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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-.

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TP63, TP73: The Guardian's Elder Brothers

, , and *.

* Corresponding Author: INSERM U590, Centre Léon Bérard, 28 rue Laennec, 69008 Lyon, France. Email: carondef@lyon.fnclcc.fr

TP73 and TP63 recently emerged as sharing overall architectural similarities with TP53. Phylogeny indicates that these three genes derive from a common ancestor, thus defining a new gene family. All three genes bind similar DNA consensus sequences in the promoters of many genes and regulate common generic aspects of growth control, survival, DNA repair or differentiation. However, their regulation patterns are distinct. While p53 is an ubiquitous, stress-response protein regulated at the post-translational level, p63 and p73 are expressed in a tissue and differentiation-specific manner and are also regulated at the transcriptional level. This regulation results in isoforms generated by alternative splicing or by the use of different promoters. They differ from each other in their C-terminus (which contains important regulatory domains) and, most strikingly, in their N-terminus. Thus, the major forms of p63 and p73 in many normal tissues are ΔN isoforms, which lack the transactivation domain, and can behave as repressors of the genes normally regulated by transactivation-competent (TA) forms of the protein. Control of the balance between levels of TA and ΔN forms of p63 and p73 is important in differentiation. Mice lacking TP63 or TP73 are not predisposed to cancer, but show developmental defects. In particular, TP63 knock-out mice have major defects in cranial and limb morphogenesis, and in the formation of squamous epithelia. These defects are partially recapitulated in human subjects with germline mutation in TP63. There is growing evidence that p63 and p73 are involved in carcinogenesis through several mechanisms. For instance, amplification of TP63 in squamous cancers results in overexpression of a ΔN protein that may counteract suppression by p63 as well as other family members. On the other hand, some mutant p53 can bind and inactivate p63 or p73, providing a mechanism for mutant p53 “gain-of-function” effect.

Introduction

In 1993, the p53 protein was awarded the title of “molecule of the year”, acknowledging its rise to fame as the “guardian of the genome”.1,2 Four years later, it emerged that the guardian, who was considered as an orphan until now, had two big brothers who were living much less glamorous and dangerous lives, TP63 and TP73.3-7 Since then, we have become much more familiar with the complex structure, expression patterns and biology of TP63 (3q27.29) and TP73 (1p36). As a result, their lives now appear much more exciting, in particular since they are expressed as multiple isoforms that play important roles in differentiation and morphogenesis, and since their interactions with p53 are emerging as potentially important events in tumorigenesis.

Note that all amino acid positions cited in the text for p53, p63 or p73, refer to human sequences, except when the species is mentioned.

The p53 family members have a typical structure of transcription factors, with a N-terminal transactivation domain, a central domain carrying the DNA-binding properties and a C-terminal domain needed for the oligomerization. The TP53 gene is the most frequently mutated gene in a wide variety of human tumors.8 The database of published mutations now contains 17,689 somatic mutations and 225 germline mutations (http://www.iarc.fr/p53). Lack of TP53 function predisposes mice to early and multiple tumors, demonstrating its function as a tumor suppressor.9,10 By analogy, it was thought that TP63 and TP73 would also be frequently altered in human cancers. However, the search for mutations in tumors gave very poor results, and knock out mouse models did not reveal any increase in spontaneous tumorigenesis. In contrast, these mice exhibited a wide spectrum of specific defects in the differentiation and morphogenesis of several tissues, indicating that TP63 and TP73 are acting as physiological regulators of developmental processes rather than as tumor suppressors.3,4,7 This is an unusual situation among gene families, since the members of the same family often have similar, if not overlapping functions. In the p53 family group, the odd one out is clearly p53, which differs from its two brothers by its mode of regulation, its acute inducibility, its ubiquity and its functional restriction to specific aspects of stress response.

In this review, we will present and discuss the structural and functional characteristics of the p63 and p73 proteins. In addition, we will review recent evidence on their implication in cancer, focusing on the potential role of particular isoforms, the ΔN isoforms, which lack the transactivation domain, and therefore may have growth-promoting effects.

