Frequent clones of p53-mutated keratinocytes in normal human skin

Abstract

The multiple genetic hit model of cancer predicts that normal individuals should have stable populations of cancer-prone, but noncancerous, mutant cells awaiting further genetic hits. We report that whole-mount preparations of human skin contain clonal patches of p53-mutated keratinocytes, arising from the dermal-epidermal junction and from hair follicles. These clones, 60-3000 cells in size, are present at frequencies exceeding 40 cells per cm 2 and together involve as much as 4% of the epidermis. In sun-exposed skin, clones are both more frequent and larger than in sun-shielded skin. We conclude that, in addition to being a tumorigenic mutagen, sunlight acts as a tumor promoter by favoring the clonal expansion of p53-mutated cells. These combined actions of sunlight result in normal individuals carrying a substantial burden of keratinocytes predisposed to cancer.Although skin cancers typically arise in patients aged 50-70, epidemiologic evidence indicates that much of the critical sunlight exposure is received before the age of 18 (1, 2). This early role of sunlight is supported by the finding of sunlightinduced mutations in the p53 tumor suppressor gene in actinic keratosis, the precancerous lesion for squamous cell carcinoma of the skin. In addition, p53-mutated cells are present in skin flanking human tumors and in UV-irradiated mouse skin (3-8). Mutations at particular p53 codons are present in sun-exposed normal human skin at frequencies of 10 Ϫ6 to 10 Ϫ2 (5, 9). Other human tumors for which mutation of p53 appears to be an early event include head and neck cancer and hepatocellular carcinoma (10, 11).Because keratinocytes are continuously lost through squamous differentiation and desquamation, it seems likely that the cell targeted by sunlight decades before a tumor's appearance is a stem cell. If so, the keratinocytes containing the mutations measured in normal skin would not be randomly dispersed but instead would reside in clonal patches arising from mutated stem cells. The frequency of p53 mutations measured in a biopsy of normal skin would then depend on whether the biopsy included a clone. The spatial arrangement of the cells in the clone might give clues to the geometry of early carcinogenesis, including the site of the stem cells from which skin tumors originate in humans.We therefore devised a whole-mount preparation method for human epidermis that permitted immunohistochemical analysis for stabilized p53 protein. A p53 mutation usually leads to nuclear immunopositivity (12). A large enough sample of skin might contain rare patches staining intensely for p53. We report here that such patches are not only present and contain mutations but are also frequent, indicating the existence of a large population of cells in normal skin that are presumably predisposed to skin cancer. In addition, we present evidence that sunlight can act both as a tumor initiator and as a tumor promoter for p53-mutated cells. MATERIALS AND METHODSEpidermal Whole Mounts. Fresh skin samples were obtained from discard...

Induction of basal cell carcinomas and trichoepitheliomas in mice overexpressing GLI-1

“…BCCs are unusual in that they are apparently genetically stable and no precursor lesion has been identified. In humans, p53 mutations occur very frequently in BCCs as well as in patches of morphologically normal skin, especially in UV-exposed areas (32,45). On the basis of these observations, it has been suggested that p53 mutations are an early and required event in BCC development.…”

Stochastic fate ofp53-mutant epidermal progenitor cells is tilted toward proliferation by UV B during preneoplasia

“…UVB induces a characteristic type of mutation (a C-to-T transition at a dipyrimidine site) (3). These signature mutations are commonly found in the p53 gene in sun-exposed (yet apparently normal) skin, sun-damaged epidermis, premalignant lesions known as actinic keratoses, and in squamous carcinomas, consistent with p53 mutation being an early event in the clonal evolution of UVB-induced SCC (4)(5)(6)(7). Keratinocytes lacking functional p53 have a greatly increased risk of malignant transformation from UVB irradiation, suggesting that the size of the p53 mutant population in epidermis will determine cancer risk (8).…”

Induction of KLF4 in basal keratinocytes blocks the proliferation–differentiation switch and initiates squamous epithelial dysplasia

“…The established role of p53 in the genesis of SCC suggested that deficiency of this tumor suppressor could promote the KLF4-induced phenotype (Boyle et al, 1993;Kemp et al, 1993;Ziegler et al, 1994;Jonason et al, 1996;Brash and Ponten, 1998;Jonkers et al, 2001). We identified settings in which p53 deficiency was important or essential for KLF4-induced dysplasia (Figures 5 and 6).…”