br Introduction The development of
Introduction The development of non-melanoma skin cancer (NMSC) is a complex process characterized by the acquisition of altered proliferation and invasiveness. These changes are classically defined as occurring in stages, i.e., initiation, promotion, and progression. In the two-stage model of initiation and promotion in mouse skin, initiation is due to DNA mutations, conferred by low dose carcinogen, while promotion involves increased proliferation and inflammation. A high dose of carcinogen, including exposure to ultraviolet light (UV) alone can accomplish both initiation and promotion. One of the most common features of exposure to chemical carcinogens or UV is the induction of inflammation, which is characterized by the production and release of cytokines, growth factors and arachidonic sirtuin 1 metabolites, particularly the prostaglandins (PGs) (Rundhaug and Fischer, 2010). The enzymes responsible for the production of PGs are referred to as the PGH synthases, of which there are two isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Although both forms exhibit similar COX activity with regard to arachidonic acid metabolism, they are differentially regulated such that COX-1 is normally constitutively expressed in murine epidermis, while COX-2, which is usually expressed at only very low levels, is highly inducible by a large number of irritating agents, including the tumor promoter 12-tetradecanoylphorbol-13-acetate (TPA) (Fischer et al., 2007, Maldve and Fischer, 1996, Rundhaug and Fischer, 2010). Both pharmacological and genetic approaches have shown that PGs are critical for NMSC development. Non-steroidal anti-inflammatory drugs that target both COX-1 and COX-2, as well as selective COX-2 inhibitors, have strong chemopreventive activity against either chemical- or UV-induced skin carcinogenesis (Fischer et al., 1999, Muller-Decker, 2011, Tober et al., 2006). The role of PGs from COX-2 in NMSC was further verified using mice in which one or more alleles of COX-2 were knocked out (Rundhaug et al., 2007, Tiano et al., 2002). The clear demonstration that the induction of COX-2 and its primary product PGE2 are involved in NMSC raised questions concerning the mechanisms by which PGE2 promotes the growth of NMSC. PGE2 binds to and activates four G-protein-coupled E receptors, referred to as EP1, EP2, EP3 and EP4. Each EP receptor is linked to Gα subunits, such that activation of the different receptors leads to activation of different signal transduction pathways (Narumiya, 2009, Sugimoto and Narumiya, 2007). This led to investigations identifying which receptor is responsible for the tumor promoting action of PGE2. Studies on the EP1 receptor, which activates phospholipase C and subsequently protein kinase C, the target of the tumor promoter TPA, showed that mice over-expressing the EP1 receptor in the epidermis developed very aggressive squamous cell carcinomas (SCC) within a few weeks after treatment with 7,12-dimethylbenz[a]anthracene (DMBA) (Surh et al., 2012). Over-expression of the EP2 receptor, which is linked to the cAMP-protein kinase A pathway (Sugimoto and Narumiya, 2007), significantly increased the number of both papillomas and SCCs after two-stage DMBA-TPA treatment (Sung et al., 2006). Conversely, knockout of the EP2 receptor reduced skin tumor numbers by 50% (Sung et al., 2005). Knockout of the EP3 receptor, of which there are multiple variants that inhibit cAMP production (Namba et al., 1993), had no effect on two-stage DMBA-TPA skin carcinogenesis (Sung et al., 2005). Like the EP2 receptor, EP4 activates adenylate cyclase, but also has been reported to activate the PI3K-Akt pathway as well (Fujino et al., 2003). However, the contribution of the EP4 receptor to skin carcinogenesis has not been previously studied. Here we describe the effect of over-expressing the EP4 receptor, under control of a keratin 5 promoter, on both two-stage DMBA-TPA and single treatment DMBA- or UV-induced skin tumor development. With all protocols, the number of SCCs was significantly higher in BK5.EP4 compared to wild type (WT) mice, which had very few tumors, suggesting that elevated EP4 levels confer endogenous tumor promoting, and especially progression, activity. A comparison of transcriptomes between WT and BK5.EP4 mice treated with PGE2 and BK5.EP4 mice treated with either EtOH or PGE2 was carried out. Ingenuity Pathway Analysis (IPA) revealed differentially expressed transcripts to be part of several networks that include keratinocyte differentiation- and inflammation-associated genes, e.g., many keratins, Csf2rb, Coro2A, GPX4 and IL-20, which further supports a pro-tumorigenic role for the EP4 receptor.