.

Results

Three p53 mRNA isoforms (FSp53, p53I2 and p53I4) were analysed by qRT-PCR in a panel of 17 short-term cultures of melanoma cell lines and in 40 melanoma lesions categorized in 4 groups of 10 cases according to the extent of metastatic spread. Figure 1 shows a schematic representation of the transcripts analysed and the position of the primers used (sequences listed in Table 1). Figure 2 shows the levels of expression in melanoma cell lines using as reference the levels of expression in two primary cultures of normal melanocytes. It was considered that normal levels of expression encompassed a 3-fold variation relative to this control. Using this threshold, increased expression of FSp53 mRNA was observed in 8 cell lines, 5 of which also show enhanced expression of p53I2 mRNA. In contrast, increased expression of p53I4 mRNA was detected in only 1 cell line which did not show enhanced expression of FSp53 or p53I2 mRNA, and decreased expression was detected in 6 cell lines. These results suggest that changes in levels of expression of p53 mRNA isoforms occur among melanoma cell lines. Enhanced expression of p53I2 tended to be coordinated with enhanced expression of FSp53, while changes in expresssion of p53I4 mRNA appeared to be independent of p52I2 and/or FSp53. This observation is compatible with the notion that FSp53 and p53I2 are transcribed from the same, main p53 promoter whereas p53I4 mRNA is transcribed from a distinct, internal promoter located in intron 4

(see Figure 1).

Next, the same qRT-PCR strategy was used to analyse levels of p53 mRNA isoforms expression in cutaneous melanoma samples, sub-grouped according to the extent of metastatic spread. The mean of DeltaCT values in primary, stage I or II melanoma (PM) was used as reference. Compared to this reference, levels of expression of p53 mRNA isoforms were observed to be higher in Nodal (NM) or Distant (DM) metastatic lesions than in Regional Dermal (RD) lesions (Figure 3). Figure 4 shows the expression status of each RD, NM and DM cases in the form of a « heat map », in relation with their previously described BRAF, NRAS and TP53 mutation status. Of a total of 30 metastatic melanomas, 21 (70%)

showed increased expression of at least 3 fold in at least one of the p53 mRNA isoforms.

Among a total of 20 NM and DM lesions,only 3 (sample numbers 28,34,37) showed no change or decrease in expression mRNA isoforms. Among these three cases, sample 28 contained a mutation in TP53 (p.R342X) whereas the TP53 mutation status of samples 34 and 37 was not available. Only one of the 21 samples with enhanced expression of p53 mRNA isoforms was found to contain a TP53 mutation (sample 23, p.R273H).This sample showed a modest increase in only p53I2 mRNA. The differences between numbers of cases with enhanced p52I2 or p53I4 expression in NM or DM lesions compared to PM and RD lesions were significant (P<0.01, Fisher-Exact). When comparing only metastatic lesions, enhanced expression of either p53I2 or p53I4 was more frequent in nodal (9/10) and distant (8/10) than in regional dermal (4/10) lesions (p=0.044, Fisher-Exact).

Of the different mRNA isoforms, FSp53 mRNA was the least frequently enhanced (8/30 cases, all NM or DM) and was systematically associated with enhanced expression of p53I2 mRNA. The latter was detected in 14 cases (47%) and enhanced expression of p53I4 was detected in 18 cases (60%). Of note, enhanced expression of p53I4 mRNA alone was detected in 6 of the 20 metastatic melanoma samples (30%). Overall, these results suggest that expression of p53 mRNA isoforms encoding proteins with different N-terminal parts is often deregulated in melanoma in the absence of inactivating TP53 mutation.

Discussion

Cutaneous melanoma is a highly metastatic form of cancer which is extremely difficult to treat using conventional cytotoxic protocols, suggesting that in these tumours the pathways of apoptotic response to DNA-damaging signals are extremely inefficient despite the fact that they contain, in their vast majority, wild-type TP53 (Avery-Kiejda et al., 2008;Gembarska et al., 2012). This observation has led to suggest that alternative mechanisms were at work to inactivate p53 protein function in melanoma. Enhanced expression of isoforms of p53 lacking the N-terminus such as Delta40p53 and Delta133p53 may represent a plausible mechanism since these isoforms are unable to transactivate p53 target genes and have been shown to inhibit transactivation of such genes by wild-type p53 in a dominant-negative manner [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF]. In this study, we have taken advantage of an existing, well characterized series of melanoma cases sub-grouped according to metastatic status to analyse the levels of expression of mRNA encoding TAp53 (FSp53 mRNA), Delta40p53 (p53I2 mRNA) or Delta133p53 (p53I4 mRNA) using a qRT-PCR strategy. Our results show that levels of expression of these mRNA isoforms are increased in the majority of metastatic melanoma cases (70%). Moreover, enhanced expression was significantly higher and more frequent among melanoma with nodal (NM) or distant (DM) metastasis than among regional dermal (RD) or non-metastatic melanoma. In NM and DM combined, 17/20 tested cases (85%) showed enhanced expression of p53I2 and/or p53I4 mRNA. Among these cases, TP53 mutation status was available of 9 cases and only one was found to be mutant (case 23, p.R273H). Among the 3 cases without enhanced p53I2 or p53I4 mRNA expression, one contained mutant TP53 (p.R342X) whereas the mutation status of the two other cases was not available. FSp53 and p53I2 mRNA show an overlapping pattern of enhanced expression, whereas p53I4 mRNA expression is enhanced in cases that show neither FSp53 or p53I2 mRNA expression. These results are compatible with the fact that FSp53 and p53I2 mRNA are alternative splicing variants expressed from the same promoter, whereas p53I4 mRNA is controlled by a distinct promoter located in intron 4 (Aoubala et al., 2011;Bourdon et al., 2005;[START_REF] Marcel | p53 regulates the transcription of its Delta 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]. Thus, more than one mechanism may contribute to enhanced expression of p53 mRNA isoforms in metastatic melanoma. Overall, these results show that deregulation of expression of p53 mRNA encoding N-terminal truncated isoforms is common in nodal or distant metastatic melanoma lesions, suggesting that this phenomenon may contribute to functional inactivation of p53 during the spread of melanoma.

Small molecular weight variants of p53 have been reported to be frequently expressed in human melanoma cell lines but not in primary melanocytes (Avery-Kiejda et al., 2008). Using The notion that p53 suppressor function may be functionally inactivated in melanoma in the absence of TP53 mutation has been recently demonstrated in a recent study on the role of Mdm4 overexpression in cutaneous melanoma (Gembarska et al., 2012). This study reported that levels of expression of Mdm4, a critical co-regulator of p53 protein stability cooperating with Mdm2 to induce p53 protein degradation, was consistently overexpressed in a majority of malignant melanoma. Functional studies in cultured cells and in a mouse model of human melanoma revealed that expression of Mdm4 enhanced the survival and growth of melanoma cells by antogonizing p53 function. Inhibition of Mdm4-p53 interaction restored p53 function and increased responses to different forms of treatments including cytoxic agents such as doxorubicin or specific inhibitor of activated BRAF oncogene, suggesting that restoring p53 may represent an interesting approach to enhance and extend the effects of targeted therapy.

Since the Mdm2-Mdm4 complex induce p53 degradation by binding to a motif in the Nterminal portion (residues 17-29), its action is expected to result in the elimination of TAp53

but not of the p53 isoforms lacking the N-terminus such as Delta40p53 or Delta133p53. Both of these isoforms have been shown to escape Mdm2-dependent degradation and to have an extended half-life as compared to TAp53 (Courtois et al., 2002;[START_REF] Marcel | p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]. In initial studies, Delta40p53 was found to accumulate in response to overexpression of Mdm2 (Yin et al., 2002), perhaps as a result of accumulation of this degradation-resistant form of the protein. Therefore, overexpression of N-terminal isoforms may cooperate with Mdm4 overexpression to antagonize p53 suppressor function. Since the present study was conducted using the same melanoma specimens as those used to assess Mdm4 expression, we compared our data on p53 isoforms with those previously published on Mdm4 expression status (Gembarska et al., 2012). Overall, compared to mean expression levels in PM (stage I and II), Mdm4 mRNA levels were enhanced in 0/10 RD, 6/10 NM and 3/10 DM lesions.

Among these 9 cases, 5 also showed enhanced FSp53 expression and all showed enhanced expression of either p53I2 and/or p53I4 mRNA. These results are compatible with the hypothesis that overexpression of Mdm4 synergizes with enhanced expression of p53 isoforms, Mdm4 inducing the elimination of TAp53 by degradation and N-terminal isoforms neutralizing the suppressor activity of residual p53 through dominant-negative inhibition.

