I will use our novel transgenic mouse model, Krt1- 15CrePR1;R26R, to determine the contribution of hair follicle bulge stem cells to cutaneous neoplasms in mice resulting from skin exposure to ultraviolet light. We will employ two strategies to attack this problem. First, we will determine the contribution of Krt1-15-expressing hair follicle stem cells to tumors when irradiated with ultraviolet light. We will transiently induce the Cre recombinase before irradiation. Upon induction, the Cre recombinase will recognize its loxP sites in the disrupted, ubiquitously expressed transgene for beta-galactosidase and reconstitute an intact, beta-galactosidase that can be histochemically stained blue. Prior to irradiation, the blue cells will be found only in the hair follicle bulge at the site of Krt1-15-expression. During the course of irradiation, we will determine whether the blue cells that are the progeny of the Krt1-15-expressing cells are found in the developing papillomas and carcinomas. Second, we will determine whether the blue-stained cell vs. unstained cell components of the resulting tumors bear signature mutations in the p53 gene. This approach will employ laser capture microdissection of blue-stained and unstained regions of the tumors followed by DNA isolation, PCR amplification of regions bearing possible signature mutations, and sequencing of those regions. We anticipate that keratinocyte stem cells from the hair follicle bulge will play a significant role in the pathogenesis of UVB-induced skin tumors. Based on ongoing studies using the chemically induced model of skin cancer, we expect that hair follicle stem cells may not be the only targets in skin cancer. We expect these proposed studies will lead to novel insight into the origins and pathogenesis of non-melanoma skin cancer.

We will also determine the role of bone marrow derived cells in skin cancer development. In the multistage model of cutaneous carcinogenesis, a single subtumorigenic exposure to a carcinogen (initiation) and subsequent chronic regenerative epidermal hyperplasia of sufficient magnitude (promotion) can induce benign and malignant neoplasms of the cutaneous epithelium. Tumor initiation is thought to convert selected epithelial cells into latent neoplastic cells, whereas promotion elicits expression of the neoplastic change leading to tumor formation. Although such cancers are widely believed to originate from the transformation of epidermal or hair follicle stem cells, the recent demonstration that gastric cancer originates from bone marrow-derived cells challenges this dogma. A further objective of this investigation is to determine the relative contributions of hair follicle stem cells and bone marrow derived cells (BMDC) to skin tumors. Our hypothesis is that the chronic tissue damage characteristic of skin tumor promotion recruits BMDC into ensuing papillomas and carcinomas. First, we will determine the relative contributions of BMDC and hair follicle stem cells in the formation of skin tumors. Our method of approach will be to transplant genetically labeled BMDC to lethally irradiated Krt1-15CrePR1;R26R recipients subjected to chemically-induced skin cancer and where the Cre-recombinase is induced before exposure to the carcinogen. We will then assess developing epithelial skin lesions for cells of donor origin (expressing EFGP and the Y chromosome) vs. hair follicle stem cell origin (blue X-gal staining cells) in skin sections. Second, we will determine in the Krt1-15CrePR1;R26R transgenic mice engrafted with genetically marked BMDCs whether labeled keratinocytes from the Krt1- 15-expressing stem cells from the hair follicle bulge vs. those keratinocytes derived from genetically marked BMDC express the signature A>T mutation in codon 61 of the Ha-ras gene. At intervals during skin tumor promotion, we will use Laser Capture Microdissection followed by mutation analysis to compare the tumor Krt1-15 progeny vs. BMDCs-derived keratinocytes to determine the presence of the initiated lesion. These experiments will determine the contributions of BMDCs and will have significant implications for the multistage model of skin cancer and for the development of anticancer therapies in that nonmelanoma skin cancer, and indeed other epithelial cancers, may need to be viewed as systemic rather than local diseases.

Our third new area of research is to identify specific keratinocyte stem cell regulatory genes. Keratinocyte stem cells have an unquestioned role in maintaining the normal structure and function of the epidermis and hair follicles and are thought to be important players in inherited and acquired skin disease. Hence, identification of genes regulating their number and proliferative potential is a critical problem in cutaneous biology. We propose here a novel strategy for identifying genes involved in keratinocyte stem cell regulation. This strategy takes advantage of several recent important advances made in our laboratory: 1) identification of a selectable marker of hair follicle stem cells, 2) development of a sensitive and quantitative in vitro assay for clonogenic keratinocyte stem cells, 3) genetic mapping of several loci with linkage to stem cell number, and 4) analysis of keratinocyte stem cell gene expression. The objective of the proposed research is to identify major genes regulating the number of keratinocyte stem cells. Our hypothesis is that there are specific genes and pathways that regulate the number of keratinocyte stem cells that may be different from those regulating transit-amplifying cells. We first plan to use genetic tools to refine the linkage intervals we have already identified. Next, we will use a candidate gene approach for stem cell gene identification. Finally, we will use a complementary global genomic approach to identify associated molecular pathways, and to assess regulatory polymorphisms causing differences in gene expression in the absence of coding sequence differences within the quantitative trait locus. This investigation will enable the identification of major genes and regulatory pathways in keratinocyte stem cells. This research will impact the fields of cutaneous biology and stem cell research and should provide new insight into the mechanism of skin carcinogenesis. Identification of stem cell regulatory genes is important for gene therapy as well as for the design of new therapeutic modalities for chronic hyperproliferative skin disease, for wounds that do not heal, and for skin cancer.