p63 is a suppressor of tumorigenesis and metastasis interacting with mutant p53

Facts

• p63 containing the transactivation domain (TAp63) and amino-deleted p63 isoforms (ΔNp63) exert distinct (often opposite) functions on stemness, cycle arrest, mobility and invasion (epithelial–mesenchymal transition, EMT) and senescence.

• TAp63 induces cell death and cell cycle arrest with tumor-suppressor features.

• ΔNp63 exerts oncogenic properties and is generally overexpressed in cancer.

• TAp63 and ΔNp63 (and their ratio) regulates chemosensitivity.

• Mutant p53 binds to, and inhibits the activity of TAp63.

• p63 (mainly the TA isoform) suppresses metastasis formation by decreasing mobility and invasion.

Open Questions

• Which isoform is responsible for each distinct function: stemness, cycle arrest, mobility and invasion (EMT), senescence?

• In vivo identification of the molecular targets and determinants for each function.

• Relative control, and potential therapeutic manipulation, of the relative expression of TAp63 and ΔNp63 in individual cancers. How are they related to diagnosis and prognosis V体育ios版.

p63 is a member of the p53 family, which also includes p73. The birth of p63 was a dystocial delivery VSports最新版本. Although the gene was formally described in 1998,1 it was originally isolated from rat tissues as ket in 19972 and the human sequence was reported in 1998 by different groups who variously referred to the encoded protein as p40,3 p63,1 p73L4 or p51A. 5 Fortunately, this confusion of names was soon clarified by adopting the p63 classification, following a paper that provided the correct context to understand its function. 1 The identification of p63, as well as that of p73 a year earlier, was unexpected as it came after 20 years of intensive studies on p53,6, 7 and also because its physiological role is mainly developmental rather than tumor suppressive, despite its striking amino acid identity with both p53 and p73. 8 For recent reviews, see refs 9,10,11,12 and 13.

Like other members of the p53 family, the TP63 gene is expressed as multiple isoforms with distinct properties, including a full length and an amino-deleted isoform, named TAp63 and ΔNp63, respectively14, 15 (Figure 1). TAp63 contains a transcription domain and can induce cell cycle arrest and apoptosis,16 linking this protein to the DNA damage response function that is commonly associated with the p53 family V体育平台登录. At the physiological level, TAp63 seems to be expressed predominantly in oocytes, although it has been detected at low levels in other tissues including the epidermis, especially following stress, and functions to protect them from toxic insults;17, 18 consequently, TAp63 has been called the ‘guardian of the female germline'. 9 Conversely, ΔNp63, the shorter isoform without the N-terminal TA that is transcribed from a separate second promoter, is expressed primarily in the epidermis, and is involved in epithelial development. 19, 20 Indeed, the full knockout of p63 is lethal owing to the absence of the epidermis,19, 20 suggesting that the prime developmental role of ΔNp63 is in the formation of the epidermis and its appendages, such as hairs and sebaceous glands. 21, 22, 23 This epithelial role by p63, and particularly the underlying molecular mechanisms, has been the subject of animated yet still partially unresolved debate throughout the last decade.

Several major issues are responsible for these controversies. First, as TP63 is expressed from two distinct promoters, each being able to produce at least five alternative 3′ splicing isoforms, it remains unclear which isoform is responsible for each specific phenotype. Second, it is becoming clear that p63 regulates an impressive array of genes V体育官网入口. 24, 25 A recent, whole-genome tiled array analysis of ΔNp63 target genes revealed that nearly 5800 gene promoters were directly bound by endogenous p63 in human cells, of which approximately 1000 showed expression changes in response to p63 expression. 26 These target genes include, for example, 200 transcription factors, a large number of adhesion molecules, and a functionally diverse set of signaling molecules. Thus, p63 may be directly affecting nearly 7% of the coding genes in the genome, suggesting highly complex interactions with a large number of pathways. In addition, p63 regulates the expression of a number of non-coding regulatory RNAs such as micro-RNAs (miRs), as well as Dicer,27 an enzyme essential for miR processing. The presence of so many isoforms, Figure 1, with distinct and extremely powerful transactivation properties, makes the conclusion about function and underlying mechanisms of p63 difficult to elucidate. Third, it is becoming clear that the roles of p63 in development and in adulthood, particularly in adult cancers, are quite distinct. In particular, while the major developmental role seems to be epithelial, the role of p63 in cancer only becomes evident in adulthood.

