UV-induced Skin Carcinogenesis Research Articles

Chemoprevention of Skin Cancer: Effect of Lawsonia inermis L. (Henna) Leaf Powder and its Pigment Artifact, Lawsone in the Epstein- Barr Virus Early Antigen Activation Assay and in Two-Stage Mouse Skin Carcinogenesis Models

In continuation of our studies with chemoprevention potential of plant-derived naphthoquinone derivatives, leaf powder of the medicinal plant Lawsonia inermis L, commonly known as 'henna', was evaluated by its inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Lawsone (2-hydroxy- 1,4-naphthoquinone), the reddish orange pigment artifact formed during the extraction or preparation of the dye from henna leaves and believed to be the active component, was also assessed in this in vitro assay. Both showed a profound inhibition (>88%) of EBV-EA activation. In the in vivo two-stage mouse skin carcinogenesis study using UV-B radiation for initiation and TPA for tumor promotion, oral feeding of henna (0.0025%) in drinking water ad libitum decreased tumor incidence by 66% and multiplicity by 40% when compared to the positive control at 10 weeks of treatment. Similarly, in the above mouse model, orally fed lawsone (0.0025%) decreased tumor incidence by 72% and multiplicity by 50%. The tumor inhibitory trend continued throughout the 20-week test period. Similar antitumor activities were observed when henna (0.5 mg/ml) was applied topically on the back skin in the UV-B initiated, TPA promoted and peroxynitrite initiated, TPA promoted mouse skin carcinogenesis models. Topically applied lawsone (0.015 mg/ml) also exhibited similar protection against tumor formation in the 7,12-dimtehylbenz(a)anthracene induced and TPA promoted skin cancer in mice. Also, there was a delay of 1 to 2 weeks in tumor appearance in both henna and lawsone treated groups compared to control in all three test models. This study ascertains the skin cancer chemopreventive activity of henna leaf powder and lawsone when administered by either oral (through drinking water) or topical (by application on the back skin) routes. Further, it emphasizes the need for the evaluation of these henna-derived green chemopreventive candidates in combination with currently used sunscreen agents for complementary anticancer potential against UV-induced skin carcinogenesis.

No Acceleration of UV-Induced Skin Carcinogenesis from Evenly Spread Dietary Intake of Cyclosporine in Contrast to Oral Bolus Dosages

Organ transplant recipients using the immunosuppressant cyclosporine have an increased risk for developing nonmelanoma skin cancer. Disparate effects of cyclosporine have, however, been reported on UV-induced skin carcinogenesis in mouse experiments. Therefore, we set out to compare three experimental protocols using mice, with the aim to emulate most closely the increased skin cancer risk in organ transplant recipients. UV carcinogenesis was performed in hairless SKH-1 mice by three protocols: dietary cyclosporine and daily UV exposures, dietary cyclosporine after a period of UV exposures, and bolus dosing cyclosporine by gavage and repeated UV exposures. Using chronic UV exposure, continuous dietary administration of cyclosporine was shown to inhibit tumor formation. Dietary cyclosporine after a period of UV exposures did not affect ensuing UV carcinogenesis. However, in contrast with dietary cyclosporine, bolus dosages of cyclosporine by gavage, resulting in strongly varying blood levels of cyclosporine, increased tumor development in chronically UV-exposed mice. There was no difference in tumor development between mice UV-irradiated during peak or trough levels of cyclosporine in the blood. Time-averaged levels in these mice were similar to those with cyclosporine in the diet. Cyclosporine in bolus doses appears to increase skin cancer development, whereas cyclosporine administration more evenly spread over time does not. Extrapolation to transplant patients suggests that the mode of administrating cyclosporine may be crucial for the increased skin cancer risk and that this risk might be lowered with a more steady release of cyclosporine in the body.

