The incidence of skin cancer is higher than all other cancers and continues to increase, with an average annual cost over 8 billion in the United States. As a result, identifying molecular pathway alterations that occur with UV exposure to strategize more effective preventive and therapeutic approaches is essential. To that end, we evaluated phosphorylation of proteins within the PI3K/Akt and MAPK pathways by immunohistochemistry in sun-protected skin after acute doses of physiologically relevant solar-simulated ultraviolet light (SSL) in 24 volunteers. Biopsies were performed at baseline, 5 minutes, 1, 5, and 24 hours after SSL irradiation. Within the PI3K/Akt pathway, we found activation of Akt (serine 473) to be significantly increased at 5 hours while mTOR (serine 2448) was strongly activated early and was sustained over 24 hours after SSL. Downstream, we observed a marked and sustained increase in phospho-S6 (serine 235/S236), whereas phospho-4E-BP1 (threonines 37/46) was increased only at 24 hours. Within the MAPK pathway, SSL-induced expression of phospho-p38 (threonine 180/tyrosine 182) peaked at 1 to 5 hours. ERK 1/2 was observed to be immediate and sustained after SSL irradiation. Phosphorylation of histone H3 (serine 10), a core structural protein of the nucleosome, peaked at 5 hours after SSL irradiation. The expression of both p53 and COX-2 was increased at 5 hours and was maximal at 24 hours after SSL irradiation. Apoptosis was significantly increased at 24 hours as expected and indicative of a sunburn-type response to SSL. Understanding the timing of key protein expression changes in response to SSL will aid in development of mechanistic-based approaches for the prevention and control of skin cancers. Cancer Prev Res; 8(8); 720–8. ©2015 AACR. An estimated 3 million nonmelanoma skin cancer (NMSC) cases are diagnosed in the United States yearly with an estimated 4% yearly increase in the Medicare population (1). Cancer registries do not require NMSCs to be reported, and as such, precise estimations of NMSC numbers are problematic. Approximately 75% to 80% of NMSCs diagnoses are basal cell carcinomas and 18% are squamous cell carcinomas (SCC). This high incidence of skin cancer is directly related to chronic solar radiation exposure (2). SCCs result from the malignant transformation and proliferation of squamous cells, the most abundant cell in the epidermis. Although NMSCs have a fairly low metastatic potential, the morbidity associated with NMSCs is high and available treatments can be disfiguring as well as expensive (3). In Medicare recipients older than 65 years, approved physician charges for treatment of NMSC totaled at least285 million per year in 2001, while 2011 estimates indicate an annual cost in both Medicare and non-Medicare populations at over \$8 billion (3, 4). This striking massive increase in treatment costs further illustrates the growing problem of skin cancer. Current primary skin cancer prevention strategies, including sun avoidance and UV protection, have had limited success (5). Therefore, a need exists for new mechanism–based approaches to prevent and treat solar radiation–induced skin cancers. To that end, determining the effect of solar radiation on skin in both the acute and chronic settings is essential.

UV radiation has been shown to be a complete carcinogen by way of mutations, many of which are signature UV mutations, in the DNA of critical genes. In addition, UV can act as a promoter through the activation of signal transduction pathways (6). The UV spectrum that reaches the earth's surface is made up of approximately 95% UVA (320–400 nm) and approximately 5% UVB (280–320 nm; ref. 7). The majority of studies on the role of UV in activation of signaling pathways and skin carcinogenesis have focused on UVA (8, 9) or UVB (10–12) alone, and in some cases UVC, which does not reach the earth's surface in appreciable amounts (13). Fewer studies have focused on the entire spectra of UVA and UVB combined in a ratio that mimics the solar spectrum (13). Strong experimental evidence indicates that exposure of epidermal cells to UV results in the activation of numerous signal transduction pathways that include the PI3K/Akt and MAPK cascades (7).

The PI3K/protein kinase B (PI3K/Akt) pathway is an effector of cell survival and proliferation (14). Stimulation of the PI3K/Akt pathway results in the activation of downstream kinases that include mTOR, p70 ribosomal protein S6 kinase 1 (p70S6K1), and 4E-binding protein 1 (4E-BP1). Phosphorylation of 4E-BP1 by mTOR inhibits its activity, which in turn leads to the activation of cellular translational machinery through inhibition of eIF4E by phosphorylated 4E-BP1. Inhibition of 4E-BP1 results in the inhibition of eIF4E, which ultimately results in the activation of translation machinery. Also, mTOR can directly activate p70S6K1, which can then activate a downstream target, ribosomal protein S6 (S6), leading to the initiation of protein synthesis (15). Studies have shown that mTOR is activated by UVB, and further aberrant activation of the mTOR pathway has been implicated in the development of SCC (6, 16). mTOR is known to coordinate cell-cycle progression, cell survival, and growth in response to genetic, epigenetic, and environmental conditions, including UV-mediated cellular stress (17).

