PTEN Positively Regulates UVB-Induced DNA Damage Repair · predisposing factor for UVB-induced skin...

Tumor and Stem Cell Biology

PTEN Positively Regulates UVB-Induced DNA DamageRepair

Mei Ming1, Li Feng2, Christopher R. Shea1, Keyoumars Soltani1, Baozhong Zhao2, Weinong Han1,Robert C. Smart3, Carol S. Trempus2, and Yu-Ying He1

AbstractNonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet

B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetictargets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is apredisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cellcarcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, therewas a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a keyrole for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity ofglobal genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-inducedmutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolongedUVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NERprotein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels inPTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essentialgenomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNAdamage. Cancer Res; 71(15); 5287–95. �2011 AACR.

Introduction

Skin cancer is the most common type of cancer in theUnited States. Each year more than 1 million new cases of skincancer are diagnosed in the United States alone, accountingfor 40% of all newly diagnosed cancer cases (1, 2). Theincidence of skin cancers continues to increase each year.The nonmelanoma skin cancers (NMSC) derived from theepidermal basal layer account for approximately 96% of allskin malignancies. The major risk factor for NMSC is ultra-violet (UV) B radiation in sunlight, which causes the formationof 2 major DNA-damaging products [i.e., cyclobutane pyrimi-dine dimers (CPD) and pyrimidine(6-4)pyrimidone dimers(6-4PP); ref. 3].

Insight into the genetics of skin cancer has come fromstudies of a rare familial form of early-onset skin carcinogen-esis (4–6). Patients with this rare inherited disorder, xero-derma pigmentosum (XP), are highly sensitive to sun exposureand have a risk of developing skin cancer about 1,000 timesthat of the general population with an NMSC onset about 50years earlier (7, 8). Molecular defects in global genome nucleo-tide excision repair (GG-NER), a subpathway that removesUVB-induced CPDs and 6-4PPs throughout the genome, incarriers of XP are responsible for increased skin cancer riskunder UV challenge. Seven NER-deficient genetic complemen-tation groups of XP (XP-A to -G) have been identified, and allof the corresponding genes have now been cloned (4–6).Accumulating evidence indicates that the XP group C(XPC) protein plays an essential role in damage recognitionfor GG-NER (9–11) and the XPC gene is found to be deleted ormutated in human squamous cell carcinoma (SCC; ref. 12).

PTEN is a negative regulator of AKT signaling and func-tions as a tumor suppressor (13). In many human cancers,the functionality of PTEN is lost, placing PTEN in a mechan-istically critical position. PTEN has been shown to be acritical tumor suppressor in mouse skin by using a chemicalcarcinogenesis model (14). Although extensive biochemicaland genetic analyses have characterized the function ofPTEN in considerable detail, much remains to be elucidatedwith regard to its direct role in UVB-induced skin tumor-igenesis and the precise molecular mechanisms in damageresponses of UVB. Here we present data showing that PTEN

Authors' Affiliations: 1Section of Dermatology, Department of Medicine,University of Chicago, Chicago, Illinois; 2Laboratory of Toxicology &Pharmacology, National Institute of Environmental Health Sciences,NIH, Research Triangle Park, North Carolina; 3Cell Signaling and CancerGroup, Department of Environmental and Molecular Toxicology, NorthCarolina State University, Raleigh, North Carolina

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Yu-Ying He, Section of Dermatology, Depart-ment of Medicine, University of Chicago, Chicago, IL 60637. Phone: 773-795-4696; Fax: 773-702-8398; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4614

�2011 American Association for Cancer Research.

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Published OnlineFirst July 19, 2011; DOI: 10.1158/0008-5472.CAN-10-4614

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downregulation in epidermal keratinocytes is a predisposingfactor for UVB-induced skin tumorigenesis. In humans,PTEN is significantly downregulated in both premalignantand malignant skin lesions. PTEN inhibition impairs GG-NER capacity through suppressing the expression of XPC.The PTEN/AKT/p38 axis seems to be critical for regulatingXPC levels and thus for affecting GG-NER capacity. Thesefindings support the essential role of PTEN in relevanthuman skin tumor suppression.

