DNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, DNp63a protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of DNp63a overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of DNp63a in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca 2 þ ], abrogates Ca 2 þ -induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that DNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. DNp63a blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, DNp63a enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal DNp63a-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of DNp63a appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support DNp63a as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

The p53 homologue #Np63A is overexpressed and inhibits apoptosis in a subset of human squamous cell carcinomas (SCC). Here, we report that in normal keratinocytes overexpressing #Np63A and in human squamous carcinoma cells, #Np63A physically associates with phosphorylated, transcriptionally active nuclear c-Rel, a nuclear factor-KB family member, resulting in increased c-Rel nuclear accumulation. This accumulation and the associated enhanced proliferation driven by elevated #Np63A are attenuated by c-Rel small interfering RNA or overexpression of mutant IKBAM, indicating that c-Rel-containing complex formation is critical to the ability of elevated #Np63A to maintain proliferation in the presence of growth arresting signals. Consistent with a role in growth regulation, #Np63A-c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human squamous carcinoma cells and normal keratinocytes overexpressing #Np63A. The relationship between #Np63A and activated c-Rel is reflected in their strong nuclear staining in the proliferating compartment of primary head and neck SCC. This is the first report indicating that high levels of #Np63A interact with activated c-Rel in keratinocytes and SCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers.

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and DeltaN. DeltaNp63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, DeltaN- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. DeltaNp63alpha declines with Ca2+-induced differentiation, while a smaller DeltaN-form, DeltaNp63s, persists, suggesting unique functions of the two DeltaN-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, DeltaNp63alpha, DeltaNp63p40, TAp63alpha, TAp63gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of DeltaNp63p40, which lacks the entire alpha-tail present in DeltaNp63alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length DeltaNp63alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of DeltaNp63alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the DeltaN domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The DeltaN-terminus is sufficient to maintain keratinocytes in a proliferative state, as both DeltaN forms block Ca2+-mediated p21WAF1 induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63alpha or TAp63gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

The p63 gene is a member of the p53/p63/p73 family of transcription factors and plays a critical role in development and homeostasis of squamous epithelium. p63 is transcribed as multiple isoforms; ΔNp63α, the predominant p63 isoform in stratified squamous epithelium, is localized to the basal cells and is overexpressed in squamous cell cancers of multiple organ sites, including skin, head and neck, and lung. Further, p63 is considered a stem cell marker, and within the epidermis, ΔNp63α directs lineage commitment. ΔNp63α has been implicated in numerous processes of skin biology that impact normal epidermal homeostasis and can contribute to squamous cancer pathogenesis by supporting proliferation and survival with roles in blocking terminal differentiation, apoptosis, and senescence, and influencing adhesion and migration. ΔNp63α overexpression may also influence the tissue microenvironment through remodeling of the extracellular matrix and vasculature, as well as by enhancing cytokine and chemokine secretion to recruit pro-inflammatory infiltrate. This review focuses on the role of ΔNp63α in normal epidermal biology and how dysregulation can contribute to cutaneous squamous cancer development, drawing from knowledge also gained by squamous cancers from other organ sites that share p63 overexpression as a defining feature.