Smad4 Loss in Mouse Keratinocytes Leads to Increased Susceptibility to UV Carcinogenesis with Reduced Ercc1-Mediated DNA Repair

Smad4 loss occurs frequently in human skin squamous cell carcinoma (SCC), but it is unknown if this loss increases ultraviolet-induced (UV) carcinogenesis, a major etiological factor in skin cancer. In the present study, mice with keratinocyte-specific Smad4 deletion (K14.Smad4−/−) and wildtype (WT) littermates were chronically UV-irradiated. Compared to WT, K14.Smad4−/− mice exhibited increased DNA damage and increased susceptibility to UV-induced skin cancer. Among genes involved in repairing UV-induced DNA damage, Excision repair cross-complementation group1 (Ercc1) mRNA was significantly reduced in UV treated K14.Smad4−/− skin compared to WT skin. Further analysis revealed that Smad4 loss confers reduced Snail binding to the Ercc1 regulatory elements, resulting in reduced Ercc1 transcription. Consistently, transient transfection of Snai1 into Smad4−/− keratinocytes led to increased repair of UV-induced DNA lesions. Transfection of Ercc1 into Smad4−/− keratinocytes restored repair of UV-induced DNA damage. Further, immunostaining revealed that the presence of Smad4 protein is associated with the presence of Snail and Ercc1 proteins in human skin SCC and precancerous actinic keratoses (AK). Collectively, Smad4 loss associated Snail reduction compromises Ercc1-mediated DNA repair, contributing to increased UV-induced skin carcinogenesis. Thus we identified a role for Snail in UV-induced DNA repair.

Ultraviolet light exposure stimulates HMGB1 release by keratinocytes

The primary cause of non-melanoma skin cancer is ultraviolet (UV) light from the sun. Many studies have demonstrated that cutaneous inflammation resulting from UV exposure is important for the development of skin cancer. In fact, anti-inflammatory drugs have been shown to be effective in preventing skin cancer in animal models and in clinical trials. One new class of inflammatory mediators that could regulate UV-induced inflammation and skin carcinogenesis is alarmins. Alarmins are endogenous molecules that act as potent pro-inflammatory mediators when they are released by cells or accumulate extracellularly. The purpose of the current studies was to examine the expression and release of the alarmin high mobility group box 1 (HMGB1) after acute and chronic UV irradiation. Acute UV exposure stimulated the release of HMGB1 in cultured human keratinocytes and epidermal keratinocytes in murine skin. HMGB1 release correlated with pro-inflammatory cytokine production in vitro and inflammatory cell infiltration in vivo. HMGB1 was also examined in tumors arising in chronically irradiated murine skin. HMGB1 protein expression in low grade, benign papillomas was similar to adjacent skin. However, HMGB1 staining was more widespread with a higher number of HMGB1-positive cells observed in high grade papillomas and malignant tumors. Overall, the data suggest that HMGB1 may be an important regulator of UV-induced cutaneous inflammation and tumor formation. Additional studies are needed to assess whether targeting HMGB1 would be a useful strategy to prevent tumors from developing in response to chronic UV exposure.

Abstract 4264: PI3K/Akt/mTOR signaling modulation in solar UV-induced skin carcinogenesis.