Materials and Methods

Human normal and tumor samples,immunohistochemistry, and sun damage

All human specimens were studied after approval by theUniversity of Chicago Institutional Review Board. Formalin-fixed, paraffin-embedded tissue blocks were obtained from thearchives in the tissue bank of the Section of Dermatology(Department ofMedicine, University of Chicago). PTEN immu-nohistochemistry (IHC) was performed by the University ofChicago ImmunohistochemistryCoreFacility, usinganti-PTEN(clone 6H2.1; Upstate). The PTEN levels were evaluated inkeratinocytes in interfollicular epidermis. For the hematoxy-lin–eosin (H&E) slides, sun damage was analyzed by a board-certified dermatopathologist by using a solar elastosis indexwith a scale from 0 to 3 as described previously (15, 16). Absentsolar elastosis was classified as having a 0 score, a low level as 1,moderate as 2, anda solid elastoticmass replacing collagenas 3.

Cell cultureHuman HaCaT keratinocytes (obtained from Prof. N. Fuse-

nig) were maintained in a monolayer culture in 95% air/5%CO2 at 37�C in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% FBS, 100 units/mL penicillin, and 100mg/mL streptomycin (Invitrogen). HaCaT cell line was cul-tured for less than 20 passages. Normal human epidermalkeratinocytes (NHEK) cells were obtained from Clonetics(Lonza) and cultured in KGM Gold BulletKit medium (Clo-netics, Lonza) according to the manufacturer's instructions.NHEK cells were cultured for less than 4 passages. Noauthentication was done.

UVB radiationUVB irradiation was carried out as described previously

(17). Our UVB radiation was monitored every other week tomeasure the exposure output and dose. Our UVB system doesnot emit UVC radiation.

Animal treatmentsAll animal procedures have been approved by the University

of Chicago Institutional Animal Care and Use Committee.K14-Cre and Ptenfl/flmice were obtained from Jackson Labora-tories. All mice used have a B6 background to minimizevariations in mouse background. Littermates were used ascomparison controls. Mice (n¼ 15) were shaved 1 day prior tothe initial UVB irradiation and later as needed. Shaved micewere exposed to UVB (30 mJ/cm2, dose selected to avoidvisible sunburn) dorsally or sham-irradiated, 3 times a week

for 25 weeks and then kept for an additional 20 weeks tomonitor tumor formation and growth. The PTEN proteinlevels were determined by IHC by the University of ChicagoImmunohistochemistry Core Facility by using anti-PTEN(138G6; Cell Signaling). Mice were housed 5 animals per cage,and there was no evidence of dorsal wounds caused byfighting or sunburn.

Statistical analysesStatistical analyses were done using Prism 5 (GraphPad

Software). Data were expressed as the mean of 3 independentexperiments and analyzed by Student's t test, ANOVA, theMann–Whitney U test, and the Kruskal–Wallis test (for datafrom human specimens). Statistical analysis for BrdUrd-labeled cells in the epidermis was done using ANOVA followedby Scheffe's post hoc test. Kaplan–Meier survival estimates andlog-rank tests were used to evaluate the tumor onset inmice. AP value of less than 0.05 was considered statistically significant.

The details for in vivo cell proliferation assay, siRNA trans-fection, retroviral infection, and adenoviral infection, Westernblotting, luciferase reporter assays, chromatin fractionation,determination of 2 major forms of UVB-induced DNA damagein genomic DNA by ELISA, and in vitro cell proliferation assaycan be found in the Supplementary Information.

Results

PTEN downregulation is a predisposing factor for UVB-induced skin tumorigenesis

To analyze whether PTEN downregulation in epidermalkeratinocytes plays an active role in skin tumorigenesis inwhich UVB is the major risk factor for humans, we comparedUVB-induced tumor formation between mice with normalPTEN (þ/þ) and mice with PTEN hemizygosity (þ/�). Wedeleted 1 allele of the Pten gene in the epidermal keratinocytesby crossbreeding mice expressing Cre recombinase under thecontrol of the keratin 14 promoter (K14-Cre) with miceexpressing a LoxP-flanked (floxed) Pten gene (Ptenfl/fl) togenerate K14-Cre;Ptenfl/þ (Ptenþ/�). To mimic the commonclinical scenario of daily, low-level exposure to UV radiation,we elected to use a suberythemogenic dose of UVB radiation,not exceeding 30 mJ/cm2 per exposure (SupplementaryFig. S1A and B), which did not cause apoptosis. The shavedmice were either exposed to this dose of UVB or protectedfrom UV radiation (sham).