The aim of this review is therefore to set this new evidence, occasionally with my personal views, that links p63 to tumorigenesis and metastasis within the context of its long conserved other functions.

p63 and ‘La Famiglia' p53

p63 is the most recently discovered but the most ancient member of the p53 family8, 9, 10, 11, 12, 13, 14, 15, 28 (Figure 1). As indicated above, p63 is transcribed from two promoters, giving rise to proteins that may (TAp63 isoforms) or may not (ΔNp63 isoforms) contain the TA,1 and alternative splicing at the 3′-end produces additional proteins (α–ɛ isoforms),29 although there is no in vivo evidence for two latter isoforms. Whereas the TAp63 proteins are capable of transactivation, the ΔNp63 forms can also act in a dominant-negative fashion to counteract the transcriptional activity of the TAp63 isoforms and p53. 8, 15 In addition, a comprehensive and systematic investigation of the transcriptional and growth inhibition activities of all the p63 isoforms identified a novel activation domain – TA2 – within exons 11 and 12 and a transcription repression (TI) function encoded in the C-terminus of the longest TAp63α isoform. The importance of this regulatory domain is also evident from the identification of mutations in this TAp63α-C-terminus in human patients. 30 Further investigation of the mechanism of inhibition suggested that the TI domain binds to the N-terminal TA and inhibits the transcriptional activity by masking important amino acids. The active molecule is a tetramer, like p53 and p73. The ability of different isoforms to tetramerize, not only within the p63 members, but also with p53 or p73, drastically expands the complexity of the system V体育2025版. 31, 32, 33 p63 protein is targeted for degradation by the E3 ligase ITCH, which belongs to the HECT family. 34 In addition to regulating p63, ITCH also targets p73, JUN, JUNB, Notch and FLIP (also known as CFLAR) for degradation. 35 This predicts not only a drastic effect of ITCH inhibitors, but predicts that ITCH may be an important therapeutic target in cancer.

In marked contrast to p53, TP63 is not a classical tumor-suppressor gene, but is instead important in embryonic development. The phenotype of patients with a p63 mutation and that of Trp63^−/− mice has revealed a key role for p63 in development, particularly of epithelia, and these phenotypes are summarized in Table 1. Mice devoid of both copies of Trp63 are born lacking limbs as well as skin and its appendages, such as hair shafts, follicles and sebaceous glands,^{19, 20, 36, 44} see later; in zebrafish also ΔNp63 is required for epidermal development.⁴⁶ Trp63^−/− animals also lack tooth primordia, mammary tissues, prostate, bladder, eyelids and a wide range of other tissues. All of these defects, described in several mouse models (Table 1), have been observed in ectodermal dysplasia patients carrying heterozygous (dominant) p63 mutations.³⁰ Most commonly, human p63 mutations (basically localized in every domain, but particularly in the DNA-binding domain (DBD), similar to the distribution of mutations described in p53) found in ectodermal dysplasia syndrome patients give rise either to amino acid substitutions in the DBD that abolish p63 DNA-binding ability, or to shifts of the reading frame, which specifically truncate the TAp63α isoform.^{13, 30} However, recent data have shown that p63 also has an important role in the development and progression of cancers.

