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Hepatocyte growth factor-overexpressing mice that harbor a deletion of the Ink4a/p16 locus (HP mice) form melanomas with low metastatic potential in response to UV irradiation. Here we report that these tumors become highly metastatic following hemizygous deletion of the Nme1 and Nme2 metastasis suppressor genes (HPN mice). Whole-genome sequencing of melanomas from HPN mice revealed a striking increase in lung metastatic activity that is associated with missense mutations in eight signature genes (Arhgap35, Atp8b4, Brca1, Ift172, Kif21b, Nckap5, Pcdha2, and Zfp869). RNA-seq analysis of transcriptomes from HP and HPN primary melanomas identified a 32-gene signature (HPN lung metastasis signature) for which decreased expression is strongly associated with lung metastatic potential. Analysis of transcriptome data from The Cancer Genome Atlas revealed expression profiles of these genes that predict improved survival of patients with cutaneous or uveal melanoma. Silencing of three representative HPN lung metastasis signature genes (ARRDC3, NYNRIN, RND3) in human melanoma cells resulted in increased invasive activity, consistent with roles for these genes as mediators of the metastasis suppressor function of NME1 and NME2. In conclusion, our studies have identified a family of genes that mediate suppression of melanoma lung metastasis, and which may serve as prognostic markers and/or therapeutic targets for clinical management of metastatic melanoma.

NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours. Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma. Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes. The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

Hepatocyte growth factor-overexpressing mice that harbor a deletion of the Ink4a/p16 locus (HP mice) form melanomas with low metastatic potential in response to UV irradiation. Here we report that these tumors become highly metastatic following hemizygous deletion of the Nme1 and Nme2 metastasis suppressor genes (HPN mice). Whole-genome sequencing of melanomas from HPN mice revealed a striking increase in lung metastatic activity that is associated with missense mutations in eight signature genes (Arhgap35, Atp8b4, Brca1, Ift172, Kif21b, Nckap5, Pcdha2, and Zfp869). RNA-seq analysis of transcriptomes from HP and HPN primary melanomas identified a 32-gene signature (HPN lung metastasis signature) for which decreased expression is strongly associated with lung metastatic potential. Analysis of transcriptome data from The Cancer Genome Atlas revealed expression profiles of these genes that predict improved survival of patients with cutaneous or uveal melanoma. Silencing of three representative HPN lung metastasis signature genes (ARRDC3, NYNRIN, RND3) in human melanoma cells resulted in increased invasive activity, consistent with roles for these genes as mediators of the metastasis suppressor function of NME1 and NME2. In conclusion, our studies have identified a family of genes that mediate suppression of melanoma lung metastasis, and which may serve as prognostic markers and/or therapeutic targets for clinical management of metastatic melanoma.

Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP-dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well-described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV-induced melanoma. C57BL/6-Mc1r+/+-HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra-follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA-induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6-Mc1re/e animals which have a non-functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6-Mc1re/e-HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6-Mc1r+/+-HGF mice, hyperpigmentation was not observed and there were few extra-follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6-Mc1re/+-HGF mice were black and hyperpigmented and, although extra-follicular melanocytes and skin melanin content were similar to C57BL/6-Mc1r+/+-HGF animals, they developed UV-induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma and postulate that this pathway is involved in human melanoma.

Non-thermal nanoelectroablation therapy completely ablates UV-induced murine melanomas. C57/BL6-HGF/SF transgenic mice were exposed to UV radiation as pups and began to develop visible melanomas 5-6 months later. We have treated 27 of these melanomas in 14 mice with nanosecond pulsed electric field (nsPEF) therapy delivering 2000 electric pulses each 100 ns long and 30 kV/cm at a rate of 5-7 pulses per second. All nanoelectroablated melanoma tumors began to shrink within a day after treatment and gradually disappeared over a period of 12-29 days. Pyknosis of nuclei was evident within 1 h of nsPEF treatment, and DNA fragmentation as detected by TUNEL staining was evident by 6 h after nsPEF treatment. In a melanoma allograft system, nsPEF treatment was superior to tumor excision at accelerating secondary tumor rejection in immune-competent mice, suggesting enhanced stimulation of a protective immune response by nsPEF-treated melanomas. This is supported by the presence of CD4(+) -T cells within treated tumors as well as within untreated tumors located in mice with other melanomas that had been treated with nanoelectroablation at least 19 days earlier.

