p63 – an Important Player in Epidermal and Tumour Development

Summary

p63 is a transcription factor which plays an important role in epidermal development, diffe­rentiation and tumourigenesis. p63 belongs to the p53 protein family and at least six isoforms were identified to date. p63 isoforms play contrary roles during the development and formation of the epidermis as well as in cancer. p63 participates in epithelial development, where it affects proliferation and differentiation of epidermal cells. Inherited mutations in the TP63 gene generate different developmental defects and p63 knockout in mice results in the absence of epidermis. Another important role of p63 is the control of cell-cell adhesion, where it regulates desmosomes. The loss of proliferation and cell-cell adhesion control are important for tumourigenesis and overexpression of p63 can enhance tumour growth and inhibit apoptosis. This review briefly summarises the roles of p63 in epithelial development, cellular proliferation, adhesion and migration and reveals its share in tumourigenesis and metastasis.

Key words:
p63 – cell development – cell proliferation – cell adhesion – tumourigenesis – epidermis

Introduction

The epidermis is a peripheral skin layer that provides the human body with a natural physical protection from dehydration and pathogens. It is composed of proliferating basal and differentiated suprabasal keratinocytes [1]. The self-renewing ability of the epidermis requires an appropriate proliferation and differentiation program in the basal layer [2]. Basal stem cells proliferate to produce daughter transit amplifying cells whose proliferative capacity is more limited compared with basal stem cells. After the set of cellular divisions, TA cells migrate through the epidermis layers and begin the process of differentiation towards the formation of external skin components [3]. Recent studies have established an important develop­mental role for p63 in stratified tissue formation. p63 regulates transcription of multiple genes which encode transcription factors, adhesion and signalling molecules, proteins involved in the cell cycle, apoptosis and also tissue specific proteins such as keratins, involucrin and loricrin [4]. The importance of p63 in skin development was mainly supported by observations in knockout mice, that after birth lacked multilayered epithelia and any skin appendages, such as teeth, hair follicles and mammary glands which indicates that p63 defines the stem cell compartment for this tissue [5]. In this article we would like to summarise the role of p63 in the development of epidermis and demonstrate the link between p63 expression and tumourigenesis.

p63 Isoforms and Their Transactivation Properties

p63 protein is one of the most significant transcription factors engaged in the growth of keratinocytes and skin development (Fig. 1) [6,7]. Protein p63 belongs to the p53 family [8,9] and like the other family members it contains three typical domains: amino-terminal transactivation domain (TA), DNA-binding domain and carboxy-terminal oligomerisation domain (OD) (Fig. 2) [8]. p63 is encoded by the TP63 gene. The expression of TP63 is directed from two distinct promoters, resulting in protein iso­forms either containing (TA) or lacking (∆N) the N-terminal transactivation domain [8]. Furthermore, these transcripts can then be subjected to alternative splicing to generate at least three diffe­rent C-terminal isoforms termed α, β and γ [10] and newly described δ and ε isoforms [11]. Only the α isoforms possess a sterile alpha motif (SAM) – a protein-protein interaction domain – and a transcription inhibitory domain (TI) (Fig. 2) [12,13]. ∆Np63 isoforms are recognised as a dominant negative inhibitor of TAp63 isoforms and other p53 family members [8]. Despite the fact that ∆Np63 lacks the transactivation domain prevalent in TA isoforms, it can demonstrate transactivation through the transactivation domain present in its N-terminal end [14].

The Role of p63 in Proliferation and Differentiation Processes in Epidermal Cells

Though TAp63 isoforms appear earlier than ∆Np63 during embryogenesis, ∆Np63 is much more abundant in the embryo [15,16]. Transgenic mice with overexpressed TAp63α in the epidermis revealed hyperplasia and inhibited diffe­rentiation [15] indicating that the correct development of epidermis needs an adequate balance between TA and ∆N isoform levels.

p63 with its‘ transactivation and inhibition capacities is required for the proliferation of epidermal cells [17] and the level strictly correlates with the proliferative abilities of keratinocytes in vitro [18]. Experiments on developing zebrafish embryos with down-regulated ∆Np63 displayed a total loss of epidermal proliferation. Later studies in p63 knock­down epidermis showed that most of the cells ceased in G1 phase followed by an additional decrease of cells in S and G2/M phase. This phenomenon was accompanied by a reduced level of the proliferation marker Ki67 and by increased levels of cell cycle inhibitors such as WAF1 or p16. These findings thus clearly indicate that p63 knockdown cells are growth-arrested [17].

