p63: The Phantom of the Tumor Suppressor

[Cell Cycle 6:9, 1062-1071, 1 May 2007]; ©2007 Landes Bioscience

Review

p63: The Phantom of the Tumor Suppressor Lee E. Finlan1,* Ted R. Hupp2

Abstract

2Cell Signalling Unit CRUK Laboratories; Division of Oncology; The University of Edinburgh; Edinburgh, Scotland UK

*Correspondence to: Lee E. Finlan; Chromosomes and Gene Expression Laboratory; MRC Human Genetics Unit; Crewe Road; Edinburgh EH4 2XU UK; Tel.: +44.0131.467.8400 ext. 2305; Fax: +44.0131.467.8456; Email: Lee. [email protected]

p53, p63, cancer stem cells, proliferation, differentiation, therapeutic targets, phosphorylation, ATM, mapk

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=4162

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Original manuscript submitted: 03/05/07 Revised manuscript submitted: 03/14/07 Manuscript accepted: 03/19/07

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and Gene Expression Laboratory; MRC Human Genetics Unit; Edinburgh, Scotland UK

The last twenty years of research into p53 function has revealed some fascinating discoveries into the orchestration of tumor suppressor pathways with a multitude of putative drug targets being investigated. However, it was not until 1998 that the ancestral mother of p53 was documented. The eldest evolutionary conserved homolog of the p53 family is known today as p63. Originally, it was thought p63 was another tumor suppressor that could function in a similar capacity to p53. However, elegant demon� strations of the divergent roles that p63 plays as a key transcriptional regulator of the proliferation and differentiation cascade in stratified epithelia are documented. These data link DNp63a to adult tissue stem cell regulation and possibly “cancer stem cells”. p63 lacks mutation in cancer development, which is in stark contrast to the classically high mutation status of p53 in a large compendium of cancer types. Perhaps suggesting a selective preference for p53 mutation. Why is p63 rarely mutated despite being part of the same gene family? Interestingly, p63 is often over‑expressed and amplified in cancer, thus revealing a paradox. Is p63 required to provide cancer cell populations with a selec� tive advantage as much as a loss of p53 function by mutation? Has p53 been masking a “phantom” with promising features as a target for drug development? Can we exploit the biochemical know how gained from the mass of p53 research to further elucidate DNp63a gene function? In this review, we will summarise the emerging advances that are elucidating DNp63a as a promising drug target.

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Cancer progression is driven in part by mutations in oncogenes and tumor suppressor genes that give rise to enhanced survival of the developing tumor. Selective pressures in the microenvironment that drive clonal evolution include intrinsic stresses such as genome instability, growth factor‑mediated activation of oncogenic pathways, metabolic stresses including hypoxia, and exogenous damage induced by exposure to mutagenic agents. The tumor suppressor protein p53 plays a central role in the cellular response to these diverse agents by activation of the cellular repair or apoptotic machinery, depending in part on the cell type.1 p53 protein is a gene that is normally held in check by E3‑ligases that mediate the ubiquitination and subsequent degradation of p53. It has been found that p53 is mutated in over 50% of all human cancers and silenced by other mechanisms in a further 20% of all human cancers.2,3 However, the most recently identified family member; p63 is rarely mutated in cancer but is often over expressed and/or amplified. This has created a wide interest towards p63 recently. The discovery of the p53 homologs p63 and p73 holds new excitement to the p53 field. Identification of homologous genes coding for several proteins with similar and contrasting properties to p53 has been both intriguing and confusing. A multitude of properties have been attributed to these new homologs. This review will focus on the functional and biological aspects of one p53 family member, p63. Although the most ancient member of the p53 family,4 p63 is the most recently discovered and the least is known about this family member. In contrast to p53, whose protein expression is not readily detectable in epithelial cells unless they are exposed to various stress conditions,5 DNp63a is expressed in distinct epithelial cells at high levels under normal conditions. DNp63a is highly expressed in embryonic ectoderm and in the nuclei of basal regenerative cells of many epithelial tissues in the adult including skin, breast myoepithelium, oral epithelium, prostate and urothelia.6 Overexpression of select p63 splice variants is observed in many squamous carcinomas suggesting that p63 may act as an oncogene.7 The use of various

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Lee E. Finlan is a Medical Research Council (MRC) Career Development Fellow. Ted R. Hupp is supported by Cancer Research UK (CRUK) Programme grant.

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p63: The Phantom of the Tumor Suppressor

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could generate identical daughter cells that retained their in vivo developmental potential, indicating that these cells were maintained by self‑renewal.18 Prostate stem cells are p63 positive too.19 p63 is present in isolate lung stem cells and is often over expressed in lung cancer.20‑22 Genetic studies, have been used extensively to determine the functional importance of p63, and have shown that p63 is crucial to preserve the regenerative proliferative structures seen within epithelia. p53‑/‑ mice develop normally, but possess an accelerated occurrence of cancer.23 �� p53+/‑ mice also develop highly penetrant tumors, although with a delayed onset in comparison with that of p53‑/‑ deficient mice.24 Mice lacking p63 (p63‑/‑) displayed severe defects in limb, cranio‑facial and epithelial development. Additionally, the skin was hypoplastic and was shown to have undergone a program of non-regenerative differentiation. Consistent with the murine phenotype, these disorders are characterized by regenerative failure of epithelial structures. These studies depict that p63 functions to maintain the regenerative capacity of stratified epithelial structures throughout the body. They also support a model in which disruption of p63 expression or activity represents an early step in cellular differentiation.25 Interestingly, transgenic mice strains developed to over express DNp63a in the epidermis were �� found exhibit an accelerated aging phenotype in the skin characterized by striking wound healing defects, decreased skin thickness, decreased subcutaneous fat tissue, hair loss, and decreased cell proliferation.26,27 They found that aging in DNp63a transgenic mice and other mouse models correlated with levels of Sirt1 and that increased DNp63a expression induced cellular senescence that could be rescued by Sirt1�.26 This suggests that p63 also has a key role in the control of the aging process. DNp63a has been found to be over‑expressed in several different human cancers often as a results of gene amplification,28‑30 and thus may be important for maintaining cancer cells immortal status, clearly marking DNp63a as a key target of cancer research and possibly therapeutic invention.12 Interestingly, two contrasting papers have recently been reported regarding p63+/‑ mice and their abilities to develop tumors. A recent paper suggested that p63+/‑ were susceptible to tumor development, but the results were less than dramatic in comparison to p53+/‑ studies.31 However, contradictory findings to this report were recently presented that exemplified p63+/‑ �� are not prone to spontaneous or chemically induced tumors.32 Developmental reactivation of p63 gene was found to predispose mice to increased spontaneous tumor development.33 �� It was also demonstrated by the same research group previously that inducible expression of TAp63a in lung and skin epithelia caused metaplasia and hyperplasia.34 �� Furthermore, mice carrying inactive alleles of p63 or p73 do not develop spontaneous tumors thus, concluding that p53 homologs do not contribute to p53 tumor suppressor activity in lymphoma development.35 Considering the vast array of information pertaining to DNp63a acting as a key regulator of the proliferation and differentiation cascade and as a stem cell associated factor, its becoming difficult to believe that p63 simply acts as an alternative p53 tumor suppressor in the light of the facts that p63 is rarely mutated9 and is often over expressed,36 but still fails to amount a significant tumor suppressive response regardless of p53 status. �� Furthermore, DNp63a can oppose p53‑mediated transactivation, growth arrest, and apoptosis.4 �� Recent data has suggested that the over‑expression of p63 and alteration of p53 regulation are not mutually exclusive in melanoma, which perhaps may be predicted if the only functions of p63 isoforms were to counteract p53 transcriptional responses by repression.37 Although, while epithelial progenitors must resist