Without radiation, Ptenþ/þ and Ptenþ/� mice did not growany tumors throughout the study period. UVB did not causetumor formation in Ptenþ/þ mice. However, Ptenþ/� micestarted to develop skin tumors at 21 weeks of UVB irradiation(Fig. 1A; n ¼ 15). A log-rank test showed that PTEN hemi-zygosity significantly accelerates skin tumorigenesis followinga low dose of UVB radiation (P < 0.0001). Pathologic analysis ofthe tumors by using similar criteria for hairless mice (18)showed that in UVB-exposed Ptenþ/� mice, 40% (8 of 20) oftumors were papillomas and 60% (12 of 20) were SCCs. In bothpapillomas and SCCs, PTEN levels were reduced as comparedwith sham-irradiated epidermis (Fig. 1B and C). Thus, PTENdownregulation is a predisposing factor for UVB-induced skin

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tumorigenesis, and further PTEN loss following chronic UVBradiation is correlated with skin tumor formation.

PTEN downregulation in human skin tumors and itsassociation with sun damageTo further investigate the specific function of PTEN in

human skin cancer, we evaluated PTEN protein levels in 69human skin samples fromU.S. patients.We focused on types ofskin neoplasia that are associated with chronic sun exposure,including actinic keratosis (AK), considered to be either apremalignant lesion or a very early stage in the developmentof SCC and invasive SCC. These lesions were all located in sun-exposed regionsof the skin, suchas thehead, face, ear, andhanddorsum. All samples were from fair-skinned individuals, thepopulation at highest risk for developing skin cancer. We usedimmunohistochemical analysis to determine the differences inPTEN protein levels in human skin tumors as compared withnormal nonlesional skin (brown staining; Fig. 2A and B andSupplementary Fig. S2). To exclude the contribution of endo-genousbrownpigmentationdue tomelanin,wealsocarriedoutH&E staining and immunohistochemical analysis using alka-line phosphatase–anti-alkaline phosphatase (pinkish staining;data not shown). The PTEN levels were reduced (score 0 or 1) in92%ofAK (23of 25) and94%of invasive SCC lesions (26 of 28) ascompared with none of the normal skin samples (0 of 16;Fig. 2C). This reduction was statistically significant as analyzedby the Mann–Whitney U test (P < 0.0001 for AK and invasiveSCC vs. normal skin). When we compared PTEN levels inspecimens with different levels of chronic sun damage asmeasured by solar elastosis (15, 16), we found that PTENdownregulation was significantly associated with increasingsun damage (P < 0.001, Kruskal–Wallis test; SupplementaryTable S1), suggesting that UV damage specifically downregu-lates PTEN in human skin during carcinogenesis.

PTEN is essential for efficient GG-NER of UVB-inducedDNA damage

To determine the molecular basis for increased tumorigen-esis caused by the interaction between PTEN downregulationand UVB irradiation, we investigated the hypothesis thatPTEN plays an important role in repairing UVB-induced

Figure 1. PTEN hemizygosity is apredisposing factor for skintumorigenesis following low-levelUVB radiation. A, percentage oftumor-free mice (n ¼ 15). B,immunoblot analysis of PTEN andb-actin in normal skin (both þ/þand þ/�), papillomas (PAP), andSCCs. C, immunohistochemicalanalysis of PTEN in sham-irradiated normal skin and UVB-induced tumor samples fromPtenþ/� mice using an anti-PTENantibody. Scale bar, 20 mm.

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Figure 2. PTEN expression is significantly reduced in human skin tumors.A and B, representative immunohistochemical analysis of PTEN proteinlevels (brown) in normal skin and SCCs. EP, epidermis; HF, hair follicle; T,tumor. Scale bar, 100 mm. C, percentage of tumors (in stacked columnformat) for each score of PTEN expression.

PTEN in UVB-Induced DNA Damage Repair

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DNA damage. To determine the percentage of repair inparental and PTEN-inhibited cells, we measured the percen-tage of CPDs or 6-4PPs remaining at different intervals post–UVB irradiation.

