- Email: [email protected]
UVA Induces Lesions Resembling Seborrheic Keratoses in Mice with Keratinocyte-Specific PTEN Downregulation
M Ming et al. SK in UVA-PTEN Interaction in Mice
Pollock PM, Harper UL, Hansen KS et al. (2003) High frequency of BRAF mutations in nevi. Nat Genet 33:19–20 Rodeck U, Melber K, Kath R et al. (1991) Constitutive expression of multiple growth factor genes by melanoma cells but not normal melanocytes. J Invest Dermatol 97:20–6
arrest mediated by p21Cip1. Mol Cell Biol 17:5598–611
Ryu B, Kim DS, Deluca AM et al. (2007) Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression. PloS One 2:e594
Zhang Q, Thomas SM, Xi S et al. (2004) SRC family kinases mediate epidermal growth factor receptor ligand cleavage, proliferation, and invasion of head and neck cancer cells. Cancer Res 64:6166–73
Woods D, Parry D, Cherwinski H et al. (1997) Rafinduced proliferation or cell cycle arrest is determined by the level of Raf activity with
UVA Induces Lesions Resembling Seborrheic Keratoses in Mice with Keratinocyte-Specific PTEN Downregulation Journal of Investigative Dermatology (2011) 131, 1583–1586; doi:10.1038/jid.2011.33; published online 10 March 2011
PTEN +/+ +/– PTEN β-Actin
Tumor-free mice (%)
TO THE EDITOR Seborrheic keratoses (SKs) are among the most common benign tumors in humans, occurring in 80–100% of people over 50 years of age (Yeatman et al., 1997; Kwon et al., 2003). Although not life-threatening, SKs may become irri-
tated 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 fibroblast growth factor
receptor 3 (FGFR3) (Logie et al., 2005; Hafner et al., 2006a, b, 2007a, b, c) and oncogenic mutations of the p110a subunit (PIK3CA) of phosphatidylinositol 3-kinase (PI3K) in an independent distribution (Hafner et al., 2007d, 2008).
+/+/Sham +/+/UVA +/–/Sham 100 80 60
+/–
SK
SCC
Sham
0
0
UVA
4/6
2/6
+/–/UVA 40 20
30 40 Weeks
50
Figure 1. PTEN (phosphatase and tensin homolog deleted on chromosome 10) hemizygosity is a predisposing factor for skin tumorigenesis following UVA irradiation. (a) Immunoblot analysis of PTEN protein levels in the epidermis from K14Cre;Pten þ / þ ( þ / þ ) and K14Cre;Ptenfl/ þ ( þ /) mice. (b) Percentage of tumor-free mice (n ¼ 15). (c) Incidence of seborrheic keratosis (SK) and squamous cell carcinoma (SCC). (d) A mouse with an SK (indicated by a red arrow) developing in its skin. (e–h) Hematoxylin and eosin staining of the mouse specimens with SK and SCC. (e) SK at 2.5 magnification. (f) SK at original magnification 10. (g) SCC at original magnification 10. (h) SCC at original magnification 10, with a smaller field of view. (e) Scale bar ¼ 400 mm and (f–h) scale bar ¼ 100 mm. The green arrow in (h) indicates a multinucleated cell. Abbreviations: AKT, a serine–threonine kinase, downstream of PI3K, also called protein kinase B; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SCC, squamous cell carcinoma; SK, seborrheic keratosis; þ / þ , Pten þ / þ , K14Cre, Pten þ / þ ; þ /, Pten þ /, K14Cre, Ptenfl/ þ
www.jidonline.org 1583
M Ming et al. SK in UVA-PTEN Interaction in Mice
At the molecular level, PI3K signaling, upstream of AKT (a serine–threonine kinase downstream of PI3K, also called protein kinase B) activation, is negatively regulated by the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) (Di Cristofano and Pandolfi, 2000; Suzuki et al., 2003; Backman et al., 2004). Our recent studies demonstrated that PTEN is downregulated by exposure to both UVB (Ming et al., 2010) and UVA (He et al., 2006).