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model systems and the study of human disease should continue to lead to rapid advances in our understanding of the role of p63 in development, epithelial cell maintenance and tumorigenesis. Several groups independently identified the third member of the p53 family, p63 also known as p51, KET, p40, p73L, p53CP, and NBP.4,8,9 p63 was later shown to be crucial in the development of all epithelial tissues.6 The p63 gene locus at chromosome 3q28 (ensembl gene ID ENSG00000073282) bears strong homology to the tumor suppressor p53 and to the related gene, p73. p63 is actually the eldest evolutionary conserved member of the p53 family �� phylogenetically��. First identified in C. elegans as a p53 homolog with a Sterile alpha motif (SAM) domain, which is absent in the true p53 gene that was first established by gene duplication.10 It has been �� speculated that, during the course of evolution, p63 and p73 first pursued broader ranges of activity in control of proliferation and differentiation of tissues during development. p53 later became specialized in genome maintenance in higher species.10 The expression profile of p63aDN isoform was demonstrated to represent the holoclone population of cells present within the epidermis of humans.11 p63 exhibits a rather tissue specific distribution pattern and was found to be highly expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signaling that direct morphogenetic patterning of the underlying mesoderm.12 Embryonic epidermis of p63‑/‑ mice undergo unusual processes of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including epidermis, mammary, lachrymal, prostate, lungs, gut and salivary glands.12 p63 is expressed in the ectoderm prior to stratification during embryonic development. In normal mammals, as the epidermis matures p63 becomes confined to the stratum basal.12 More recently, the most predominantly expressed isoform DNp63a was demonstrated to be a consistent marker of keratinocyte stem cells within the epidermis.11 It was also revealed that integrin 1b and DNp63a were markers representing the same cell populations within the human epidermis.13,14 One could postulate that DNp63a may actually control integrin 1b expression and in turn, integrin 1b may regulate signals exerted from the extracellular matrix (stem cell niche) into the p63 transcriptional cascade. It was recently demonstrated that �� stratification occurs through asymmetric cell divisions where the mitotic spindle orients perpendicularly to the basement membrane. However, p63‑/‑ cells lack this mechanism consistent with its function in maintaining stem cells by asymmetric cell division while replenishing cells entered into the stratification program.15,16 It was reported that a member of the integrin family; the integrin a3 subunit is indeed under the transcriptional control of DNp63 isoforms.13,14 p63 was found to be critical for cell adhesion by maintaining Integrin4b.17 This provides a molecular basis for the hypothesis that the p63 family are essential for epidermal‑mesenchymal interactions controlling stem cell fate. The development of the epidermis requires an orchestrated progression of events, which regulate proliferation and differentiation of keratinocytes. The p63 family of proteins has been strongly identified to be the key regulator behind such a series of events in keratinocytes. Evidence of how p63 interacts with other signaling factors maybe useful in gathering information on how stem cell markers are critically involved in anti‑tumorigenic responses; such as cross‑talk between the tumor suppressor p53, which has evolutionary functions to prevent and/or eliminate the creation of abnormal cells with the proliferative potential to form tumors. Isolation of Mammary stem cells were found to be p63 positive and these �� cells www.landesbioscience.com

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The tumor suppressor protein p53 is controlled by the balance between E3‑ligase mediated p53 degradation and kinase‑mediated assembly of p53:p300 and p53:DNA transcription complexes. Genetic studies in mice have shown that mutation of the CK2 phospho‑acceptor site (ser392) in p53 increases UV‑induced skin cancer formation, suggesting an unexpected role for p53 phosphorylation in mediating p53‑dependent tumor suppression. p53 plays a role in modulating UV induced skin cancer suppression48 and identifying modifiers of p53‑mediated tumor suppression is a goal of cancer research. Recent research has highlighted the role of the p53 homologs, p73 and p63, in cooperating with p53 in the DNA‑damage response.39 p63 is particularly relevant with respect to cancer control, since it is crucial for the development of epithelial tissues, although how this protein crosstalks with p53 is not well defined. One possible mechanism for crosstalk of p63 and p53 is through transcription dependent mechanisms as p63aDN functions as both a positive and a negative transcriptional regulator and blocks differentiation.49 It was found that p63/p73 are required for p53 activation after DNA damage.39 DNp63a is also expressed in basal cells of human skin, where cells coexpress both DNp63 isoforms and CK2‑phosphorylated p53 after UV‑irradiation. It was not simply that DNp63 or the phosphorylated p53 were marking actively cycling cells, since Ki67 and DNp63 generally revealed mutually exclusive expression after UV‑exposure, suggesting that p53 phosphorylation events are somehow restrained to p63 progenitor cells in human skin.50 DNp63 isotypes were shown to be able to transcriptionally regulate heat shock protein 70 (hsp70) expression, multi drug resistant gene 1 (MDR1) thought to be crucial in cancers becoming resistant to classical cancer therapeutics, the epidermal growth factor receptor (EGFR), and interestingly the putative epidermal stem cell markers; the integrin family.43,51‑53 Reciprocally, EGFR depletion has been demonstrated to culminate in a marked down regulation of DNp63a expression, whereas TAp63g (opposing effects to DN isoforms associated with induction of terminal differentiation) actually represses EGFR.54 This gives another key insight into p63 being a key regulator of replenishing tissues and that may be a key axis potentiated in an array of cancer cell types of epithelial origin to give selective growth advantages.53 Similar to the p53:p300 association,55 p63 has been found to recruit the histone acetyltransferase (HAT) p300 that is essential for modification of the chromatin structure by acetylation, which facilitates the access of transcription factors and transcriptional machinery to the promoter and initiation site.56 JAG1 encodes a ligand for Notch receptors; Notch signaling is critical for cell fate determination, and influences limb and craniofacial development. Jagged1 (JAG1) is directly regulated by p63 transcription, but not p53,57 suggesting that p63 regulation of Notch signaling exists. Key genes involved within the epidermal differentiation cascade have been observed to be regulated by p63; such as the differentiation markers loricrin and involucrin.44 However, whether these are direct target genes have not been established. Glyoxalase II (GLX2), a detoxifying enzyme of glycolysis byproduct Methylglyoxal is a transcriptional target of p63 and p73, and acts as critical pro‑survival factor role in normal development and the pathogenesis of various human diseases, including