Structure of the p63 and p73 Isoforms

Structural Homology between the Family Members

TP63 and TP73 are expressed as multiple isoforms, which all conserve the DNA-binding domain but differ by their N- or C-terminal regions (fig. 1). The highest percentage of homology between these proteins is observed in the central domain (65% between p53 and p63 or p73, 85% between p63 and p73).3,7 All the residues that play an important role in the folding of the DNA-binding domain and in the contact with DNA are well conserved, suggesting that the overall shape of the domain is similar in the three family members. This similarity accounts for their capacity to bind and to transactivate many of the same promoters as those activated by p53. Furthermore, the DNA-binding domain alone is responsible for the specificity of transactivation, the other domains providing either generic (transactivation, oligomerization) or regulatory functions.11

Figure 1. Structural comparison and homology between p53, p63 and p73.

Figure 1

Structural comparison and homology between p53, p63 and p73. The schematic structure of TAα isoforms of p63 and p73 is presented, including the transactivation domain (TA), the DNA-binding domain and the oligomerization domain (oligo). SAM: sterile (more...)

The C-terminal isoforms are generated by an alternative splicing between exons 10 and 15. Six different forms have been identified for p73 (α, β, γ, δ, ε and ζ)12-14 and three for p63 (α, β and γ)7 (fig. 2A,B). The p63α and p73α isoforms contain a sterile alpha motif (SAM) domain, usually implicated in development.15 This structural feature has no equivalent in p53.

Figure 2. Structure of mammalian p63 and p73 isoforms.

Figure 2

Structure of mammalian p63 and p73 isoforms. The proteins are represented with an invariant, DNA-binding domain, that combines with variable N- (TA or Δ) and C-terminal (greek letters) segments generated by alternative splicing and/or by the use (more...)

The N-terminal isoforms show differences in the transactivation domain. They are generated either by alternative splicing or by the use of an internal promoter in intron 3 (P2). These N-truncated forms lack the main transactivation domain and are termed “Δ” forms, as opposed to “TA”, transactivation-competent forms. However, Δ forms may retain some transcriptional capacity through secondary domains located in the C-terminus or residual N-terminus.16,17 Several types of Δ forms are distinguished according to their exact divergence with TA counterparts. For p73, Δ2, Δ3, Δ2/3 arise by alternative splicing of, respectively, exons 2, 3, or both. ΔNp73 lacks exons 1, 2 and 3 and is generated from the internal promoter in intron 3 (P2), resulting in the use of an initiation codon located in an alternative exon 3'.18 ΔN'p73 corresponds to a protein identical to ΔNp73, but translated from a mRNA initiated at the first promoter and incorporating the 3' portion of the alternative exon 3'.19 For p63, only ΔN isoforms, initiated at the P2 promoter, have been identified so far.7 However, a different, transcription-competent N-terminal variant, TA*p63, has been characterized in the mouse but not (yet) in other species.7 This variant contains 39 additional amino acids resulting from the use of an in-frame AUG located upstream of the major initiation site. This particular AUG is not conserved in human TP63.

The Proline-Rich Domain in the p53 Family

The N-terminal part of the p53 protein contains a proline-rich region suspected to bind a number of cellular proteins (fig. 3). Human p53 contains five repeats of PXXP motif (P = proline, X = any amino acid) between amino acids 61 and 94, whereas human TAp63 and TAp73 contain only two repeats (between amino acids 60 and 130 and 80 and 120, respectively). The PXXP motifs are known to form a left-handed polyproline type II helix, which creates a binding site for the SH3 domains present in many signal transduction proteins. Such motifs play a role in p53-mediated signal transduction.20 In particular, the murine p53 polyproline domain, which contains two PXXP motifs, has been shown to be a docking site in the transmission of Gas1-dependent anti-proliferative signals21 and to be required to activate apoptosis, but not growth arrest.22 Although the biological role of the proline-rich domains of p63 and p73 has not been demonstrated experimentally, it is tempting to speculate that they serve as protein-binding sites in p63 or p73-mediated signal transduction.

Figure 3. Comparison between the proline-rich domains of p53, p63 and p73 from various species.

Figure 3

Comparison between the proline-rich domains of p53, p63 and p73 from various species. The amino acid sequence of the N-terminal domain of p53, p63 and p73 from selected species were aligned using Clustalw program. Proline residues are colored in blue; (more...)

