UVA induces lesions resembling seborrheic keratoses in mice with keratinocyte-specific PTEN downregulation.

TO THE EDITOR Seborrheic keratoses (SK) are among the most common benign tumors in human, occurring in 80–100% of people over 50 years (Kwon et al., 2003; Yeatman et al., 1997). Although not life-threatening, SK may become irritated and itchy, are often unattractive and disfiguring, and may have a significantly negative psychological impact as daily reminders of aging. Several recent studies of the pathogenesis of SK have identified the importance of somatic activating mutations of the fibroblast growth factor receptor 3 (FGFR3) (Hafner et al., 2007a; Hafner et al., 2007b; Hafner et al., 2007c; Hafner et al., 2006a; Hafner et al., 2006b;Logie et al., 2005) and oncogenic mutations of the p110α subunit (PIK3CA) of phosphatidylinositol 3-kinase (PI3K) in an independent distribution (Hafner et al., 2007d; Hafner et al., 2008). At the molecular level, PI3K signaling, upstream of AKT activation, is negatively regulated by the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) (Backman et al., 2004; Di Cristofano and Pandolfi, 2000; Suzuki et al., 2003). Our recent studies demonstrated that PTEN is down-regulated by exposure to both UVB (Ming et al., 2010) and UVA (He et al., 2006). Although SK occur at both sun-exposed and non-sun-exposed skin, cumulative exposure to sunlight and aging have been proposed as independent risk factors for their development (Kwon et al., 2003; Yeatman et al., 1997). Here we report that in mice with a targeted PTEN down-regulation in their epidermis, UVA causes the formation of tumors resembling human SK, as well as of squamous cell carcinoma (SCC). To determine the role of PTEN in UVA-induced skin tumorigenesis, we compared UVA-induced tumor formation between mice with normal PTEN (+/+) and mice with PTEN hemizygosity (+/−). All animal procedures have been approved by Institutional Animal Care and Use Committee. We deleted one allele of the Pten gene in the epidermal keratinocytes by crossbreeding mice expressing Cre recombinase under the control of the keratin 14 promoter (K14-Cre) with mice expressing a LoxP-flanked (floxed) Pten gene (Ptenfl/fl) to generate K14-Cre;Ptenfl/+ (Pten+/−) (Figure 1a). To mimic the scenario of low-level UVA exposure that occurs in daily life for most people, we determined that the maximal dose of UVA that did not cause erythema was 15 J/cm2 and used that dose for all UVA experiments. The shaved mice were exposed to UVA or protected from UVA (Sham) for 25 weeks and then monitored for tumor development. Figure 1 PTEN hemizygosity is a predisposing factor for skin tumorigenesis following UVA irradiation. a, immunoblot analysis of PTEN protein levels in epidermis from K14Cre;Pten+/+ (+/+) and K14Cre;Ptenfl/+ (+/−) mice. b, percent (%) of tumor-free mice ... In the absence of UVA, Pten+/+ and Pten+/− did not grow tumors throughout the study period. UVA did not cause tumor formation in Pten+/+ mice. However, Pten+/− mice started to develop skin tumors at 37 weeks of UVA irradiation (Figure 1b). A log-rank test showed that PTEN hemizygosity significantly accelerated skin tumorigenesis following UVA irradiation (P < 0.0001). Histopathologic analysis of the tumors, using similar criteria for mice (Benavides et al., 2009; Logie et al., 2005), showed that in UVA-exposed Pten+/− mice 67% of tumors were of SK type, whereas 33% were SCC (Figure 1c and d). Thus, following UVA radiation PTEN down-regulation is a predisposing factor for skin tumorigenesis. Similar to the transgenic mouse model with an activating FGFR3 mutation (Logie et al., 2005), the skin lesions observed in our mouse model share several histological features in common with human SK including acanthosis, hyperkeratosis, papillomatosis, and keratin-filled structures (horn cysts and pseudo-horn cysts) (Figure 1e and f) while lacking significant cytologic atypia, whereas the SCCs are characterized by the presence of atypical nuclei, mitotic figures, and multinucleated cells (Figure 1g–h). To determine the molecular mechanisms by which UVA causes SK and SCC in Pten+/− mice, we carried out immunohistochemical analysis of sham-irradiated epidermis, UVA-irradiated nontumor epidermis, SK, and SCC, and investigated whether UVA further reduces the PTEN dose during tumorigenesis (Figure 2a and b). In Pten+/− epidermis, UVA irradiation further reduced epidermal PTEN levels (Figure 2c), consistent with our previous findings (He et al., 2006). In SK and SCCs, PTEN levels were further reduced as compared to non-lesional epidermis exposed to UVA or sham-irradiated (Figure 2d and e). Immunoblot analysis showed that PTEN was significantly down-regulated in both UVA-induced SK and SCC (Figure 2f and g). As compared with normal Pten+/− skin, AKT phosphorylation was significantly increased in AK and SCC (Figure 2f and h). However, there was no significant difference in either PTEN or AKT phosphorylation between SK and SCC (Figure 2f–h). These findings suggest that PTEN loss, in which UVA plays an active role, is an early event in the development of AK and SCC. Figure 2 The PTEN/AKT pathway is involved in UVA-induced tumorigenesis in Pten+/− mice. a–e, immunohistochemical analysis of PTEN in non-tumor and tumor samples using an anti-PTEN antibody. Scale bar: 50 µm. a and b, sham-irradiated normal ... To determine the mechanisms by which UVA causes SK in Pten+/− mice, we examined the role of the glioma tumor-suppressor candidate region gene 2 (GLTSCR2), a nuclear protein that binds and stabilizes PTEN (Okahara et al., 2004; Yim et al., 2007) and is reduced in SK (Kim et al., 2010). Real-time RT-PCR analysis showed that there were no significant differences in GLTSCR2 mRNA levels between +/+, +/−, and SKs (Figure 2i), suggesting that GLTSCR2 is not involved in our SK mouse model. To determine the molecular impact of PTEN down-regulation, we examined the phosphorylation of AKT and ERK in parental and PTEN-down-regulated epidermis. We found that phosphorylation of AKT and ERK in Pten+/+ skin was increased at 6 and 24 h after 15 J/cm2 UVA irradiation (Figure 2j and s1). PTEN down-regulation significantly increased AKT phosphorylation at both 6 h and 24 h following 15 J/cm2 UVA (Figure 2j and s1A). However, it only significantly increased ERK phosphorylation at 24 h following UVA (Figure 2j and s1B). Furthermore, we found that UVA irradiation inhibited cell growth in vitro and in vivo (Figure 2k and s2). Constitutive PTEN reduction in epidermis delayed cell exit from UVA-induced growth arrest (Figure s2). While acute re-expression of PTEN in PTEN+/− keratinocytes had little effect on cell growth post-UVA in vitro, it significantly inhibited cell growth in sham-irradiated cells (P < 0.05) (Figure 2k). These data indicate that PTEN reduction plays a critical role in UVA-induced development of SK as well as SCC, likely through the AKT and ERK pathways.