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The p63 gene locus possesses two transcription start sites that create two functionally distinct protein isoforms, namely Transactivation domains (TA)and deltaN (DN)isoform. DNp63 lack the NH2‑terminal transactivation (TA‑1) domain and were originally thought to function as “dominant negative” proteins, e.g., DNp63a, DNp63b and DNp63g, that block the function of the corresponding full‑length proteins. However, it has been discovered that DNp63 isotypes were shown to be able to function as bona fide transcription factors in their own right.42,43 The reported presence of a second intrinsic transactivation domain (TA‑2) is thought to be critical for this function40 (Fig. 1). In addition to these NH2 differences, COOH terminal splice forms exist for p63, three additional forms a, b and g result from the use of an alternative splicing. Therefore, there are at least six p63 isoforms with a complex array of similarities and differences in their structural domains and cellular functions.4,44 Only DNp63a and TAp63 possess a sterile alpha motif (SAM domain) that is developmentally implicated in protein‑protein interactions. It is becoming increasingly clear that the six isoforms of p63 play pivotal roles in the induction of a stratification program during development whilst maintaining pluripotent stem cell populations in mature epidermis.34 A survey of genes upregulated by p63 shows that p63 can regulate a wide range of downstream gene targets with various cellular functions, including cell cycle control, stress, signal transduction, and development.43 p73 and p63 show much greater molecular complexity than p53 because they are expressed both as multiple alternatively spliced C‑terminal isoforms, and as N‑terminally deleted, dominant‑negative proteins that show reciprocal functional regulation. p73 shares significant structural peculiarities to p63: the presence of an extended non-conserved C‑terminus containing a sterile alpha motive (SAM), typical of developmental proteins, and the presence of number of different splicing isoforms differing in the N‑terminus or in the absence of the transactivation domain (DN isoforms).45 �� Over expression of p73 in cells can induce apoptosis and p21(waf1/cip1) mediated G1 cell cycle arrest.46 Intimate biochemical cross‑talk among family members suggests a functional network that might influence many different aspects of individual gene action. The most interesting part of this family network derives from the fact that the p63 and p73

The p63/p53/p73 Network

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genes are said to be based on the “two‑genes‑in‑one” idea, encoding both agonist and antagonist in the same open reading frame.47

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developmental apoptosis, it follows that under conditions of genotoxic stress,38 this resistance would be overridden to allow for the destruction of damaged cells that were potentially transformable due their attributes of high proliferative capacity associated with the stem cell phenotype and also a key hallmark of tumorigenesis. p63 and p73 are known to contribute to mediating apoptotic cell kill in response to genotoxic stress in a wild type p53 background using mouse embryonic fibroblasts.39 However, TA‑p63 and DNp63 isoforms predominate in mediating apoptotic effects.40 Although great rewards are to be gained from comparison of p63 to its sibling p53, we need to break the moulds of our thoughts regarding the trinity of the p63/p73/p53 network. This may allow for new insights into the lives of these master transcriptional regulators to be truly elucidated rather than narrowly perceiving them as “tumor suppressors” end of story. Of course, a network of co-operation between these regulators comprehensively maintains genomic integrity. However, it is just not simply a matter of p63/p73 fulfilling the same roles that p53 controls. p63 regulates events that p53 has not been reported to be involved in.41

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that can be activated and bound by p63 but not p53, although the protective function of GPX2 is p53 dependent, suggesting that co-operation between p63 and p53 exists to regulate this factors activity.60 TAp63b and TAp63g, but not DNp63a could activate the skin‑specific promoter of bullous pemphigoid antigen 1 (BPAG‑1),52 thereby further exemplifying a novel molecular mechanism by which a gene is regulated p63 and not other p53 family members (Fig. 2). The PKCD promoter harbors at least three p53-like response elements that were identified to be transactivatable by DNp63a and TAp63a.61 Activation of p53 in response to DNA damage occurs through an ATM‑dependent pathway leading to enhanced multi‑site phosphorylation of p53 at key regulatory sites.62 An association between p53 phosphorylation mediated activation and p63 positive stem cells in human skin following UVB irradiation has recently been established, suggesting that DNp63a cells preferentially possess a active p53 pathway by possibly maintaining high levels of specific kinases involved in DNA damage responses. A Genome wide study of p63 reguFigure 1. (A) p63a structural/functional �� domains and post‑translational modifications. p63a is comprised of a lated genes was recently reported Transactivation domain 1 (TA‑1) is present only in TA‑p63 isoforms, transactivation domain 2 (TA‑2), poly‑proline using chromatin immunoprecipirich domain (PRD), DNA binding domain, tetramerisation domain (Tet) C‑terminal transactivation domain 3 (CTA‑3), tation from keratinocytes (KCs) and a sterile alpha motif domain (SAM). The GPS kinase module has predicted several putative phosphorylation coupled to microarray technology. sites within p63, which possess similarities to p53 phosphorylation site clustering. TA‑1 and TA‑2 seem to be much focused on for phospho‑regulatory signals from cell cycle, stress induced, growth, and survival signaling pathways.� They found that p63 gene family control many more targets than (B) �� p53 structural/functional domains and post‑translational modifications I) An illustration depicting the important previously published.63 Several functional domains of the tumour suppressor p53 defined by biochemical structure-function analyses. p53 is com� prised of at least four distinct domains that are involved in the modulation of it DNA binding and transcriptional interesting hits are of interest functions: The transactivation domain possesses key binding sites for positive and negative regulators of p53 such to stem cell biologists including as MDM 2 and p300 respectively. Regulatory phosphorylation sites within the N‑terminal protein‑protein interaction transforming growth factor beta domain at Ser 15, Thr 18, and Ser 20 are critical functional sites for the transactivation functions of p53. The DNA regulators (TGFb pathway), sequence specific binding domain contains most of the mutations found in cancer, and essential tetramerisation motif sequences that enables functional properties for DNA binding. p300 possesses specific acetyltransferase HOXC4, fibroblast growth factor activity to lysines 320, 373 and 382, which inhibit mdm2 mediated degradation and stimulates DNA binding. II) (FGF) and Sonic hedgehog (Sh) pathways. Diagram shows the evolutionary conserved domains within p53. (Box I to V), which reside within regions that have shown to be important in the post‑translational activation and sequence specific DNA binding.119 These data taken together exemplify that the p63 family possess distinct functional roles cancer, diabetes, and neurodegenerative diseases.58 The pigment from those of p53 and p73, but may act in a complex cooperaepithelium derived factor (PEDF) and vitamin D receptor (VDR) tive manner to orchestrate and maintain the overall homeostasis by are other genes recently identified as a target of p63, but is not under protecting cells potentially at risk of transformation. Evidence that the transcriptional control of p53.57,59 GPX2, which encodes a gluta- the p63 isoform, p63aDN, is a marker of unipotent “stem” cells thione peroxidase, is also up regulated by p63 but not p53. A unique within epidermal tissue highlights the interest in understanding how responsive element was found in the promoter of the GPX2 gene this p63 isoform, which has evolved functions to prevent and/or www.landesbioscience.com