Abstract Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most common types of cancers. Since most BCC and SCC develop on sun-exposed areas of the body, chronic sun exposure is considered the main etiologic factor of these tumors. In vitro and in vivo studies have identified PI3K/Akt/mTOR signaling pathway as a solar light-activated pathway. However, little work has been done on studying the expression profiles of this pathway in humans. In the study herein, we evaluate the modulation of solar light-activated PI3K/Akt/mTOR signaling pathway in normal human skin and solar simulated light (SSL)-irradiated human skin to determine the expression profiles of upstream and downstream phosphorylated proteins Akt (Ser473), mTOR (Ser2448), S6 ribosomal protein (Ser235/236), and 4E-BP1 (Thr37/46). The study population consisted of individuals 18 years or older in general good health with skin types II-III. SSL irradiation at 2, 2.5, and 3 times minimal erythema dose (MED) were applied to unexposed skin. Punch biopsies were taken at baseline, 5 minutes, 1 h, 5 h, and 24 h post-irradiation and immediately fixed in 10% formalin. Tissues were evaluated by immunohistochemistry for the expression of phosphorylated proteins. Proliferating cell nuclear antigen (PCNA) and cleaved caspase 3 (Asp175) were evaluated as markers for keratinocyte proliferation and apoptosis, respectively. Induction of phosphorylated Akt was seen at 5 hours with a 2-fold increase in expression when compared to baseline. Expression of phosphorylated mTOR gradually increased with a peak at 24 hours (3-fold). Significant induction of phosphorylated S6 ribosomal protein was seen at 1 h (3-fold), 5 h (7-fold), and 24 h (10-fold) post SSL irradiation. Induction of phosphorylated 4E-BP1 was limited to the 24 h time point with an increase of 2-fold when compared to baseline. Our data corroborate in vitro and in vivo studies finding, which allow for better understanding of the expression profile patterns of phosphorylated proteins in the PI-3 kinase/Akt/mTOR pathway during exposure of physiological relevant solar-simulated light. Identification of key proteins in this pathway could lead to the use of these proteins as biomarkers and/or specific targets for the prevention and control of skin tumors. Citation Format: Yira Bermudez, Steve P. Stratton, Clara Curiel-Lewandrowski, Chengcheng Hu, George T. Bowden, Kathylynn Saboda, David S. Alberts, Janine G. Einspahr. PI3K/Akt/mTOR signaling modulation in solar UV-induced skin carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4264. doi:10.1158/1538-7445.AM2013-4264

Dual role for mammalian DNA polymerase ζ in maintaining genome stability and proliferative responses

DNA polymerase ζ (polζ) is critical for bypass of DNA damage and the associated mutagenesis, but also has unique functions in mammals. It is required for embryonic development and for viability of hematopoietic cells, but, paradoxically, skin epithelia appear to survive polζ deletion. We wished to determine whether polζ functions in a tissue-specific manner and how polζ status influences skin tumorigenesis. Mice were produced in which Rev3L (the catalytic subunit of polζ) was deleted in tissues expressing keratin 5. Efficient epidermal deletion of Rev3L was tolerated but led to skin and hair abnormalities, accompanied by evidence of DNA breaks. Unchallenged mice developed tumors in keratin 5-expressing tissues with age, consistent with the chromosomal instability accompanying a polζ defect. Unexpectedly, mice with the Rev3L deletion were much more sensitive to UVB radiation than mice defective in other DNA repair genes. Following irradiation, polζ-defective mice failed to mount skin-regenerative responses and responded to stress by mobilizing melanocytes to the epidermis. However, they did not develop skin tumors after chronic UVB irradiation. To determine the proliferative potential of polζ-deficient skin epithelia, keratinocytes were isolated and examined. These keratinocytes harbored chromosomal gaps and breaks and exhibited a striking proliferation defect. These results can be unified by a model in which slowly dividing cells accumulate replication-associated DNA breaks but otherwise survive Rev3L deletion, but functional polζ is essential for responses requiring rapid proliferation, both in cell culture and in vivo. The results reveal a biological role for mammalian polζ in tolerating DNA damage and enabling proliferative responses in vivo.

Enhanced UV-Induced Skin Carcinogenesis in Transgenic Mice Overexpressing Proprotein Convertases

The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice.

Dietary Immunosuppressants Do Not Enhance UV-Induced Skin Carcinogenesis, and Reveal Discordance between p53-Mutant Early Clones and Carcinomas