To inhibit PTEN in keratinocytes, we infected humanHaCaT cells with a retroviral vector expressing short hairpinRNA targeting PTEN (shPTEN) or transfected the cells withsiRNA targeting PTEN (siPTEN; Fig. 3A and B). In bothmodels,inhibition of PTEN significantly inhibited repair of CPDs(Fig. 3C and D; P < 0.05, 2-way ANOVA). In comparison,inhibition of PTEN significantly reduced the repair of 6-4PPs at 6 hours post–UVB irradiation (Fig. 3E; P < 0.05,Student's t test), whereas complete repair was detected at24 hours post–UVB irradiation, indicating that the inhibitionof 6-4PP repair by PTEN loss was less profound than that ofCPD repair. Our findings showed that loss of PTEN impairsDNA repair in UVB-induced CPDs and to some extent in 6-4PPs, indicating that PTEN is required for efficient GG-NER.

PTEN downregulation delayed exit from growth arrestpost–UVB irradiation

UVB-induced damage to DNA activates checkpoint path-ways and thereby induces cell-cycle arrest (19, 20), a criticalmechanism for facilitating proper DNA repair. To determinethe role of PTEN downregulation in DNA damage checkpointfunction in vivo, we measured differences in percentage ofBrdUrd-labeled (BrdUrdþ) keratinocytes between Ptenþ/þ andPtenþ/� epidermis by IHC. In the absence of UVB treatment,the percentage of BrdUrdþ cells in Ptenþ/þ epidermis was

similar to that in Ptenþ/� counterparts (Fig. 4A). At 6 hourspost–UVB irradiation, the percentage of BrdUrdþ cellsdecreased in both Ptenþ/þ and Ptenþ/� epidermis, showingan arrest in cell proliferation. At 24 to 72 hours post–UVBirradiation, however, the percentage of BrdUrdþ cellsremained decreased in Ptenþ/� epidermis but not in itsPtenþ/þ counterparts, implying that Ptenþ/þ cells but notPtenþ/� cells exited from UVB-induced growth arrest(Fig. 4A). Similarly, PTEN knockdown reduced BrdUrd incor-poration post–UVB irradiation (Supplementary Fig. S3A).These findings indicate that PTEN downregulation impairsthe exit from UVB-induced growth arrest.

DNA damage–mediated growth arrest operates through theactivation of Chk1 and Chk2 and subsequently p53 accumula-tion (19, 21, 22). Recent studies have shown that in cellstreated with ionizing or UVC radiation, PTEN loss inhibitsChk1 activation through the AKT pathway and thus causesgenomic instability (23, 24). To determine the role of PTEN inUVB-induced checkpoint function at the molecular level, weexamined the phosphorylation of Chk1 and Chk2 and theformation of g-H2AX, a marker for persistence of DNA damage(25–27), in parental and PTEN-downregulated keratinocytes.In HaCaT cells, UVB irradiation (5 or 20 mJ/cm2) dramaticallyinduced Chk1 phosphorylation at serine 345 (p-Chk1) andChk2 phosphorylation at threonine 68 (p-Chk2) at 1.5 and 6hours (Fig. 4B). Post–UVB irradiation (5 mJ/cm2), p-Chk1 inshPTEN cells was lower than in negative control (NC) cells,whereas post–UVB irradiation (20 mJ/cm2), p-Chk1 was simi-lar to that in NC cells at 0.5 hours (Fig. 4B and SupplementaryFig. S3B). At 1.5 and 6 hours post–UVB irradiation (5 or 20 mJ/cm2), however, p-Chk1 in shPTEN cells was significantlyhigher than in NC cells. In comparison, p-Chk2 in shPTENcells was similar to that in NC cells (Fig. 4B). In NHEK cells,PTEN knockdown increases Chk1 phosphorylation, g-H2AXformation, and p53 accumulation, but not Chk2 phosphoryla-tion, similar to the response of HaCaT cells and primarymouse keratinocytes (Fig. 4C and Supplementary Fig. S3B–D). However, activating AKT by expressing constitutivelyactive AKT (Aþ) in NHEK cells did not resemble the effectof PTEN knockdown (Fig. 4D). Inhibiting AKT signaling byLY294002 (10 mmol/L) did not reverse the effect of PTENdownregulation (Fig. 4E). These data indicated that, followingUVB irradiation, the increased checkpoint activation causedby PTEN loss is independent of AKT activation.