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origins of the cell population that has to be transformed (obtained mutations in key tumor suppressors) to induce cancer development.

p53 Family Post‑Translational Modifications

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p53 modifications include phosphorylation, acetylation, glycosylation and sumolation, and have been shown to affect the activity of p53 as a transcription factor and tumor suppressor. �� Protein phosphorylation is an important reversible post‑translational modification of proteins, and it orchestrates a variety of cellular processes. To exemplify, p53 undergoes �� phosphorylation at Ser392 that leads to enhanced DNA‑binding,64 phosphorylation at Ser20 that stabilizes p300 binding65 and which mediates DNA‑dependent acetylation of p53,66 recruitment of transcriptional components, and the induction of p53‑dependent genes that mediate its tumor suppressor activity.67 There are many other sites of covalent modification of p53, although a precise biochemical mechanism is usually undefined. For example, other p53 sites of covalent modification exist: p53 protein is also phosphorylated at Ser6, Ser9, Ser15, Ser33, Ser37, Ser46, Thr18 and Thr55 in response to UV light and IR, whereas in the C‑terminus Ser315, �� Ser360, Thr365, Ser370 and Thr377�� are phosphorylated, Lys386 is sumolated, and Lys320, Lys373 and Lys382 are acetylated in response to DNA damage. The physiological significance of these phosphorylation sites in vivo has been rarely determined. For example, although phosphorylation at Thr18 can inhibit MDM2 binding,68 and VRK‑1 can phosphorylate this site and stabilize p53 protein, the Ala18 p53 mutant is still stabilized by VRK‑1.69 Thus the significance of some sites of covalent modification is unclear. Further, although the C‑terminal lysines residues have been implicated as ubiquitination sites,70 mutation of these sites do not prevent ubiquitination.71 Rather, these sites seem to specifically Figure 2. (A) Cross‑talk between p63 and p53. The basic transcriptional control of ATM kinase and HSP 70 are at least partially under the control of the p63 gene family. p53 be involved in mediating p53 transactivation by driving 72 phosphorylation and protein folding is primarily brought about by ATM kinase and HSP70 acetylation control of p53. However, of all the sites of respectively. Latent ATM is subsequently activated by double strand DNA breaks and/or modification that have been reported, it has been shown heat shock culminating in ATM autophosphorylation at ser 1981, which leads to subse� that Ser15 and Ser392 phosphorylation on p53 can occur quent targeting of downstream targets (involved in DNA repair, cell cycle and tumour in the spleen of animals exposed to whole body ionizing suppression), including phosphorylation of p53 at ser 9,15 and 46, Phosphorylation radiation.73 Further, Ser15 and Ser392 phosphorylation promotes the dissociation of MDM2/Pirh2 E3 ligases, and the formation of stable p53 can occur in vivo after UVB irradiation of human skin tetramers required full transcriptional activity. The GPS kinase results suggest that ATM is with phosphorylation of p53 confined to basal layer or also signalling into p63 to either stabilize the formation of active tetramers or destabilize p63 protein and augment protein turnover by the protein degradation machinery but this stem cell populations.5 These latter data suggest a possible remains undefined (Grey dashed line). Sumo‑1 and the E3 ligase Itch/AIP4 target p63 cross talk between the p63 and the kinases that activate for proteosomal/lysosomal degradation, which is distinct from the mdm2/Pirh mediated the p53 response to DNA damage. degradation of p53. (B) p63aDN transcriptionally controls key factors involved in growth However, little has been reported with regards to signaling (EGFR, Jagged and PERP), survival (HSP 70), drug resistance (MDR‑1), and p63 phosphorylation events insofar. A first confirmation DNA repair (ATM). Some of these markers have been documented to be over‑expressed came that at least DNp63a is a phosphorylated protein in a large number of solid epithelial tumours and are the focus of drug development and in neonatal human epidermal keratinocytes.74 A compreinhibitor screening. hensive characterization of the sites phosphorylated and the specific kinases involved remains elusive, although eliminate the creation of abnormal cells with the proliferative poten- there is experimental evidence that report several kinases involved in tial to form tumors. The potential implications of elucidating that cell signaling, stress responses and proliferation which impinge upon DNp63a augments the p53 mediated response by regulating the p63 stability and function.53,54,75,76 The regulation of p73 by phosphorylation has been reported.77 �� The levels of key kinase pathways in normal adult tissue stem cells could be insightful because it poses interesting questions about the true first identified example of phosphorylation of p73 by a non-receptor 1066