Besides playing roles in cellular proliferation processes, p63 performs an important function in the regulation of differentiation. Epidermis created from pan-p63 knockdown keratinocytes showed defects in stratification and differentiation and the expression of characteristic differentiation markers was not observed in the absence of p63. On the contrary, the loss of p63 led to the expression of markers of simple epithelium (keratins 8 and 18) which normally are not present in stratified tissues [17]. A proposed model for p63 function in the differentiation process is based on the correct balance between protein levels of p63 isoforms [15]. In the basal layers of the epidermis, ∆Np63 is the predominantly expressed isoform, but its abundance is reduced in the suprabasal layer of stratified epithelium. In contrast, increased levels of TAp63 can be observed in the upper parts of the epithelium inhibiting terminal differentiation [8,15]. While ∆Np63 seems to play a major role in stratification as well as in differentiation of epithelium, TAp63 appears to be important in its’ late differentiation [17].

Recent studies revealed that differentiation of epidermal cells begins through asymmetric cell division in the basal layer [19]. This process was not observed in p63-null keratinocytes. In addition, p63 activity has been related to many genes significant in differentiation such as Notch, inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) [20] or keratin 14 which are only expressed in basal cells within stratified epithelia [21]. IKKα plays an important role in the proliferation and differentiation process and mice with IKKα deletion displayed incorrect proliferation and differentiation of skin cells. p63 isoforms can directly transactivate IKKα by attaching to the p53-like sequence on its promoter or via transcription factor protein C-ets1 (Ets-1) [20,22].

Notch signalling is a major pathway that fosters stem cells to begin the process of differentiation towards keratinocytes [23,24]. One of the ligands for Notch receptor is jagged 1 protein (JAG-1), which was found to be a p63 target [25]. Notch activation can suppress p63 expression in keratinocytes while permanent p63 function inhibits the ability of Notch receptor to induce cell cycle arrest. Moreover, promoting cell cycle arrest by Notch signalling involves induc­tion of WAF1 protein, which can be controlled by p63 [24].

The differentiation program is based on the cooperation between specific sets of genes being part of the epidermal differentiation complex (EDC) within mouse chromosome 3 and encoding components of the cornified layer [26]. Chromatin architecture within the tissue-specific EDC locus can be remodelled by special AT-rich sequence-bind­ing protein-1 (Satb1). Satb1 functions as a genome organiser, directing chromatin-remodelling enzymes and trans­cription factors and therefore playing an important role in the regulation of tissue-specific gene expression programs [27,28]. It was shown that Satb1 stimulates the differentiation of progenitor cells in the basal layer towards keratinocytes and can be directly regulated by p63 [26]. Satb1 is co-expressed with p63 in basal cells during embryonic and postnatal development and mice that have lost expression of p63 exhibited severely decreased levels of Satb1 and also loricrin (a marker of differen­tiated keratinocytes). Additionally, Satb1 knockout mice showed diminished epidermal cell proliferation and their skin appeared to be much thinner in comparison with wild-type Satb1 mice [29].

p63 and Skin Appendages Development

Skin appendages which include teeth, hairs and glands are derived from ectodermal and mesodermal tissues [30]. The first step of skin appendage formation is very similar for all types, when the placodes start to form a bud. However, the following events differ among them and depend on the kind of skin derivative [30,31]. Genes which control the development of skin derivatives are highly conserved across species [31]. The essential role of p63 in skin appendage develop­ment was observed in the p63-knock­out mice where animals lacking p63 expression died perinatally and demonstrated a dramatic phenotype. Their epithelium stayed single-layered and lacked all the appendages such as hair follicles, teeth, whiskers and glands [5]. Analogically, mutations in p63 are related to such phenotype in humans as ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome (EEC), limb-mammary syndrome (LMS), ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC), acro-dermato-ungual-lacrimal-tooth syndrome (ADULT) or Rapp-Hodgkin syndrome (RHS) [32]. When developing hair, teeth and vibrissae during embryogenesis, p63 is initially expressed through­out the epithelium. With the progressive development the level of p63 decreases within the inner layers [16,33]. At the beginning, as well as during the whole process of ectoderm development, only ∆N iso­forms were detected, whereas the expression of TA isoforms was observed at later stages [16]. Research in zebrafish revealed ∆Np63α to be the most abundantly expressed isoform during embryonic development [34] and mutations in p63-associated syndromes are mostly found within regions specific for the α iso­form [35]. These observations suggest that ∆Np63α is the essential isoform participating in the development of skin and skin appendages.

