Role of COX-2 in UVB-induced beta-catenin signaling in keratinocytes

描述(申请人提供)：在美国，每年有超过一百万例由紫外线诱导的非黑色素瘤皮肤癌的新病例被诊断出来。细胞对UVB的反应导致环氧合酶-2(COX-2)的诱导，COX-2是花生四烯酸转化为前列腺素的关键酶，COX-2的过度表达与包括皮肤癌在内的许多癌症有关。我们的实验室和其他人已经报道了UVB照射后角质形成细胞和皮肤中COX-2的表达。我们的合作研究员苏珊·费舍尔已经证明，COX-2和前列腺素的产生在小鼠皮肤癌的发生中起关键作用。-连环蛋白信号通路是参与多种组织肿瘤发生的另一个重要信号通路。最近，Castellone et al.报道称，COX-2及其代谢产物PGE2通过连环蛋白信号转导促进结肠癌细胞生长。据我们所知，COX-2和？-catenin信号之间的这种联系，其中 COX-2是由UVB辐射诱导的，在正常的表皮中尚未被证实。我们实验室已获得的初步证据表明，经UVB辐射处理的正常人表皮角质形成细胞(NHEKs)可导致信号转导形式-连环蛋白(Active-Catenin)水平升高，同时COX-2表达升高。鉴于UVB辐射诱导COX-2表达和PGE2产生，以及我们的初步证据表明PGE2治疗NHEKs导致活性-连环蛋白表达增加，我们建议检验这一新的假设，即暴露于UVB辐射导致皮肤中依赖于COX-2表达的连环蛋白信号增加，并促进角质形成细胞的不适当增殖，从而导致皮肤癌的发生。我们建议在使用原代小鼠和人类角质形成细胞的基于细胞的研究中，以及使用COX-2+/-杂合子小鼠和EP2-/-敲除小鼠的活体小鼠表皮和人类皮肤肿瘤样本来验证这一假说。此外，我们将采用一种新的经皮传递连环蛋白抑制剂ICAT的策略，以测试抑制连环蛋白信号是否减弱UVB诱导的增殖。目的研究UVB对体外培养的小鼠和人角质形成细胞β-连环蛋白信号的影响，以确定UVB对活性连环蛋白的诱导是否具有转录功能，是否依赖于COX-2的表达，以及是否为正反馈回路的一部分。目的#2研究UVB辐射对连环蛋白信号转导的影响以及随后对体内表皮增殖的影响。这一目标还将确定UVB诱导的小鼠皮肤中的连环蛋白信号是否依赖于体内COX-2的表达，以及体内是否存在连环蛋白和COX-2之间的正反馈回路。目的#3研究β-连环蛋白信号转导通路在UVB诱导的皮肤癌中的作用，并确定抑制β-连环蛋白信号转导通路是否可以预防UVB诱导的肿瘤。证实表皮中存在UVB/COX-2/β-连环素轴，并确定其在皮肤癌发生中的作用，将在正常皮肤生物学和皮肤癌领域产生重大影响。

DESCRIPTION (provided by applicant): Over one million new cases of UVB-induced non-melanoma skin cancer are diagnosed yearly in the US. Cellular response to UVB results in induction of cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandins, and overexpression of COX-2 is implicated in many forms of cancer, including skin cancer. Our laboratory and others have reported that COX-2 expression is induced in keratinocytes and skin following UVB exposure. COX-2 and prostaglandin production have been shown by our Co-investigator Susan Fischer to be critical for murine skin carcinogenesis. The ¿-catenin signaling cascade is another important signaling pathway involved in tumorigenesis in a variety of tissues. Recently, Castellone et al. reported that COX-2 and its metabolite PGE2 promoted the growth of colon cancer cells by signaling through ¿-catenin. To the best of our knowledge, this link between COX-2 and ¿-catenin signaling, where COX-2 is induced by UVB exposure, has yet to be identified in normal epidermis. Our laboratory has obtained preliminary evidence that treatment of normal human epidermal keratinocytes (NHEKs) with UVB radiation resulted in increased levels of the signaling form of ¿-catenin (active ¿-catenin), concurrent with elevated COX- 2 expression. Given that UVB radiation induces COX-2 expression and PGE2 production, and our preliminary evidence that PGE2 treatment of NHEKs results in increased expression of active ¿-catenin, we propose to test the novel hypothesis that exposure to UVB radiation results in increased ¿-catenin signaling in the skin, which is dependent on COX-2 expression, and contributes to inappropriate proliferation of keratinocytes, leading to skin carcinogenesis. We propose to test this hypothesis in cell-based studies using primary mouse and human keratinocytes, as well as in mouse epidermis in vivo using COX-2+/- heterozygote mice and EP2-/- knockout mice and in human skin tumor samples. Furthermore, we will employ a novel strategy for transdermal delivery of the ¿-catenin inhibitor, ICAT, to test whether inhibition of ¿-catenin signaling attenuates UVB-induced hyperplasia. Aim #1 will investigate the effect of UVB on ¿-catenin signaling in cultured mouse and human keratinocytes to determine if UVB-induction of active ¿-catenin is transcriptionally functional, is dependent on COX-2 expression, and is part of a positive feedback loop. Aim #2 will investigate the effect of UVB radiation on ¿-catenin signaling and subsequent effects on epidermal proliferation in vivo. This Aim will also establish whether UVB-induced ¿-catenin signaling in mouse skin is dependent on COX-2 expression in vivo and whether a positive feedback loop between ¿-catenin and COX-2 exists in vivo. Aim #3 will investigate the involvement of ¿-catenin signaling in UVB-induced skin cancer and determine if inhibition of ¿-catenin signaling can prevent UVB-induced tumors. Demonstrating the existence of a "UVB/COX-2/¿-catenin axis" in epidermis and identifying its contribution to skin carcinogenesis would have significant impact in the fields of normal skin biology and skin cancer.