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thus demonstrating that p73 and p63 families are involved in surveillance of genome integrity also. Experimental identification of phosphorylation sites and kinases regulating p63 is much needed to truly aid our understanding of the p63 pathway. In a bid to instigate some interest and to highlight some of the putative kinases signaling into p63 and the respective peptide consensuses they hit, the use of the GPS kinase prediction module was employed (http://973‑proteinweb.ustc.edu. cn/gps/gps_web/predict.php). These results offer limited scope, but they do reveal some interesting hits for consideration and potential investigation. GPS offers satisfactory sensitivity (94%) and specificity (97%).82,83 The parameters of the GPS scan were set to be stringent and the hit scores accepted as being acceptable for interest were double the cut‑off point score generated by the GPS module, hence they are highly likely to be precise hits (Fig. 3). Further to this, experimental data exists which supports that some of the kinases determined by the GPS scan are already implicated in regulating the steady state levels of p63 for three of the six kinases identified. The six kinases uncovered to potentially regulate p63 are listed in Figure 3A. Multiple phosphorylation sites were found to exist for each kinase respectively. Also, three of the kinases have previously been demonstrated to regulate the accumulation, tetramerization, and transcriptional activation of p53 by phosphorylation. These were ATM, cyclin dependent kinases (CDKs), and MAPK.5 This gives some plausible but not definitive leads to which signaling pathways feed into the p63 transcriptional cascade. These data linking key kinase pathways to p63 Figure 3. Group based phosphorylation identifies several interesting kinases signalling into the p63 pathway. (A) The sequence of the p63a protein with highlighted motifs depicting potential regulation have been discussed, but the actual sites phosphorylated within p63 remain elusive. The phosphorylation sites within p63. Roughly four clusters of potential sites regulated by kinase GPS data augment these findings GPS to provide modulation have been identified using the GPS phosphorylation prediction module. Of note, cluster I resides within transactivation domain 1 (TA‑1 of p63 that devoid in p63DN isoforms, some stimulation of future directions and to reveal whereas cluster II is present within transactivation 2 (TA‑2). Differential regulation of these two distinctions from the p53 pathway. It was found sites may exist that gives rise to contrasting effects upon the protein stability and transcriptional that treatment of keratinocytes with epidermal activities of the TA/DN isoforms respectively, that may lead to different biological outcomes growth factor results in an increase in DNp63a associated with the different isoforms of p63. (B) The table outlines the GPS kinase prediction results and the respective sites targeted in p63. Columns are designated to provide information expression at the mRNA level, which is abrogated of whether the respective kinases have previously been found to modulate either p53 and/or by inhibition of phosphoryl inositide‑3 kinase (PI‑3K) but not mitogen‑activated protein kinase p63 by experimentation. (MAPK) signaling.75 Although PI‑3K was not found to phosphorylate p63 by the GPS module, a member of the phosphatidylinositol 3‑kinase (PI3K)‑like tyrosine kinase, c‑Abl, at Tyr99 in response to DNA‑damaging ATM is�� PI3K‑IA inhibits the activation of agents,78 which led to an increase in p73 stability.79 Overexpression kinase family,84 and suppression of �� of c‑Abl also induces phosphorylation of p73 on threonine residues ATM that is caused by either ionizing radiation or high NaCl concenadjacent to prolines, an effect that is blocked by dominant nega- trations that induce double strand DNA breaks.85 This suggests that tive inhibitors of p38(MAPk). p38(MAPk) is able to phosphorylate PI‑3K inhibition gives an indirect effect, by depleting ATM activap73 at threonine 99 and activate p73 without the c‑Abl.80 Both tion through the PI‑3K pathway. These results demonstrate that p63 phosphorylation pathways enhance the stability and transcriptional is likely to modulated by phosphorylation events that are either stabiIt has been proposed that ATM inhibition activity of p73. The activities of p73 are also regulated by acetylation. lizing or degrading p63. �� DNA‑damage‑dependent acetylation of p73 at residues 321, 327 and would cause cellular radio‑ and chemosensitization. The compound 331 by p300 and dictates its promoter specificity.81 A non-acetylat- KU‑55933 inhibits ATM kinase activity with an excellent IC(50) able p73 is transcriptionally ineffective. It has been shown that the of 13 nmol/L and a Ki of 2.2 nmol/L. KU‑55933 shows specificity combined loss of p63 and p73 results in the failure of cells containing with respect to inhibition of other phosphatidylinositol 3’‑kinase‑like functional p53 to undergo apoptosis in response to DNA damage,39 kinases,86 but its remains untested if this compound effects p63 steady www.landesbioscience.com

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be degraded as the mutants DNp63a K193E and DNp63a K194E fail to be degraded as efficiently in Saos‑2 transfection experiments, although degradation is not totally lost.88 p73 can be degraded by Itch/AIP4 also, but p53 is not a suitable substrate.92,93 It is interesting that this ubiquitin/lysosomal mediated degradation pathway is distinct from p53 mdm2/Pirh2 mediated degradation of p53,94,95 thereby providing evidence that the p63 regulatory network is distinct from but co-operative with the p53 tumor suppression network.

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What an ironic revelation it would be for a key member of the p53 tumor suppressor family to be actually acting as a critical cancer‑promoting factor by “inadvertently” maintaining and propagating genetically damaged adult tissue stem cells in the face of p53. These data would suggest that p53 gene evolution from the p63 ancestral gene might have occurred in metazoans to counteract the proliferative capacity of p63. Such an opera of “the phantom masked in the shadows of the fallen angel” is perhaps elegantly akin to the reality of p63/p53 paradigm. The use of various model systems and the study of human disease should continue to lead to rapid advances in our understanding of the role of p63 in development, epithelial cell maintenance and tumorigenesis. The existence of cancer stem cells have been demonstrated in many tumors classes of different tissue origins. A study in human skin cancers revealed basal cell carcinomas (BCCs) possess p63 expression and while squamous cell carcinoma (SCCs) were found to have heterogeneous p63 expression with negativity in terminally differentiated squamous cells.96 All pre-neoplastic epidermal lesions showed p63 expression in all cell layers.96 In addition a correlation between hTERT, survivin expression was seen with p63 expression. By RT‑PCR, it was shown that DNp63a is the predominant isoform that highly over‑expressed in cell lines from squamous cell carcinomas (SCC) of the head and neck, confirming immunochemical observations.36 A correlation between high DNp63a expression and poor prognosis in head and neck squamous cell carcinoma was recently reported.30,97 Protein over‑expression in primary lung tumors was limited to squamous cell carcinoma and tumors known to harbor a high frequency of p53 mutations.20,98 A role for amplification and over expression has been implicated in lung cancers. High expression of p63 in Rat 1a cells led to an increase in soft agar growth and tumor size in mice.28 Micro‑satellite analysis revealed that 14 of 26 (54%) primary head and neck squamous cell carcinoma (HNSCC) had allelic imbalance in at least one of the seven microsatellite loci. However, FISH analysis with a p63 gene probe showed that a majority of HNSCC had an increased copy number of the locus regardless of allelic status.30 Despite an abundance of reports of p63 over‑expression in many tumors,98‑103 it is clear that p63 is very rarely mutated in cancers.41,104 The mass of data now documented exemplify that the stem cell associated factor DNp63a is in fact an proto‑oncogene in direct contrast to its family member p53. It is likely that the tumor suppression phenotype sometimes attributed to p63 comes from TAp63 and not DNp63 isoforms.40 Recent reports have suggested that metastatic potential of certain cancers do not correlate with high p63 expression.105 These studies focus on p63 as a single entity, which can very be misleading. Other explanations are that only very low levels of DNp63 expression is required to maintain stem cell survival and that DNp63 can be attenuated to facilitate mesenchymal transition processes required in metastasis. Perhaps DNp63 could be up‑regulated post metastasis to