PTEN regulates GG-NER through XPCXPC is essential for repairing UVB-induced DNA damage

(10, 28). To examine the mechanism by which PTEN partici-pates in GG-NER, we investigated the role of PTEN in XPCregulation. We compared the protein level of XPC in NC cellswith that in shPTEN cells by immunoblotting and found thatthe protein level of XPC in HaCaT cells transfected withshPTEN was significantly lower than in NC cells whereasthe levels of DDB1 and DDB2 were not affected (Fig. 5A).Similarly the XPC protein level in HaCaT cells transfected withsiPTEN was significantly reduced as compared with HaCaTcells transfected with negative control siRNA (NC; Fig. 5B).Furthermore, XPC levels were lower in the Ptenþ/� mouse

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Figure 3. PTEN is required for efficient GG-NER. A and B, immunoblotanalysis of PTEN and b-actin (equal loading control) in NC and shPTENcells (A) as well as in NC and siPTEN cells (B). C–E, ELISA of percent repairof CPDs (C and D) and 6-4PPs (E) at intervals post–UVB irradiation (20 mJ/cm2). C, HaCaT cells infected with a retroviral vector expressing NC orshPTEN. D and E, HaCaT cells transfected with NC or siPTEN. Error barsshow SE. *, P < 0.05, significant differences between shPTEN and NC cellsor between PTEN and NC cells.

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epidermis than in its Ptenþ/þ counterparts (Fig. 5C). Thesedata indicate that PTEN positively regulates XPC proteinlevels.To determine whether reduced XPC expression in PTEN-

inhibited cells impacts its recruitment to chromatin uponDNA damage, we determined the protein levels of XPC thatbound to chromatin with or without UVB irradiation in NCcells and shPTEN cells. We found that the basal levels ofchromatin-bound XPC in shPTEN cells were significantlylower than those in NC cells without UVB irradiation (Sup-plementary Fig. S4A and B; P < 0.05, Student's t test). In NCcells, UVB irradiation increased chromatin-bound XPC levelsat 10 minutes (Supplementary Fig. S4A and B; P < 0.05,Student's t test), consistent with the crucial role of XPC inDNA damage recognition and initiation of NER (10, 29). InshPTEN cells, however, XPC levels bound to chromatin weresignificantly lower than in NC cells whereas the levels ofchromatin-bound DDB1 and DDB2, 2 other critical recogni-tion factors for GG-NER (30), were not affected (Supplemen-tary Fig. S4A and B). Kinetic analysis further showed that at 5,10, and 30 minutes post–UVB damage the chromatin-boundXPC levels were significantly reduced in shPTEN cells as

compared with NC cells (Fig. 5D and E; P < 0.05, Student'st test for each time point and 2-way ANOVA for all timepoints). These data indicate that PTEN loss significantlydownregulates the recruitment to chromatin of XPC inresponse to UVB-induced DNA damage.

To further investigate how PTEN regulates XPC proteinlevels, we asked whether PTEN loss decreases XPC expressionat the transcription level. Using the promoter reporter assay,we found that the transcriptional activity of the XPC promoterin shPTEN cells was significantly lower than in NC cells(Fig. 5F; P < 0.05, Student's t test). XPC immunoblot analysisshowed that infection with an adenoviral vector expressingfunctional PTEN (Ad-PTEN) in shPTEN cells increased theprotein levels of XPC (Fig. 5G). Similarly, inhibiting AKTactivation by the phosphoinositide 3-kinase (PI3K) inhibitorLY294002 (10 mmol/L) increased the XPC protein levels in bothHaCaT and NHEK cells (Fig. 5H and I), indicating that AKTactivation mediates XPC suppression caused by PTEN inhibi-tion. To determine whether XPC downregulation is essentialfor impaired GG-NER mediated by inhibition of PTEN, weoverexpressed XPC in cells transfected with siPTEN (Fig. 5J)and then determined GG-NER by measuring the repair of