Immunosuppressive drugs are thought to cause the dramatically increased risk of carcinomas in sun-exposed skin of organ transplant recipients. These drugs differ in local effects on skin. We investigated whether this local impact is predictive of skin cancer risk and may thus provide guidance on minimizing the risk. Immunosuppressants (azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil, and rapamycin) were assessed on altering the UV induction of apoptosis in human skin models and of p53 mutant cell clones (putative tumor precursors) and ensuing skin carcinomas (with mutant p53) in the skin of hairless mice. Rapamycin was found to increase apoptosis (three-fold), whereas cyclosporine decreased apoptosis (three-fold). Correspondingly, a 1.5- to five-fold reduction (P = 0.07) or a two- to three-fold increase (P < 0.001) was found in cell clusters overexpressing mutant p53 in chronically UV-exposed skin of mice that had been fed rapamycin or cyclosporine, respectively. Deep sequencing showed, however, that the allelic frequency (∼5%) of the hotspot mutations in p53 (codons 270 and 275) remained unaffected. The majority of cells with mutated p53 seemed not to overexpress the mutated protein. Unexpectedly, none of the immunosuppressants admixed in high dosages to the diet accelerated tumor development, and cyclosporine even delayed tumor onset by approximately 15% (P < 0.01). Thus, in contrast to earlier findings, the frequency of p53-mutant cells was not predictive of the incidence of skin carcinoma. Moreover, the lack of any accelerative effect on tumor development suggests that immunosuppressive medication is not the sole cause of the dramatic increase in skin cancer risk in organ transplant recipients.

Taxifolin Suppresses UV-Induced Skin Carcinogenesis by Targeting EGFR and PI3K

Skin cancer is one of the most commonly diagnosed cancers in the United States. Taxifolin reportedly exerts multiple biologic effects, but the molecular mechanisms and direct target(s) of taxifolin in skin cancer chemoprevention are still unknown. In silico computer screening and kinase profiling results suggest that the EGF receptor (EGFR), phosphoinositide 3-kinase (PI3K), and Src are potential targets for taxifolin. Pull-down assay results showed that EGFR, PI3K, and Src directly interacted with taxifolin in vitro, whereas taxifolin bound to EGFR and PI3K, but not to Src in cells. ATP competition and in vitro kinase assay data revealed that taxifolin interacted with EGFR and PI3K at the ATP-binding pocket and inhibited their kinase activities. Western blot analysis showed that taxifolin suppressed UVB-induced phosphorylation of EGFR and Akt, and subsequently suppressed their signaling pathways in JB6 P+ mouse skin epidermal cells. Expression levels and promoter activity of COX-2 and prostaglandin E(2) (PGE(2)) generation induced by UVB were also attenuated by taxifolin. The effect of taxifolin on UVB-induced signaling pathways and PGE(2) generation was reduced in EGFR knockout murine embryonic fibroblasts (MEF) compared with EGFR wild-type MEFs. Taxifolin also inhibited EGF-induced cell transformation. Importantly, topical treatment of taxifolin to the dorsal skin significantly suppressed tumor incidence, volume, and multiplicity in a solar UV (SUV)-induced skin carcinogenesis mouse model. Further analysis showed that the taxifolin-treated group had a substantial reduction in SUV-induced phosphorylation of EGFR and Akt in mouse skin. These results suggest that taxifolin exerts chemopreventive activity against UV-induced skin carcinogenesis by targeting EGFR and PI3K.

Abstract 1590: Taxifolin suppresses UV-induced skin carcinogenesis by targeting EGFR and PI3-K

Abstract Skin cancer is one of the most commonly diagnosed cancers in United States. Taxifolin (3, 3′, 4′, 5, 7-pentahydroxy-flavanone, (2R,3R)-dihydroquercetin), which is found in many citrus fruits such as grapefruits and oranges, has been studied for multiple biological effects. However, the molecular mechanisms and direct target of taxifolin in skin cancer chemoprevention are unclear. Here we show that taxifolin suppresses UV-induced skin carcinogenesis by directly targeting epidermal growth factor receptor (EGFR) and phosphoinositide 3-kinase (PI3-K). In silico computer screening results suggest that EGFR and PI3-K are potential targets for taxifolin. In vitro and ex vivo pull-down assay data revealed that taxifolin indeed directly interacts with EGFR and PI3-K at the ATP-binding pocket, and inhibited their kinase activities in vitro. UVB-induced phosphorylation of EGFR and Akt were substantially suppressed by taxifolin in mouse skin epidermal JB6 P+ cells. Taxifolin also suppressed UVB-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and cyclooxygenase-2 (COX-2) expression. Importantly, topical treatment with taxifolin to the dorsal skin significantly suppressed tumor incidence, multiplicity and volume in a solar UV-induced skin carcinogenesis mouse model. Further analysis of the solar UV-exposed mouse skin showed that the taxifolin-treated group had a substantial reduction in solar UV-induced phosphorylation of EGFR and Akt. These results suggest that taxifolin exerts potent chemopreventive activity against UV-induced skin carcinogenesis by targeting EGFR and PI3-K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1590. doi:1538-7445.AM2012-1590