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state levels or phosphorylation that would truly test if ATM impinges upon stem cell signaling into DNp63a. Transfection of the EGFR increases the levels of DNp63a. �� The ZD1839 �� (Iressa) is an adenosine triphosphate‑competitive inhibitor specific to the EGFR tyrosine kinase currently under evaluation as a chemotherapeutic agent was evaluated in�� tumors of the head and neck for �� inhibition of epidermal growth factor receptor signaling decreases p63 expression.53,75 This identifies a potential molecular relationship between EGFR signaling and p63 and could provide insight into the mechanism of action of ZD1839 (Iressa). �� The p63 isoform associated with terminal differentiation and apoptosis TAp63g has been found to repress EGFR, suggesting that switching off growth signaling is important in the differentiation response.54 It was also recently demonstrated that the abl specific kinase inhibitor �� Imatinib (Gleevec) reduces TAp63/ DNp63 expression in a dose‑dependent manner in tumors of the head and neck (HNSCC) and overrides p63 protein induction by DNA‑damaging agents. Over‑expression of c‑Abl in the absence of Imatinib (Gleevec) results in higher levels of p63 than those treated with Imatinib (Gleevec), implicating c‑Abl kinase activity as a regulator of p63 protein stability. Imatinib (Gleevec) down regulates TAp63/DNp63 levels in HNSCC in a dose‑dependent manner under both normal and DNA‑damaging conditions. This down regulation can be explained by Gleevec’s inhibition of c‑Abl, which destabilizes p63.76 Interesting observations that the putative phosphorylation target sites in p63 for c‑Abl, ATM, EGFR and MAPK are clustered in both cluster I that is only present within the transactivation domain 1 (TA‑1), which is exists only in p63‑TA isoforms and also in Cluster II present in the Transactivation domains 2/Polyproline domain (TA‑2/PRD). �� Experimental evidence remains elusive regarding if these 3 kinases CDKs, S6K and PKB identified by GPS truly do regulate p63. Although there is evidence that p53 is targeted by CDK2 and phosphorylates at Ser 315,87 giving minor evidence that its not out of the question that these kinases do regulate these sites in p63. �� Unfortunately, no report exists that reveals if phosphorylation of the TA or DN preferentially stablizes one isoform over the other. As an example, if ATM phosphorylated DNp63a and TAp63a after a genotoxic insult, does ATM phosphorlyate both isoforms? Are the effects of this phosphorylation different upon the steady state levels and transcriptional activities of these isoforms respectively? Do the phosphorylation sites of the TA‑1 (Cluster I) act in a dominant fashion over the TA‑2 (Cluster II) and proline rich domain (Cluster III) phosphorylation sites? It poses a potential mechanism of how the p63 isoforms function differently to one another as part of their role in the epithelial stratification program.34 Together with these reports, there is enough reasoning to suggest that the GPS module has likely identified bona fide kinases that regulate p63 by phosphorylation and also revealed that the kinase/receptor inhibitors are perhaps effecting cancer cell proliferation and cell survival by modulating stem cell fate through blocking essential input signals into the p63 cascade. Further to phosphorylation, it was recently exemplified that p63 is targeted for ubiquitin mediated degradation by a HECT (homologous to the E6‑associated protein C terminus) E3‑ubiquitin ligase Itch/AIP4 and SUMO‑1.40,88‑91 Itch/AIP4 binds, ubiquitinylates, and promotes the degradation of p63. The physical interaction occurs close to the SAM (sterile alpha motif ) domain; a single Y504F mutation significantly affects p63 degradation. Elegantly, Itch and p63 are co-expressed in the epidermis and in primary keratinocytes, where Itch controls the p63 protein steady‑state levels.89 It has been suggested that lysines 193 and 194 are important for p63 to 1068

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Elucidating the tapestries of the p53 families operational network has already revealed so much to the cause of cancer research, but a long awaited therapy from this research has been elusive.118 The future is arguably brighter with the focus now shifting towards p63 as a putative therapeutic target. The amount of information documenting p63 function and how novel therapeutics are impinging on its activities is encouraging but much remains unsolved. All of these attributions are leading to novel insights of how the p53 family evolved to govern the integrity of the genome over millions of years, not just by tumor suppression but by orchestrating a myriad of events important in contributing to the maintenance of the overall homeostasis in cell hierarchies that make up epithelial tissues and ultimately organisms. References