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Figure 4. PTEN inhibition delays exit from growth arrest post–UVB irradiation. A, quantification of percentage of BrdUrdþ epidermal keratinocytes in Ptenþ/þ

or Ptenþ/� mouse epidermis at different times post–sham irradiation or UVB irradiation. Error bars show SE. *, P < 0.05, significant difference betweenPtenþ/þ and Ptenþ/�mouse epidermis. B, immunoblot analysis of PTEN, p-Chk1, Chk1, p-Chk2, Chk2, g-H2AX, and b-actin at 0.5, 1.5, and 6 hours post–UVBirradiation (5 or 20 mJ/cm2) in HaCaT cells stably infected with a retroviral vector expressing NC or shPTEN. C, immunoblot analysis of PTEN, p-AKT(serine 473), AKT, p-Chk1, Chk1, p-Chk2, Chk2, g-H2AX, p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in NHEK cells transfected with NCor siPTEN. D, immunoblot analysis of p-AKT, AKT, p-Chk1, Chk1, p-Chk2, Chk2, g-H2AX, p53, and GAPDH in NHEK cells infected with an adenoviralvector expressing empty vector (EV) or constitutively active AKT (Aþ). E, immunoblot analysis of p-AKT, AKT, p-Chk1, Chk1, p-Chk2, Chk2, g-H2AX, p53,and GAPDH in vehicle or LY294002 (LY; 10 mmol/L)-treated NHEK cells transfected with siPTEN.






CPDs. Although inhibition of PTEN reduced GG-NER, increas-ing XPC to levels comparable with those in parental cellsrestored GG-NER (Fig. 5K). These data indicate that PTEN lossimpairs GG-NER through downregulating the expression ofXPC at the transcriptional level.

PTEN enhances NER through suppressing AKT-dependent inhibition of p38 pathway

Activation of p38 plays an important role in UVB responsesand has been implicated in regulating NER in human fibro-blasts and HeLa cells (1, 31, 32). To determine the mechanismsby which PTEN/AKT regulates XPC function, we examined thehypothesis that in keratinocytes, PTEN/AKT regulates NERthrough modulating the p38 pathway. To determine the effectof PTEN loss on p38 activation post–UVB irradiation, wedetermined p38 phosphorylation at different times post–UVB irradiation in NC and shPTEN cells. In NC cells, p38phosphorylation increased at 5 to 30 minutes post–UVBirradiation, whereas in shPTEN cells, it was abolished(Fig. 6A). To determine whether AKT is necessary to inhibitUVB-induced p38 activation, we infected HaCaT cells withan adenoviral vector expressing constitutively active AKT

(Myr-AKT; refs. 33, 34). Expression of active AKT inhibitedp38 phosphorylation post–UVB irradiation (Fig. 6B). Tofurther examine the regulation of p38 by AKT in the kerati-nocyte response to UVB, we investigated the effect of repeatedUVB irradiation on p38 as it relates to PTEN inhibition andAKT activation, because our recent studies indicate that UVBradiation activates AKT and suppresses PTEN expression (17).Compared with cells without previous UVB exposure, basaland UVB-induced p38 phosphorylation was inhibited in cellswith previous UVB exposure, in parallel with increased AKTphosphorylation and decreased PTEN expression (Fig. 6C).Inhibition of the AKT pathway by the PI3K inhibitor LY294002increased phosphorylation of p38 in shPTEN cells (Fig. 6D).Consistent with this finding, inhibiting p38 with its specificinhibitor SB203580 (10 mmol/L) in parental HaCaT cellsreduced the XPC levels (Fig. 6E). The presence of SB203580significantly inhibited CPD repair (Fig. 6F). As compared withnormal skin, human SCC exhibited increased AKT phosphor-ylation and decreased p38 phosphorylation and XPC proteinlevels (Fig. 6G). Taken together, these findings suggest thatAKT-dependent inhibition of p38 signaling plays an active rolein compromising GG-NER caused by PTEN loss.