Abstract 1591: Green tea polyphenols reactivate silenced tumor suppressor genes, p16INK4 a and Cip1/p21, by reducing DNA methylation and increasing histone acetylation in UV-irradiated mouse skin and skin tumors

Abstract Overexposure of the human skin to solar ultraviolet (UV) radiation is the major etiologic factor for the development of skin cancers, and UV-induced epigenetic modifications have been implicated in the risk of this disease. Epigenetic alterations can be inherited without changing the DNA sequence and are believed to play a crucial role in carcinogenesis including photocarcinogenesis. Epigenetic inactivation of genes by promoter hypermethylation has been recognized as an important mechanism by which tumor suppressor genes are shut down during development of tumors. For the development of effective chemopreventive agents and appropriate strategies for the prevention of skin cancers, the consideration of their effects on UV-induced epigenetic modifications is important. Earlier, we have shown that administration of green tea polyphenols (GTPs) in drinking water of SKH-1 hairless mice inhibited UVB-induced skin tumor development; however it is unclear how GTPs affect UV-induced epigenetic modifications during the prevention of UV-induced skin carcinogenesis. The present study was designed to investigate whether administration of GTPs in drinking water of mice would reactivate the expression of tumor suppressor genes in UV-exposed skin and skin tumors, if it is so then what is the underlying mechanism? For this purpose, SKH-1 hairless mice were exposed to UVB (180 mJ/cm2) with and without the administration of GTPs (0.2%, w/v) in drinking water of mice three times per week for 24 weeks. At the termination of the experiment, skin tumors and tumor-uninvolved UV-exposed mouse skin biopsies were harvested and subjected to the analysis of epigenetic biomarkers using immunohistochemistry, western blot analysis, enzymatic activity assays and real-time PCR. Our study shows that the administration of GTPs decreased UVB-induced global DNA methylation levels in UVB-exposed skin. GTPs decreased the levels of UVB-enhanced 5-methylcytosine (5-mC), DNA methyltransferase (DNMT) activity, messenger RNA (mRNA) and protein levels of Dnmt1, Dnmt3a and Dnmt3b. GTPs inhibited UVB-induced histone deacetylation (HDAC) activity and increased levels of acetylated lysine 9 on histone H3 and acetylated lysine 5 and 16 on histone H4. Administration of GTPs resulted in reactivation of the mRNA and proteins of UVB-induced silenced tumor suppressor genes, p16INK4a and Cip1/p21. Identical chemopreventive effects of GTPs were found when UVB-induced skin tumors from mice treated with or without GTPs were analyzed for tumor suppressor genes, the levels of Dnmts, and HDAC activity, etc. Together, these data provide insight into the epigenetic mechanism of action of GTPs against UV radiation effects that may contribute to the chemoprevention of photocarcinogenesis and may have important implications for epigenetic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1591. doi:1538-7445.AM2012-1591

SKHIN/Sprd, a new genetically defined inbred hairless mouse strain for UV‐induced skin carcinogenesis studies