1. Levine AJ, Hu W, Feng Z. The p53 pathway: What questions remain to be explored? Cell Death Differ 2006; 13:1027‑36. 2. Lane DP, Hupp TR. Drug discovery and p53. Drug Discov Today 2003; 8:347‑55. 3. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88:323‑31. 4. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, McKeon F. p63, a p53 homolog at 3q27‑29, encodes multiple products with transactivating, death‑inducing, and dominant‑negative activities. Mol Cell 1998; 2:305‑16. 5. Hupp TR, Lane DP, Ball KL. Strategies for manipulating the p53 pathway in the treatment of human cancer. Biochem J 2000; 352(Pt 1):1‑17. 6. McKeon F. p63 and the epithelial stem cell: More than status quo? Genes Dev 2004; 18:465‑9. 7. Taniere P, Martel‑Planche G, Saurin JC, Lombard‑Bohas C, Berger F, Scoazec JY, Hainaut P. 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. Br J Cancer 2001; 85:721‑6. 8. Augustin M, Bamberger C, Paul D, Schmale H. Cloning and chromosomal mapping of the human p53‑related KET gene to chromosome 3q27 and its murine homolog Ket to mouse chromosome 16. Mamm Genome 1998; 9:899‑902. 9. Osada M, Ohba M, Kawahara C, Ishioka C, Kanamaru R, Katoh I, Ikawa Y, Nimura Y, Nakagawara A, Obinata M, Ikawa S. Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nat Med 1998; 4:839‑43. 10. Blandino G, Dobbelstein M. p73 and p63: Why do we still need them? Cell Cycle 2004; 3:886‑94. 11. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, De Luca M. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 2001; 98:3156‑61. 12. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999; 398:714‑8. 13. Tiberio R, Marconi A, Fila C, Fumelli C, Pignatti M, Krajewski S, Giannetti A, Reed JC, Pincelli C. Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl‑2 dependent manner. FEBS Lett 2002; 524:139‑44. 14. Kurata S, Okuyama T, Osada M, Watanabe T, Tomimori Y, Sato S, Iwai A, Tsuji T, Ikawa Y, Katoh I. p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche. J Biol Chem 2004; 279:50069‑77. 15. Lechler T, Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 2005; 437:275‑80. 16. Koster MI, Roop DR. Asymmetric cell division in skin development: A new look at an old observation. Dev Cell 2005; 9:444‑6. 17. Carroll DK, Carroll JS, Leong CO, Cheng F, Brown M, Mills AA, Brugge JS, Ellisen LW. p63 regulates an adhesion program and cell survival in epithelial cells. Nat Cell Biol 2006; 8:551‑61. 18. Deugnier MA, Faraldo MM, Teuliere J, Thiery JP, Medina D, Glukhova MA. Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the Comma‑Dbeta cell line. Dev Biol 2006; 293:414‑25. 19. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, Yang A, Montironi R, McKeon F, Loda M. p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 2000; 157:1769‑75. 20. Massion PP, Taflan PM, Jamshedur Rahman SM, Yildiz P, Shyr Y, Edgerton ME, Westfall MD, Roberts JR, Pietenpol JA, Carbone DP, Gonzalez AL. Significance of p63 amplification and overexpression in lung cancer development and prognosis. Cancer Res 2003; 63:7113‑21.

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If stem cell survival factors were the targets of therapeutic strategy it would not necessarily have to be a case of trying to reactivate mutated genes, use of receptor antagonists, or even kinase inhibition. Although some of these drugs have proven very useful in cancer treatment, perhaps the targeting of key transcription factors such as DNp63a that act as critical survival factors often overamplified and highly expressed in the seeding populations of many tumors may offer more control and specificity of desired outcome for cancer therapy. If we could try to disarm cancer stem cells of their inherited mechanisms of protection they unintentionally gained from parental normal adult stem cells; such as protection against chemotherapeutic agents used to treat cancer and implicated in chemoresistance (ATM, MDR1, ALDH), survival signals (HSP 70, GLX2), and growth signaling pathways (EGFR, Jagged2) with “one shot” before targeting cancers with radio or conventional chemotherapy. With this strategy we may pose a more serious strategic threat to cancer progression. The problem that exists with singularly targeting kinase pathways such as Abl (Iatinib Gleevec), ATM (�� KU‑55933), �� and growth receptors such as�� EGFR�� (Iressa) is the phenomena of compensation in the regulatory signals by cancer cells,114‑117 thereby allowing cells to bypass key secondary messengers or signal through a different growth receptor, which culminates in the inhibitor becoming easily resisted and ineffective. More novel approaches for targeting and inhibiting p63 function would be to use oligonucleotide based inhibitors of sequence‑specific DNA‑binding or peptidomimetic inhibitors of p63‑specific interactions with transcription machinery. In particular, peptide therapeutics is now a relatively advanced field and stabilizing modifications in vivo permit effective peptide‑ligand delivery to cancers. It is plausible but in no way definitive that a transcription factor like p63aDN is not so easily compensated for in cancer. Solid tumors rarely possess a mutated form of the p63 gene suggesting it may be a regulator that is selected out by its ability to sustain genetically damaged cells, hence, p63 wild type (p63+/+) cancer cells survive more proficiently. We regard key transcription factors such as p63 as a fundamental source of future therapeutic targets that may be more effective than growth receptor antagonists and/or kinase inhibitors

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seed a secondary tumor. Much remains to be discovered in the area. The prognostic implications for high expression of DNp63a in breast cancer,106‑109 lung cancer20,98 and ovarian cancer also exist.100 The aggressive malignant �� Adenoid cystic/basal cell carcinoma �� ACBCC of the prostate is highly p63 positive and is associated with a poor prognosis.110 Pancreas tumors are p63 positive.111 p63 is present in isolate lung stem cells and is often over expressed in lung cancer.20‑22 �� It has been proposed that p63aDN expression profiling could be useful in the classification of the differentiation stages of lung cancers, with poorly differentiated cancers staining strongly for p63 and being associated with a poor prognosis.22 The �� malignant transformation of non-tumorigenic human prostatic epithelial cell line correlates with high DNp63a positivity.112 �� Immunohistochemical expression of p63 in endometrial polyps has provided evidence that a basal cell immunophenotype is maintained.113 �� These data combined, suggest that a selective program of events in cancer development takes place that somehow protects p63 from mutation, and potentiates its expression while actively targeting p53 for inactivation by mutation, thus providing cancer stem cells like “weed seeds in the garden”.