Pten+/+
Although SKs occur on both sunexposed and non-sun-exposed skin, cumulative exposure to sunlight and aging have been proposed as independent risk factors for their development (Yeatman et al., 1997; Kwon et al., 2003). Here we report that in mice with a targeted PTEN downregulation in their epidermis, UVA causes the formation of tumors resembling human SKs as well as of squamous cell carcinomas (SCCs). To determine the role of PTEN in UVA-induced skin tumorigenesis, we
Pten+/–
Pten+/–; UVA
compared UVA-induced tumor formation between mice with normal PTEN ( þ / þ ) and mice with PTEN hemizygosity ( þ /). All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago. 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 LoxPflanked (floxed) Pten gene (Ptenfl/fl) to
SK
SCC
EP
HF
EP T EP D
D
S
D T S
120
p-AKT
80
AKT
+/+ + 6
p-ERK
40
ERK 0 +/+
+/–
SK
PTEN β-Actin
+ 24
–
+/– + + 6 24
Sham/+/–/EV Sham/+/–/Ad-PTEN UVA/+/–/EV UVA/+/–/Ad-PTEN 2.5 2 1.5 1 0.5
C SC –/ E +/ V –/ +/ Ad+/ P EV TE N
NS
UVA – Hours
+/
% GLTSCR2 mRNA
NS
N
GAPDH
NS
NS
2 1.6 1.2 0.8 0.4 0 SK
AKT
*
N
p-AKT
2.5 2 1.5 1 0.5 0
*
*
C
PTEN (a.u.)
PTEN
*
p-AKT/AKT (a.u.)
SCC
SK
SK
SC
+/–
Cell growth (a.u.)
+/+
* * 0
24 Hours
PTEN β-Actin
48
Figure 2. The PTEN (phosphatase and tensin homolog deleted on chromosome 10)/AKT (a serine–threonine kinase) pathway is involved in UVA-induced tumorigenesis in Pten þ / mice. (a–e) Immunohistochemical analysis of PTEN in nontumor and tumor samples using an anti-PTEN antibody. Scale bar ¼ 50 mm. (a, b) Sham-irradiated normal epidermis from (a) Pten þ / þ and (b) Pten þ / mouse. (c) UVA-irradiated nontumor epidermis, (d) seborrheic keratosis (SK), and (e) squamous cell carcinoma (SCC) from Pten þ / mouse. (f) Immunoblot analysis of PTEN, p-AKT (serine 473), AKT, and GAPDH (equal loading control) in þ / þ , þ /, SK, and SCC. (g) Quantification of PTEN protein levels in (f). (h) Quantification of the ratio of p-AKT/AKT in (f). N in (g, h), normal Pten þ / skin. (i) Real-time RT-PCR analysis of the GLTSCR2 mRNA levels in þ / þ , þ /, and SK (n ¼ 3). (j) Immunoblot analysis of p-AKT, AKT, p-ERK (extracellular signal-regulated kinase), ERK, PTEN, and b-actin (equal loading control) in individual þ / þ and þ / mouse epidermis (n ¼ 3) at 6 and 24 h post-UVA (15 J cm2). (k) Cell growth analysis of neonatal primary Pten þ / keratinocytes infected with empty vector (EV) alone or wild-type PTEN adenovirus (Ad-PTEN) at a multiplicity of infection of 50 at different times post sham or UVA irradiation (5 J cm2) (n ¼ 3). The right panel shows immunoblot analysis of PTEN and b-actin. Error bars show standard error (SE). (g, h) *Po0.05, significant difference between comparison groups. NS, not statistically significant, P40.05. (k) *Po0.05, significant difference between sham-irradiated þ //EV and þ //Ad-PTEN groups. þ / þ , K14Cre;Pten þ / þ ; þ /, K14Cre;Ptenfl/ þ ; EP, epidermis; D, dermis; HF, hair follicle; S, stroma; T, tumor.
