Exposure to UV instead of DMBA also
Exposure to UV instead of DMBA also produced a sloughing and angiogenic response in transgenic, but not WT, mice, indicating that the epidermis of the BK5.EP4 mice has a much lower apoptosis threshold than WT mice, as suggested by others (Chun and Langenbach, 2007). Overall, these studies suggest that a very strong regenerative response is responsible for the subsequent development of tumors. To assess whether the EP4 receptor was activating the signaling pathways reported by others (Yoshida et al., 2013), we examined the levels of cAMP, PI3K/Akt and ERK1/2 activation and the upregulation of COX-2. Although we found increased levels of cAMP in the epidermis of transgenic mice, we did not find activation of PI3K/Akt, ERK1/2 or Stat3, or the upregulation of COX-2 (Suppl. Figure 1 and data not shown). To elucidate possible mechanisms through which EP4 promotes the development and progression of skin tumors, the RNAseq approach was used to assess differences in gene expression in BK5.EP4 and WT mice, with and without PGE2 treatment. This is an unbiased approach, however, we expected to see changes in the expression of genes associated with inflammation because EP4 was reported to be involved in the activation of NFkB in keratinocytes (Soontrapa et al., 2011) and in the regulation of the proliferative and pro-inflammatory genes TNFα, PGE2 synthase and sennoside sale D1 (Fujino et al., 2003). Even though these specific genes were not found to be significantly upregulated, several gene changes associated with inflammation and tumor development were noted. Among the genes that were the most highly upregulated in EP4 transgenic mice treated with PGE2, compared to PGE2-treated WT mice (Table 1A), were A130040M12Rik and interleukin-20 (IL-20). A130040M12Rik belongs to the VL30 family of murine retroelement noncoding RNAs and has been shown to regulate proto-oncogene transcription and tumorigenesis (Wang et al., 2009a, Wang et al., 2009b). Interestingly, IL-20 transcripts were below the limits of detection in WT mice but were highly upregulated in the BK5.EP4 mice. IL-20 has been shown to play a role in skin inflammation, along with several related cytokines (Uto-Konomi et al., 2012). The regenerative hyperplasia seen in psoriasis also has been linked to IL-20 (Wang et al., 2012, Wegenka, 2010); this may be relevant to the robust regenerative response of BK5.EP4 mice to DMBA-induced cell death, which in turn resembles the regenerative cell cycle progression in keratinocytes following TPA treatment (Kirkhus et al., 1992). Some of the other upregulated genes, including Kcnk2 (TREK-1), have been linked to ovarian and prostate cancer (Innamaa et al., 2013, Voloshyna et al., 2008). Serbinb6 is an inhibitor of kallikrein-8 that modulates proliferation and differentiation of keratinocytes and has been implicated in the pathogenesis of psoriasis (Kishibe et al., 2007). Small proline-rich protein (Sprr) family members are antioxidants and are expressed by migrating keratinocytes in healing wounds (Vermeij and Backendorf, 2010) and thus might be expected to be elevated in tissues that are susceptible to regeneration and tumor development. The down-regulation of S100a8 in the keratinocytes of PGE2-treated BK5.EP4 mice was surprising because it is a pro-inflammatory mediator and is elevated in a variety of cancers (Gebhardt et al., 2006). Because the biological function for S100a8 (and its partner S100a9) is unknown, it is difficult to determine the relationship between its down-regulation and increased sensitivity to tumor development in BK5.EP4 mice. A comparison of differentially expressed transcripts between BK5.EP4 mice treated either with PGE2 or with EtOH also showed that several of the genes that were upregulated after PGE2 treatment are related to cancer development. Csf2rb is the common beta chain of the high affinity receptor for IL-3, IL-5 and GM-CSF (Woodcock et al., 1994), which is known to promote the progression of tumors in a number of organs including murine skin (Aziz et al., 2006, Gutschalk et al., 2013). Metallothionein-4 (MT-4) is involved in differentiation of stratified squamous epithelium (Vasak and Meloni, 2011) suggesting that EP4 is involved in keratinocyte differentiation. Pmepa1 expression has not been previously reported in keratinocytes, however, it is over-expressed in colorectal, breast and ovarian cancer (Giannini et al., 2003, Shi et al., 2012) and promotes proliferation via suppression of Smad3/4 signaling (Liu et al., 2011), suggesting a possible mechanism/pathway through which EP4 promotes tumor development.