Cutaneous malignant melanoma is rapidly increasing in the developed world and continues to be a challenge in the clinic. Although extensive epidemiologic evidence points to solar UV as the major risk factor for melanoma, there is a significant gap in our knowledge about how this most ubiquitous environmental carcinogen interacts with the largest organ of the mammalian body (skin) at the microenvironmental and molecular level. We review some recent advances that have started to close this gap. Cancer Res; 72(7); 1591–5. ©2012 AACR.

OBJECTIVE. The SunWise School Program is a school-based sun safety education program that was developed by the US Environmental Protection Agency and aims to teach children how to protect themselves from overexposure to the sun. The objectives of this study were to assess the health benefits of the SunWise School Program and use economic analysis to determine the program's net benefits and cost-effectiveness. METHODS. Standard cost/benefit and cost-effectiveness analysis methods were used. Intervention costs were measured as program costs estimated to be incurred by the US government, which funds SunWise, using 3 funding scenarios. Health outcomes were measured as skin cancer cases and premature mortalities averted and quality-adjusted life-years saved. These health outcomes were modeled using an effectiveness evaluation of SunWise based on pretest and posttest surveys administered to students who participated in the program and the Environmental Protection Agency's peer-reviewed Atmospheric and Health Effects Framework model. Costs averted were measured as direct medical costs and costs of productivity losses averted as a result of SunWise. Net benefits were measured as the difference between costs averted and program costs. RESULTS. Economic analysis indicated that if the SunWise School Program continues through 2015 at current funding levels, then it should avert >50 premature deaths, nearly 11000 skin cancer cases, and 960 quality-adjusted life-years (undiscounted) among its participants. For every dollar invested in SunWise, between approximately $2 and $4 in medical care costs and productivity losses are saved, depending on the funding scenario. CONCLUSIONS. From a cost/benefit and cost-effectiveness perspective, it is worthwhile to educate children about sun safety; small to modest behavioral impacts may result in significant reductions in skin cancer incidence and mortality.

Ultraviolet B (UVB, 290-320 nm) radiation initiates in vivo a dose- and wavelength-dependent down regulation of cell-mediated immunity. An action spectrum for UV-induced immunosuppression indicated that the photoreceptor for this effect is urocanic acid (UCA), which undergoes a trans to cis isomerization in the stratum corneum on UV exposure. An accumulation of evidence has supported this conclusion. However, evidence has also been presented that formation of thymine dimers in DNA is responsible for initiation of UV-induced immunosuppression. Because photobinding of UCA to DNA in vitro forming cyclobutane-type adducts has been shown, we sought to resolve this dilemma by investigating if UCA photobinds to DNA in vivo. The [14C]cis-UCA, [14C]trans-UCA or [3H]8-MOP (8-methoxypsoralen) was applied topically to BALB/c mice that were then irradiated with a dose of UV previously shown to cause systemic suppression of contact hypersensitivity. The DNA was prepared from epidermal cells by phenol extraction immediately after in vivo irradiation and bound radioactivity determined. Although photobinding of [3H]8-MOP was readily demonstrable under these conditions (0.9 nmol/mg DNA), no significant binding of either isomer of UCA to DNA (between 1.2 x 10(-3) and 2.1 x 10(-3) ng/mg DNA) could be detected. Uptake studies in keratinocytes prepared from epidermis of untreated animals indicated that [3H]8-MOP was taken up with a rate constant of 4.2 x 10(-3) pmol/s/mg protein/mumol/L. In contrast, uptake of [14C]cis-UCA was not statistically significant from zero and uptake of [14C]trans-UCA was negligible (0.8 x 10(-3) +/- 0.08 x 10(-3) pmol/s/mg protein/mumol/L). There was no significant difference between uptake of UCA isomers, but uptake of [3H]8-MOP was significantly greater than that of either UCA isomer (P < 0.01). These studies indicate that the photobinding of UCA to DNA does not play a role in UV-induced immunosuppression.

Vitamin D production is initiated by exposure of 7-dehydrocholesterol in the skin to the UVB (280–320 nm) component of sunlight, resulting in the formation of photoproducts, which are subsequently metabolically activated to biologically active moieties in a series of dark reactions as described elsewhere in this symposium. Irradiation of the skin with UVB has, however, other effects not all of which are beneficial. Most notable is the initiation of skin cancer. Non-melanoma skin cancer is clearly initiated by UVB but for the most lethal of the skin cancers, cutaneous malignant melanoma, although associated with sunlight exposure, the wavelengths responsible have not been clearly identified. Using a mouse model for UV-induced melanoma, we have recently shown that UVB, not UVA (320–400 nm), is also responsible for melanoma initiation. A balance therefore needs to be struck between the healthy effects of exposure to UVB in sunlight—vitamin D formation—and the deleterious effects of which the most potentially serious is melanoma initiation. A powerful tool in determining this balance would be an understanding of the action spectra or wavelength dependence for each of these effects. Here we describe methodologies, approaches and potential pitfalls for action spectra determination illustrated by our experience with the HGF/SF transgenic mouse model for UV-induced melanoma.