1584 Journal of Investigative Dermatology (2011), Volume 131
M Ming et al. SK in UVA-PTEN Interaction in Mice
generate K14-Cre;Ptenfl/ þ (Pten þ /) (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 group) for 25 weeks and then monitored for tumor development. In the absence of UVA, Pten þ / þ and Pten þ / did not grow tumors throughout the study period. UVA did not cause tumor formation in Pten þ / þ mice. However, Pten þ / mice began 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 (Po0.0001). Histopathological analysis of the tumors, using similar criteria for mice (Logie et al., 2005; Benavides et al., 2009), showed that in UVAexposed Pten þ / mice 67% of the tumors were of the SK type, whereas 33% were SCCs (Figure 1c and d). Thus, following UVA radiation, PTEN downregulation becomes 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 with human SKs, including acanthosis, hyperkeratosis, papillomatosis, and keratin-filled structures (horn cysts and pseudo–horn cysts) (Figure 1e and f), while lacking significant cytological atypia, whereas the SCCs are characterized by the presence of atypical nuclei, mitotic figures, and multinucleated cells (Figure 1g and h). To determine the molecular mechanisms by which UVA causes SK and SCC in Pten þ / mice, we carried out immunohistochemical analysis of sham-irradiated epidermis, UVA-irradiated nontumor epidermis, SK, and SCC to investigate whether UVA further reduces the PTEN dose during tumorigenesis (Figure 2a and b). In Pten þ / epidermis, UVA irradiation further reduced epidermal PTEN levels (Figure 2c), consistent with our previous findings (He et al., 2006). In SKs and
SCCs, PTEN levels were further reduced as compared with nonlesional epidermis exposed to UVA or sham-irradiated (Figure 2d and e). Immunoblot analysis showed that PTEN was significantly downregulated in both UVA-induced SK and SCC (Figure 2f and g). As compared with normal Pten þ / skin, AKT phosphorylation was significantly increased in SK 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 has an active role, is an early event in the development of SK and SCC. To determine the mechanisms by which UVA causes SK in Pten þ / mice, we examined the role of the glioma tumor-suppressor candidate region gene 2 (GLTSCR2), a nuclear protein that binds to 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 þ / þ , þ /, and SK (Figure 2i), suggesting that GLTSCR2 is not involved in our SK mouse model. To determine the molecular impact of PTEN downregulation, we examined the phosphorylation of AKT and ERK (extracellular signal-regulated kinase) in parental and PTENdownregulated epidermis. We found that phosphorylation of AKT and ERK in Pten þ / þ skin was increased at 6 and 24 h after 15 J cm2 UVA irradiation (Figure 2j and Supplementary Figure S1 online). PTEN downregulation significantly increased AKT phosphorylation at both 6 and 24 h following 15 J cm2 UVA irradiation (Figure 2j and Supplementary Figure S1A online). However, it significantly increased ERK phosphorylation only at 24 h following UVA irradiation (Figure 2j and Supplementary Figure S1B online). Furthermore, we found that UVA irradiation inhibited cell growth in vitro and in vivo (Figure 2k and Supplementary Figure S2 online). Constitutive PTEN reduction in the epidermis delayed cell exit from UVA-induced growth arrest (Supplementary Figure S2 online). While acute re-expression
of PTEN in PTEN þ / keratinocytes had little effect on cell growth postUVA irradiation in vitro, it significantly inhibited cell growth in sham-irradiated cells (Po0.05) (Figure 2k). These data indicate that PTEN reduction exerts a critical role in UVA-induced development of SK as well as SCC, probably through the AKT and ERK pathways. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This work was supported by NIH grant ES016936 (YYH) and the NIH/NIEHS intramural program. Further support was provided by the UC Friends of Dermatology Fund. We thank Ramon Parsons (Departments of Pathology and Medicine, Columbia University, New York) for kindly providing us with the adenoviral PTEN vector and Terri Li for PTEN immunohistochemistry. This paper is dedicated to the memory of Colin F. Chignell.