Signaling cascades that lead to activation of MAPKs are also important in UV-induced cutaneous SCC (18), particularly, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), c-Jun N-terminal kinases (JNK), and p38 (12, 13). The MAPK signaling pathways are activated by growth factors and stress stimuli and they play central a role in transducing extracellular signals to target proteins involved in cell growth and proliferation (19, 20). Activated B-Raf phosphorylates and activates ERK 1/2, which in turn phosphorylates several substrates, including members of the 90-kDa ribosomal S6 kinase (RS6K; refs. 19, 20). These signaling pathways are regulated by a complex network that includes crosstalk and feedback mechanisms.

The objective of this study was is to determine whether acute doses of solar light [using a solar simulator to generate a measured erythemal dose of solar-simulated light (SSL)] would activate the expression of key proteins/phosphoproteins within the PI3K/Akt/mTOR and MAPK signaling pathways in normally sun-protected skin of healthy volunteers.

### Study population

Study participants were recruited from a pool of subjects who had been previously screened and/or participated in previous skin chemoprevention trials and had agreed to be recontacted for future studies. The eligibility criteria for participants from this pool included age of 18 years or older and Fitzpatrick skin types II (burns easily, tans poorly) or III (burns moderately, tans gradually). Exclusion criteria included immunosuppression, serious concurrent illness, invasive cancer (including any type of skin cancer) within the past 5 years, and baseline serum chemistry values outside of normal limits. In addition, those using photosensitizing medications or topical medications on the test area during the past 30 days were ineligible. Individuals taking mega doses of vitamins were not eligible [i.e., more than five times the Recommended Daily Allowance (RDA), more than five capsules of multivitamins, 400 IU of vitamin E, 200 g of selenium, and 1 g of vitamin C]. Additional exclusion criteria included individuals with a history of sun exposure to the buttocks within 30 days of randomization and participants must have agreed to avoid sun exposure during the study period. Finally, individuals with a known allergy to lidocaine were ineligible. The University of Arizona Institutional Review Board approved the study and written informed consent was obtained from all study participants.

### Minimal erythema dose

The minimal erythema dose (MED) of SSL was determined for each individual using a Multiport UV Solar Simulator Model 600 (Solar Light Co.). The spectrum of light generated by the Solar Simulator consisted of 8.7% UVB and 91.3% UVA (21). The dose of emission was precisely regulated to include the UVA and UVB range (290–390 nm). MED was defined as the smallest dose of energy necessary to produce confluent erythema with distinct borders at 22 to 26 hours after exposure. MED was determined on a buttock area normally protected from sunlight. Each test area was subdivided into six subsites (each 1 cm2) corresponding to the liquid light guide pattern on the solar simulator. The solar simulator was calibrated prior to each use and a series of six increasing SSL radiation exposures were administered concurrently at each site area. Following exposure, the test sites were covered until evaluations were completed (22–26 hours).

### Administration of 2 to 3 MED

After determination of the MED for each individual, the contralateral buttock was exposed to one of the following: 2, 2.5, or 3 times the MED. A 6-mm skin punch biopsy was collected from one buttock at baseline prior to SSL exposure and additional 6 mm punch biopsies were removed at 5 minutes, 1, 5, and 24 hours after SSL irradiation. Biopsy sites were then sutured and subjects returned to the clinic for suture removal in approximately 1 week. A dose of 2 MED of SSL was applied to sun-protected skin of 12 subjects. Two additional groups of 6 subjects each received doses of 2.5 and 3 MED SSL.

### Immunohistochemistry

Biopsies were immediately fixed in 10% neutral buffered formalin for 24 hours then transferred to 70% ethanol prior to routine processing and paraffin embedding. Tissue sections (5 μm) were deparaffinized and rehydrated. All tissue sections were subjected to antigen retrieval using Diva buffer in a Decloaking Chamber Pro (Biocare) for 30 seconds. Immunohistochemical staining was performed using a Vectastain avidin biotin–based peroxidase kit with a Novared substrate (Vector Laboratories, PK-6100 and SK-4800) or a Vectastain ABC-AP kit and a Vector red alkaline phosphatase substrate (AK-5000 and SK-5100) depending on the antibody, and a hematoxylin counterstain (Leica Microsystems, Inc.). Positive and negative controls were included for each antibody. Antibodies included those to detect phospho-Akt (p-Akt serine 473, #3787) at 1:100 overnight, p-mTOR (serine 2448, #2976) at 1:300 overnight, p-4E-BP1 (threonine 37/46, #2855) at 1:100 overnight, phosphorylated S6 protein (serine 235/236, #2211) at 1:200 overnight, p-p38 (threonine 180/tyrosine 182, #4511) at 1:800 for 1 hour, p-ERK 1/2 (threonine 202/tyrosine 204, #4376) at 1:100 overnight, p-histone H3 (serine 10, #9701) at 1:100 overnight, and cleaved caspase-3 (#9661) at 1:1,000 overnight and all purchased from Cell Signaling Technology. Antibodies to detect p53 (#OP43) at 1:100 for 1 hour and PCNA at 1:800 for 1 hour were obtained from EMD Chemicals, Inc. (#NA03). The antibody to detect COX-2 at 1:100 for 1 hour was from Cayman Chemical (#160112).