To assess the premise that genetically determined differences in susceptibility to UV-induced immunosuppression are reflected in UV carcinogenesis, we investigated UV skin cancer induction in two strains of reciprocal F1 hybrid mice CB6F1 males with high susceptibility to UV immunosuppression and a BALB/c X-chromosome and B6CF1 males with low susceptibility to UV immunosuppression and a C57BL/6 X-chromosome. Four experimental groups comprising both strains treated three times weekly with two UV regimens (daily doses incremented from 2.25 to 6 or 4.5 to 12 kJ per m2) were monitored for skin tumor development. Survival without a skin tumor differed over the four groups (p< 0.0001) and differed according to UV regimen within each strain (p

Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well-described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV-induced melanoma. C57BL/6-Mc1r+/+-HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra-follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA-induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6-Mc1re/e animals which have a non-functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6-Mc1re/e-HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6-Mc1r+/+-HGF mice, hyperpigmentation was not observed and there were few extra-follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6-Mc1re/+-HGF mice were black and hyperpigmented and, although extra-follicular melanocytes and skin melanin content were similar to C57BL/6-Mc1r+/+-HGF animals, they developed UV-induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma and postulate that this pathway is involved in human melanoma.

Dear Sir, Cutaneous malignant melanoma incidence has more than doubled over the past 25 yrs, and this trend continues across all age groups at a rate of over 3% per yr (Linos et al., 2009). Exposure to the ultraviolet (UV) portion of sunlight is strongly implicated in melanoma etiology and considered the major environmental risk factor. Higher melanoma risk is associated with burning UV doses, both intermittent and during childhood (Gandini et al., 2005). Alarmingly, melanomas are the most prevalent cancer in 25–29 yr old females, and a link to commercial tanning sunlamps use is suggested (Purdue et al., 2008). Melanoma prevention recommendations include avoiding mid-day sun and artificial UV light, wearing protective clothing and hats, and using sunscreen with a Sun Protective Factor (SPF) of 15 or higher (American Cancer Society, 2010). However, the SPF sunscreen rating describes erythema or sunburn protection; sunscreens are not rated for the prevention of melanoma or other skin cancers (FDA, 1999). Melanoma incidence reduction through sunscreen use has not yet been proven, and in fact has been controversial (Saraiya et al., 2003). We here present data that demonstrate for the first time significant sunscreen prevention in UV-dependent, melanoma-prone transgenic mice, and propose that when applied properly should be preventive in people. Human epidemiologic studies of melanoma prevention are limited by recall bias, insufficient statistical power, and non-uniform estimations of sun exposure and sunscreen use. Therefore, sunscreen use and melanoma risk reduction or mortality prevention are difficult to accurately assess and remain inconclusive (Bastuji-Garin and Diepgen, 2002; Saraiya et al., 2003). We evaluated sunscreen use as a risk factor for melanoma in a large case-control study with 717 non-Hispanic white, invasive melanoma patients and 945 matched controls (Fears et al., 2002). The univariate analyses of ever sunscreen use or regular use of sunscreen of SPF ≥ 8 indicated minimal risk associated with sunscreen use (relative odds 1.05 and 1.11, respectively). This association is suggested to reflect users’ sun sensitivity (burning and tanning tendencies), and not a property of sunscreen exposure per se (Dennis et al., 2003). A matched logistic regression analysis of sunscreen use adjusted for average UVB intensity of residences, adult hours outdoors, tan-type, number sunburns, age-group, study-site and gender (Appendix S1), showed that sunscreen use was not associated with melanoma (OR 0.90, 95% CI 0.70–1.16; Table 1). When evaluating sunscreen users and examining risk in those who burned easily and persons who did not burn in a single model, the relative odds for those who burned easily decreased slightly to 0.85 (0.62–1.19) and for those who did not burn was 0.91 (0.70–1.19) with