Delta40p53 and Delta133p53 differ by their functional properties. While they both appear to retain the capacity to oligomerize with TAp53, Delta40p53 has an intact DNA-binding

Results |3. Expression of ∆40p53 and ∆133p53, two isoforms of p53 lacking the N-terminus, in cutaneous melanoma 161 domain (residues 96-306) and specifically binds to a p53 Response Element (RE) in vitro. In contrast, Delta133p53 lacks the proximal part of the DNA-binding domain, does not bind to specific DNA in vitro, and prevents TAp53 from binding to its cognate RE (Aoubala et al., 2011;[START_REF] Marcel | p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF]. Although the inhibitory capacity of these two isoform has not been systematically compared, Delta133p53 appears to have a stronger inhibitory effect than Delta40p53. Indeed, we have shown that, depending upon expression levels relative to TAp53, as well as cell context, Delta40p53 could exert either activating or inhibiting effects on TAp53. When expressed in excess compared to TAp53, its effect is however clearly antagonistic, preventing growth arrest induced by wild-type p53 in vitro.

Our study has strong limitations in the fact that it does not provide a direct assessment of p53 protein status. Indeed, thus far, there is no specific antibody that specifically recognizes Delta40p53 or Delta133p53 isoforms. Since their amino-acid sequence is a part of TAp53 sequence, isoforms are recognized by antibodies that also react with TAp53, making it cumbersome to analyse p53 isoform expression using histochemistry. In contrast, TAp53 can be identified through its differential reactivity with antibodies recognizing epitopes in the N-terminus of the protein, allowing for identification through differential western blotting.

Another limitation is that our study does not address the expression status of the C-terminus isoforms p53beta and p53gamma, which may interfere with p53 functions through a different mechanism than inhibition of transactivation [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF]. Further studies will be needed to assess the levels of p53 protein isoforms and to understand the dynamics of interactions between TAp53 and p53 isoforms in melanoma cells that overexpress Mdm4

or not.

Table 1. Primers used for qRT-PCR analysis of FSp53, p53I2 and p53I4 mRNA and high (>3, red) expression, with reference to mean value in non-metastatic melanomas).

The gene mutation status of BRAF, NRAS and TP53 is given (from (Gembarska et al., 2012)).

Discussion

DISCUSSION

The p53 tumour suppressor protein has an extremely complex pattern of regulation at transcriptional and post-translational levels, leading to describe p53 as a prototype of "massively regulated" transcription factor [START_REF] Hollstein | Massively regulated genes: the example of TP53[END_REF]. As highlighted in our Introduction review, the discovery of p53 isoforms has added another layer of complexity to the many mechanisms that regulate p53 functions (Marcel and Hainaut, 2009). However, so far, there is limited understanding of the patterns of expression and of the functions of each of the potentially existing 12 p53 isoforms. In this Thesis, we have focused on the two major p53

N-terminal truncated isoforms, 40p53 and 133p53. We have analysed their patterns of interactions with wild-type TAp53 (the full-length form which retains an intact transactivation domain) and we have investigated whether their expression could be deregulated in melanoma, a cancer type in which TP53 mutations are infrequent (Chin et al., 2006;Tsao et al., 2012). Our results show that both 40p53 and 133p53 entertain complex interactions with TAp53, with different effects despite the fact that the two isoforms have generic capacity to operate as dominant-negative inhibitors of p53.

At the onset of this project, 40p53 had been characterized (Courtois et al., 2002;Yin et al., 2002) but the existence of 133p53 was still a question of debate (Bourdon et al., 2005).

From a structural viewpoint, 40p53 is a counterpart of Np63 and Np73 isoforms, which both lack a segment of the N-terminus that encompasses the main transactivation domain [START_REF] Ishimoto | Possible oncogenic potential of DeltaNp73: a newly identified isoform of human p73[END_REF][START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF]. 40p53 corresponds to a form of p53 which is truncated at a point located between two clearly structurally defined domains, removing a segment of the protein containing binding sites for the basal transcriptional machinery, for p300/CBP acetylase and for the Hdm2/Hdm4 protein complex, the main regulator of p53 protein stability [START_REF] Dornan | The proline repeat domain of p53 binds directly to the transcriptional coactivator p300 and allosterically controls DNA-dependent acetylation of p53[END_REF][START_REF] Ohki | Dissecting functional roles of p53 N-terminal transactivation domains by microarray expression analysis[END_REF]. 40p53 is predicted to retain a folded central DNA-binding domain and a C-terminal oligomerisation domain. In contrast, 133p53 represents a form of the protein which is truncated within the structurally coherent, globular DNA-binding domain. Removal of the first 132 residues encompasses the interaction domains listed for 40p53 and a number of additional domains, including the proline-rich domain containing SH3 binding sites for the c-Abl protein [START_REF] Brown | c-abl is involved in the association of p53 and trk A[END_REF], the minor transactivation domain that selectively modulates the transcriptional activation of genes involved in apoptosis and a beta-sheet motif that is an integral part of the DNA-binding domain [START_REF] Cho | Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations[END_REF]. Together with a loop-helix motif encoded by residues 266 to 290, this beta-sheet constitutes the motif that binds in the major grove of target DNA [START_REF] Cho | Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations[END_REF]. Thus, 133p53 is predicted to have a crippled and at least partially unfolded DNAbinding domain. Based on these considerations, it is expected that the two N-terminal isoforms may have different biological effects and regulation mechanisms.

40p53: titrating TAp53 transactivation capacity while enhancing its stability

40p53 is expressed in an apparently p53-independent manner, using the same pre-mRNA as TAp53 and either alternative splicing or alternative codon initiation usage (Marcel and Hainaut, 2009). Our results show that the resulting protein forms mixed oligomers with TAp53 which retain DNA-binding capacity. When expressed at excess levels, 40p53 competes with TAp53 for its incorporation into tetramers and inhibits the transactivation capacity towards a standard reporter gene. However, when expressed at lower levels in which TAp53 and 40p53 may exist in roughly equivalent amounts, 40p53 appears to have a paradoxical effect which depend upon cell context. In H1299 cells, we found that 40p53 exerted an inhibitory effect on transactivation. In contrast, in Saos-2 cells, 40p53 exerted a "two-phases" effect, with preserved and even enhanced p53 transcriptional activity at low levels of 40p53, followed by an inhibitory effect at higher levels. These results suggest that the interactions of 40p53 with TAp53 could not be simply rationalized to a competition between the two proteins for oligomerization and DNA-binding. Depending upon other factors, 40p53 may exert some kind of positive effect on TAp53 functions.

We reasoned that, in addition to the lack of a transactivation domain, a major difference between 40p53 and TAp53 was the lack in the former of the Hdm2 binding domain (residues 17-29) [START_REF] Bottger | Molecular characterization of the hdm2-p53 interaction[END_REF]. Compatible with this hypothesis, our analysis of 40p53 expression in two cell lines that constitutively express detectable amounts of this isoform, either alone or together with TAp53, detected that 40p53 was more stable than TAp53. This observation raised the question of knowing whether, in mixed oligomers containing both 40p53 and TAp53, the degradation behaviour would be driven by the TAp53 or the 40p53 isoform. By co-transfecting Hdm2 together with both isoforms, we have shown that, in Saos-2 cells, 40p53 could at least partially protect TAp53 from rapid degradation. This result suggests that, in certain conditions, 40p53 may cooperate with TAp53 in forming mixed complexes which retain stable expression of 40p53 together with transactivation capacity of TAp53 (Figure 23). This model provides a rationale to understand the effects in both Saos-2 and in H1299 cells. In H1299 cells, we speculate that 40p53 has only a small effect on TAp53 stability. Thus, the "dominant" effect even at low concentration of 40p53 is the competitive inhibition of transactivation. With increasing concentrations of 40p53, however, this inhibition effect is partially compensated by the effect on stabilization and activity, leading to a progressive shift towards lower inhibition efficiency. In contrast, in Saos-2 cells, 40p53 can enhance stability and activity in a range of concentrations where the inhibitory effect on transactivation is, at best, limited. In such conditions, the biological effect is enhanced transactivation capacity, followed by rapid decrease at higher 40p53 concentrations. This model postulates that H1299 and Saos-2 cells differ in their intrinsic expression of factors that regulate p53 protein stability.

Figure 23. "Two-phases" model of regulation of TAp53 by 40p53

We postulate here that p53 biological response depends on the ratio 40p53/TAp53 isoforms are expressed and the cellular context. In one hand, 40p53 has only a small effect on TAp53 stability, thus a dominant negative role on TAp53 transactivation. On the other hand, 40p53 can enhance stability and activity in a range of concentrations where 40p53 has only a limited inhibitory effect on TAp53.