Immunohistochemically stained tissue sections were measured using the ImagePro Plus (Media Cybernetics) software system, a Leica DMR microscope, and a Sony 3CCD color video camera. The mean positive nuclear or cytoplasmic area per 40× field was determined for each biopsy. The number of apoptotic cells was assessed on the basis of morphology (i.e., condensed and/or pyknotic nuclei, eosinophilic cytoplasm, formation of apoptotic bodies) per 100 basal keratinocytes on hematoxylin and eosin (H&E)-stained sections.

### Statistical analysis

The percent positive area was calculated for each marker with the exception of the apoptotic markers (cleaved caspase-3 and morphologic apoptosis). Apoptosis was expressed as the number per 100 basal keratinocytes. As seen in Table 1, mean and SEs were used to describe the data at each time point, where the clustered robust SE was used to adjust for possible correlation of multiple measurements per study participant. The primary research question was to compare the expression level of each IHC marker between baseline and at each of the four time points after SSL exposure. Distribution of the level of each marker was checked graphically and the appropriate transformation, if needed, was applied to achieve approximate normality. The comparison of each maker (possibly after transformation) was performed using the generalized estimating equations (GEE) models, which adjusted for any potential correlation between multiple measurements of the same study subject. For any marker where no appropriate transformation is available to remove heavy skewness the rank was used as the response variable of the GEE models. The three MED groups were adjusted for in the models as confounders. The change in each IHC marker was also compared between the three MED groups using a Wald test based on fitted GEE models (data presented in Supplementary Data). GEE models were also used to conduct a test for trend across the five time points, with adjustment for MED levels. All analyses were conducted using Stata version 13 (Stata Corporation). All reported P values were not adjusted for multiple comparisons.

Table 1.

Means ± SE for markers

BiomarkerNBaseline5 MinutesPa1 hourPa5 HoursPa24 HoursPaTime trend P
p-Aktb 24 12.3 ± 2.0 10.8 ± 1.7 0.502 13.9 ± 2.1 0.383 17.8 ± 2.2 ↑0.010 12.7 ± 1.7 0.658 0.108
p-mTORb 24 21.4 ± 4.3 23.2 ± 3.9 0.340 36.0 ± 6.0 ↑0.020 46.7 ± 5.9 ↑<0.0001 68.2 ± 5.5 ↑<0.0001 ↑<0.0001
p-4E-BP1b 24 23.9 ± 4.2 30.6 ± 4.4 0.075 29.1 ± 4.7 0.092 28.7 ± 4.2 0.177 49.2 ± 4.7 ↑<0.0001 ↑<0.0001
p-S6b,c 24 4.9 ± 1.1 4.7 ± 1.1 0.519 14.0 ± 3.1 ↑<0.0001 35.7 ± 5.8 ↑<0.0001 50.7 ± 5.0 ↑<0.0001 ↑<0.0001
p-p38b 24 7.0 ± 1.1 7.4 ± 1.1 0.663 18.3 ± 1.7 ↑<0.0001 18.7 ± 2.4 ↑<0.0001 14.8 ± 2.1 ↑<0.0001 ↑<0.0001
p-ERK 1/2c,d 24 4.2 ± 1.0 6.0 ± 1.3 ↑0.012 16.0 ± 4.7 ↑0.004 22.0 ± 4.1 ↑<0.0001 23.8 ± 3.3 ↑<0.0001 ↑<0.0001
p-histone H3c,d 24 1.5 ± 0.4 2.2 ± 0.7 0.816 8.4 ± 2.0 ↑<0.0001 29.5 ± 5.7 ↑<0.0001 9.1 ± 2.3 ↑<0.0001 ↑<0.0001
COX-2d 24 0.3 ± 0.1 0.1 ± 0.03 0.116 0.4 ± 0.2 0.590 2.0 ± 0.3 ↑<0.0001 17.1 ± 1.4 ↑<0.0001 ↑<0.0001
p53d 22 1.3 ± 0.3 1.4 ± 0.4 0.837 1.1 ± 0.3 0.844 4.5 ± 1.2 ↑<0.0001 26.1 ± 2.8 ↑<0.0001 ↑<0.0001
PCNAb 24 25.6 ± 2.5 20.8 ± 2.2 ↓0.023 26.2 ± 3.4 0.930 30.1 ± 3.3 0.214 21.0 ± 2.2 0.114 0.832
Cleaved caspase-3c,d 22 0.0 ± 0.00 0.05 ± 0.04 0.103 0.0 ± 0.00 0.973 0.04 ± 0.03 0.111 4.15 ± 1.14 ↑<0.0001 ↑<0.0001
Morphologic apoptosis 24 0.09 ± 0.02 0.04 ± 0.06 ↓0.016 0.02 ± 0.04 ↓<0.0001 0.24 ± 0.25 ↑0.011 10.4 ± 6.9 ↑<0.0001 ↑<0.0001
BiomarkerNBaseline5 MinutesPa1 hourPa5 HoursPa24 HoursPaTime trend P
p-Aktb 24 12.3 ± 2.0 10.8 ± 1.7 0.502 13.9 ± 2.1 0.383 17.8 ± 2.2 ↑0.010 12.7 ± 1.7 0.658 0.108
p-mTORb 24 21.4 ± 4.3 23.2 ± 3.9 0.340 36.0 ± 6.0 ↑0.020 46.7 ± 5.9 ↑<0.0001 68.2 ± 5.5 ↑<0.0001 ↑<0.0001
p-4E-BP1b 24 23.9 ± 4.2 30.6 ± 4.4 0.075 29.1 ± 4.7 0.092 28.7 ± 4.2 0.177 49.2 ± 4.7 ↑<0.0001 ↑<0.0001
p-S6b,c 24 4.9 ± 1.1 4.7 ± 1.1 0.519 14.0 ± 3.1 ↑<0.0001 35.7 ± 5.8 ↑<0.0001 50.7 ± 5.0 ↑<0.0001 ↑<0.0001
p-p38b 24 7.0 ± 1.1 7.4 ± 1.1 0.663 18.3 ± 1.7 ↑<0.0001 18.7 ± 2.4 ↑<0.0001 14.8 ± 2.1 ↑<0.0001 ↑<0.0001
p-ERK 1/2c,d 24 4.2 ± 1.0 6.0 ± 1.3 ↑0.012 16.0 ± 4.7 ↑0.004 22.0 ± 4.1 ↑<0.0001 23.8 ± 3.3 ↑<0.0001 ↑<0.0001
p-histone H3c,d 24 1.5 ± 0.4 2.2 ± 0.7 0.816 8.4 ± 2.0 ↑<0.0001 29.5 ± 5.7 ↑<0.0001 9.1 ± 2.3 ↑<0.0001 ↑<0.0001
COX-2d 24 0.3 ± 0.1 0.1 ± 0.03 0.116 0.4 ± 0.2 0.590 2.0 ± 0.3 ↑<0.0001 17.1 ± 1.4 ↑<0.0001 ↑<0.0001
p53d 22 1.3 ± 0.3 1.4 ± 0.4 0.837 1.1 ± 0.3 0.844 4.5 ± 1.2 ↑<0.0001 26.1 ± 2.8 ↑<0.0001 ↑<0.0001
PCNAb 24 25.6 ± 2.5 20.8 ± 2.2 ↓0.023 26.2 ± 3.4 0.930 30.1 ± 3.3 0.214 21.0 ± 2.2 0.114 0.832
Cleaved caspase-3c,d 22 0.0 ± 0.00 0.05 ± 0.04 0.103 0.0 ± 0.00 0.973 0.04 ± 0.03 0.111 4.15 ± 1.14 ↑<0.0001 ↑<0.0001
Morphologic apoptosis 24 0.09 ± 0.02 0.04 ± 0.06 ↓0.016 0.02 ± 0.04 ↓<0.0001 0.24 ± 0.25 ↑0.011 10.4 ± 6.9 ↑<0.0001 ↑<0.0001