Phylogeny of the TP53 Family

During the late eighties, the TP53 gene was identified in all vertebrate species tested, but not in invertebrates or lower organisms, such as yeast or Drosophila. In the early nineties, genes showing homologies with mammalian TP53 were isolated from mollusks (Loligo forbesi, Mya arenaria).23 The later discovery of TP63 and TP73 led to realize that the TP53-like genes of mollusks were, in fact, very similar to TP63/TP73, in particular with the presence of a SAM domain. The current view on the phylogeny of the family is that all three members derive from a common ancestor, which may correspond to the gene identified in mollusks (fig. 4). This unique ancestor probably underwent duplication in chordates. The earliest duplication resulted in the specialization into two related genes that have evolved nearly simultaneously, TP63 and TP73. TP53, in contrast, may have appeared later through a distinct duplication event that implied the loss of exons coding for the SAM domain. As a result of this evolution, the three family members are present only in vertebrates. It seems counter-intuitive that TP53, which is the most recently evolved gene, is much less complex than TP63 and TP73. This paradox is also observed at the level of protein functions. The p53 protein exerts multiple functions in stress response and DNA repair, that are more similar to the ones of the unique, prototypic gene product present in invertebrates, than to the complex, development-related functions of p63 and p73.

Figure 4. Phylogeny of TP53, TP63 and TP73.

Figure 4

Phylogeny of TP53, TP63 and TP73. A simplified view of the family's phylogeny is proposed, based on Clustalw alignment. Although sequence alignment suggests that TP63 might be the most ancestral gene, there is evidence that the functions of the gene present (more...)

Functions of p63 and p73 Isoforms

Lessons from Knock-Out Mouse Models

Inactivation of TP53 by homologous recombination in the mouse is developmentally viable, although 25% of female embryos develop exencephaly and die in utero. After birth, TP53-/- mice show normal growth, but exhibit a dramatic susceptibility to early tumors and die before one year of age.9,24

Mice lacking all p63 isoforms reach term, but die within one day, owing to desiccation and maternal neglect (fig. 5).25,26 They show cranofacial abnormalities, absence or truncation of the limbs and absence of an epidermis. Products of epidermal-mesenchymal interactions, such as hair follicles, teeth and mammary glands, are absent. All squamous epithelia show a lack of stratification and mature into a monolayer of epithelial cells that lack the typical squamous differentiation markers. Impaired squamous development results in abnormalities in many organs including skin, tongue, esophagus, cervix and bladder. This defect is interpreted as the consequence of a fundamental role of p63 in regulating the asymmetric division of precursor cells in squamous epithelia (see below). Heterozygous, TP63+/- mice are viable and do not show any major developmental defects, or increased susceptibility to tumorigenesis.

Figure 5. Phenotype of TP63 and TP73 knock-out mice.

Figure 5

Phenotype of TP63 and TP73 knock-out mice. TP63 and TP73 knock-out mice are shown, next to their wild-type counterpart. TP63 knock-out mice (upper panel) exhibit dramatic defects in craniofacial and epidermal development and die within the first day of (more...)

The TP73-/- mice have a complex phenotype characterized by hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation, gastro-intestinal hemorrhages, as well as abnormalities in the pheromone sensory pathway (fig.5).27TP73 deficient males lack both a sexual interest in females and aggressiveness in response to other males, implying an altered hormonal or sensory pathway. TP73 deficient females do not get pregnant when mated with wild-type males, indicating a defect in conceiving or maintaining embryos. All this data clearly indicates that p73 plays an important role in the development of the brain and many epithelial tissues. However, p73 does not act as a tumor suppressor in mouse, since after 15 months of follow up, these mice do not develop spontaneous tumors.

Inherited TP63 Mutations in Humans

In humans, TP63 maps to chromosome 3q27, a region involved in several malformation syndromes, such as EEC (Ectrodactily Ectodermal dysplasia and facial Clefting), SHFM (Split-Hand/split-Foot Malformation), Hay-Wells syndrome, LMS (Limb Mammary Syndrome), ADULT syndrome (Acro-Dermato-Ungual-Lacrimal-Tooth), CLEPD1 (cleft lip/palate ectodermal dysplasia).28 Genetic analysis reveals that these syndromes, all characterized by limb development and/or ectodermal dysplasia, actually result from heterozygous missense or frameshift mutations in TP63.