Mei Ming1, Christopher R. Shea1, Li Feng2, Keyoumars Soltani1 and Yu-Ying He1 1 Section of Dermatology, Department of Medicine, University of Chicago, Chicago, Illinois, USA and 2Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA E-mail: [email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
REFERENCES Backman SA, Ghazarian D, So K et al. (2004) Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten. Proc Natl Acad Sci USA 101: 1725–30 Benavides F, Oberyszyn TM, VanBuskirk AM et al. (2009) The hairless mouse in skin research. J Dermatol Sci 53:10–8 Di Cristofano A, Pandolfi PP (2000) The multiple roles of PTEN in tumor suppression. Cell 100:387–90 Hafner C, Hartmann A, Real FX et al. (2007a) Spectrum of FGFR3 mutations in multiple intraindividual seborrheic keratoses. J Invest Dermatol 127:1883–5 Hafner C, Hartmann A, van Oers JM et al. (2007b) FGFR3 mutations in seborrheic keratoses are already present in flat lesions and associated with age and localization. Mod Pathol 20:895–903 Hafner C, Hartmann A, Vogt T (2007c) FGFR3 mutations in epidermal nevi and seborrheic keratoses: lessons from urothelium and skin. J Invest Dermatol 127:1572–3
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Hafner C, Lopez-Knowles E, Luis NM et al. (2007d) Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc Natl Acad Sci USA 104:13450–4 Hafner C, van Oers JM, Hartmann A et al. (2006a) High frequency of FGFR3 mutations in adenoid seborrheic keratoses. J Invest Dermatol 126:2404–7 Hafner C, Vogt T, Hartmann A (2006b) FGFR3 mutations in benign skin tumors. Cell Cycle (Georgetown, TX) 5:2723–8 Hafner C, Vogt T, Landthaler M et al. (2008) Somatic FGFR3 and PIK3CA mutations are present in familial seborrhoeic keratoses. Br J Dermatol 159:214–7 He YY, Pi J, Huang JL et al. (2006) Chronic UVA irradiation of human HaCaT keratinocytes induces
malignant transformation associated with acquired apoptotic resistance. Oncogene 25:3680–8 Kim JY, Kim HS, Lee S et al. (2010) The expression of GLTSCR2, a candidate tumor suppressor, is reduced in seborrheic keratosis compared to normal skin. Pathol Res Pract 206:295–9 Kwon OS, Hwang EJ, Bae JH et al. (2003) Seborrheic keratosis in the Korean males: causative role of sunlight. Photodermatol Photoimmunol Photomed 19:73–80
Okahara F, Ikawa H, Kanaho Y et al. (2004) Regulation of PTEN phosphorylation and stability by a tumor suppressor candidate protein. J Biol Chem 279:45300–3 Suzuki A, Itami S, Ohishi M et al. (2003) Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation. Cancer Res 63:674–81
Logie A, Dunois-Larde C, Rosty C et al. (2005) Activating mutations of the tyrosine kinase receptor FGFR3 are associated with benign skin tumors in mice and humans. Hum Mol Genet 14:1153–60
Yeatman JM, Kilkenny M, Marks R (1997) The prevalence of seborrhoeic keratoses in an Australian population: does exposure to sunlight play a part in their frequency? Br J Dermatol 137:411–4
Ming M, Han W, Maddox J et al. (2010) UVBinduced ERK/AKT-dependent PTEN suppression promotes survival of epidermal keratinocytes. Oncogene 29:492–502
Yim JH, Kim YJ, Ko JH et al. (2007) The putative tumor suppressor gene GLTSCR2 induces PTEN-modulated cell death. Cell Death Differ 14:1872–9
1586 Journal of Investigative Dermatology (2011), Volume 131