NOTE: Arrows before P values show the direction of change.

aP values are for the difference between baseline and each time point.

by = √x.

cMED was controlled for in the model.

dRanks were used in the model.

The study included 12 males and 12 females with an average age of 68.3 ± 9.4 (mean ± SD) years for males and 61.1 ± 8.1 years for females. Eighty-three percent (20/24) of the subjects listed themselves as Caucasian, 12.5% (3/24) listed themselves as having more than one race, and 1.7% (1/24) as other. Forty-two percent (10/24) of the subjects had Fitzpatrick skin type II and 58% (14/24) skin type III. The solar simulator provided 8.7% of UVB and 91.3% of UVA (21). The aim of the study was to expose subjects to low-dose SSL. To that end, 12 participants were exposed to 2 MED with an average (± SE) dose of 3.6 ± 0.5 J/cm2 UVA and 51.6 ± 7.0 mJ/cm2 UVB. After the initial group of 12 subjects, the study was expanded to add 2.5 and 3 MED exposures. Six participants were exposed to 2.5 MED with an average dose of 4.8 ± 0.5 J/cm2 UVA and 70.0 ± 6.8 mJ/cm2 UVB and 6 participants were exposed to 3 MED with an average dose of 5.6 ± 0.8 J/cm2 UVA and 81.4 ± 11.9 mJ/cm2 UVB. Our primary question was to determine whether there was modulation of key proteins/phosphoproteins within the PI3K/Akt/mTOR and MAPK signaling pathways after SSL, not to compare MED levels. The comparison of protein expression by MED level is presented as Supplementary Figs. S1–S3. To account for the 3 MED levels, statistical models adjusted for MED level as a confounder.