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49. King KE, Ponnamperuma RM, Yamashita T, Tokino T, Lee LA, Young MF, Weinberg WC. DeltaNp63alpha functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes. Oncogene 2003; 22:3635‑44. 50. Finlan LE, Nenutil R, Ibbotson SH, Vojtesek B, Hupp TR. CK2‑site phosphorylation of p53 is induced in deltaNp63 expressing basal stem cells in UVB irradiated human skin. Cell Cycle 2006; 5:2489‑2494. 51. Wu G, Osada M, Guo Z, Fomenkov A, Begum S, Zhao M, Upadhyay S, Xing M, Wu F, Moon C, Westra WH, Koch WM, Mantovani R, Califano JA, Ratovitski E, Sidransky D, Trink B. DeltaNp63alpha upregulates the Hsp70 gene in human cancer. Cancer Res 2005; 65:758‑66. 52. Osada M, Nagakawa Y, Park HL, Yamashita K, Wu G, Kim MS, Fomenkov A, Trink B, Sidransky D. p63‑specific activation of the BPAG‑1e promoter. J Invest Dermatol 2005; 125:52‑60. 53. Matheny KE, Barbieri CE, Sniezek JC, Arteaga CL, Pietenpol JA. Inhibition of epidermal growth factor receptor signaling decreases p63 expression in head and neck squamous carcinoma cells. Laryngoscope 2003; 113:936‑9. 54. Nishi H, Senoo M, Nishi KH, Murphy B, Rikiyama T, Matsumura Y, Habu S, Johnson AC. p53 Homolog p63 represses epidermal growth factor receptor expression. J Biol Chem 2001; 276:41717‑24. 55. Finlan L, Hupp TR. The N‑terminal interferon‑binding domain (IBiD) homology domain of p300 binds to peptides with homology to the p53 transactivation domain. J Biol Chem 2004; 279:49395‑405. 56. MacPartlin M, Zeng S, Lee H, Stauffer D, Jin Y, Thayer M, Lu H. p300 regulates p63 transcriptional activity. J Biol Chem 2005; 280:30604‑10. 57. Sasaki Y, Ishida S, Morimoto I, Yamashita T, Kojima T, Kihara C, Tanaka T, Imai K, Nakamura Y, Tokino T. The p53 family member genes are involved in the Notch signal pathway. J Biol Chem 2002; 277:719‑24. 58. Barbieri CE, Tang LJ, Brown KA, Pietenpol JA. Loss of p63 leads to increased cell migration and up‑regulation of genes involved in invasion and metastasis. Cancer Res 2006; 66:7589‑97. 59. Kommagani R, Caserta TM, Kadakia MP. �� Identification of vitamin D receptor as a target of p63. Oncogene 2006; 25:3745‑51. 60. Yan W, Chen X. GPX2, a direct target of p63, inhibits oxidative stress‑induced apoptosis in a p53‑dependent manner. J Biol Chem 2006; 281:7856‑62. 61. Ponassi R, Terrinoni A, Chikh A, Rufini A, Lena AM, Sayan BS, Melino G, Candi E. p63 and p73, members of the p53 gene family, transactivate PKCdelta. Biochem Pharmacol 2006. 62. Wahl GM, Carr AM. The evolution of diverse biological responses to DNA damage: Insights from yeast and p53. Nat Cell Biol 2001; 3:E277‑86. 63. Vigano MA, Lamartine J, Testoni B, Merico D, Alotto D, Castagnoli C, Robert A, Candi E, Melino G, Gidrol X, Mantovani R. New p63 targets in keratinocytes identified by a genome‑wide approach. Embo J 2006; 25:5105‑16. 64. Hupp TR, Lane DP. Two distinct signaling pathways activate the latent DNA binding function of p53 in a casein kinase II‑independent manner. J Biol Chem 1995; 270:18165‑74. 65. Dornan D, Hupp TR. Inhibition of p53‑dependent transcription by BOX‑I phospho‑peptide mimetics that bind to p300. EMBO Rep 2001; 2:139‑44. 66. Dornan D, Shimizu H, Perkins ND, Hupp TR. DNA‑dependent acetylation of p53 by the transcription coactivator p300. J Biol Chem 2003; 278:13431‑41. 67. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408:307‑10. 68. Craig AL, Burch L, Vojtesek B, Mikutowska J, Thompson A, Hupp TR. Novel phosphorylation sites of human tumor suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers. Biochem J 1999; 342(Pt 1):133‑41. 69. Lopez‑Borges S, Lazo PA. The human vaccinia‑related kinase 1 (VRK1) phosphorylates threonine‑18 within the mdm‑2 binding site of the p53 tumor suppressor protein. Oncogene 2000; 19:3656‑64. 70. Rodriguez MS, Desterro JM, Lain S, Lane DP, Hay RT. Multiple C‑terminal lysine residues target p53 for ubiquitin‑proteasome‑mediated degradation. Mol Cell Biol 2000; 20:8458‑67. 71. Shimizu H, Saliba D, Wallace M, Finlan L, Langridge‑Smith PR, Hupp TR. Destabilizing missense mutations in the tumor suppressor protein p53 enhance its ubiquitination in vitro and in vivo. Biochem J 2006; 397:355‑67. 72. Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, Anderson CW, Appella E. DNA damage activates p53 through a phosphorylation‑acetylation cascade. Genes Dev 1998; 12:2831‑41. 73. Wallace M, Coates PJ, Wright EG, Ball KL. Differential post‑translational modification of the tumor suppressor proteins Rb and p53 modulate the rates of radiation‑induced apoptosis in vivo. Oncogene 2001; 20:3597‑608. 74. Westfall MD, Mays DJ, Sniezek JC, Pietenpol JA. The Delta Np63 alpha phosphoprotein binds the p21 and 14‑3‑3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay‑Wells syndrome‑derived mutations. Mol Cell Biol 2003; 23:2264‑76. 75. Barbieri CE, Barton CE, Pietenpol JA. Delta Np63 alpha expression is regulated by the phosphoinositide 3‑kinase pathway. J Biol Chem 2003; 278:51408‑14. 76. Ongkeko WM, An Y, Chu TS, Aguilera J, Dang CL, Wang‑Rodriguez J. Gleevec suppresses p63 expression in head and neck squamous cell carcinoma despite p63 activation by DNA‑damaging agents. Laryngoscope 2006; 116:1390‑6.

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21. Ling TY, Kuo MD, Li CL, Yu AL, Huang YH, Wu TJ, Lin YC, Chen SH, Yu J. Identification of pulmonary Oct‑4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS‑CoV) infection in vitro. Proc Natl Acad Sci USA 2006; 103:9530‑5. 22. Wang BY, Gil J, Kaufman D, Gan L, Kohtz DS, Burstein DE. P63 in pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors. Hum Pathol 2002; 33:921‑6. 23. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery Jr CA, Butel JS, Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature 1992; 356:215‑21. 24. Harvey M, McArthur MJ, Montgomery Jr CA, Bradley A, Donehower LA. Genetic background alters the spectrum of tumors that develop in p53‑deficient mice. Faseb J 1993; 7:938‑43. 25. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homolog required for limb and epidermal morphogenesis. Nature 1999; 398:708‑13. 26. 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