While the molecular mechanisms leading to decreased transactivation are easy to understand, and supported by numerous published studies using N-terminal truncated p53 (Courtois et al., 2002;[START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF]Yin et al., 2002), the mechanisms of enhanced

transactivation are an open question. The N-terminus contains, in addition to the Hdm2binding site, the binding sites for Histone Acetyl Transferases (HATs) such as p300/CBP and for components of the basal transcriptional machinery. Thus, several scenarios can be considered. First, incorporation of 40p53 may decrease the binding of Hdm2/Hdm4 complexes, thus decreasing degradation and inducing the accumulation of the heterooligomer. Second, it may somehow modulate the interaction of the hetero-oligomer with HATs and with components of TBP. A third scenario is that post-translational modifications such as phosphorylation, acetylation and ubiquitination of p53 may depend upon the numbers of TA domains in the hetero-oligomer. Dissecting these mechanisms is a task of considerable complexity. Our results implicate a possible role for Hdm2, although we have not been able to detect a measurable change in steady-state p53 levels. Interestingly, a wealth of evidence show that TAp53 is not degraded by Hdm2 when in a monomeric form and that at least dimer formation is needed for targeting by Hdm2 and degradation [START_REF] Kubbutat | Regulation of p53 stability by Mdm2[END_REF]. These observations add weight to our hypothesis that inclusion of one or two subunits of 40p53 within a tetramer may affect its targeting by Hdm2/Hdm4.

133p53: a novel p53 auto-regulatory loop implicating p53 isoforms

133p53 is expressed using a different mRNA than TAp53 and 40p53. This mRNA is transcribed from a promoter overlapping with a region of the TP53 gene extending from intron 1 to exon 5. The transcription start site is located in intron 4 and AUG 1+ corresponds to methionine codon at position 133 in exon 5. The existence of 133p53 was postulated by JC Bourdon and his collaborators in 2005, on the basis of their identification of a specific mRNA predicted to encode this protein (Bourdon et al., 2005). In 2010, in collaboration with Virginie Marcel, we provided a demonstration of the existence of this protein by showing that a siRNA targeted specifically to sequences derived from intron 4, which are present in 133p53 mRNA but not in TAp53 mRNA, reduced by 70% the expression of a p53 product of about 34 kDa, compatible with the predicted molecular weight of 133p53 [START_REF] Marcel | p53 regulates the transcription of its Delta 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF].

The main mechanism of Np63 isoform expression is transcriptional activation through a self-regulated internal promoter. This observation prompted us to investigate whether p53 may also directly regulate its own internal P2 promoter. Close examination of promoter sequence coupled with promoter dissection experiments identified 4 potential p53 Response Elements (p53RE). Two of these elements lied in close vicinity to each other near the transcription start site. Their deletion reduced the level of promoter activity and abrogated its regulation by TAp53. Site-directed mutagenesis of critical residues within these p53RE decreased, but not totally abrogated, promoter activity, suggesting that this promoter retains activity even in a degenerated form. The fixation of p53 on a fragment of the promoter encompassing these two p53REs was confirmed by ChIP assay.

Consistent with its expected protein structure, we found that 133p53 did not bind to specific DNA in vitro. When co-expressed with TAp53, it counteracted DNA-binding. In contrast with 40p53, super-shifting experiments did not show evidence that mixed oligomers could bind to DNA. As it has been described that TAp53 and 133p53 can form heterooligomers [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF], our DNA-binding data were consistent with the hypothesis that 133p53, when incorporated in a p53 oligomer, may scramble its conformation and prevent stable interaction with DNA. As a result, 133p53 is unable to transactivate target genes and, in a clonogenic assay, was shown to counteract TAp53-induced growth suppression when expressed in excess as compared to TAp53. Overall, these results support the hypothesis that, although sharing some functional similarities with 40p53, 133p53 is involved in a distinct regulatory system and is exerting different effects on TAp53 than its sister N-terminal truncated isoform. While the expression of 40p53 is driven by the same main P1 p53 promoter as TAp53, the expression of 133p53 is driven by its own, distinct promoter, and is regulated by p53 itself. Thus, the regulation of 133p53 by p53 defines a novel autoregulatory feedback loop in which p53 regulates the expression of an isoform acting as dominant-negative inhibitor of its own function (Figure 24). As shown in the result section, this feedback loop can reach further complexity by considering that excess levels of 40p53 may suppress the effect of TAp53 on the internal P2 promoter. On the other hand, it should be considered that 133p53 may bind to 40p53 and antagonize its effects by scrambling its capacity to bind to DNA. Thus, despite obeying to different regulatory mechanisms, the two N-terminal isoforms may entertain a form of mutual equilibrium.

Figure 24. Auto-regulatory feedback loop of p53 and its isoforms

TAp53 and D40p53 are expressed from the P1 promoter, while D133p53 expression is driven by its own promoter P2. TAp53 is able to induce the expression of 133p53 on its own internal P2 promoter.

In turn, 133p53 has the capacity to inhibit TAp53 activity, suggesting an auto-regulatory negative control of p53 on its own function. Moreover, we illustrate here that 40p53 has a suppressive effect on the TAp53 capacity to induce the P2 promoter. Finally, it should be considered that 133p53 may bind to 40p53 and antagonize its effects by scrambling its capacity to bind to DNA (dashed red line).

Green arrows: positive effects (increased expression and/or activity). Red lines: negative effects (decreased expression and/or activity).

Interactions between TAp53 and N-terminal isoforms: physiological relevance

The physiological role of N-terminal p53 isoforms is far from clear. First, although small molecular weight isoforms are commonly detected in cultured cells and in tissues, they are generally expressed together with TAp53 and in amounts which are inferior or, at best, equal to basal TAp53 levels. Second, in contrast to TAp53, N-terminal isoforms do not accumulate in response to genotoxic stress or other forms of stress that activate p53 suppressor functions.

Therefore, upon activation, TAp53 becomes the overwhelmingly dominant form of p53, accumulating at levels much higher than the basal levels of N-terminal p53 isoforms. Neither 40p53 nor 133p53 have been shown to transactivate gene expression on their own. In contrast, their biochemical capacity to antagonize TAp53 activity is well established.

Together, these observations suggest that the main effect of N-terminal isoforms is to modulate the activity of basal TAp53 levels and perhaps control its capacity to selectively regulate different promoters in response to low-levels of stress signals.

In 2009, Virginie Marcel in our laboratory has proposed a model suggesting that Nterminal isoforms could operate as "buffers" to control basal p53 levels and activity in normal conditions (Marcel and Hainaut, 2009). The central assumption of this model is that cells are equipped with mechanisms that prevent inappropriate or untimely activation of p53 in response to low levels of stress signals such as those generated as by-product of physiological processes. For example, basal cell metabolism generates intracellular reactive oxygen species that are normally detoxified by redox regulatory systems but may also induce low levels of DNA damage which may activate p53 and therefore turn on cell-cycle arrest, senescence or apoptosis [START_REF] Hafsi | Redox Control and Interplay Between p53 Isoforms: Roles in the Regulation of Basal p53 Levels, Cell Fate, and Senescence[END_REF]. To avoid turning these responses on as undesired consequence of normal metabolic processes, N-terminal p53 isoforms may provide a molecular "buffer" for TAp53 activity, setting thresholds above which TAp53 should accumulate in order to exert defined suppressive effects. According to this hypothesis, isoform levels may define incremental thresholds which set the limits for activation of p53 towards specific promoters, and thus for the induction of distinct physiological responses. For example, at intermediate levels of activation, TAp53 may partially override N-terminal isoforms to activate essentially cell-cycle arrest, DNA repair and growth regulatory responses that preserve cell survival. At high levels of activation, however, TAp53 may completely override the "buffer" provided by N-Terminal isoforms to activate promoters that induce cell senescence or apoptosis and therefore, death or permanent growth arrest. Our results are compatible with this model and provide further refinements (Figure 25). First, they suggest that the buffer provided by N-terminal isoforms results from a complex interplay between the two N-terminal isoforms and TAp53 with multiple cross-talks as illustrated in Figure D2.

Second, our results with 40p53 suggest that changes in the levels of this isoform might actually provide a dynamic system to set the levels of basal p53 activity.

Figure 25. 40p53, a "buffer" regulating the p53 response

To avoid uncontrolled response of p53 to low levels of stress signals, 40p53 could act as a molecular "buffer" for p53 activation, setting thresholds above which p53 should accumulate and exert its functions (dark blue line). For example, at high level of activation (light blue line), p53 may override the "buffer" provided by 40p53 and induce target genes involved in cell cycle arrest or apoptosis (A) (dotted light blue line). On the other hand, at low to intermediate levels of activation, TAp53 may not be able to override the threshold provided by 40p53, resulting in the absence of p53 response (B).

One of the predictions of this model is that levels of 40p53 may be higher in cells that retain a high proliferative capacity such as stem cells and progenitor cells, thus setting up a high basal threshold for activation of TAp53. This would, in turn, preserve the stem/progenitor status by preventing these cells from activating anti-proliferative responses such as differentiation, senescence and apoptosis. This prediction is compatible with results demonstrated by Ungewitter and Scrable (2010) [START_REF] Ungewitter | Delta40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs[END_REF]. These authors have found that 40p53, in addition to being highly expressed in embryonic stem cells (ESCs), was the major p53 isoform during early stages of embryogenesis in the mouse.

Furthermore, haploinsufficiency for 40p53 caused a loss of pluripotency in ESCs and acquisition of a cell cycle progression profile typical of differentiated cells (with longer G1 and G2 phases, in contrast with the rapid succession of S/M phases that characterizes ESCs).