Copyright of Journal of Investigative Dermatology is the property of Nature Publishing Group and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Pollock PM, Harper UL, Hansen KS et al. (2003) High frequency of BRAF mutations in nevi. Nat Genet 33:19–20 Rodeck U, Melber K, Kath R et al. (1991) Constitutive expression of multiple growth factor genes by melanoma cells but not normal melanocytes. J Invest Dermatol 97:20–6
arrest mediated by p21Cip1. Mol Cell Biol 17:5598–611
Ryu B, Kim DS, Deluca AM et al. (2007) Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression. PloS One 2:e594
Zhang Q, Thomas SM, Xi S et al. (2004) SRC family kinases mediate epidermal growth factor receptor ligand cleavage, proliferation, and invasion of head and neck cancer cells. Cancer Res 64:6166–73
Woods D, Parry D, Cherwinski H et al. (1997) Rafinduced proliferation or cell cycle arrest is determined by the level of Raf activity with
UVA Induces Lesions Resembling Seborrheic Keratoses in Mice with Keratinocyte-Specific PTEN Downregulation Journal of Investigative Dermatology (2011) 131, 1583–1586; doi:10.1038/jid.2011.33; published online 10 March 2011
PTEN +/+ +/– PTEN β-Actin
Tumor-free mice (%)
TO THE EDITOR Seborrheic keratoses (SKs) are among the most common benign tumors in humans, occurring in 80–100% of people over 50 years of age (Yeatman et al., 1997; Kwon et al., 2003). Although not life-threatening, SKs may become irri-
tated 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 fibroblast growth factor
receptor 3 (FGFR3) (Logie et al., 2005; Hafner et al., 2006a, b, 2007a, b, c) and oncogenic mutations of the p110a subunit (PIK3CA) of phosphatidylinositol 3-kinase (PI3K) in an independent distribution (Hafner et al., 2007d, 2008).
+/+/Sham +/+/UVA +/–/Sham 100 80 60
+/–
SK
SCC
Sham
0
0
UVA
4/6
2/6
+/–/UVA 40 20
30 40 Weeks
50
Figure 1. PTEN (phosphatase and tensin homolog deleted on chromosome 10) hemizygosity is a predisposing factor for skin tumorigenesis following UVA irradiation. (a) Immunoblot analysis of PTEN protein levels in the epidermis from K14Cre;Pten þ / þ ( þ / þ ) and K14Cre;Ptenfl/ þ ( þ /) mice. (b) Percentage of tumor-free mice (n ¼ 15). (c) Incidence of seborrheic keratosis (SK) and squamous cell carcinoma (SCC). (d) A mouse with an SK (indicated by a red arrow) developing in its skin. (e–h) Hematoxylin and eosin staining of the mouse specimens with SK and SCC. (e) SK at 2.5 magnification. (f) SK at original magnification 10. (g) SCC at original magnification 10. (h) SCC at original magnification 10, with a smaller field of view. (e) Scale bar ¼ 400 mm and (f–h) scale bar ¼ 100 mm. The green arrow in (h) indicates a multinucleated cell. Abbreviations: AKT, a serine–threonine kinase, downstream of PI3K, also called protein kinase B; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SCC, squamous cell carcinoma; SK, seborrheic keratosis; þ / þ , Pten þ / þ , K14Cre, Pten þ / þ ; þ /, Pten þ /, K14Cre, Ptenfl/ þ
www.jidonline.org 1583
M Ming et al. SK in UVA-PTEN Interaction in Mice
At the molecular level, PI3K signaling, upstream of AKT (a serine–threonine kinase downstream of PI3K, also called protein kinase B) activation, is negatively regulated by the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) (Di Cristofano and Pandolfi, 2000; Suzuki et al., 2003; Backman et al., 2004). Our recent studies demonstrated that PTEN is downregulated by exposure to both UVB (Ming et al., 2010) and UVA (He et al., 2006).