p63 Expression in Cancer

p63 has added a new dimension to the role of the p53 family in cancer biology owing to its effects in the DNA damage response and in the regulation of cell cycle arrest,^{1, 47, 48} apoptosis,^{16, 47, 49} stemness⁵⁰ and tumorigenesis.^{39, 51, 52, 53, 54, 55} p63 has been detected in several cancers, including pre-cancerous conditions of the oral mucosa,⁵⁶ and cancers of the prostate,^{57, 58, 59} bladder,^{60, 61, 62, 63} thyroid,⁶⁴ lung,^{65, 66, 67, 68, 69, 70} cervix^{71, 72} and breast,^{73, 74, 75, 76} see Table 2. However, the detailed mechanisms of its role in tumorigenesis remain partially obscure at the molecular level and particularly the relative contribution of the distinct pathways (functions) exerted by p63.⁸¹

Mutations of p63 are extremely rare in human cancers, indicating it is not a canonical tumor suppressor. Most tumors (>80% of primary head and neck squamous cell carcinomas (HNSCCs), as well as other squamous cell epithelial malignancies and non-small cell lung cancer) retain p63 expression, where it is often overexpressed and occasionally amplified.^{78, 80, 82, 83} (Table 2) Frequently, tumors have simultaneous transcriptional upregulation of both TAp63 and ΔNp63 isoforms, with ΔNp63 being predominant at the protein level.^{78, 80, 82} This would be in accordance with the anti-apoptotic and proliferative (oncogenic) effects of the ΔNp63 isoform described above. Moreover, ΔNp63α expression directly correlates with a poor clinical response to cisplatin in HNSCC.⁷⁹ DNA damage by chemotherapy in HNSCC causes a decrease in ΔNp63-mediated transcriptional repression by blocking responsive elements sites to TAp63 or sequestering TAp63 in less active hetero-tetramers, together with increased expression of p73, and allows TAp73-mediated cell death.⁸⁴ Together, these data suggest that it is not necessarily the levels of individual p53 family members, but rather the ratio between TA (transcriptionally highly active; showing tumor-suppressor functions) and ΔN (transcriptionally less active, and acting as dominant negative over the TA isoforms; showing oncogenic properties) isoforms that determines the biological outcome. Clearly, however, although this may be true for HNSCC and other squamous cell epithelial tumors, this cannot be the whole story in cancers in general as malignant lymphomas seem to preferentially express TAp63.^{77, 85}

Do Trp63 genetically modified mice develop cancer? The analysis of Trp63^−/− mice has led to conflicting results with regard to its role in tumorigenesis. Although some Trp63^+/− mice are cancer-prone,⁵¹ this appears to depend on other factors like for example the genetic background, as Trp63^+/− mice of a different inbred strain show premature aging but no cancer,⁸⁶ even when p53 is compromised.⁸⁷ A note of caution is required when using Brdm2 mice,⁸⁶ because exon skipping could occasionally result in wanton expression of the p63γ isoform, resulting in a partial knockout.⁴⁵ Although these conflicting results appear difficult to reconcile, the ratio between the different p63 isoforms (and also other p53 family members) may in theory cause strain-specific differences. In this respect, p63 and p73 are required for full p53 transactivation of apoptotic transcriptional targets such as Bax but not for cell cycle-related targets such as p21, thus suggesting that the interplay between members of the family may have a big impact on tumorigenesis.^{39, 49} The strain-specific differences also suggests that genes outside the p53 family may also influence the behavior of p53 family members, adding further complexity to the story. Clearly, however, more work is needed to understand how the different genetic background could influence tumorigenesis.

p63 is a Suppressor of Metastasis

More aggressive, metastatic tumors lose p63 expression, suggesting that p63 loss accelerates tumorigenesis and metastatic spread^{61, 88} (Figure 2). Correspondingly, disruption of p63 in squamous cell lines results in upregulation of genes associated with increased invasiveness and metastasis in tumors,⁶² see also below. This suggests that p63 is a marker of non-invasive epithelial tumors,⁷² such as ductal carcinoma in situ of the breast or prostatic intraepithelial neoplasia. Indeed, sclerosing adenosis or small foci of dense fibrosis with distortion of the normal acinar architecture, remain p63 positive. This highlights the potential value of p63 as a differential diagnostic marker of tumors with more benign properties.