Study results are summarized in Table 1, which shows the mean ± SE for each marker and for each time point. As noted earlier, biomarker expression was averaged over all subjects (12 subjects at 2 MED, 6 at 2.5 MED, and 6 at 3 MED). In Table 1, data are expressed as the percent positive area for all markers with the exception of apoptosis and cleaved caspase-3, which are expressed as the number of apoptotic cells per 100 basal cells. Representative immunohistochemically stained micrographs of each biomarker are shown in Figs. 1–5. We previously reported that in human sun-protected skin, 4 MED of UV resulted in the phosphorylation of MAPKAPK2, CREB, c-JUN, p38, GSK-3b, and p53, leading to markedly increased levels of c-FOS, COX-2, and apoptosis in the epidermal cells (12). In the current study, we investigated the impact of a lower dose of SSL on key proteins within the PI3K/Akt and MAPK pathways by immunohistochemistry. We found that a single dose of 2–3 MED of SSL activated the PI3K/Akt signaling pathway with a small but statistically significant increase in cytoplasmic and nuclear staining of p-Akt at serine 473 (Fig. 1A–D, 1.4-fold increase at 5 hours after SSL irradiation; P = 0.01). We next evaluated mTOR, a direct substrate of activated Akt, for phosphorylation at serine 2448. p-mTOR was present in both the cytoplasm and nucleus of epidermal cells (Fig. 1E–H) and its expression was significantly increased from 1 to 24 hours (1.7-fold; P = 0.02, 2.2-fold; P < 0.001, and 3.2-fold; P < 0.001 at 1, 5, and 24 hours, respectively). Another PI3K/Akt signaling protein that is downstream of both mTOR and p70S6K1 is S6. We found that p-S6 at serines 235 and 236 showed both cytoplasmic and nuclear staining (Fig. 2A–D) and its expression was significantly increased from 1 to 24 hours (2.9-fold; P < 0.001, 7.3-fold; P < 0.001, 10.3-fold; P < 0.001 at 1, 5, and 24 hours, respectively). We then examined p-4E-BP1 (threonines 37/46), a downstream target of activated mTOR, and found statistically significant increased primarily nuclear immunostaining (Fig. 2E–H) at 24 hours (2.1-fold; P < 0.001).

Figure 1.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. Phospho-Akt (serine 473), at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). Phospho-mTOR (serine 2448) expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Images, 400× magnification; scale bar, 50 μm. Red nuclear and cytoplasmic staining is positive and blue is negative.

Figure 1.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. Phospho-Akt (serine 473), at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). Phospho-mTOR (serine 2448) expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Images, 400× magnification; scale bar, 50 μm. Red nuclear and cytoplasmic staining is positive and blue is negative.

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Figure 2.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. Phospho-S6 (serine 235/236) expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). Phospho-4E-BP1 (threonine 37/46) expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Images, 400× magnification; scale bar, 50 μm. Red nuclear and cytoplasmic staining is positive and blue is negative.

Figure 2.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. Phospho-S6 (serine 235/236) expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). Phospho-4E-BP1 (threonine 37/46) expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Images, 400× magnification; scale bar, 50 μm. Red nuclear and cytoplasmic staining is positive and blue is negative.

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Figure 3.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. Phospho-p38 (threonine 180/tyrosine 182) expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). Phospho-ERK 1/2 (threonine 202/tyrosine 204) expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Phospho-histone H3 (serine 10) expression at baseline (I), 1 hour (J), 5 hours (K), and 24 hours (L). Images, 400× magnification; scale bar, 50 μm. Red (phospho-ERK 1/2 and phospho-histone H3) or brown (phospho-p38) nuclear and cytoplasmic stains represent positive and blue stain represents negative staining.

Figure 3.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. Phospho-p38 (threonine 180/tyrosine 182) expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). Phospho-ERK 1/2 (threonine 202/tyrosine 204) expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Phospho-histone H3 (serine 10) expression at baseline (I), 1 hour (J), 5 hours (K), and 24 hours (L). Images, 400× magnification; scale bar, 50 μm. Red (phospho-ERK 1/2 and phospho-histone H3) or brown (phospho-p38) nuclear and cytoplasmic stains represent positive and blue stain represents negative staining.

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Figure 4.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. COX-2 expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). p53 expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Images, 400× magnification; scale bar, 50 μm. Brown nuclear and cytoplasmic stains represent positive and blue stain represents negative staining.

Figure 4.

Examples of immunohistochemically stained proteins in sun-protected skin after 2 to 3 MED of SSL irradiation. COX-2 expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D). p53 expression at baseline (E), 1 hour (F), 5 hours (G), and 24 hours (H). Images, 400× magnification; scale bar, 50 μm. Brown nuclear and cytoplasmic stains represent positive and blue stain represents negative staining.

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Figure 5.

Examples of proliferation and apoptosis in sun-protected skin after 2 to 3 MED of SSL irradiation. PCNA expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D) after SSL irradiation. Cleaved caspase-3 at baseline (A) and 24 hours (B). Apoptotic cells on H&E-stained slides at baseline (C) and at 24 hours with inset in the lower left at a magnification of 1,000× (D). Images, 400× magnification; scale bar, 50 μm. Brown nuclear stain is positive for PCNA (A–D). Red stain is positive and blue is negative for cleaved caspase-3 (E and F). Arrows point to apoptotic cells present at 24 hours after SSL irradiation.

Figure 5.