Role of p63 in Cellular Adhesion

Among other things, p63 plays an important role in the regulation of processes mediating cell-cell adhesion within the epidermis. Reduced p63 expression in cultured mammary cells caused impaired cell adhesion and decreased expression of desmosomal components [36,37]. Furthermore, p63 was stated to directly regulate the expression of membrane protein Perp – one of the most important elements for desmosome development [38]. Desmosomes are cell to cell junction protein complexes, necessary to provide skin with the strength needed to withstand mechanical stress. They attach cell surface proteins to the intermediate cytoskeletal filaments [39,40]. Beaudry et al showed that Perp-deficient mouse epithelium had a decreased number of desmosomes and was filled with blisters leading to postnatal lethality [41]. Furthermore, they revealed that Perp acts as a significant tumour suppressor in UVB-induced squamous cell carcinoma (SCC). It plays an important role as a mediator of p53-induced apoptosis. In vivo experiments performed in mice confirmed that loss of Perp protein promotes tumourigenesis and contributes to tumour progression.

Studies in squamous cell carcinoma of head and neck (SCCHN) cell lines, revealed that p63 regulates genes responsible for cellular adhesion which plays a crucial role in cell invasiveness and metastatic potential of this cancer type [42] and the loss of p63 results in increased cell migration [43].

p63 in Tumourigenesis

p63 participates in the cellular signalling processes following DNA damage by controlling cell cycle arrest and apoptosis and therefore it is also important in cancer development. Probably, it does not function as a basic tumour suppressor because it is rarely mutated in human cancers. In most cases, tumours maintain p63 expression and moreover the TP63 locus is sometimes amplified and thus p63 is overexpressed [9,44]. High level of p63 was found in more than 80% of SCCHN and other squamous epithelial malignancies [45,46]. As ∆Np63 induces proliferation it can also enhance tumour growth [47,48]. ∆Np63 binds to and suppresses p73 activity that results in the inhibition of apoptosis [45,49] and also functions as a transcriptional repressor of Bcl-2 family members, as it was found to bind to their promoters [49,50]. p63 is a key survival factor for SCC, because its inhibition by interfering RNA induces apoptosis. It is also degraded after cisplatin treatment in SCC, which seems to be an important chemotherapy response [49,51]. What is more, it was found that ∆Np63 is a novel ATM regulator, which controls p53 serine-15 phosphorylation through transcription of the ATM kinase. This research proved that, loss of ∆Np63 results in reduction of ATM-dependent phosphorylation and inversely overexpressed ∆Np63 stimulates ATM signalling. These observations suggest that ∆Np63 isoform may play a significant role in response to DNA damage [52]. Regarding TAp63 isoforms, they are expressed in some malignant lymphomas, while ∆Np63 isoform is not present [53]. However, the role of particular isoforms of p63 in cancer is still not clear and needs further investigation.

As mentioned above, p63 can regulate expression of desmosomes components. Some studies suggest that a decrease in desmosomes components occurs during cancer progression in humans and can be correlated with tumour metastasis [54,55]. Furthermore, the loss of Perp expression, a direct p63 target, can promote tumour initiation [41].

Barbieri et al showed that disruption of p63 expression in squamous cell lines led to a decrease of transcripts specific for squamous tissues and significant modifications in keratinocytes differentiation. Furthermore, it resulted in the upregulation of non-epithelial tissues markers, where many of these proteins were associated with an increased invasiveness and metastatic potential in tumour cells [43]. Nevertheless, the role of p63 in tumourigenesis is not fully elucidated to date and remains the subject of promising cancer research.

Future Perspectives

Animal models and cell culture studies indicate that p63 plays an important role in epithelial formation, especially in the control of proliferation, differen­tiation and adhesion of basal stem cells. It also plays an important role in cancer progression through cell cycle arrest and the regulation of apoptosis. TAp63 and ∆Np63 isoforms may collaborate together to maintain the program of stratification and differentiation, with ∆Np63 apparently predominant in the regeneration process. Further studies into the role of p63 are crucial for better under­standing of epidermis creation and cancer cell development.

This study was supported by grant of G rant Agency Czech Republic No. P301/10/P431 and by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101).

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers.

Jitka Holcakova, M.Sc., Ph.D.

Masaryk Memorial Cancer Institute

Regional Centre for Applied Molecular Oncology

Zluty kopec 7

656 53 Brno

Czech Republic

e-mail: holcakova@mou.cz

Submitted: 28. 9. 2012

Accepted: 24. 10. 2012