The fact that a majority of cancers harbor p53 mutations does not necessarily mean that p53 is absent or inactive because p53 mutants may gain new properties not present in the wild-type protein.^{92, 93, 94} Recently, a novel mechanism has been reported whereby mutant p53 (e.g., 175H; 273H), independently of its function in the response to DNA damage, physically interacts with p63, resulting in the loss of the anti-metastatic properties of p63^{89, 90} (Figure 2). Distinct mechanisms of mutant p53 (175H; 273H)–p63 interaction have been described. First, transforming growth factor-β (TGFβ)-dependent expression of SMAD2 creates a platform for p53R175H – phosphorylated by NRAS – to interact with p63, creating a ternary complex (of SMAD2-p53R174H-p63).⁹⁰ This results in reduced expression of two downstream metastasis-suppressor genes, BHLHE41 (which encodes SHARP1) and cyclin G2 (CCNG2), and patients with low expression of these two genes are at higher risk of metastasis and show reduced survival.⁹⁰

In the second mechanism, in H1299 human cells lacking TGFβ receptors, a direct interaction between mutant p53 (175H; 273H) and p63 seems to occur, resulting in inhibition of p63-mediated regulation of integrin α5β1 and epidermal growth factor receptor endocytosis cycling.^{12, 89} It is unclear whether this effect is common to all p53 mutants or confined to a particular subset, but this would be important information to refine the clinical predictive value of p53 genotyping.

The molecular interaction between mutant p53 and p63 involves both the formation of hetero-aggregates between the two proteins, and the tetramerization via the oligomerization domain domains to form large aggregates.⁹⁵ This suggests an increased propensity of the conformationally mis-structured mutant p53 to aggregate both as homotetramers (p53) and heterotetramers (p63), resulting in a gain of function, Figure 3.

A further way by which TAp63 may influence metastasis is by the direct transactivation of DICER1 and miR-130B, two genes that also inhibit metastasis.²⁷ Whether this is also inhibited by mutant p53 is currently unknown, but would be expected because mutant p53 seems to repress p63 transcription^{89, 90} and also DICER1/miR-130B are dependent on p63 transcription to affect metastasis.²⁷ DICER1 expression is inhibited by miR-103 and miR-107, thus also conferring migratory properties and empowering metastatic dissemination of cancer cells by inducing an EMT,⁹¹ a signature that is important in metastasis formation,⁹⁶ see also the section below on ‘Epithelial Organizer'.

Which of these mechanisms is pivotal as a suppressor of metastasis? And how does this reconcile with the notion that TAp63 acts as a tumor suppressor while ΔNp63 as an oncogene? Possibly these latter effects are more important in tumorigenesis and expansion of the primary, while a migratory effect (mediated by any of the above mechanisms) is involved in invasion and metastasis. These new data,^{27, 89, 90} while revealing a crucial role for p63 as a metastasis suppressor involving an adhesion program, also emphasizes that it is the expression and interaction of the different isoforms of all p53 family members (p53, mutant p53, TAp63, ΔNp63) that contributes to the tumor phenotype. It also suggests new factors that may be used both as predictors of tumor behavior and as novel therapeutic targets.

Clinical Implications

The involvement of p63 in tumorigenesis and metastasis has evident clinical implications.

V体育官网入口 - Possible Molecular Mechanisms Involving p63 Function in Cancer

Several mechanisms have been proposed for p63 functions that may have a crucial role in cancer (Figures 5 and 6). However, we have to remember that, since much of the p63 research has been performed in epithelial models, there could be similarities, but also differences when applied in a tumor context.