Pten+/+
Although SKs occur on both sunexposed and non-sun-exposed skin, cumulative exposure to sunlight and aging have been proposed as independent risk factors for their development (Yeatman et al., 1997; Kwon et al., 2003). Here we report that in mice with a targeted PTEN downregulation in their epidermis, UVA causes the formation of tumors resembling human SKs as well as of squamous cell carcinomas (SCCs). To determine the role of PTEN in UVA-induced skin tumorigenesis, we
Pten+/–
Pten+/–; UVA
compared UVA-induced tumor formation between mice with normal PTEN ( þ / þ ) and mice with PTEN hemizygosity ( þ /). All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago. 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 LoxPflanked (floxed) Pten gene (Ptenfl/fl) to
SK
SCC
EP
HF
EP T EP D
D
S
D T S
120
p-AKT
80
AKT
+/+ + 6
p-ERK
40
ERK 0 +/+
+/–
SK
PTEN β-Actin
+ 24
–
+/– + + 6 24
Sham/+/–/EV Sham/+/–/Ad-PTEN UVA/+/–/EV UVA/+/–/Ad-PTEN 2.5 2 1.5 1 0.5
C SC –/ E +/ V –/ +/ Ad+/ P EV TE N
NS
UVA – Hours
+/
% GLTSCR2 mRNA
NS
N
GAPDH
NS
NS
2 1.6 1.2 0.8 0.4 0 SK
AKT
*
N
p-AKT
2.5 2 1.5 1 0.5 0
*
*
C
PTEN (a.u.)
PTEN
*
p-AKT/AKT (a.u.)
SCC
SK
SK
SC
+/–
Cell growth (a.u.)
+/+
* * 0
24 Hours
PTEN β-Actin
48
Figure 2. The PTEN (phosphatase and tensin homolog deleted on chromosome 10)/AKT (a serine–threonine kinase) pathway is involved in UVA-induced tumorigenesis in Pten þ / mice. (a–e) Immunohistochemical analysis of PTEN in nontumor and tumor samples using an anti-PTEN antibody. Scale bar ¼ 50 mm. (a, b) Sham-irradiated normal epidermis from (a) Pten þ / þ and (b) Pten þ / mouse. (c) UVA-irradiated nontumor epidermis, (d) seborrheic keratosis (SK), and (e) squamous cell carcinoma (SCC) from Pten þ / mouse. (f) Immunoblot analysis of PTEN, p-AKT (serine 473), AKT, and GAPDH (equal loading control) in þ / þ , þ /, SK, and SCC. (g) Quantification of PTEN protein levels in (f). (h) Quantification of the ratio of p-AKT/AKT in (f). N in (g, h), normal Pten þ / skin. (i) Real-time RT-PCR analysis of the GLTSCR2 mRNA levels in þ / þ , þ /, and SK (n ¼ 3). (j) Immunoblot analysis of p-AKT, AKT, p-ERK (extracellular signal-regulated kinase), ERK, PTEN, and b-actin (equal loading control) in individual þ / þ and þ / mouse epidermis (n ¼ 3) at 6 and 24 h post-UVA (15 J cm2). (k) Cell growth analysis of neonatal primary Pten þ / keratinocytes infected with empty vector (EV) alone or wild-type PTEN adenovirus (Ad-PTEN) at a multiplicity of infection of 50 at different times post sham or UVA irradiation (5 J cm2) (n ¼ 3). The right panel shows immunoblot analysis of PTEN and b-actin. Error bars show standard error (SE). (g, h) *Po0.05, significant difference between comparison groups. NS, not statistically significant, P40.05. (k) *Po0.05, significant difference between sham-irradiated þ //EV and þ //Ad-PTEN groups. þ / þ , K14Cre;Pten þ / þ ; þ /, K14Cre;Ptenfl/ þ ; EP, epidermis; D, dermis; HF, hair follicle; S, stroma; T, tumor.