Strains of mice vary in their susceptibility to ultra-violet (UV) radiation-induced skin tumors. Some strains of hairless mice (homozygous for the spontaneous Hr(hr) mutation) are particularly susceptible to these tumors. The skin tumors that develop in hairless mice resemble, both at the morphologic and molecular levels, UV-induced squamous cell carcinomas (SCC) and their precursors in human. The most commonly employed hairless mice belong to the SKH1 stock. However, these mice are outbred and their genetic background is not characterized, which makes them a poor model for genetic studies. We have developed a new inbred strain from outbred SKH1 mice that we named SKHIN/Sprd (now at generation F31). In order to characterize the genetic background of this new strain, we genotyped a cohort of mice at F30 with 92 microsatellites and 140 single nucleotide polymorphisms (SNP) evenly distributed throughout the mouse genome. We also exposed SKHIN/Sprd mice to chronic UV irradiation and showed that they are as susceptible to UV-induced skin carcinogenesis as outbred SKH1 mice. In addition, we proved that, albeit with low efficiency, inbred SKHIN/Sprd mice are suitable for transgenic production by classical pronuclear microinjection. This new inbred strain will be useful for the development of transgenic and congenic strains on a hairless inbred background as well as the establishment of syngeneic tumor cell lines. These new tools can potentially help elucidate a number of features of the cutaneous response to UV irradiation in humans, including the effect of genetic background and modifier genes.

Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G(2)-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-κB during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

Epigenetic Alterations in Ultraviolet Radiation‐Induced Skin Carcinogenesis: Interaction of Bioactive Dietary Components on Epigenetic Targets†

The importance of epigenetic alterations in the development of various diseases including the cancers has been realized. As epigenetic changes are reversible heritable changes, these can be utilized as an effective strategy for the prevention of cancers. DNA methylation is the most characterized epigenetic mechanism that can be inherited without changing the DNA sequence. Although limited available data suggest that silencing of tumor suppressor genes in ultraviolet (UV) radiation-exposed epidermis leads to photocarcinogenesis and is associated with a network of epigenetic modifications including alterations in DNA methylation, DNA methyltransferases and histone acetylations. Various bioactive dietary components have been shown to protect skin from UV radiation-induced skin tumors in animal models. The role of bioactive dietary components, such as, (-)-epicatechins from green tea and proanthocyanidins from grape seeds has been assessed in chemoprevention of UV-induced skin carcinogenesis and underlying epigenetic mechanism in vitro and in vivo animal models. These bioactive components have the ability to block UV-induced DNA hypermethylation and histone modifications in the skin required for the silencing of tumor suppressor genes (e.g. Cip1/p21, p16(INK4a) ). This information is of importance for understanding the role of epigenetic modulation in UV-induced skin tumor and the chemopreventive mechanism of bioactive dietary components.

Protection from UV-induced skin carcinogenesis by genetic inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase

Multiple human epidemiologic studies link caffeinated (but not decaffeinated) beverage intake with significant decreases in several types of cancer, including highly prevalent UV-associated skin carcinomas. The mechanism by which caffeine protects against skin cancer is unknown. Ataxia telangiectasia and Rad3-related (ATR) is a replication checkpoint kinase activated by DNA stresses and is one of several targets of caffeine. Suppression of ATR, or its downstream target checkpoint kinase 1 (Chk1), selectively sensitizes DNA-damaged and malignant cells to apoptosis. Agents that target this pathway are currently in clinical trials. Conversely, inhibition of other DNA damage response pathways, such as ataxia telangiectasia mutated (ATM) and BRCA1, promotes cancer. To determine the effect of replication checkpoint inhibition on carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive background, Xpc(-/-). Unlike caffeine, this genetic approach was selective and had no effect on ATM activation. These transgenic mice were viable and showed no histological abnormalities in skin. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and twofold augmentation of apoptosis after UV exposure (P = 0.006). With chronic UV treatment, transgenic mice remained tumor-free for significantly longer (P = 0.003) and had 69% fewer tumors at the end of observation of the full cohort (P = 0.019), compared with littermate controls with the same genetic background. This study suggests that inhibition of replication checkpoint function can suppress skin carcinogenesis and supports ATR inhibition as the relevant mechanism for the protective effect of caffeinated beverage intake in human epidemiologic studies.