In contrast, increased dosage of 40p53 contributed to extend pluripotency and to inhibit the progression to a more differentiated state. However, in both cases, the activity of 40p53 was dependent upon the presence of TAp53. It is important to note that, in these experiments, haploinsufficiency for 40p53 actually promoted p53-dependent transcriptional control, with activation of the p21WAF1 promoter and suppression of promoters regulating genes characterizing the stem cell status such as Nanog. Increased dosage of 40p53 led to a transcriptional switch with increased expression of Nanog and decreased expression of p21 WAF1 . These observations cannot be explained by a simple, dosage-dependent antagonistic effect of 40p53 on TAp53 activity. Rather, they suggest that when present at low levels relative to TAp53, 40p53 can activate basal p53 function. Conversely, when expressed at higher levels, 40p53 can titrate TAp53 function. These observations in ESCs are therefore fully compatible with our results and with our "two-phases" model of regulation of TAp53 by 40p53.

Our interpretation sheds new light on the effects of 40p53 in animal models. Two such models have been developed, in mouse [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF] and in Zebrafish [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]. The mouse model is a transgenic model expressing an N-terminal truncated p53 matching 40p53 (p44) in either p53-null or in wild-type p53 background. In a p53-null background, no significant phenotype was observed. Expression of 40p53 in the absence of TAp53 (and of other p53 isoforms) appeared to have no effect on development, differentiation, physiological fitness or aging. These mice show the same susceptibility to early tumourigenesis as the parental, p53-null mice. In contrast, when 40p53 was expressed in a p53-competent background, a phenotype of early aging was detected. At 4 months of age, mice showed a physiological decline with loss of bone density, decreased skin renewal, hair graying and loss of memory. Further studies have shown that mice overexpressing 40p53 in a p53-competent background developed a neurological deficiency reminiscent of Alzheimer's disease [START_REF] Pehar | Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death[END_REF]. Interestingly, Western Blot analysis of p53 protein levels revealed an increase in TAp53 protein in p53-competent mice overexpressing 40p53 (Figure 26).

This observation is compatible with our results in transfected cells showing that expression of 40p53 at low levels compared to TAp53 may promote the stabilization of the latter.

Increased levels of basal p53 activity may largely explain the aging phenotype, which may result from the suppression of adult stem cells and the subsequent loss of tissue renewal capacity. Alternatively, mice overexpressing 40p53 may have constitutively high levels of oxidative stress that induces the stabilization of p53 through DNA damage. While such an increase in oxidative stress is not documented in vivo, we consider this possibility as unlikely since mice overexpressing 40p53 in a p53-null background do not show increased cancer risk as compared to the parental p53-null controls. Indeed, higher levels of oxidative stress would be expected to result in accelerated tumourigenesis in the absence of a functional p53 protein. [START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF] The Zebrafish model offers a biological parallel with the mouse model. After cloning of the Zebrafish 40p53 homolog, Rodeck and his collaborators have forced the expression of either human or Zebrafish 40p53 by micro-injecting their respective mRNAs into Zebrafish embryos [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]. They observed that ectopic expression of both species' 40p53 caused hypoplasia and malformation of the head, eyes and somites, yet partially counteracted the lethal effects caused by concomitant forced expression of TAp53. Moreover, TAp53 expression was required for the developmental aberrations caused by 40p53 expression. Strikingly, knockdown of p21WAF1 expression markedly reduced the severity of developmental malformations associated with forced expression of 40p53, suggesting that these malformations were indeed driven by an increase in basal p53 transcriptional capacity. Thus, in both animal models, the main effect of 40p53 appears to consist into increasing the basal level and activity of TAp53, rather than impairing p53-dependent growth suppression.

We speculate that this effect is due to the fact that these organisms co-express TAp53 and 40p53 isoforms at respective levels which are compatible with increased stability and activity of TAp53 by interaction with 40p53.

The general expression patterns of p53 into TAp53 and N-terminal isoforms appears to be conserved in evolution. In Drosophila, two forms of p53 mRNA have been described, encoding respectively TAp53 (Dp53) and an isoform lacking the first 110 residues (D Np53) (Bourdon et al., 2005;[START_REF] Jin | Identification and characterization of a p53 homologue in Drosophila melanogaster[END_REF][START_REF] Ollmann | Drosophila p53 is a structural and functional homolog of the tumor suppressor p53[END_REF]. To date, there is no animal model for 133p53. In Zebrafish, Davidson et al. have shown that the ortholog of 133p53 is a protein truncated for its first 113 residues ( 113p53) [START_REF] Davidson | Differential regulation of p53 function by the N-terminal DeltaNp53 and Delta113p53 isoforms in zebrafish embryos[END_REF]. Forced expression of TAp53 in Zebrafish embryos induced the expression of 113p53, in agreement with our in vitro results showing that TAp53 regulates the internal P2 promoter. In a p53-competent background, overexpression of 40p53 also induced an increase in 113p53 mRNA, consistent with the idea that 40p53 could increase the basal activity of endogenous TAp53. However, forced expression of 113p53 could not reverse the malformation phenotype induced by 40p53, suggesting that in this context 113p53 is rather inefficient in antagonizing 40p53 effects.

Pathological relevance of N-terminal isoforms

Summarizing our results as well as the observations in vivo discussed above, we propose that expression of N-terminal isoforms may be critical for the maintenance of stem cell status.

This, together with the fact that excess expression of N-terminal isoforms antagonizes p53 suppressor activity, suggests that overexpression of N-terminal isoforms may represent a mechanism for functional inactivation of p53 during tumourigenesis. Inactivation of p53 by somatic mutation of the TP53 gene is common in human cancer and is generally associated with highly metastatic behaviour. A curious exception to this rule is cutaneous melanoma, in which TP53 mutations are very rare (about 5% of the cases) despite a highly metastatic phenotype. It has long been proposed that, in melanoma, wild-type p53 protein may be inactivated by alternative mechanisms [START_REF] Chin | The genetics of malignant melanoma: lessons from mouse and man[END_REF]. Such mechanisms have been identified in several cancers, such as cervical and oral cancers in which the inhibition of p53 activity is caused by rapid degradation through Human Papilloma Virus (HPV) antigen E6 acting as component of an ubiquitin-ligase system [START_REF] Dyson | The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product[END_REF][START_REF] Scheffner | The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53[END_REF], and in sarcomas with amplifications of the Hdm2 gene, also leading to increased degradation (Muthusamy et al., 2006;[START_REF] Reifenberger | Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations[END_REF].

A previous report in melanoma cell lines has identified that most of them expressed small molecular weight variants of p53 (Avery-Kiejda et al., 2008). However, their precise identity remains obscure. At mRNA level, there is evidence that these cells express high levels of p53I2 (encoding 40p53) and/or p53I4 (encoding 133p53) mRNA. At protein level, expression of TAp53 , a C-terminal isoform, has been documented while the expression of N-terminal isoforms has not been analysed in sufficient details. We have taken advantage of a collaboration with Chris Marine, University of Ghent, Belgium, to analyse the relative levels of p53I2 and p53I4 mRNA in primary melanoma sub-grouped according the extent of metastatic dissemination. This series of cases has been recently analysed for Hdm2 and Hdm4 expression by Gembarska et al., leading to the identification of Hdm4 overexpression as a plausible mechanism inducing rapid p53 turnover and inactivation in metastatic cutaneous melanoma (Gembarska et al., 2012). We found that expression of either p53I2 or p53I4 mRNA was substantially increased in 17/20 nodal and distal metastatic lesions. Only one of those had a TP53 mutation, whereas 1 in 3 lesions with no significant change in p53I2 or p53I4 mRNA levels had a TP53 mutation (the mutation status of the two other cases being unknown). The FS (Fully-Spliced) p53 mRNA encoding TAp53 was often up-regulated together with p53I2, consistent with their common origin from the same pre-mRNA. Tumours with increased FSp53 levels tended to match those with increased Hdm4 levels (Gembarska et al., 2012). Overall, our results suggest that deregulation of wild-type p53 mRNA expression is a common occurrence in cutaneous melanoma. Moreover, levels of p53I2 and p53I4 seem to be correlated with nodal and distant metastatic behaviour. Thus, in melanoma cells, increased expression of Hdm4, 40p53 and 133p53 cooperate to reduce p53 protein activity and turn off p53 suppressor function (Figure 27).

Figure 27. Alternative mechanisms for TP53 inactivation in human melanoma

TP53 is rarely mutated in human melanoma, suggesting alternative mechanisms to switch off p53 functions. When TP53 is mutated (A), mutant p53 inactivates p53 growth suppressive functions, and Hdm4, 40p53 and 133p53 expression levels are decreased. In contrast, when TP53 gene is not mutated (B), Hdm4, 40p53 and 133p53 have an increased expression, suggesting an interplay of these proteins to inhibit p53 activity and turn off p53 suppression functions.