no statistically significant difference in risks (Table 1). The changes in risks observed after appropriate adjustment likely reflect confounding, a spurious relationship, as persons using sunscreens may be more susceptible to melanoma. These data demonstrate the challenges of assessing complex behaviors, such as sunscreen use, outside of a prospective randomized study. Even in our large investigation, difficulty in addressing these challenges illustrates the urgent need for appropriate animal models, employed experimentally, to better assess the value of sunscreen use in melanoma prevention. Table 1 Melanoma in persons with no sunscreen use, and those who used any sunscreen, among sunscreen users by tendency to burn or sun sensitivity The availability of the Hepatocyte Growth Factor/Scatter Factor (HGF/SF) genetically engineered mouse, which develops neonatal UV-dependent skin lesions highly reminiscent of cutaneous malignant melanoma (Noonan et al., 2001), provides a favorable platform to demonstrate experimentally the capabilities of sunscreen in a melanoma prevention study. This mouse model has been used to support the notion that childhood sunburn constitutes a major melanoma risk factor (Noonan et al., 2001). Therefore, we hypothesized that blocking UV radiation-induced erythema with current dermatologist recommended SPF15 sunscreen would significantly decrease the incidence of melanoma in this relevant murine melanoma model. Neonatal mice were exposed to a single dose of UV radiation with prior application (15 min) of either vehicle-control lotion or SPF15 sunscreen containing FDA-approved active agents (Appendix S1). The UV dose corresponded to a human erythemally-weighted UV dose of 2.3 kJ/m2 or 23 Standard erythemal dose units, equivalent to a sun-burning dose in people (Noonan et al., 2001). The primary endpoint was melanoma development in the dorsal application area, confirmed by histopathology and positivity for melanocytic antigens. The SPF15 sunscreen-treated animals developed significantly fewer melanomas than the vehicle-control group (P = 0.043; Figure 1). In the sunscreen-treated group (n = 97), one mouse (1%) developed two dorsal melanomas, while the eight mice (7%) in the vehicle-control group (n = 118) developed 18 melanomas (Figure 1; Table S1). The multiplicity of melanomas per animal arising in the sunscreen-treated group was also lower, with two per animal versus up to seven per animal in the vehicle controls. Animals from both groups equally developed melanoma outside of lotion-treated areas (tails and/or hindquarters): two in vehicle controls and one in the SPF15 group. Figure 1 UV-induced melanomas and UV-induced DNA damage are significantly inhibited in sunscreen-treated compared to vehicle control-treated mice. Melanoma incidence per group and the average ratio of TT-dimer-containing cells to no TT-dimer-containing cells are ... The molecular mechanisms by which sun and artificial UV light initiate and/or promote melanoma development are being elucidated (Zaidi et al., 2008). Clearly, both direct and/or indirect DNA damage mechanisms, including thymine dimer and genotoxic reactive oxygen species formation, may play a mechanistic role. We therefore quantified DNA damage in skin cells of UV-treated control and SPF15 sunscreen-treated animals using an antibody to thymine-thymine (TT) dimers, a type of damage initiated by UVB irradiation. Cells were scored ‘positive’ if double-stained for TT dimers and nuclear counter-stain at seven minutes post-UV. The vehicle control lotion-treated skin averaged greater than five cells with nuclear DNA damage to each non-DNA damaged cell (Figure 2; Table S1). In sunscreen-treated skin, the TT:no-TT ratio was

Cleland and Kreevoy recently advanced the idea that a special type of hydrogen bond (H-bond), termed a low-barrier hydrogen bond (LBHB), may account for the “missing” transition state stabilization underlying the catalytic power of many enzymes, and Frey et al. have proposed that the H-bond between aspartic acid 102 and histidine 57 in the catalytic triad of serine proteases is an example of a catalytically important LBHB. Experimental facts are here considered regarding the aspartic acid–histidine and cis– urocanic H-bonds that are inconsistent with fundamental tenets of the LBHB hypothesis. The inconsistencies between theory and experiment in these paradigm systems cast doubt on the existence of LBHBs, as currently defined, within enzyme active sites.