Abstract 3673: Expression profile of phosphorylated proteins from the mTOR and Fyn/RSK2 signaling pathways in solar UV-induced skin carcinogenesis

Abstract Chronic sun exposure is considered the main etiologic agent of non-melanoma skin cancer (NMSC). Inactivation of tumor suppressor p53 and activation of key oncogenic signaling pathways important in cell proliferation and transformation are considered crucial steps in the development of NMSC. Therefore, our overall hypothesis is that topically administered small molecule drugs can modulate specific targets in ultraviolet (UV)-induced signaling cascades resulting in attenuation or reversal of carcinogenic events in human skin. In the study herein, we evaluate the modulation of solar light-activated mTOR and Fyn/RSK2 signaling pathways in normal human skin and solar simulated light (SSL)-irradiated human skin to determine the expression profiles of upstream and downstream phosphorylated proteins p38 (Thr180/Tyr182), p44/42 MAPK (Thr202/Tyr204), histone H3 (Ser10), p90RSK (Thr359/Ser363), Akt (Ser473), mTOR (Ser2448), S6 ribosomal protein (Ser235/236), and 4E-BP1 (Thr37/46). The study population consisted of individuals 18 years or older in general good health with skin types II-III. SSL irradiation at various doses ranging from two to four times the minimal erythema dose (MED) was applied to unexposed skin. Punch biopsies were taken at baseline, 5 minutes, 1 h, 5 h, and 24 h post-irradiation and immediately fixed in 10% formalin. Tissues were evaluated by immunohistochemistry for the expression of phospho-proteins. Proliferating cell nuclear antigen (PCNA) and cleaved caspase 3 (Asp175) were evaluated as markers for keratinocyte proliferation and apoptosis, respectively. Marked SSL-induced changes in human epidermis were seen in the expression of phosphorylated histone H3, S6 ribosomal protein, and 4E-BP1 at 1 h, 5 h, and 24 h post-SSL as compared to control. A linear increase in phosphorylated p44/42 MAPK expression was observed at 1 h, 5 h, and 24 h post-SSL as compared to control. Phosphorylated Akt expression increased post-SSL, and appears dependent on duration and intensity of SSL exposure. Ultimately, the expression profile patterns of phosphorylated proteins in the mTOR and Fyn/Rsk2 pathways will be used to determine activity of new and selective chemoprevention agents being developed to target these SSL-induced skin carcinogenesis pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2011-3673

Abstract 1859: Targeting APE-1/Ref-1 activity for prevention of non-melanoma skin cancer

Abstract Apurinic/apyrimidinic endonuclease-1/Redox Factor-1 (APE-1) is a dual function protein and novel target for chemoprevention of ultraviolet (UV) light-induced skin carcinogenesis. Each of the primary functions of APE-1, base excision repair of DNA damaged by oxidation and transcription factor reduction after exposure to an oxidizing stimulus like UV, is performed through the activity of a unique functional domain and plays a potential role in skin cancer promotion and progression. Our hypothesis was that APE-1 activity would correlate with survival after UV irradiation due to enhanced AP-1 activity and DNA repair. Preliminary experiments with HaCaT keratinocytes, an initiated cell line with characteristic UV-signature hotspot mutations in p53, using APE-1 siRNA demonstrated that downregulation of APE-1 increased cell sensitivity to UV-induced apoptosis. We then PCR amplified APE-1 from reverse transcribed HaCaT cell RNA and cloned it into an EGFP-tagged expression vector to generate two stable APE-1 overexpressing HaCaT cell lines, HGA9 (high expressing) and HGA10 (low expressing) cells. As expected, HGA9 and HGA10 cells were less sensitive than HaCaT cells to UV-induced apoptosis by 49.6 and 43.6%, respectively. Collectively, these data indicate that APE-1 activity does in fact play a significant role in the cellular response to UV and suggest that inhibiting APE-1 activity could be an effective means of eliminating initiated cells from the skin and preventing tumor development and growth. To further evaluate APE-1 as a therapeutic target, a library of molecules was screened for their ability to dock within one of two known binding pockets on the surface of APE-1 using an in silico docking algorithm. The first pocket screened lies within the AP endonuclease domain of APE-1. Initially, five individual molecules were chosen because they were predicted to bind APE-1 better than resveratrol, a known inhibitor that has been shown to interact with APE-1 directly. These molecules are predicted to have a higher specificity for APE-1 than resveratrol and have been assessed for their ability to inhibit DNA repair in vitro using recombinant human APE-1. Novel molecules that inhibit APE-1 activity could prove valuable in preventing UV-induced skin carcinogenesis by promoting the death of initiated keratinocytes that would otherwise undergo clonal expansion and progress to skin tumors. This work was supported by NIH grants: 5P01CA027502 and 5P30CA023074 and by a generous gift from Alliance Beverage Distributing Company. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1859. doi:10.1158/1538-7445.AM2011-1859