It is striking to note that, in this cancer context, our model proposes that the main effect of N-terminal isoforms is to inactivate TAp53 functions, although we propose that in animal models, the main effect is to stabilize and increase basal p53 levels. This paradox can be resolved by considering that, in melanoma, N-terminal isoforms are expressed at much higher levels than TAp53, whereas in animal models they are expressed at roughly equivalent levels.

This speculation needs to be confirmed by systematic protein studies. Nevertheless, one of the factors that may tilt the balance towards a strong inhibiting effect of N-terminal isoforms is overexpression of Hdm4, which decreases p53 protein levels and therefore displaces the TAp53/N-terminal isoform ratio towards conditions in which p53 activity is strongly antagonized.

The notion that high levels of N-terminal p53 isoforms in melanoma are correlated with metastatic behaviour is interesting from a cancer stem cell perspective. Indeed, high levels of N-terminal isoforms may represent a "signature" for cancer cells which retain stem cell status and are prone to undergo Epithelial to Mesenchymal Transition (EMT), a critical process for dissemination of cancer cells to distant sites. Tumours with wild-type p53 but high levels of N-terminal isoforms may tend to have more stem cells, or stem-cell-like properties, than other tumours, thus providing a basis to explain their aggressiveness. Thus, restoring p53 activity may represent an elegant mechanism to prevent tumour cells to retain and accumulate highly malignant stem cells. This principle is supported by studies by JC Marine and collaborators (Gembarska et al., 2012) who have shown that inhibition of Hdm2/Hdm4 complexes in melanoma cells could prevent progression in mouse xenografts. Whether a similar effect can be achieved by decreasing N-terminal isoforms or by blocking the formation of heterooligomers between TAp53 and N-terminal isoforms, remain to be demonstrated.

The isoform paradigm may apply to many other forms of aggressive cancer which retain wild-type TP53. Several reports have noted high levels of mRNA encoding different p53 isoforms in squamous cell carcinomas of the head and neck (Boldrup et al., 2007) and of the breast (Moore et al., 2010) but it is not clear whether there is any correlation with the persistence of wild-type TP53. In this respect, our study is the most detailed to date although it has many shortcomings such as the lack of data at protein level. Another context in which N-terminal p53 isoforms may have an impact on tumour development is the Li-Fraumeni Syndrome, a disease of familial aggregation of multiple early cancers due to inheritance of a germline TP53 mutation [START_REF] Palmero | Tumor protein 53 mutations and inherited cancer: beyond Li-Fraumeni syndrome[END_REF]. In subjects who inherit one copy of mutant TP53, levels of 40p53 (and perhaps 133p53) may be critical for determining the basal level of p53 activity and thus the efficiency of p53 responses to multiple stimuli during life. In 2009, Marcel et al. have shown that a common polymorphism located in intron 3 of p53 (a 16 bp motif present as one or two copies) was associated with early cancer risk in a Brazilian cohort of carriers of a specific germline TP53 mutation, R337H (Marcel et al., 2009).

Individuals with two copies of the 16bp motif on the wild-type allele had a much reduced risk of early cancer (before age 30) compared to carriers of a wild-type allele with only one copy of the 16bp motif. In vitro studies have shown that this polymorphism falls into a segment of intron 3 that generates the formation of G-quadruplex structures in p53 pre-mRNA, and that these G-quadruplexes could regulate the splicing of the pre-mRNA to produce either FSp53 or p53I2 (Marcel et al., 2011c). Therefore, the level of 40p53 may vary according to TP53 polymorphic status, providing a potent intragenic modifier of cancer risk in carriers of a germline TP53 mutation.

Conclusions and perspectives

CONCLUSIONS AND PERSPECTIVES

In this Thesis, we claim as primary contributions [START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF] the demonstration that 40p53 can modulate TAp53 function with a bi-phasic effect that provides a basis to reconcile the apparent paradox between its biochemical (inhibitory) and physiological (apparently activating) effects on TAp53 and (2) the demonstration that mRNAs encoding N-terminal isoforms are often over-expressed in highly metastatic melanoma when compared to noninvasive forms, suggesting that N-terminal isoforms contribute to functionally inactivate p53.

A secondary contribution is the analysis of the complex loops of interactions that link TAp53, 40p53 and 133p53 isoforms, leading to the concept that p53 function depends on the dynamic fluctuations of a protein network rather than on the expression of a single molecule.

Our work has however major limitations. The main ones are (1) the absence of proof of effect for N-terminal isoforms in non-transformed, non-transfected experimental cell systems; [START_REF] Avery-Kiejda | Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin[END_REF] the lack of comparison between mRNA and protein expression studies in melanoma, and [START_REF] Boldrup | Expression of p53 isoforms in squamous cell carcinoma of the head and neck[END_REF] the lack of consideration for other p53 isoforms such as C-terminal isoforms. Indeed, the mechanisms identified here are bound to be dependent upon the expression of C-terminal p53 isoforms, since they depend upon the formation of hetero-oligomers between N-terminal isoforms and TAp53. C-terminal isoforms differ from full-length p53 by a variable portion of the C-terminus. While the p53 isoform retains dimerisation, but not tetramerization motifs, p53 has a truncation that encompasses the whole oligomerization domain. Thus, the rate of complex formation between isoforms may be critically dependent upon the status of Cterminal isoforms.

Our results are calling for further analysis of the role of p53 isoforms in the maintenance of stem cell status. Indeed, control of stemness may represent the main conserved biological activity of p53 family members. For example, Np63 isoforms are commonly expressed as the major, if not the only, type of p63 isoform in epidermal stem cells (ESC) and in squamous cell progenitors [START_REF] Medawar | DeltaNp63 is essential for epidermal commitment of embryonic stem cells[END_REF]. In contrast, in squamous epithelia, TAp63 expression is restricted to differentiated cells [START_REF] Nylander | Differential expression of p63 isoforms in normal tissues and neoplastic cells[END_REF]. Knocking out p63 or Np63 alone results in impaired development of many tissues including skin, breast and prostate, and leads to major morphogenetic defects due to impaired epithelium to mesenchyme interactions. These defects result from the rapid exhaustion of pools of stem cells in these tissues, which are unable to retain their stem status in the absence of Np63. The functions of 40p53 appear to have similarities with those of Np63. Further studies are needed to determine whether p53, p63 and perhaps p73 operate in parallel pathways of stem cell maintenance, or are all part of a complex, highly redundant regulatory network.

Finally, our results call to a reassessment of the roles of p53 in normal conditions and in stress responses. Since its identification as "guardian of the genome" in 1992 [START_REF] Lane | CANCER -P53, GUARDIAN OF THE GENOME[END_REF], the idea has prevailed that p53 had no significant function in cells unless it accumulates in response to a wide range of stress signals. Regulation through N-terminal isoforms sheds light on the fact that basal p53 levels are indeed important for the dynamics of stem cells and for their renewal. It follows that N-terminal isoforms of p53 might have a considerable impact not only on the expansion and dissemination of tumour stem cells, but also the pathogenesis of chronic diseases characterized by stem cell exhaustion as well as on normal aging. It would be very interesting to determine whether subtle changes in N-terminal isoform levels may contribute to increase the lifespan of organisms, or to improve the regeneration of aging tissues.

__________________________________________________________________________________ ABSTRACT:

The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslational levels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms that regulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N-and C-terminus due to alternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns of expression and of the functions of each of these isoforms.

In this Thesis, we have focused on the two major p53 N-terminal isoforms, 40p53 and 133p53. We have analysed their patterns of interactions with the full-length p53 and we have investigated whether their expression could be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that 40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control its levels and activity. Moreover, we demonstrate that the internal P2 promoter produces 133p53 and is regulated by p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control the expression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminal isoforms are often over-expressed in highly metastatic melanoma when compared to non-invasive forms, suggesting that N-terminal isoforms contribute to functionally inactivate p53.

Thus, we propose that 40p53 and 133p53 modulate p53 functions within dynamic fluctuations of a protein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functional inactivation of p53 in cancer cells.

___________________________________________________________________________

TITRE en français :

Isoformes du domaine N-terminal du suppresseur de tumeur p53: effets sur l'activité transcriptionnelle de p53 et expression dans les mélanomes cutanés

__________________________________________________________________________________ RESUME en français :

La protéine suppresseur de tumeur p53 est soumise à de complexes régulations transcriptionnelles et posttraductionnelles. La découverte d'isoformes de p53 a introduit un degré de complexité supplémentaire aux mécanismes de régulation des fonctions de p53. On dénombre à ce jour douze isoformes qui diffèrent de p53 dans leurs domaines N-et C-terminal. Cependant, les modes d'expression et de fonction de ces isoformes restent à être clarifiés.