The clinical features of these syndromes clearly correlate with the phenotype of the TP63 knock-out mice, therefore implicating p63 in ectodermal development. Interestingly, the mutations associated with the different syndromes cluster in different domains of the p63 protein, resulting in a genotype-phenotype correlation as illustrated in Figure 6.29

Figure 6. Clustering of TP63 mutations found in human developmental disorders.

Figure 6

Clustering of TP63 mutations found in human developmental disorders. The domain structure of p63 is shown and the regions where germline mutations cluster are indicated by boxes, corresponding to different syndromes. EEC, Ectrodactily Ectodermal dysplasia (more...)

There is no known human syndrome associated to loss of TP73 or inheritance of a TP73 mutation. The chromosomal region that contains TP73 on 1p36 is the centre of somatic allelic loss in many cancers,30 but there is no evidence that the TP73 locus is the specific target of these losses.31-33 In the case of TP53, inherited missense mutations have been linked to a rare autosomal dominant syndrome, Li-Fraumeni Syndrome (LFS), characterized by high susceptibility to multiple tumors at an early age, including tumors of the adrenal gland, brain, breast, as well as sarcomas.34 Although the molecular basis of this tissue-specificity is not well understood, this phenotype is consistent with the impairment of the tumor suppressive properties of p53.

A comparison between the location of “hot spot” TP53 mutations in human cancer and of TP63 mutations in malformation syndromes reveals that these mutations often target the same positions (fig. 7). This striking concordance has a structural basis, as illustrated in Figure 8, which shows the position of these residues in the crystal structure of the DNA-binding domain of the p53 protein. This domain consists in a sandwich of two beta-sheets that support several flexible loops and alpha helices. Common TP53 mutations in cancer and TP63 mutations in malformation syndromes affect residues in direct contact with DNA (amino acid 248, 273 in p53), as well as residues involved in the folding of the molecule (amino acid 175, 249 in p53). Thus, the folding of the two proteins may depend on the same, conserved amino acids, providing a rationale for the existence of concordant “hot spot” mutations that abrogate sequence-specific DNA-binding. It is quite surprising, therefore, to observe that these mutations have opposite biological effects. While TP53 mutation results in tumorigenesis due to loss of suppressor function (and perhaps gain of promoting function, see chapter “TP53 mutations in human cancers: selection versus mutagenesis”), mutation of TP63 impairs morphogenesis and results in the under-development of specific organs and tissues.

Figure 7. Comparison of ”hot spot” Mutation Codons in p53 (from tumors) and in p63 (from Developmental Disorders).

Figure 7

Comparison of ”hot spot” Mutation Codons in p53 (from tumors) and in p63 (from Developmental Disorders). A portion of the p53 DNA-binding domain (156-293), containing most of the common mutation hot spots, is aligned with the corresponding (more...)

Figure 8. ”Hot spot” codons on the three-dimensional structure of p53.

Figure 8

”Hot spot” codons on the three-dimensional structure of p53. Two views of the 3-D structure of the central region of p53 in contact with DNA. Zinc atom: orange. Yellow: residues most conserved between p53, p63 and p73. The position corresponding (more...)

Expression of p63 and p73

The expression of the p63 and p73 isoforms is tissue and cell-specific. The distribution pattern of TA and ΔN isoforms depends on the differentiation status of cells.35-37 In squamous epithelia, ΔN isoforms of p63 are mainly expressed in proliferating cells of the basal layers, whereas TA isoforms are preferentially detected in differentiated cells of the upper layers (fig. 9). This distribution is consistent with the notion that p63 plays an active role in generating the “molecular gradient” that drives the maturation of squamous epithelia. The switch from ΔN to TA isoforms is thought to result from differential use of P1 (TA forms) and P2 (ΔN forms) promoters. However, the nature and regulation of the transcription factors that control these promoters remain to be analyzed.

Figure 9. Expression of TA and ΔNp63 isoforms in normal squamous epithelium.

Figure 9

Expression of TA and ΔNp63 isoforms in normal squamous epithelium. Transversal representation of a squamous epithelium. The expression of ΔNp63 isoform is restricted to basal and suprabasal layers (including proliferating stem cells). (more...)