Impact of Nrf2 on UVB‐induced skin inflammation/photoprotection and photoprotective effect of sulforaphane

Ultraviolet (UV) of sunlight is a complete carcinogen that can burn skin, enhance inflammation, and drive skin carcinogenesis. Previously, we have shown that sulforaphane (SFN) inhibited chemically induced skin carcinogenesis via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and others have shown that broccoli sprout extracts containing high SFN protected against UV-induced skin carcinogenesis in SKH-1 hairless mice. A recent study showed that there was no difference between Nrf2 knockout (Nrf2 KO) and Nrf2 wild-type (WT) BALB/C mice after exposing to high dose of UVB. Since Nrf2 plays critical roles in the anti-oxidative stress/anti-inflammatory responses, it is relevant to assess the role of Nrf2 for photoprotection against UV. In this context, the role of Nrf2 in UVB-induced skin inflammation in Nrf2 WT and Nrf2 KO C57BL/6 mice was studied. A single dose of UVB (300 mJ/cm(2)) resulted in skin inflammation in both WT and Nrf2 KO (-/-) mice (KO mice) at 8 h and 8 d following UVB irradiation. In the WT mice inflammation returned to the basal level to a greater extent when compared to the KO mice. SFN treatment of Nrf2 WT but not Nrf2 KO mice restored the number of sunburn cells back to their basal level by 8 d after UVB irradiation. Additionally, UVB-induced short-term inflammatory biomarkers (interleukin-1β and interleukin-6) were increased in the KO mice and UVB-induced apoptotic cells in the KO mice were significantly higher as compared to that in the WT. Taken together, our results show that functional Nrf2 confers a protective effect against UVB-induced inflammation, sunburn reaction, and SFN-mediated photoprotective effects in the skin.

Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

The Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-ras(Ha) transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser(280). Since Akt phosphorylates Chk1-Ser(280), the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism.

Misuse of suncare products: Toward a protocol for sunscreen application

1548 Background: Increasing awareness of the UV-induced skin carcinogenesis has led to a significant demand for improved photo-protection both in UVB and UVA ranges. Sunscreen products are subjected to strict efficacy controls relying on standardized UV protection measurements and are widely recommended by oncologists and dermatologists in order to prevent skin cancers. However, sunscreen effectiveness depends not only on the quality of the product itself but also on the way people use it in real life. Misuse of suncare products might even lead to opposite deleterious effects. We conducted three successive studies in order to validate a sunscreen application protocol. Methods: Three studies were done on healthy volunteers who were asked to apply a sunscreen product as they usually do without any guidance or following a protocol of product application. Spontaneous application was evaluated in the first study (N = 17). Two application protocols were successively tested (N = 31) where both quantity of product and quality of sunscreen spreading were assessed (without and with the protocol) by three independent evaluators using photos taken under UVA light. Results: Spontaneous application of sunscreen resulted in an heterogeneous application of only 27% of the quantity recommended following COLIPA guidelines. Two application protocols giving practical informations concerning the quantity and the way to spread the product on each body anatomical sites were successively evaluated. Use of the application protocols improved both the quantity of suncare product and the homogeneity of product spreading with an highly significant improvement resulting from the use of the final and improved application protocol tested in the third study (p &lt; 0.02). Conclusions: The correct use of sunscreens is a public health issue about which the public still requires considerable education. We propose that, in addition to the usual photoprotection recommendations (i.e., clothing protection and avoidance of middle day exposure), population should be trained to use validated protocols for sunscreen application. [Table: see text]