Fase de transición

p63 is part of the p53 family and was first discovered by Yang et al. (1998) to be expressed in the basal epithelial cells in the epidermis of the breast, skin, cervix, urothelium and prostate, amongst others. Initial studies into p63 generated a lot of confusion due to seemingly conflicting roles of p63 in various cellular processes, including cancer. It has since been discovered that p63 is able to play conflicting roles in a variety of cancers due to its many different isoforms (Su et al., 2013).

1.4.2. Structure & location

p63 is encoded by the Tp63 gene which is located on chromosome 3q27-28. Like all the members of the p53 family, the p63 isoforms have the same core structures in common: a transactivation domain, a DNA binding domain and oligomerization domain, and are able to form tetramers via their oligomerization domains to enable their stability (Figure 1.11) (Yang et al., 1998).

p63 is found in many different isoforms as a result of alternative splicing and alternative promoters. Two different promoters give rise to two main subgroups of p63: ΔNp63 and TAp63. The TAp63 subgroup contains a transactivation domain, a DNA binding domain, and oligomerization domain. The ΔNp63 subgroup lacks the transactivation domain present in the other members of the family (Murray-Zmijewski et al., 2006).

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Figure 1.11. Structure of p63 isoforms. (A) The human p63 gene structure encoded by 16 exons with

alternative splicing promoter sites (P1 and P2) giving rise to TA and ΔN isoforms and alternative splicing sites giving rise to splice variants. (B) The two subgroups of p63; TAp63 and ΔNp63 containing DNA binding domain (DBD), oligomerisation domain (OD), a second transactivation domain (TA2), sterile alpha motif (SAM), and a post-inhibitory domain (PID). Figure adapted from Murray-Zmijewski et al. (2006).

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TAp63 and ΔNp63 have very different functions, with TAp63 playing a predominantly tumour suppressive role and ΔNp63 playing an oncogenic role (Su et al., 2013). Moreover ΔNp63 can actually antagonize TAp63, p73 and p53 and block their activity (Yang et al., 1998; Liefer et al., 2000; Ratovitski et al., 2001; Rocco et al., 2006; Marcel et al., 2012). In addition to the two subgroups, further subcategories are created as a result of alternative splicing on the 3’ end which can generate α, β, γ, δ and ε isoforms. All of these isoforms contain the DNA binding domain and the oligomerization domain (Yang et al., 1998; Mangiulli et al., 2009). In addition, the α and β forms contain a second transactivation domain and the α forms contain a sterile alpha motif and a post-inhibitory domain, which respectively are required for protein-protein interactions and to mask the transactivation domain of TAp63α preventing its activation (Thanos & Bowie, 1999; Straub et al., 2010).

p63 is predominantly a nuclear protein, and thus is expressed in the nucleus in the basal cells of the epidermis and also in a variety of cancers, including squamous cell carcinomas (Di Como et al., 2002). Cytoplasmic p63, however, has also been detected in certain cancers, for example, in melanoma and prostate cancer (Dhillon et al., 2009; Matin et al., 2013).

1.4.3. Function

1.4.3.1. Embryonic tissue

p63 null mice are unable to survive long after birth due to severe dehydration and display truncated limbs and deformed craniofacial structures (Mills et al., 1999; Yang et al., 1999). One of the key functions of the skin is to control fluid loss from the body. The skin of these mice, however, was unable to differentiate leaving it in an unstratified state. Although initial studies using p63 null mice had reported similar phenotypes, their explanation as to the role of p63 in the developmental process of the skin differed and still remains a controversial topic (Mills et al., 1999; Yang et al., 1999). Subsequent studies have been performed to try to understand this difference.

Candi et al., (2006) created p63 null mice expressing either ΔNp63α or TAp63α to investigate the potentially diverse roles of the two promoter variants. From these data, and previous data from one of the initial mouse models, it was found that mice expressing ΔNp63α were able to develop a basal layer of skin while mice expressing TAp63α were unable to form the skin. ΔNp63 was expressed in the basal layers and was able to activate early differentiation

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markers and TAp63 was expressed in the suprabasal layers and activated late stage markers (Figure 1.12). These data, and data from Laurikkala et al. (2006), suggest that ΔNp63α is key to the development and proliferation of the epidermis and that only subsequent TAp63α expression allows differentiation of the cell.

These data have, however, been criticsed by Koster et al. (2007) due to the inability of the researchers to recover the normal phenotype when expressing both ΔNp63α and TAp63α. Koster et al. (2004) had previously claimed their model demonstrated that the expression of TAp63 was the initial key stage of epidermal development. They showed that expression of only TAp63 in single layered lung epithelia was sufficient to initiate stratification and that overexpression of TAp63 prevented the differentiation of the cells. At present this is still a debated topic (Koster et al., 2007; Candi et al., 2008).

Figure 1.12. p63 and the development of the skin. ΔNp63 is expressed in the basal layers of the skin

and is involved in the proliferation of the basal cells. TAp63 is expressed later and aids the expression of proteins involved in differentiation. Figure adapted from Candi et al. (2006).

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In adult tissue, ΔNp63 is the predominant isoform expressed in the mature epidermis and ensures its maintenance (Parsa et al., 1999). ΔNp63 is found in the basal cells of the adult epidermis and loses expression as the cells differentiate towards the surface. In cancer it is also ΔNp63 which behaves as an oncogene. In addition to cancer, p63 has been found to play a role in cellular senescence and ageing. Mice p63+/- _{display signs of accelerated ageing e.g.,}

skin lesions, alopecia and severe degenerative disc disease of the spine and thus live for a shorter period of time (Flores et al., 2005; Keyes et al., 2005).

Further investigation into this phenomenon showed evidence for the involvement of both isoforms. TAp63 conditional knockout mice age prematurely, developing both blisters and skin ulcerations and also give rise to senescence of hair follicle-associated dermal and epidermal cells (Su et al., 2009). Supporting the notion of opposing roles ΔNp63α overexpression resulted in premature ageing correlating with decreased expression of Sirt1, a protein known to promote longevity in mice (Sommer et al., 2006).