Examples of proliferation and apoptosis in sun-protected skin after 2 to 3 MED of SSL irradiation. PCNA expression at baseline (A), 1 hour (B), 5 hours (C), and 24 hours (D) after SSL irradiation. Cleaved caspase-3 at baseline (A) and 24 hours (B). Apoptotic cells on H&E-stained slides at baseline (C) and at 24 hours with inset in the lower left at a magnification of 1,000× (D). Images, 400× magnification; scale bar, 50 μm. Brown nuclear stain is positive for PCNA (A–D). Red stain is positive and blue is negative for cleaved caspase-3 (E and F). Arrows point to apoptotic cells present at 24 hours after SSL irradiation.

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We next explored the effect of 2–3 MEDs of SSL on the MAPK signaling pathway. To that end, we examined p38 for dual phosphorylation at threonine 180 and tyrosine 182 and found predominately nuclear expression (Fig. 3A–D) from 1 to 24 hours (2.6-fold; P < 0.001, 2.7-fold; P < 0.001, and 2.1-fold; P < 0.001 at 1, 5, and 24 hours after SSL irradiation, respectively). Another key MAPK pathway protein, ERK 1/2, was evaluated for dual phosphorylation on threonine 202 and tyrosine 204. Nuclear expression of p-ERK 1/2 (Fig. 2D–H) was presented early (1.4-fold at 5 minutes; P = 0.012) and increased from 1 through 24 hours (3.9-fold; P = 0.004 at 1 hour, 5.2-fold at 5 hours; P < 0.001, and 5.6-fold at 24 hours; P < 0.001). Subsequently, we found that nuclear expression of p-histone H3 at serine 10 (Fig. 2I–L) was significantly increased from 1 to 24 hours with a peak at 5 hours (5.6-fold; P < 0.001 at 1 hour, 19.7-fold; P < 0.001 at 5 hours, and 6.1-fold; P < 0.001 at 24 hours).

In our evaluation of downstream events, we next assessed expression of COX-2, p53, PCNA, cleaved caspase-3, and morphologic apoptosis on H&E. Cytoplasmic expression of COX-2 (Fig. 4A–D) was negligible at baseline but was significantly increased starting at 5 hours (2.3-fold; P < 0.001) and was maximal at 24 hours (13.9-fold; P < 0.001) after SSL irradiation with expression primarily in the basal layer. Statistically significant total p53 nuclear expression (Fig. 4E–H) was observed beginning at 5 hours (3.4-fold; P < 0.001) and was maximal at 24 hours (20.1-fold; P < 0.001) after SSL irradiation.

As shown in Fig. 5A–D, PCNA expression yielded little change over time after SSL irradiation with the exception of a small decrease at 5 minutes after SSL irradiation (P = 0.023). Apoptosis was measured by two methods, which included cleaved caspase-3 and a count of morphologically apoptotic cells (sunburn cells) as observed by H&E. The number of cleaved caspase-3–positive cells (Fig. 5E and F) was significantly increased (5-fold; P < 0.001) at 24 hours after SSL irradiation. The number of morphologically apoptotic cells as visualized by H&E (Fig. 5G and H) showed a statistically significant 10.3-fold (P < 0.001) increase at 24 hours after SSL irradiation.

Analysis of the effect of MED on biomarker expression is shown in Supplementary Figs. S1–S3. There was not a statistically significant difference in expression of p-Akt (Fig. 1A), p-mTOR (Fig. 1B), p-4E-BP1 (Fig. 1D), p-p38 (Fig. 2A), COX-2 (Fig. 2D), p53 (Fig. 3A), or morphologic apoptosis (Fig. 3C) by MED level. For p-S6, there was a statistically significant difference (Fig. 1C, P < 0.001) between the three MED levels. The three SSL levels followed the same pattern over time (baseline, 1, 5, and 24 hours) but the 2 MED dose showed overall higher p-S6 expression. This was also the case for p-ERK 1/2 (Fig. 2B, P = 0.009) and p-histone H3 (Fig. 2C, P = 0.007). In the case of cleaved caspase-3 (Fig. 3B, P = 0.007), it appeared that 2.5 MED had higher expression.

Previously we reported that 4 MED of UV resulted in activation of proteins within the MAPK, PI3K, p53, and JNK pathways (12). In the current study, we investigated the impact of a lower and more applicable dose of SSL on key proteins on MAPK signaling. We then expanded the work to assess the impact of acute low dose SSL on key proteins within the PI3K/Akt pathway, an essential pathway for which there are limited data with regard to the modulations that occur after both acute and chronic exposure to solar radiation.

Because skin is exposed to daily low dose UV over an individual's lifetime, the investigation of signaling pathway alterations that result from acute exposures of normal skin to SSL may have relevance to skin carcinogenesis. We expect to find both significant overlap, as well as significant differences, in the pathways that are modulated as a result of chronic solar light exposure (i.e., AK's and SCCs) compared with acute exposures. Although chronic exposure to sunlight is necessary to acquire the critical combination of gene mutations and altered cell signaling in skin for the development of UV-induced SCC, we propose that the study of acute effects of UV may serve as a model for investigation of the effects of SSL on this complex array of signal transduction pathways that are also likely involved in UV-induced carcinogenesis. The study of both acute and chronic solar light exposure may also serve as a model whereby critical signaling pathways and individual proteins can be identified as potential biomarkers for use as endpoints in clinical trials or as companion biomarkers. Furthermore, the study of both acute and chronic solar light exposure may allow for the development of interventions such as targeted therapies in a much shorter amount of time than required for studies using cancer as an endpoint.