Dans cette thèse, nous nous sommes intéressés aux deux isoformes 40p53 et 133p53, en analysant leur interaction avec p53 et en mesurant leur expression dans les mélanomes, un type de cancer où p53 est très rarement mutée. Nous montrons que 40p53 peut moduler l'activité de p53 avec un effet bi-phasique, tantôt activateur ou répresseur du niveau d'expression et des fonctions de p53. 133p53 est produite par un promoteur P2 localisé dans le gène TP53. Nous avons montré qu'en réponse à un stress génotoxique, l'expression de 133p53 est régulée par p53, qui se lie au promoteur P2. Ceci suggère une boucle d'auto-régulation par p53, qui est capable de contrôler l'expression d'une isoforme inhibant ses propres fonctions. Enfin, les isoformes 40p53 et 133p53 sont surexprimées dans les tumeurs métastatiques de mélanomes comparées aux tumeurs noninvasives, suggérant à ces isoformes un rôle dans l'inactivation de p53 dans les cancers.

Ainsi, 40p53 et 133p53 interagissent avec p53 de façon complexe, avec des effets plus contrastés que la simple inhibition de l'activité suppressive de p53. Les isoformes de p53 jouent ainsi un rôle majeur dans les activités basales de p53, ainsi que dans l'inactivation fonctionnelle de p53 dans les cancers.

Faculté

  Chimie Physique Electronique Institut Universitaire de Technologie de Lyon 1 Institut Universitaire de Formation des Maîtres Institut de Science Financière et d'Assurances Directeur : M. le Professeur F. De MARCHI Directeur : M. le Professeur F. FLEURY Directeur : Mme le Professeur H. PARROT Directeur : M. N. SIAUVE Directeur : M. le Professeur S. AKKOUCHE Directeur : M. le Professeur A. GOLDMAN Directeur : M. le Professeur H. Directeur : M. R. BERNARD Directeur : Mme la Professeure V. MAUME-DESCHAMPS À mes parents et ma soeur, First of all, I would like to thank Dr Stéphanie Cox-Courtois, Pr Charles Dumontet, Dr Pierre Roux, Pr Klas Wiman and Pr Gérard Zalcman for accepting to be jury members of my thesis, thus enabling my work to be evaluated by very high quality researchers. I am very grateful to Dr Pierre Roux and Pr Klas Wiman for their careful review of my thesis manuscript and their valuable comments. I wish to warmly thank all the people who participated to my life in the lab during these four years of PhD: Pierre, cette thèse n'aurait jamais vu le jour sans votre confiance et votre générosité. Un grand merci pour avoir toujours été là pour moi et pour m'avoir tant appris. Vous avez toujours cru en moi et je vous remercie énormément pour cela. Pour toutes ces discussions stimulantes, ces nombreuses heures de travail acharné, mais aussi ces moments moins sérieux et ces déjeuners « soupe et fromage », MERCI du fond du coeur !

  List of figures and tables ......................................................................................................15 Foreword .............................................................................................................................17 1 INTRODUCTION ........................................................................................................... 1.1 Cancer: from society to genes .................................................................................. 1.1.1 The global burden of cancer ................................................................................ 1.1.2 The concept of the Hallmarks of Cancer ............................................................

2 . 5 C

 25 -terminal basic domain ..................................................................... 1.2.3 Regulation of p53 activation and stability .......................................................... 1.2.3.1 p53/Hdm2 feedback loop ........................................................................... 1.2.3.2 Post-translational modifications ................................................................ 1.2.4 Regulation of p53 in basal conditions .................................................................Review "Redox control and interplay between p53 isoforms: roles in the regulation of basal p53 levels, cell fate, and senescence"(p.40) 1.3 TP53 and the Hallmarks of Cancer .......................................................................... 1.3.1 Repressing proliferative signalling ..................................................................... 1.3.2 Enhancing the effects of growth suppressors ...................................................... 1.3.3 Sensitizing cells to death ..................................................................................... 1.3.4 Suppressing replicative immortality through senescence ................................... 1.3.5 Exerting anti-angiogenic effects ......................................................................... 1.3.6 Repressing invasion and metastasis .................................................................... 1.3.7 Promoting genetic and genomic stability ............................................................ 1.3.8 Controlling tumour-promoting inflammation ..................................................... 1.3.9 Regulating cell bioenergetics .............................................................................. 1.3.10 Facilitating innate and adaptive immune response ............................................. 1.4 The TP53 family ....................................................................................................... 1.4.1 TP53 homologues ............................................................................................... 3.1.4 Conclusion ........................................................................................................ 3.2 p53 regulates the transcription of its 133p53 isoform through specific response elements contained within the TP53 P2 internal promoter............................................... 3.2.1 Context .............................................................................................................. 3.2.2 Strategy ............................................................................................................. 3.2.3 Results ............................................................................................................... 3.2.3.1 Regulation of P2 promoter by TAp53 ..................................................... 3.2.3.2 Effect of 40p53 on P2 promoter activity ............................................... 3.2.4 Conclusion ........................................................................................................3.3 Expression of 40p53 and 133p53, two isoforms of p53 lacking the N-terminus, in cutaneous melanoma ..................................................................................................... 3.3.1 Context .............................................................................................................. 3.3.2 Strategy ............................................................................................................. 3.3.3 Results ............................................................................................................... 3.3.4 Conclusion ........................................................................................................ 4 DISCUSSION ................................................................................................................ novel p53 auto-regulatory loop implicating p53 isoforms ..................4.3Interactions between TAp53 and N-terminal isoforms: physiological relevance .. 4.4 Pathological relevance of N-terminal isoforms ...................................................... Biological functions of p53 isoforms through evolution: lessons from animal and cellular models ................................................................................................ 7.2 Appendix II: Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation ......................................................................................... 7.3 Appendix III: Pharmacological rescue of p53 in cancer cells: the soloist meets the PRIMA donna ....................................................................................................................

Figure 1 .

 1 Figure 1. Global incidence of cancers ...................................................................................................

Figure 2 .

 2 Figure 2. Comparison of the most common cancers in more and less developed countries in 2000 ....

Figure 3 .

 3 Figure 3. The Hallmarks of Cancer .......................................................................................................

Figure 4 .

 4 Figure 4. Emerging Hallmarks and Enabling Characteristics ...............................................................

Figure 5 .

 5 Figure 5. p53, from discovery to clinical application ............................................................................

Figure 6 .

 6 Figure 6. Structure of the full-length p53 protein and the protein-interaction associated domains ......

Figure 7 .

 7 Figure 7. p53/Hdm2 feedback loop and other regulatory networks ......................................................

Figure 8 .

 8 Figure 8. Post-translational modifications of p53 .................................................................................

Figure 9 .

 9 Figure 9. A p53-centric system in the "hallmarks of cancer" ...............................................................

Figure 10 .

 10 Figure 10. Oncogenic and anti-oncogenic roles of SnoN ......................................................................

Figure 11 .

 11 Figure 11. p53 and apoptosis .................................................................................................................

Figure 12 .

 12 Figure 12. p53 and regulation of metabolism ........................................................................................

Figure 13 .

 13 Figure 13. Homologies of the protein structures of TP53 family members ..........................................

Figure 14 .

 14 Figure 14. Representation of the structure of the human p53 isoforms.................................................

Figure 15 .

 15 Figure 15. Structural organization of p53 isoforms in animal models ..................................................

Figure 16 .

 16 Figure 16. Tumour site distribution of somatic TP53 mutations ...........................................................

Figure 17 .

 17 Figure 17. Tumour site distribution of germinal TP53 mutations .........................................................

Figure 18 .

 18 Figure 18. Properties of mutant p53 proteins ........................................................................................

Figure 19 .

 19 Figure 19. Predicted and observed inhibition capacity of 40p53 ......................................................

Figure 20 .

 20 Figure 20. Effects of 133p53 on p53 biochemical and biological properties ...................................

Figure 21 .

 21 Figure 21. P2 promoter activity is down-regulated by 40p53 ...........................................................

Figure 22 .

 22 Figure 22. Representation of expression levels of FSp53, p53I2 and p53I4 mRNAs in melanoma tissues ..................................................................................................................................................

Figure 23 .

 23 Figure 23. "Two-phases" model of regulation of TAp53 by 40p53 .................................................

Figure 24 .

 24 Figure 24. Auto-regulatory feedback loop of p53 and its isoforms .....................................................

Figure 25 .

 25 Figure 25. 40p53, a "buffer" regulating the p53 response ................................................................

Figure 26 .

 26 Figure 26. Overexpression of the 40p53 isoform of p53 in transgenic mice. ...................................

Figure 27 .

 27 Figure 27. Alternative mechanisms for TP53 inactivation in human melanoma ................................

  terminal isoforms interfere with basal p53 activity? If so, by which mechanisms? And what are the biochemical and biological consequences of this interference?

Figure 2 .

 2 Figure 2. Comparison of the most common cancers in more and less developed countries in 2000 Left panel: male; right panel: female. NHL: Non-Hodgkin lymphoma. Globocan 2003, IARC.

Figure 3 .

 3 Figure 3. The Hallmarks of Cancer Model of the 6 Hallmarks proposed by Hanahan and Weinberg in 2000 suggesting that most of the cancer cells acquire the same set of functional capabilities during their development, through various mechanistic strategies[START_REF] Hanahan | The hallmarks of cancer[END_REF].