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Figure 5. PTEN loss suppresses XPC expression through increasing AKT signaling. A, immunoblot analysis of XPC, DDB2, DDB1, PTEN, and b-actin(equal loading control) in NC and shPTEN cells. B, immunoblot analysis of XPC, PTEN, and b-actin in HaCaT cells transfected with siPTEN or NC. C,immunoblot analysis of XPC, PTEN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mouse epidermis (both þ/þ and þ/�). D, immunoblotanalysis of XPC, DDB1, DDB2, and histone H3 (loading control) in soluble and/or chromatin-bound fractions fromNC and shPTEN cells at different times (5, 10,and 30 minutes) post–UVB irradiation (20 mJ/cm2). E, quantification of XPC levels (in D). Error bars show SE. *, P < 0.05, significant difference betweenNC and shPTEN cells (in E). F, luciferase reporter assay of XPC promoter in NC and shPTEN cells. G, immunoblot analysis of XPC, PTEN, and b-actin inshPTEN cells infected with an adenoviral vector expressing wild-type PTEN (Ad-PTEN) at different multiplicity of infection (MOI). H, immunoblotanalysis of XPC, p-AKT (serine 473), AKT, and b-actin in shPTEN cells treated with vehicle or LY294002 (LY; 10 mmol/L), as well as in NC HaCaT cells.I, immunoblot analysis of XPC, PTEN, p-AKT, AKT, and GAPDH in siPTEN-transfected cells treated with vehicle or LY294002 (LY; 10 mmol/L),as well as in NC-transfected NHEK cells. J, immunoblot analysis of XPC, PTEN, and b-actin in HaCaT cells transfected with or without the siPTENand the XPC plasmid. K, ELISA of percent repair of CPDs from cells (in J) at different times post–UVB irradiation (20 mJ/cm2).

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Discussion

In this study, we investigated the specific function of PTENin UVB damage responses and skin tumorigenesis. Using lowsuberythemal UVB radiation, we showed that mice with atargeted PTEN downregulation in their epidermis are predis-posed to skin tumorigenesis. These findings support theconclusion that a 50% reduction in the PTEN level increasessusceptibility to skin tumorigenesis following UVB radiation.In human skin malignancies, PTEN is significantly down-regulated in association with sun damage. Our findings furthersupport the conclusion that the PTEN dose is critical fortumor suppression under genotoxic stress.Our findings clearly indicate that PTEN positively regulates

GG-NER by promoting XPC transcription in keratinocytes.Downregulation of XPC transcription caused by PTEN down-regulation provides a previously unrecognized mechanism ininhibiting XPC function in addition to deletion and mutationsin the XPC gene in human SCCs (12). Considering that failureto repair CPD is the principal cause of skin cancer, the role ofPTEN in GG-NER through XPC is critical for the tumor-suppressing action of PTEN for the skin.Our current studies identified the AKT/p38 pathways as

important actors in the regulation of XPC by PTEN, a knownnegative regulator of the PI3K/AKT pathway (13). PTENdownregulation increases AKT activation and enhances cellsurvival after UVB damage (17). Indeed, the regulation of thep38 pathway by AKT and its impact on cell survival have beenreported in several other models, including endothelial cells

(35), b-cell (36), and E1A-induced apoptosis (37). Although therole of the p38 pathway in UVB responses is complex (1, 31), inhuman fibroblasts and HeLa cells, p38 has recently beenshown to promote GG-NER by stabilizing DDB2 (32). In linewith these findings, our studies have shown that the reductionin the p38 pathway through increased AKT activation uponPTEN inhibition can be mimicked by the effect of biochemicalinhibition of p38 in regulating XPC levels and GG-NER,suggesting that in PTEN-downregulated keratinocytes, sup-pression of p38 by AKT signaling reduces XPC levels and thusimpairs GG-NER. In SIRT1-inhibited cells, however, PTENinhibition and AKT activation due to increased PTEN acet-ylation suppress XPC expression through increased nucleartranslocation of a transcription repressor p130 (38) but not thep38 pathway (data not shown). Further investigation is neededto reveal these different molecular mechanisms depending onSIRT1 status. Taken together, these results imply that thePTEN/AKT/p38 axis is critical for GG-NER and regulatestumor susceptibility.

