HACAT AS A MODEL FOR KERATINOCYTESTRANSFORMATION

Mutations of the tumor suppressor gene p53 occur in more than 50% of human malignancies and lead to the loss of suppressor activity. Moreover frequently p53 mutants gain novel, oncogenic properties by transcriptional activation of the genes, involved in cellular proliferation, cell survival and angiogenesis. Today’s challenge is to understand mechanisms underlying the gain-of-function of mutant p53 proteins. By the example of KLF4 promoter we demonstrate that mutant p53 can carries out its gain-of-function by interaction with another p53 family member, transcription factor p63. In the first report we provide strong evidence that KLF4 is negatively controlled by p63 in normal skin in the presence of physiological levels of wild type p53 (wtp53) and that this regulation is subverted by oncogenic mutations of p53, establishing a direct link between these TFs, commonly overexpressed in squamous cell carcinoma (SCC). These results inspired us to investigate the mechanisms of mutant p53 gain-of-function on the genome scale. In the second manuscript we demonstrate that mutant p53 HaCaT alleles are pro-growth and mutp53 have thousands of binding sites in the human genome; they affect gene expression profoundly, both by binding with p63 to consensus elements and by being tethered by other TFs to their locations. Although 2 mutant p53 alleles are definitely gain-of-function in the HaCaT, they are not sufficient to render these cells malignant. Based on the sphere-forming assay we developed novel model for the tumorigenic conversion of the HaCaT cells: while immortalized keratinocytes display transformed phenotype in vitro and not tumorigenic upon injection into nude mice, HaCaTderived spheres give rise to the SCC in vivo. Thus, this simple model can be 3 useful for the future studies of the genetic and phenotypic signatures during SCC initiation and development. There is an indication that mutant p53 can execute its gain-of-function via interaction with NF-Y transcription factor. Ongoing study is devoted to the investigation of NF-YA subunit role in the processes of cellular proliferation and apoptosis in the cells with different p53 status.