Recently, our group (22) found cell-signaling derangements in SCC compared with AK or to normal skin using reverse-phase protein microarray analysis. We found statistically significant differences in protein expression within the MEK–ERKs and Akt/mTOR signaling pathways (22). Germane to the current study, we found phosphorylated forms of p38, ERK 1/2, mTOR, p70S6K1, 4E-BP1, and histone H3 to be significantly increased in SCC compared with AK. P-4E-BP1 and p-Akt were also increased in AK compared with normal skin (22). Chen and colleagues (16) also showed that p-Akt (serine 473), p-mTOR (serine 2448), S6, and po-4E-BP1 (S65) were higher in SCC compared with AK.

Activation of the PI3K/Akt pathway is an important element in cell survival and proliferation after UV (14). In the current study, we show a small but significant increase in the phosphorylation of Akt (serine 473) at 5 hours. Previous studies of Akt activation have primarily focused on UVB or UVA alone in keratinocytes or mouse skin with fewer human studies (11, 23–26). We next evaluated p-mTOR (serine 2448), a direct substrate of activated Akt, and found early and strong sustained activation. mTOR exists in two protein complexes: rapamycin-sensitive mTOR complex 1 (mTORC1), which is associated with phospho-mTOR (serine 2448), and the rapamycin-resistant mTOR complex 2 (mTORC2), which is associated with p-mTOR (serine 2481; refs. 6, 27–29). Although there is limited information on the effect of UV on skin, both mTORC1 and mTORC2 are thought to play complementary roles in controlling proliferation and apoptosis after UVB irradiation (6, 29).

p70S6K1 is a substrate of p-mTOR and when activated it phosphorylates its downstream target, S6 to initiate protein synthesis. In the current study, we observed a marked and sustained increase in p-S6 (serine 235/S236) expression. Studies have shown that UV-irradiation is associated with increased p70S6 kinase phosphorylation, but fewer studies have addressed the activation of S6 following UV irradiation (25). The activity of p-4E-BP1, another downstream target of mTOR, is inhibited through its interaction with mTOR (7). 4E-BP1 is a translational repressor that negatively regulates the eukaryotic initiation factor, eIF-4 (30). We found that phosphorylation of 4E-BP1 at threonines 37 and T46 was significantly increased 24 hours. A limited number of studies have demonstrated activation of 4E-BP1 by UVB irradiation (18, 30).

We next explored the effect of 2 to 3 MEDs of SSL on the MAPK signaling pathway. The MAPK family includes ERKs, JNKs, and p38 kinases (18). MAPK pathways are activated as a result of growth factors or stress such as UV irradiation and have central roles in transducing extracellular signaling to intracellular target proteins involved in cell growth and proliferation (18). We previously reported that p-p38 was increased at 1 hour with a further increase at 24 hours after 4 MEDs (12). In the current study, 2 to 3 MED of SSL resulted in peak phosphorylation between 1 and 5 hours followed by a decrease at 24 hours. Our results using 2 to 3 MED of SSL are more consistent with previous studies using in vitro models (mouse or human keratinocytes), and mouse epidermis where phosphorylation of p38 occurred within minutes after UVB irradiation and returned to basal levels by 24 hours (9, 31, 32).

In the current study, SSL-induced phosphorylation of ERK 1/2, another key MAPK protein, was observed to be immediate and sustained as previously shown with UVB (33). Activation of ERK 1/2 in skin after UVA or B has been studied in vitro and in vivo using murine models with variable results (18, 24, 25). In human epidermis, acute UVB activated ERK 1/2 within 30 minutes and remained elevated for 24 hours using 2 MED (33). We found that phosphorylation of histone H3 (serine 10) peaked at 5 hours and was decreased at 24 hours, after SSL irradiation. Histone H3 is a core structural protein of the nucleosome and phosphorylation of histone H3 at serine 10 is essential for immediate-early gene expression, chromatin remodeling, and chromosome condensation during mitosis (34). ERKs and p38 kinases are mediators of UVB-induced histone H3 phosphorylation at serine 10 in mouse epidermal cells (35).

UVB-induced COX-2 expression has been shown to occur via activation of the p38 MAPK/MSK1 pathway, which in turn results in phosphorylation of histone H3 to stimulate COX-2 expression (9, 36). COX-2 is increased after UVA or UVB irradiation in human and murine skin (31, 37–40). Moreover, COX-2 expression is increased in SCCs and AK compared with normal skin (39). Increased COX-2 leads to increased PGE2, cell proliferation, and tumor promotion (39). We previously reported (12) that 4 MED of UV resulted in increased COX-2 expression at 24 hours, a finding confirmed in this current study. In addition, we found that COX-2 expression was increased as early as 5 hours after SSL irradiation.