The p73 protein plays a role in the development and survival of neuronal cells. In developing neurons, p73 is constitutively present as a truncated isoform whose levels are dramatically decreased when sympathetic neurons undergo apoptosis after withdrawal of nerve growth factor (NGF). Overexpression of the ΔNp73α protein protects these neurons from apoptosis induced by NGF withdrawal as well as by overexpression of p53. These results indicate that p73 expression is essential for the survival of neuronal cells.38

Regulation of p63 and p73 Stability

In the case of p53, post-translational stabilization through escape from proteasome degradation is the major mechanism for regulating protein level. There is evidence that p63 and p73 are also regulated through proteasome-dependent degradation. However, the effectors of this regulation are not clearly identified. It has been reported that p53 could trigger ΔNp63 degradation by the proteasome, suggesting that there are cross-talks between the degradation pathways of the various family members.39

Degradation of p53 by the proteasome involves two main cellular effectors, Mdm2 (and the parent protein Mdmx) and the inactive form of the Jun-N (amino)-terminal kinase (JNK).40,41 JNK, in its inactive form, is thought to bind in the proline-rich domain of p53 and to phosphorylate a threonine residue at position 81.42 It is interesting to note that the proline-rich domains of p63 and p73 both contain a conserved Threonine (see fig.3), raising the possibility that a form of regulation by JNK may also affect these proteins. However, there is no evidence so far that inactive JNK binds p63 or p73.

The inter-relations between Mdm2-Mdmx and p63/p73 are better documented but the data remain controversial. Protein interactions between p63 and Mdm2 or Mdmx, and their resulting effects on transactivation and protein degradation, are yet to be demonstrated.43-45 In contrast, Mdm2 or Mdmx have been shown to interact with p73, resulting in an inhibition of transactivation but not in protein degradation. Quite the opposite, there is evidence that binding of Mdm2 increases the half-life of both proteins (p73 and Mdm2), thus creating a positive feedback loop in which p73 enhances Mdm2 accumulation.46-50 In turn, accumulation of Mdm2 triggers a reduction of p53 levels, suggesting a role for high levels of p73 in controlling p53 protein levels.46 However, the molecular effectors that control the destabilization of p73 are not yet identified.

Common and Specific Target Genes of p63 and p73

The structural similarity of the DNA binding domain of p63, p73 and p53 led several groups to investigate the capacity of p63 and p73 to regulate the same target genes as p53. The TA isoforms of p63 and p73 were found to transactivate some p53-target genes, such as WAF1/CIP1, MDM2, GADD45 and PIG3, but with a lower efficiency.16,51,52 This suggests that TA isoforms of p63 or p73 could compensate for p53 deficiency. However, this capacity may just illustrate the fact that all three family members have inherited from their common ancestor a capacity to induce the expression of genes involved in cell cycle arrest, DNA repair or apoptosis. In fact, many of the genes identified so far as “p53-targets” after in vitro overexpression of the protein, might be physiological targets of p63 or p73.

The new target REDD1 (regulated in development and DNA damage responses, also known as RTP801), recently isolated by Ellisen and collaborators, provides an example of the difficulty in identifying which transcription factor is physiologically responsible of expression control.53 Initially identified as a “p53-target”, REDD1 was found to have a developmentally-regulated expression pattern that overlaps with the known localization of p63 expression. The same gene is up-regulated in adult cells in response to ionizing radiation in a p53-dependent manner. Ectopic expression of REDD1 enhances intracellular levels of reactive oxygen species (ROS) and modulates the sensitivity of cells to oxidative stress-induced apoptosis. Furthermore, in p63-/- fibroblasts, there is a marked decrease in ROS levels, suggesting that p63 plays a physiological role in maintaining the normal intracellular balance of reactive oxygen species. Modification of ROS levels may be important in the signaling of differentiation as well as of the cellular responses to genotoxic agents. It is therefore interesting that the same molecular mechanism can be triggered in distinct circumstances by either p63 or p53.

Recent studies have reported that p63 and p73 are able to specifically transactivate promoters of some genes involved in the terminal differentiation of epidermal cells. For example, p63 and p73 specifically regulate the expression of IVL (Involucrin) and LOR (Loricrin).54 So far, there is no evidence that these promoters contain a responsive element that binds p53. Thus, the mechanism by which these two promoters are transactivated remains to be identified.

