The long-term goal of this project is to understand the mechanism of the coordinated relationship between keratinocyte differentiation, and carcinogenesis in the epidermis. We recently demonstrated that IkappaB kinase alpha (IKKalpha) plays a central role in regulation of keratinocyte terminal differentiation via a novel signaling pathway independent of nuclear factor-kappaB (NF-kappaB). Mice lacking IKKalpha fail to develop the late differentiating granular and cornified cell layers in the epidermis, resulting in a dramatic increase in the thickness of the epidermis. Our preliminary results show that the immortalized IKKalpha-/- keratinocytes are able to form cell colonies in soft agar and grow into poorly differentiated spindle cell carcinomas in nude mice following subcutaneous injection. We found that the immortalized IKKalpha-/- keratinocytes carry a p53 mutation, which frequently occurs in human squamous cell carcinomas. We further found that levels of wild-type p53 and 14-3-3sigma, a p53-regulated G2/M checkpoint, are down-regulated in cultured primary IKKalpha-/- keratinocytes. Based on our findings, we hypothesize that the down-regulated negative cell cycle regulator of 14-3-3sigma may allow IKKalpha-/- keratinocytes to continuously proliferate, and the reduced G2/M checkpoint may also make keratinocytes susceptible to gene mutations. Therefore, loss of IKKa not only interferes with keratinocyte differentiation but also permits or causes neoplastic transformation. To determine the mechanism of IKKalpha-related skin carcinogenesis, we plan to: 1. Determine the mechanism of the neoplastic transformation in IKKalpha-/- keratinocytes. We plan to study how IKKalpha regulates the G2/M checkpoint through 14-3-3sigma and how IKKa regulates activities of 14-3-3sigma and p53. We will determine the effect of 14-3-3sigma on IKKa-related keratinocyte differentiation, proliferation and tumor progression. Finally we will test whether p53 mutations frequently occur in immortalized IKKalpha-/- keratinocytes. 2. Determine the stage of skin carcinogenesis at which IKKalpha is involved and analyze whether IKKalpha-related tumorigenesis is associated with p53 and 14-3-3sigma. Because IKKalpha-/- mice die soon after birth, we plan to generate epidermal-specific IKKa conditional knockout mice. We will determine whether epidermal-specific IKKalpha-/- mice develop spontaneous skin tumors. We will also use these conditional knockout mice to determine the impact of IKKa loss at specific stages of skin carcinogenesis: tumor initiation, or tumor promotion, and/or tumor progression, and to define the relationship between alterations in p53 and 14-3-3sigma and tumor development associated with loss of IKKalpha.
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