The p53 tumor suppressor gene plays an important role in UV-induced skin carcinogenesis (41). p53 is a highly regulated gene that plays a key role in skin homeostasis. p53 is normally present at low levels, but an insult such as UV irradiation can lead to increased p53 protein stability and nuclear accumulation (18, 42). This increase in p53 stability and accumulation occurs as a result of UV-induced phosphorylation of p53 through MAPKs that include p38 and ERK. p53 is frequently mutated in cutaneous SCCs and AK and in addition p53 mutations are present in sun-exposed skin providing strong evidence that there is a field effect of UV exposure on skin (43). In the current study, a significant increase in total p53 protein was observed at 5 hours with maximal expression at 24 hours. In a previous study, we found that phospho-p53 (serine 15) was present at 24 hours after 4 MED of UV irradiation (12). Activation or increased expression of p38, ERK 1/2, and p53 have been reported to dose-related and likely wavelength dependent (18, 44).

We also measured the effect of SSL on proliferation and apoptosis. PCNA expression was largely unchanged over 24 hours with the exception of a small but significant reduction at 5 minutes after SSL irradiation. As we have previously observed (12), apoptosis was significantly increased at 24 hours after SSL irradiation (e.g., cleaved caspase-3 or morphologically apoptotic cells). In our previous study using 4 MED, levels of apoptosis were higher (12). The cellular reaction to UV irradiation is complex and results in the stimulation of multiple cell signaling pathways. p53 responds to UV-induced DNA damage where cells that are too damaged for complete repair go on to apoptosis in an effort to eliminate severely damaged cells and reduce the risk of transforming mutations (45). Pathways such as the PI3K pathway push cells toward survival where activated Akt inhibits p53 and apoptosis, but there are other pathways that can stimulate or inhibit apoptosis after SSL irradiation (7, 14, 46). There are also p53-independent mechanisms for UV-induced cell death that respond to UV-generated reactive oxygen species. Ultimately, the balance of pathway activation or suppression will determine the fate of damaged cells (17).

Analyses of the effect of MED on biomarker expression are shown in Supplementary Data Figs. S1–S3. For 7 of the 12 endpoints, there was not a statistically significant difference in expression by MED level. Conversely, for p-S6, p-ERK 1/2, and p-histone H3, there was a statistically significant difference in expression by MED level. There was no attempt to make the groups comparable and the small sample size precludes drawing conclusions with regards to these difference in marker expression by MED applied.

To conclude, we confirm that there is significant overlap between the pathway modulations observed in normal skin after acute SSL to those observed in SCC and AK, which result from years of chronic exposure. We chose specific proteins/phosphoproteins based on prior experience using in vitro and in vivo models as well as from our previous protein array work that implicated these specific pathways as important in the transition of AK to SCC, as well as those reported in the literature to have relevance in UV-induced carcinogenesis. Our study adds to the understanding of how the PI3K/Akt and MAPK signaling pathways respond to physiologically relevant acute doses of SSL in sun-protected human skin. These findings are crucial to the design of future studies analyzing the activation of signaling networks and identification of molecular targets for intervention in high-risk groups, in settings of acute sunburns as well as chronic skin damage. Progress in our understanding of mechanisms by which normal skin cells respond to solar light is vital for the identification and development of mechanistic-based approaches for the prevention and control of skin tumors, a continuously expanding cancer problem with significant morbidity and economic impact in terms of healthcare costs.

No potential conflicts of interest were disclosed.

Conception and design: S.P. Stratton, C. Curiel-Lewandrowski, S.E. Dickinson, Z. Dong, D.S. Alberts, J.G. Einspahr, G.T. Bowden

Development of methodology: S.P. Stratton, C. Curiel-Lewandrowski, C. Hu, J.G. Einspahr

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Bermudez, C. Curiel-Lewandrowski, J.A. Warneke, C. Brooks, E. Petricoin III, C. Hurst, J.G. Einspahr

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Bermudez, C. Curiel-Lewandrowski, C. Hu, K. Saboda, J.G. Einspahr

Writing, review, and/or revision of the manuscript: Y. Bermudez, S.P. Stratton, C. Curiel-Lewandrowski, J.A. Warneke, C. Hu, S.E. Dickinson, Z. Dong, A.M. Bode, K. Saboda, E. Petricoin III, D.S. Alberts, J.G. Einspahr

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Curiel-Lewandrowski, D.S. Alberts

Study supervision: S.P. Stratton, C. Curiel-Lewandrowski, Z. Dong

The authors thank Mary Krutzsch and Michael Yozwiak for assistance with measurement of immunohistochemistry and image analysis.

This work was supported by NCI grants CA027502 (to Y. Bermudez, S.P. Stratton, C. Curiel-Lewandrowski, J. Warneke, C. Hu, G.T. Bowden, S.E. Dickinson, K. Saboda, C.A. Brooks, D.S. Alberts, J.G. Einspahr), CA023074 (all authors received grant), and CA023074S2 (to Y. Bermudez)

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

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