What is the function of p63 in the epidermis? See Figure 7. The first controversy surrounds which isoform determines the lack of skin development in Trp63^−/− mice: TAp63^{40, 41} versus ΔNp63.¹⁷ However, work by Thesleff,²¹ Melino²² and Khavari²³ has clearly shown that ΔNp63 is the major (99% of total Trp63 mRNA, as shown by in situ hybridization and qRT-PCR) isoform involved in epidermal development. This has also been shown using an embryonic stem cell model in vitro.¹⁰⁹ A contribution by TAp63 remains possible, as the message is indeed expressed during various stages of development as well as in adulthood, and TAp63^−/− mice have a detectable epithelial phenotype with blisters and epidermal ulcers.³⁷ Moreover, in a different animal model, TAp63 contributes to the maintenance of dermal and epidermal precursors, genomic stability, and organismal longevity;^{28, 37} mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis.³⁷ A further controversy has emerged, assigning either a stem cell regulatory role or a differentiation role to p63. Work by McKeon⁵⁰ shows that ΔNp63 is required for the maintenance of ‘stemness' of stem cells within all stratified epithelia, which includes skin, breast and prostate, among others, and that the phenotype (absence of stratified epithelial) is the consequence of tissue failure owing to depletion of the stem cell compartment. Before its role in adult stem cell regulation, ΔNp63 is essential for the initiation of epithelial commitment and the stratification cycle of the embryonic ectoderm.¹¹⁰ At this embryonic stage, ΔNp63 switches on an array of genes required for epidermal morphogenesis while repressing those involved in mesodermal lineage commitment.¹¹⁰

What is the function of p63 in cancer? In addition to the roles indicated for the epidermis, different hypotheses have been proposed (Figures 5 and 6), and particularly the idea that ΔNp63 maintains not only the normal epithelial stem cell population, but may also be involved in the persistence of cancer stem cells, especially in epithelial tumors.

Here, I will briefly review the different possibilities.

Conclusions and Perspectives

The ancestor of the p53 family is a p63-like protein that first appears in unicellular flagellates,¹²⁵ and radiation into clear p53, p63 and p73 proteins only occurs with the emergence of vertebrates.¹²⁵ In organisms such as the precursors of sea anemones, worms and flies, the principal role of this p63 precursor is the preservation of germline genomic integrity, a function that has clearly been conserved through at least 1 billion years of evolutionary time.⁹ As the family diversified, so did its functions, with the N-terminally truncated isoform of ΔNp63 having a role in maintenance of the stem cell pool, and potentially, I have argued, in the perpetuation of cancer stem cells.

Although our understanding of the role of p63 isoforms, and in particular of TAp63, remains preliminary, and of their influence on chemosensitivity by TAp63 even more so, it is likely that chemosensitivity may be determined not only by p53, but also by p73- and p63-function, and dominant-negative isoforms could have a crucial role in this scenario. The potential synergistic effects of different p53 family members in the response to chemotherapeutic drugs should be taken into consideration for the development of future anticancer strategies.

The clinical problem of human cancer is metastasis. There is now evidence that, perhaps as a reflection of its role in epithelial development and in stemness, p63 as an ‘epithelial organizer' is also important in EMT, an essential prerequisite for invasion and migration of tumor cells.¹²⁶ The present day complexity of the p53 family, with multiple isoforms now recognized for each member, allows the possibility of a whole web of inter-family interactions. This results in a highly plastic and subtle modulation of the activity of an individual isoform, and we are now beginning to see, for example, in the interaction between mutant p53 and p63, how this may influence the pathology of cancer.

Here, we highlight the important role for TAp63 and ΔNp63 in tumorigenesis and metastasis, although the underlying molecular mechanisms still require further clarification. Specifically, the issue of the differential effects of the splicing isoforms is basically untouched. Clearly, the identification of specific interactors, acting as modulators, of transcriptional targets, including miRs, and of the degradation mechanisms are still too preliminary to allow a consistent movement to develop drugs that target p63 in cancer.

The author declares no conflict of interest.