p63 and epithelial homeostasis : studies of p63 under normal, hyper-proliferative and malignant conditions

Sammanfattning: Background: The p63 gene is a member of the p53 transcription factor family and can produce six different proteins using two promoters and differential splicing. Expression of p63 is required for proper formation of epithelial tissues. Studies on the transcriptional control of specific genes involved in cell survival, proliferation, differentiation and adhesion have revealed the contributions of p63 to the continuously renewing stratified epithelium. In this thesis, the aim was to improve our understanding of the roles of p63 in epithelial homeostasis by investigating expression of p63 in normal and benign hyper-proliferative epithelia and exploring the influence of p63 deregulation on cancer progression. Materials and methods: Using quantitative real time RT-PCR and immunohistochemistry, we first examined the expression of different p63 isoforms in patients diagnosed with psoriasis - a benign hyper-proliferative and inflammatory skin disease. Afterwards, we investigated responses of p63 in psoriatic epidermis upon Narrowband-UVB (NB-UVB) phototherapy. At the same time, we studied the potential impact of p63 in carcinogenesis by searching for p63 transcriptional targets in a cell line derived from squamous cell carcinoma of the head and neck (SCCHN) - the sixth most common cancer worldwide with over-expression of the ∆Np63α protein as a common feature. p63 gene silencing and microarray were used to identify p63 regulated genes. Real time RT-PCR, western blot, immunohistochemistry, chromatin immunoprecipitation, transient transfection and reporter assays were performed to confirm specific genes as direct p63 targets. Results: Significant down-regulation of p63 mRNA levels was found in psoriatic lesions compared to patients’ own clinically normal skin. Moreover, a trend of decreased TAp63 mRNA levels was seen in patients’ normal skin compared to age- and sex-matched healthy controls. Following NB-UVB phototherapy, an effective first line therapy for psoriasis, expression of p63 was not significantly affected. However, significant changes in p53, FABP5, miR-21 and miR-125b were found. Surprisingly, location and expression levels of p63 proteins detected by immunohistochemistry were similar under all skin conditions. A direct transcriptional regulation of TRAF4 by p63 was seen in the SCCHN cell line and we further found that the localization of the TRAF4 protein was associated with histological differentiation of SCCHN cells. However, unlike its over-expression in SCCHN, similar TRAF4 mRNA expression levels were seen in psoriatic lesions as compared to healthy controls. Besides TRAF4, a total of 127 genes were identified as potentially p63 regulated in the SCCHN cell line and strikingly, about 20% of these genes are involved in cell adhesion or migration. Conclusions: Dysregulation of p63 isoforms in psoriatic epidermis, especially decreased TAp63 expression, and their resistance to NB-UVB phototherapy implicated a contribution of p63 to the psoriasis phenotype. Transcriptional regulation of genes involved in multiple biological pathways indicated that over-expression of p63 in SCCHN might account for altered cell differentiation, adhesion and migration, thus contributing to SCCHN. In conclusion, our studies have found additional mechanisms through which p63 guarded homeostasis of the established epithelium. Deregulation of p63 might play a role in distinct pathological conditions by participating in diverse cellular pathways under different microenvironments.