Regulation of p63 and p73 in Response to Stress

As described above, p63 and p73 are able to transactivate many of the reported p53-target genes, the products of which are involved in cell cycle arrest or apoptosis. The p53 protein responds to a broad range of genotoxic or nongenotoxic forms of stress, through complex post-translational modifications that have been extensively described.55 For p63, however, very little is known regarding the possible regulation in response to stress. Upon DNA damage, ΔN isoforms are down-regulated, whereas TA isoforms are up-regulated.56, 57 Using a phosphatase inhibitor, Okada and collaborators have obtained evidence that accumulation of p63 in response to DNA-damage requires phosphorylation at at Ser/Thr residues.58

Regarding p73, up-regulation has been observed in response to various DNA-damaging agents, with inconsistencies, however, that may result from heterogeneity in the cellular models, the dose and the nature of DNA damage. In response to cisplatin and ionizing radiation, p73 is regulated by the nonreceptor tyrosine kinase c-abl. This direct protein interaction occurs through the binding of the SH3 domain of c-abl to the PXXP motif of p73, and results in the phophorylation by c-abl at Tyrosine 99 of p73.59-61 This interaction requires intact ATM function to phosphorylate and activate c-abl. Recently, it was reported that the interaction of p73 with c-abl also induces the phosphorylation of threonine residues which are adjacent to proline, and that the p38 MAP kinase pathway mediates this response.62 Other documented post-translational changes of p73 include acetylation by p300, which potentiates the pro-apoptotic functions of p73.63 Sumoylation, another post-translational modification that affects p53,64 has been described for p73a, but not for β, in response to stress.65 However, the biological significance of this post-translational modification is unclear.

The p73 protein has been reported to mediate apoptosis in a p53-independent manner, in response to nongenotoxic stimuli. Oncogenic signaling by E2F-1 directly up-regulates TP73 expression, leading to the activation of p53-target genes and to apoptosis.66-68 Moreover, ΔNp73 isoforms have recently been shown to inactivate the RB tumor suppressor gene.69 Thus, p73 activation by deregulated E2F-1 activity might constitute a p53-independent, anti-tumorigenic safeguard mechanism. To add further complexity to these interactions, Flores and collaborators reported that p63 and p73 are required for p53-dependent apoptosis, in response to ectopic overexpression of oncogenes (e.g., adenovirus early region 1A: E1A) in combination with genotoxic agents.70 These results suggest that there might be two classes of p53-family target genes: a first class of genes regulated by p53 alone, in the absence of p63 or p73 (P21WAF1, MDM2) and a second class, for which genetic and biochemical data indicate that p63 or p73 are required for p53 to be recruited and to function properly (PERP, BAX and NOXA). The different pathways involving p53 and p73 in response to genotoxic or nongenotoxic stimuli are summarized in Figure 10.

Figure 10. Pathways involving p73 in response to stress signals.

Figure 10

Pathways involving p73 in response to stress signals. Upon genotoxic stress, p73 is post-translationally modified by acetylations or phosphorylations, and induces apoptosis in a p53-independent manner. In this respect, p73 can work in parallel with p53. (more...)

Involvement of TP63 and TP73 in Cancer Development

TP63 Amplification

TP63 is rarely mutated in cancer. However, fluorescent in situ hybridization (FISH) analysis of the TP63 locus revealed frequent amplification in primary squamous cell carcinoma of the lung, head and neck, and esophagus.71,72,73 This amplification was initially identified by Sidransky and his collaborators, who named this locus AIS (amplified in squamous cell carcinoma).71 The protein expressed from the amplified locus, p40AIS, lacks the transactivation domain and is equivalent to a ΔN isoform. These results are consistent with the notion that overexpression of a ΔN isoform of p63 can counteract the growth suppressive effects of TA forms of p63, p73 or p53, therefore promoting tumor development as an oncogene. In keeping with this hypothesis, overexpression of p40 (AIS) in Rat1a cells led to an increase in soft agar growth and tumor size in mice.71

TP73 Deregulation

The TP73 locus (1p36) is frequently targeted by loss of alleles in many cancers, including in particular neuroblastoma. However, mutation analysis of the remaining TP73 allele in neuroblastoma did not reveal frequent somatic mutations, indicating that TP73 is not a key tumor suppressor in these cancers.32 In several other cancers, gene silencing by hypermethylation has been reported (inflammatory breast cancers;74 oligodendroglial tumors;75 leukemias and lymphomas76,77). In contrast, overexpression of the p73 protein has also been reported in several types of cancer (lung;78,79 gastric;80 breast;81 bladder82). Tannapfel and collaborators have reported an inverse correlation between overexpression of p73 and mutation of TP53 in liver cancer, suggesting a compensatory role.83 It is important to note that the studies listed above have not determined the relative levels of expression of ΔN versus TA isoforms of p73. Since ΔNp73 has the potential to behave as an oncogene, it is possible that differential expression of ΔN versus TA isoforms may account for the apparently contradictory observations reported above.