Figure 4 .

 4 Figure 4. Emerging Hallmarks and Enabling Characteristics In 2011, Hanahan and Weinberg updated their model of the Hallmarks of Cancer from 2000 with two additional Hallmarks, the capability to modify cellular metabolism and to evade immunological destruction. The acquisition of those Hallmarks is facilitated by two characteristics of neoplasia, genomic instability and inflammation (Hanahan and Weinberg, 2011).

Figure 5 .

 5 Figure 5. p53, from discovery to clinical application Key steps in p53 research from discovery of the protein in 1979 to projected applications for 2030. Pink: early steps in the search for p53 function(1979)(1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988). Blue: definition of p53 as a tumour suppressor and transcription factor involved in growth suppression after DNA damage(1988)(1989)(1990)(1991)(1992)(1993)(1994). Green: current developments of drugs targeting p53 and prognostic studies of TP53 mutations in cancer(1995-present). Purple: projected applications of p53 research to cancer diagnosis and treatment[START_REF] Hainaut | 30 years and a long way into p53 research[END_REF].

Figure 6 .

 6 Figure 6. Structure of the full-length p53 protein and the protein-interaction associated domains p53 contains an N-terminal transactivation domain (TAD1 and TAD2), followed by a proline-rich region (PXXP), the central DNA-binding domain (DBD), the oligomerization domain (OD), and the extreme C-terminus consisting of a basic domain (BD). Nuclear Export Signal (NES), Nuclear Localization Signal (NLS) and Hdm2-binding site are indicated by a black line. Some of the proteins described to interact with these domains are listed (lower part of the figure). Tridimensional structure of the p53 protein domains is illustrated (upper part of the figure) (Swiss PDBViewer)[START_REF] Cho | Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations[END_REF].

Figure 7 .

 7 Figure 7. p53/Hdm2 feedback loop and other regulatory networks p53 levels are tightly controlled by several negative feedback loops, involving Hdm2, Pirh2 and COP1, which regulate p53 ubiquitination and are regulated by p53 at the transcriptional level (Hollstein and Hainaut, 2010).

Figure 8 .

 8 Figure 8. Post-translational modifications of p53 Specific residues are modified with phosphorylation (P) in orange, acetylation (A) in green, ubiquitination (Ub) in purple, neddylation (N) in pink, methylation (M) in blue and sumoylation (SU) in brown. Proteins responsible for these modifications are shown in matching colours. TAD: transactivation domain; PRD: proline-rich domain; DBD: DNA-binding domain; L: nuclear localization signal; 4DE: tetramerization domain; CTD: C-terminal domain[START_REF] Toledo | Regulating the p53 pathway: in vitro hypotheses, in vivo veritas[END_REF].

Figure 9 .

 9 Figure 9. A p53-centric system in the "Hallmarks of cancer"

Figure 11 .

 11 Figure 11. p53 and apoptosis p53 can induce apoptosis via several pathways: the mitochondrial pathway (A), the death receptor pathway (B), and the pathway regulating the bioavailability of survival factors (C) (adapted from Marcel V, PhD Thesis, University of Lyon, 2010).

  to severe DNA damage, p53 may stop BER to accelerate apoptosis of cells that have accumulated DNA damage beyond repair capacity.Mismatch repair (MMR) is the main mechanism that corrects DNA following DNA polymerase errors, removing mismatches in heteroduplex DNA during recombination and preventing MSH2), MutL homologue 1 (MLH1), and postmeiotic segregation 2 (PMS2) as

Figure 12 .

 12 Figure 12. p53 and regulation of metabolism p53 acts as a transcriptional regulator of genes involved in the control of energy metabolism. It participates to the inhibition of glycolysis (left) and the increase in oxidative phosphorylation (right). Green arrows: positive effects (increased expression and/or activity); red arrows: negative effects (decreased expression and/or activity) (Hafsi and Hainaut, 2011) (see paragraph 1.2.4).

Figure 13 .

 13 Figure 13. Homologies of the protein structures of TP53 family members p53, p63 and p73 proteins contain a transactivation domain (TAD), a DNA-binding domain (DBD), and an oligomerization domain (OD). p63 and p73 proteins have another protein/protein interaction domain, the sterile alpha motif (SAM). The sequences of these domains are very similar (percentage in yellow: similarity between p53 and p63/p73; in pink: similarity between p63 and p73). The truncated domains of the different p63 and p73 isoforms are also indicated.

  apoptosis and promote cell cycle progression. In response to DNA damage Np73 isoforms are degraded thus allowing the TA forms to exert their effect. The role of Np63 on p53 was studied in response to DNA damage. It has been shown that Np63 is down-regulated following exposure to UV radiation in mouse or human keratinocytes, thus allowing p53 and

Figure 14 .

 14 Figure 14. Representation of the structure of the human p53 isoforms (A) Structure of the human TP53 gene. The TP53 gene contains 11 exons and expresses several isoforms owing to usage of alternative promoters (P1 and P2), translational initiation sites (ATG1/40/133/160) and splicing sites (^). (B) Human p53 mRNA variants. FSp53 (Fully-spliced p53); I2: intron 2; I4: intron 4; *: stop codons. (C) Human p53 protein isoforms. The canonical p53protein contains a TAD (blue), a proline-rich domain (PXXP, purple), a DBD (orange) and an OD (green) that encompasses a NLS (green) and five regions conserved through evolution (I-V in grey boxes). At the C-terminal, the and isoforms have different residues (brown). The theoretical molecular weight is indicated on the right. Coloured boxes: coding sequence; grey boxes: non-coding sequence(Marcel et al., 2011a).

  activating apoptosis, but that each induced distinct RHG family members (Rpr or Hid) to inhibit DIAP1. Furthermore, they observed that D Np53 induced Wingless (wg) expression and enhanced proliferation in the wing imaginal disc, suggesting that D Np53 promotes apoptosis-induced proliferation. In contrast to D Np53, Dp53 did not induce wg expression in the absence of the endogenous p53 gene (Dichtel-Danjoy et al., 2012). However, D Np53 also exerts effects through other pathways. In particular, D Np53 may activate either apoptotic or non-apoptotic responses in photoreceptor cells depending on the cell differentiation status

  al. isolated a shorter N-terminal form produced by an internal promoter within the mouse p53 gene, M 157p53, equivalent to the human 160p53 form. Khoury et al. also showed that M 41p53 and M 157p53 can be expressed as a C-terminal AS variant. In stably transfected myeloid cells, Mp53AS is able to induce apoptosis, albeit with much slower kinetics (a 12-hour delay)

Figure 17 .

 17 Figure 17. Tumour site distribution of germinal TP53 mutations http://www-p53.iarc.fr/(Petitjean et al., 2007)

  properties including:[START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF] transactivation activities (TA) of mutant proteins on reporter genes placed under the control of p53-RE; (2) induction of cell-cycle arrest or apoptosis; (3) dominant negative effects (DNE) over the wild-type protein inducing loss of function (LOF);(4) temperature sensitivity; (5) activities of mutant proteins that are independent and unrelated to the wild -type protein (gain of function (GOF)) (Figure18).

Figure 18 .

 18 Figure 18. Properties of mutant p53 proteins Missense mutations at hot-spot codons located in the DNA binding domain of p53 are the most frequent alterations in human cancers. Three types of p53 mutants have been described: dominant negative mutants (DN) inhibit the suppressive function of the residual wild-type p53 protein; loss of function mutants (LOH) lack suppressive function; while gain of function mutants (GOF) exhibit oncogenic properties through an array of mutant specific activities including aberrant protein interactions or gene regulation. TADI/II: transactivation domain I/II; PXXP: proline-rich domain; DBD: DNA binding domain; OD: oligomerization domain; BD: basic domain (Goldstein et al., 2011).

Figure 19 .

 19 Figure 19. Predicted and observed inhibition capacity of 40p53 (A) Predicted composition and effects on transactivation capacity of different configurations of mixed TAp53/ 40p53 hetero-oligomers, assuming a linear effect of 40p53. (B) The transcriptional activities obtained in the -galactosidase experiments were plotted (large dotted lines) and compared to the predicted inhibition curves (small dotted lines). Grey triangles represent the different values obtained in three independent experiments done in triplicate in H1299 cells and grey crosses joined by dotted line, the average curve. White circles represent the different values obtained in three independent experiments done in Saos-2 cells and black crosses joined by dotted line, the average curve. *P < 0.05 and ***P < 0.001.

Figure 20 .