Interestingly, we found that, in contrast to the response toionizing or UVC radiation (23, 24), PTEN loss increases UVB-induced Chk1 activation in keratinocytes in an AKT-indepen-dent manner. It seems that AKT plays differential roles in DNArepair and checkpoint response. Therefore, the effect of PTENon checkpoint pathways may depend on the type of DNAdamage, the repair efficiency, the waveband-specific signalingpathways betweenUVB andUVC (31, 39), and/or the cell-type–specific response to PTEN inhibition, similar to the differentfindings on the regulation of Rad51 expression (40, 41). Much

A B C

p-p38

+ + + –UVB – + + +Min 5 10 30 5 10 30

NC shPTEN

p-p38

+ + –Myr–AKT –+ + + UVB –

p-p38+ + –UVB –

1st 2nd

p-AKTPTEN

AKT

p38p38

p-AKTAKT

Myr-AKT PTEN

p-AKTp38

AKT

ββ

D E80 Veh

Fβ-Actinββ-Actin

ββ-Actin

ββ-Actin

++ –+ –UVB –Veh LY VehshPTEN NC G

Normal SCC

PTEN

**

20

40

60

% R

epai

r SB

AKTp-AKT

p-p38p38

+SB –XPC

p-p38

p-AKTAKT

00 6 24

Hours post–UVBirradiation

PTEN XPC

GAPDH

p38

Figure 6. Inhibition of p38 signaling by AKT is critical for inhibiting GG-NER by PTEN downregulation. A, immunoblot analysis of p-p38, p38, PTEN, p-AKT(serine 473), and AKT in NC or shPTEN cells at different times post–UVB irradiation (20 mJ/cm2). B, immunoblot analysis of p-p38, p38, p-AKT, AKT,Myr-AKT (by HA tag), and b-actin in HaCaT cells infected with an adenoviral vector expressing constitutively active AKT (Myr-AKT) or empty vector at 1.5 hourspost–sham irradiation or UVB irradiation. C, immunoblot analysis of p-p38, p38, p-AKT, AKT, PTEN, and b-actin at 6 hours post–UVB irradiation incells that have not been exposed to UVB (1st) and in cells that have been exposed to UVB (20 mJ/cm2) once 1 week before (2nd). D, immunoblot analysisof p-p38, p38, p-AKT (serine 473), AKT, PTEN, and b-actin in shPTEN cells treated with vehicle (Veh) or LY294002 (LY; 10 mmol/L) at 1.5 hourspost–UVB irradiation in comparison with NC cells. E, immunoblot analysis of XPC and b-actin in HaCaT cells treated with vehicle or SB203580 (SB; 10 mmol/L).F, ELISA of percent repair of CPDs from cells (in E) at different times post–UVB irradiation (20 mJ/cm2). G, immunoblot analysis of PTEN, p-AKT, AKT,p-p38, p38, XPC, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in normal human skin and human SCCs.






more investigation is needed to elucidate these importantdifferences at the molecular level. Nevertheless, our findingsindicate that normal PTEN expression and AKT activation arerequired for proper XPC expression and thus better DNArepair. These data also suggest that, in keratinocyte responseto UVB-induced DNA damage, the checkpoint response ispassive, associated with the levels of unrepaired DNA damage,anddependson thePTEN levels but notAKTactivation, furtherunderscoring the importance of constitutive PTEN levels inreducing susceptibility to UVB tumorigenesis.

In summary, we showed that PTEN downregulation is apredisposing factor for UVB-induced skin carcinogenesis invivo and negatively regulates UVB-inducedDNAdamage repairthrough limiting XPC expression. Taken together, our findingssuggest that the interaction between PTEN levels and UVB iscritical for early tumorigenesis and tumor progression.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Terri Li for the PTEN immunohistochemistry, Dr. Pradip Ray-chaudhuri (University of Illinois at Chicago, Chicago, IL) for kindly providing theXPC promoter luciferase construct, and Dr. Ann Motten for critical reading ofthe manuscript.

In Memoriam

This article is dedicated to the memory of Dr. Colin F. Chignell.

Grant Support

This work was supported by NIH grant ES016936 (Y.Y. He), the University ofChicago Comprehensive Cancer Center Pilot program (P30 CA014599), theCTSA (NIH UL1RR024999), the NIH/NIEHS intramural program, and UC Friendsof Dermatology Research Funds.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 22, 2010; revised May 27, 2011; accepted May 30, 2011;published OnlineFirst July 19, 2011.

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2011;71:5287-5295. Published OnlineFirst July 19, 2011.Cancer Res Mei Ming, Li Feng, Christopher R. Shea, et al. PTEN Positively Regulates UVB-Induced DNA Damage Repair

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