1584 Journal of Investigative Dermatology (2011), Volume 131
M Ming et al. SK in UVA-PTEN Interaction in Mice
generate K14-Cre;Ptenfl/ þ (Pten þ /) (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 group) for 25 weeks and then monitored for tumor development. In the absence of UVA, Pten þ / þ and Pten þ / did not grow tumors throughout the study period. UVA did not cause tumor formation in Pten þ / þ mice. However, Pten þ / mice began 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 (Po0.0001). Histopathological analysis of the tumors, using similar criteria for mice (Logie et al., 2005; Benavides et al., 2009), showed that in UVAexposed Pten þ / mice 67% of the tumors were of the SK type, whereas 33% were SCCs (Figure 1c and d). Thus, following UVA radiation, PTEN downregulation becomes 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 with human SKs, including acanthosis, hyperkeratosis, papillomatosis, and keratin-filled structures (horn cysts and pseudo–horn cysts) (Figure 1e and f), while lacking significant cytological atypia, whereas the SCCs are characterized by the presence of atypical nuclei, mitotic figures, and multinucleated cells (Figure 1g and h). To determine the molecular mechanisms by which UVA causes SK and SCC in Pten þ / mice, we carried out immunohistochemical analysis of sham-irradiated epidermis, UVA-irradiated nontumor epidermis, SK, and SCC to investigate whether UVA further reduces the PTEN dose during tumorigenesis (Figure 2a and b). In Pten þ / epidermis, UVA irradiation further reduced epidermal PTEN levels (Figure 2c), consistent with our previous findings (He et al., 2006). In SKs and
SCCs, PTEN levels were further reduced as compared with nonlesional epidermis exposed to UVA or sham-irradiated (Figure 2d and e). Immunoblot analysis showed that PTEN was significantly downregulated in both UVA-induced SK and SCC (Figure 2f and g). As compared with normal Pten þ / skin, AKT phosphorylation was significantly increased in SK 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 has an active role, is an early event in the development of SK and SCC. To determine the mechanisms by which UVA causes SK in Pten þ / mice, we examined the role of the glioma tumor-suppressor candidate region gene 2 (GLTSCR2), a nuclear protein that binds to 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 þ / þ , þ /, and SK (Figure 2i), suggesting that GLTSCR2 is not involved in our SK mouse model. To determine the molecular impact of PTEN downregulation, we examined the phosphorylation of AKT and ERK (extracellular signal-regulated kinase) in parental and PTENdownregulated epidermis. We found that phosphorylation of AKT and ERK in Pten þ / þ skin was increased at 6 and 24 h after 15 J cm2 UVA irradiation (Figure 2j and Supplementary Figure S1 online). PTEN downregulation significantly increased AKT phosphorylation at both 6 and 24 h following 15 J cm2 UVA irradiation (Figure 2j and Supplementary Figure S1A online). However, it significantly increased ERK phosphorylation only at 24 h following UVA irradiation (Figure 2j and Supplementary Figure S1B online). Furthermore, we found that UVA irradiation inhibited cell growth in vitro and in vivo (Figure 2k and Supplementary Figure S2 online). Constitutive PTEN reduction in the epidermis delayed cell exit from UVA-induced growth arrest (Supplementary Figure S2 online). While acute re-expression
of PTEN in PTEN þ / keratinocytes had little effect on cell growth postUVA irradiation in vitro, it significantly inhibited cell growth in sham-irradiated cells (Po0.05) (Figure 2k). These data indicate that PTEN reduction exerts a critical role in UVA-induced development of SK as well as SCC, probably through the AKT and ERK pathways. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This work was supported by NIH grant ES016936 (YYH) and the NIH/NIEHS intramural program. Further support was provided by the UC Friends of Dermatology Fund. We thank Ramon Parsons (Departments of Pathology and Medicine, Columbia University, New York) for kindly providing us with the adenoviral PTEN vector and Terri Li for PTEN immunohistochemistry. This paper is dedicated to the memory of Colin F. Chignell.
Mei Ming1, Christopher R. Shea1, Li Feng2, Keyoumars Soltani1 and Yu-Ying He1 1 Section of Dermatology, Department of Medicine, University of Chicago, Chicago, Illinois, USA and 2Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA E-mail: [email protected]
SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid
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