Inactivation by Protein Interactions

Inactivation of p63 and/or p73 by interaction with mutant p53 has been recently put forward as one of the mechanisms by which mutant p53 may exert a dominant, gain-of-function effect in cancer development. Most mutations in TP53 are missense and affect residues of the DNA-binding domain, inhibiting sequence-specific DNA-binding. However, all mutations are not equivalent in their functional effects and at least some mutants have acquired the capacity to exert dominant effects. This new property is thought to result from specific changes in protein conformation induced by the mutation. There is evidence that a conformationally altered p53 DNA-binding domain can form stable complexes with p73 and p63. This interaction is mediated by the binding of the central domain of mutant p53 to the oligomerization domain of p63 or p73.84-86 Recent functional studies indicate that binding of mutant p53 down-regulates transactivation by p63 and p73.85,86 Whether this mechanism contributes to explain the persistence and accumulation of mutant p53 protein in cancer cells, remains to be elucidated. In future studies, it will be important to determine whether tumors may contain different types of TP53 mutations depending upon their TP63 or TP73 expression status.

Conclusion

The TP53 gene family provides a good illustration of how genes with very specialized functions may have evolved from a common blueprint. The three members show obvious family traits in their structure, architecture and basic biochemistry. They however have completely different lifestyles and functions, due to the fact that their modes of regulation are extremely specialized. A closer look at the family suggests that the most divergent members, in terms of function, are TP53 and TP63. Whereas TP53 is entirely specialized in stress response, TP63 plays its roles in development and differentiation. The functional divergence between these two genes is underlined by the consequence of their germline mutations (predisposing to cancer for TP53; inducing malformations for TP63) and of their somatic alterations in cancer (inactivating mutations for TP53, activating amplifications for TP63). TP73 occupies a somewhat intermediate position between the two “extremist” brothers. Although it plays basic roles in several aspects of normal differentiation (in particular in epithelial and neuronal cells), it has been implicated in response to genotoxic agents that also activate p53.

It is very likely that the current picture of the role of TP63 and TP73 in cancer is, at best, very fragmentary. Further studies will be necessary to uncover the full extent of their contribution to carcinogenesis. In this respect, a very important characteristic of these two genes is their expression as both TA and ΔN isoforms, which confers to p63 and p73 the capacity to exert opposite effects on transcriptional regulation, depending on which type of isoform may predominate in the cell. As discussed above, TA isoforms exert primarily suppressive effects, whereas ΔN isoforms can counteract this suppression and therefore hold the potential to behave as oncogenes. Deregulation of the balance between the levels of the two types of isoforms should therefore be considered as a potential mechanism of carcinogenesis (fig. 11), the demonstration of which awaits the development of knock out mice specifically lacking either TA or ΔN isoforms. Since the three family members may control a common subset of essential genes involved in growth regulation, overexpression of either ΔNp63 or ΔNp73 may block the suppressive effects of not only its TA counterpart, but also of other family members, including p53. The existence of amplifications of TP63, with concomitant overexpression of a ΔN-like protein in some squamous cancers, provides evidence that this mechanism may work in a subset of epithelial tumors. It is likely that, in cancer cells, other mechanisms than amplification may contribute to upset the balance between levels of TA and ΔN isoforms. These mechanisms may include differential promoter regulation, transcript stability, protein nuclear accumulation, and protein degradation. Further studies need to be developed to address these possibilities.

Figure 11. Dominant negative effects of ΔN isoforms on TA isoforms.

Figure 11

Dominant negative effects of ΔN isoforms on TA isoforms. Upper panel) TA isoforms (TAp63 or TAp73) bind p53-specific DNA sequences (RE for Responsive element) and recruit the transcription machinery. Middle panel) ΔN isoforms could exert (more...)

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