 20 Figure 20. Effects of 133p53 on p53 biochemical and biological properties(A) Electro-Mobility-Shift Assay (EMSA) measuring DNA-binding activity of 133p53. p53-null H1299 cells were co-transfected with a constant amount of pcDNA3-p53 vector (0.5 μg/ml) in the presence of increasing amounts of pcDNA3-133p53 vector (0.25-1 μg/ml). Nuclear extracts were incubated with a 32 P-labeled p53RE oligonucleotide, with or without PAb421 antibody, which stabilizes and super-shifts p53:DNA complexes. DNA:p53 shifts and DNA:p53:PAb421 super-shifts are indicated by arrowheads. *: non-specific band. (B) Colony formation assays were performed using p53-null H1299 cells transfected with 0.5 μg/ml of wild-type pcDNA3-p53 vector and/or -133p53 vector during 14 days under 0.5 mg/ml of neomycin selection. As compared to wild-type p53, 133p53 did not suppress cell growth and partially reverted the suppression induced by wild-type p53.

a

  combination of two-dimensional gel electrophoresis and western blotting, Avery-Kiejda et al. have shown that one of these small molecular weight isoform was compatible with p53beta, a C-Terminus variant generated by alternative splicing of exon 9(Avery-Kiejda et al., 2008). Using RT-PCR, they also detected enhanced expression of p53I2, but not p53I4, in most melanoma cell lines. By transfection of expression vectors for p53beta or for Delta40p53 in a melanoma cell line with low constitutive p53 expression, they found that these two isoforms had opposite effects on the transactivation of p53-dependent reporter genes. Delta40p53 was shown to inhibit, whereas p53beta was shown to enhance, p53dependent transcription(Avery-Kiejda et al., 2008). Our results on a different panel of melanoma cell lines are compatible with those of Avery-Kiejda et al., further supporting the notion that deregulated p53 mRNA expression is deregulated in melanoma(Avery-Kiejda et al., 2008).

Figure 1 .

 1 Figure 1. Structure of FS, p53I2 and p53I4 mRNA

  Figure 3

Figure 4 «

 4 Figure 4

Figure 26 .

 26 Figure 26. Overexpression of the 40p53 isoform of p53 in transgenic mice. (A) 40p53 protein in transgenic and non transgenic (NT) mice. Western blots of 100 μg thymus protein extracted from transgenic (P,Q) and NT (NT) tissues. The anti-p53 polyclonal antiserum CM5 detects both isoforms of p53, as indicated. TAp53 expression is increased in mice overexpressing 40p53. Biotinylated markers were run alongside the tissue extracts during SDS-PAGE; the marker corresponding to 45 kD is shown. (B) Effect of p44 on body size. Size of NT (top), hemizygous (middle), and homozygous (bottom) P mice at 3 months of age. Organ weights of representative hemizygous (black bars) and homozygous (stippled bars) transgenic mice are plotted as a percentage of NT weights. Transgenic animals were generated on a p53 +/+ background.[START_REF] Maier | Modulation of mammalian life span by the short isoform of p53[END_REF]

  Studies by B Mollereau and his collaborators have shown that in p53-competent transgenic Drosophila overexpressing either Dp53 or D Np53 in the imaginal discs, both isoforms could induce apoptosis through different pathways, whereas only D Np53 could stimulate apoptosis-induced compensatory proliferation through the release of growth factors of the Wg (Wingless) family (Dichtel-Danjoy et al., 2012) (see Appendix II). It still unclear how these effects compare with those observed in mouse and zebrafish. Moreover, further studies are needed to determine whether the vertebrate orthologs can recapitulate p53 effects in Drosophila and vice versa.

  tumeur p53, isoformes, régulation transcriptionnelle, inactivation de p53, mélanomes__________________________________________________________________________________ INTITULE ET ADRESSE DE L'U.F.R. OU DU LABORATOIRE :Centre International de Recherche sur le Cancer -Groupe de Mécanismes Moléculaires et Biomarqueurs

  1.2 p53: the molecular gatekeeper .................................................................................. 1.2.1 From oncogene to tumour suppressor gene ........................................................ 1.2.2 Structure of human p53 gene and protein ...........................................................

1.2.2.1 TP53 gene and its promoters ..................................................................... 1.2.2.2 p53 protein: domain organization and functions ....................................... 1.2.2.2.1 The amino-terminal transactivation region ........................................ 1.2.2.2.2 Polyproline domain ............................................................................ 1.2.2.2.3 DNA-binding domain ......................................................................... 1.2.2.2.4 Oligomerization domain ..................................................................... 1.2.2.

  . Patients and melanoma cases used for this study were recruited as part of the Melbourne Melanoma Project at the Peter McCallum Cancer Center, Melbourne, Australia (http://melbournemelanomaproject.com/). All

	Results |3. Expression of ∆40p53 and ∆133p53, two isoforms of p53 lacking the N-terminus,
	in cutaneous melanoma
	patients provided informed consent. From an initial group of 54 patients, a series of 40
	fresh-frozen tissues consisting of 4 matched groups of 10 cases was assembled, including
	Primary Melanomas (PM ; stage I and II), Regional Dermal metastasis (RD, stage III), Nodal
	Metastatic lesions (NM, stage III) and Distant Metastatic lesions (DM, stage IV). The samples
	used in this study are the same as those previously analysed for Mdm4 expression at mRNA
	and protein levels by
	156

senescence, activating embryonic developmental programs such as EMT, promoting tumour inflammation and avoiding immune destruction. In cells with normal p53 competence, transient activation of any of these capabilities will be compensated by p53-dependent downregulation of other capabilities, thus maintaining homeostasis.

R175, G245, R249 and R282)[START_REF] Joerger | Structure-function-rescue: the diverse nature of common p53 cancer mutants[END_REF][START_REF] Olivier | TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use[END_REF]. Outside the DBD, missense mutations represent only about 40%, the majority of mutations being

Objectives

7.2 Appendix II: Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation

7.3 Appendix III: Pharmacological rescue of p53 in cancer cells: the soloist meets the PRIMA donna

Acknowledgements

Acknowledgements

The authors are grateful to Dr JC Marine, University of Ghent, Belgium, for providing mRNA samples from primary melanoma and melanoma cell lines. This study was supported by a grant from Institut National du Cancer (INCa), France.

References

TP53 mutations

Somatic mutations are the cornerstone of cancer. The development of cancer involves the contributions of many heritable genetic events as well as a large number of epigenetic changes, but what makes the turning point between untransformed and transformed cell irreversible is the acquisition of somatic mutations, conferring to cells a selective advantage for clonal proliferation.

The TP53 gene is one of the most frequently altered genes, with over 50% of all the mutations described in human cancers (http://www-p53.iarc.fr/). Somatic TP53 gene alterations are frequent in most human cancers (Figure 16) and germline TP53 mutations predispose to a wide spectrum of early-onset cancers (Li-Fraumeni (LFS) and Li-Fraumenilike syndromes (LFL)) [START_REF] Hainaut | p53 and human cancer: the first ten thousand mutations[END_REF]Petitjean et al., 2007) (Figure 17).

Objectives

My Thesis is focused on the two N-terminal isoforms 40p53 and 133p53 and aims at better understanding their impact on overall p53 suppressor effects in vitro and in tumour tissues. Specifically, we have addressed the following objectives:

To determine whether 40p53 interferes with p53 DNA-binding and transactivation capacity in intact cells, and to analyse how 40p53 may interfere with wild-type TAp53 activity, depending upon the relative levels of expression of each of these proteins;

To analyse the mechanisms that regulate the expression of 133p53 and to assess the differences in the biochemical activities of 40p53 and 133p53;

To analyse 40p53 and 133p53 expression in melanoma, a tumour type in which inactivation of TP53 by mutation is infrequent, in order to determine whether isoform expression may represent a plausible alternative mechanism to inhibit p53 suppressor

Abstract

Cutaneous melanomas are highly malignant tumours that retain wild-type TP53 in over 90% of the cases, suggesting that p53 suppressive activity is abrogated through alternative mechanisms. We have used mRNA extracted from short-term cultures of melanoma cells and from stage I-IV melanomas to analyse the expression of FSp53 (Fully-Spliced) mRNA as well as p53I2 and p53I4 mRNA, encoding Delta40p53 and Delta133p53, respectively. These p53 isoforms lack the first 39 (Delta40p53) or 132 (Delta133p53) N-terminal residues and are unable to transactivate p53 target genes in vitro. In cultured cells, enhanced expression of FSp53, p53I2 or p53I4 compared to primary melanocytes was detected in 53% of the cases (9/17). Compared to primary stage I-II melanoma, enhanced expression of either p53I2 or p53I4 was detected in a substantial proportion of metastatic cases (70%; 21/30). When subgrouped according to metastatic status, enhanced expression of either p53I2 or p53I4 was more frequent in nodal (9/10) and distant (8/10) than in regional dermal (4/10) lesions (p=0.044, Fisher-Exact). Whereas enhanced expression of FSp53 mRNA was detected only in cases with enhanced expression of p53I2, p53I4 alone was increased in 6/30 lesions (20%). Stojic, L., Brun, R., and Jiricny, J. (2004). Mismatch repair and DNA damage signalling. DNA Repair (Amst) 3, 1091-1101. Stommel, J.M., Marchenko, N.D., Jimenez, G.S., Moll, U.M., Hope, T.J., and Wahl, G.M. (1999). A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J 18, 1660EMBO J 18, -1672. .