Solar ultraviolet-B radiation (UVB; 290-320 nm) causes skin cancer and suppresses cell-mediated immunity, preventing the rejection of UV-induced tumors. One mechanism initiating UV suppression involves the trans to cis photoisomerization of urocanic acid (UCA), a histidine derivative found in the stratum corneum. The addition of L-histidine to nonpurified mouse diet has been shown to increase skin trans-UCA levels and sensitivity to UVB immune suppression. Specially formulated L-histidine diets (0.40-64 g/kg) fed to BALB/c mice that were monitored over a 19-wk period resulted in an unexpected modulation of skin trans-UCA. ANOVA revealed a group-time interaction, providing initial evidence that the skin levels of trans-UCA were modulating up and down in all groups except the control group (6.4 g/kg diet). We observed that both high (64 g/kg diet) and low (0.4 g/kg diet) levels of dietary L-histidine resulted in the increase of skin trans-UCA to levels significantly higher than those recorded in the control group. In mice fed these histidine levels, skin trans-UCA increased to between 2.9 and 3.6 nmol/mg skin (64 g/kg diet, over 5 wk; 0.4 g/kg diet, over 8 wk) and then decreased to approximately 1.69 nmol/mg skin, the base-line level (64 g/kg diet, over 11 wk; 0.4 g/kg diet, over 17 wk). The increase in trans-UCA levels in mice with low L-histidine intake may be the result of protein malnutrition, consistent with weight loss observed in those mice. The modulation of trans-UCA levels in skin by dietary L-histidine has not been previously described; its role in skin cancer development is under investigation.

A micro-sampling procedure has been developed for studying lymphocyte secretion of biologically important peptides in low cell density cultures. The technique is based on microdialysis recovery of the analytes of interest coupled with immunoaffinity capillary electrophoresis separation of the microdialysis samples and laser-induced fluorescence detection. Although the technique is able to recover secreted materials only at the 5-10 cell level, the detection system has a limit of detection (LOD) in the attomole (10(-18) M) range. This degree of sensitivity indicates that the system has the potential to measure secreted products at the single cell level. An added advantage of this system over other sampling techniques is that the microdialysis probe allows continuous sampling over time.

The G-protein-coupled receptor, Mc1r, plays a major role in pigment production and has been reported to be important in the inflammatory response. We have investigated the effect of deficiency in Mc1r on UV-induced inflammation. Mice on the same genetic background were used - C57BL/6-c (albino), C57BL/6 (black), C57BL/6-Mc1r(e/e) deficient (yellow). FACS analysis of disaggregated skin showed a similar dose-dependent increase in Ly6G(+) and CD11b(+) cells in response to UV radiation in all groups. No differences in UV-induced edema or in DNA damage were detected between groups. The contact hypersensitivity response, neonatal immune tolerance and UV immunosuppression were all similar in C57BL/6 and C57BL/6-Mc1r(e/e) mice. We conclude that the absence of Mc1r does not impair the inflammatory response to UV radiation or the generation of immunosuppression.

Malignant melanoma of the skin (CMM) is associated with ultraviolet radiation exposure, but the mechanisms and even the wavelengths responsible are unclear. Here we use a mammalian model to investigate melanoma formed in response to precise spectrally defined ultraviolet wavelengths and biologically relevant doses. We show that melanoma induction by ultraviolet A (320–400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes. In contrast, ultraviolet B radiation (280–320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage. Thus, we identified two ultraviolet wavelength-dependent pathways for the induction of CMM and describe an unexpected and significant role for melanin within the melanocyte in melanomagenesis., Exposure to ultraviolet light is responsible for a large proportion of melanomas but the molecular mechanisms are unknown. In this study, melanoma is found to be induced in mice by UVA and UVB light in a pigment-dependent and -independent manner, respectively, resulting in different types of DNA damage.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, whose incidence continues to rise. Epidemiological studies reveal that the major etiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor (HGF/SF) transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and etiologic criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4. However, mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, and suggest that IFN-γ-R signaling represents a novel therapeutic melanoma target.

— It has been demonstrated that UVB radiation (290–320 nm) suppresses mammalian cell-mediated immunity by effecting the trans to cis isomerization of urocanic acid (UCA) in the stratum corneum, the uppermost layer of the skin. Trans-urocanic acid has been shown to be the photoreceptor for UVB-induced immune suppression and the cis-isomer has been demonstrated to be immunosuppressive. Little is known, however, about how the isomerization of UCA may affect the proximal or distal cells of the skin or the immune system. We report here that trans-UCA is biologically active in vitro in human dermal fibroblasts, inducing adenyl cyclase as measured by cAMP (adenosine 3', 5'-cyclic monophosphate) formation in a dose-dependent manner similar to the action of histamine. Trans-UCA and histamine stimulate 50% of maximum activity at concentrations of 3.3 μM end 13.8 μM respectively. Cis-UCA does not increase cAMP in these human fibroblasts but actively down regulates the increase of cAMP induced by either histamine or trans-UCA. Cis-UCA down regulated the histamine response by 75% and the trans-UCA response by 60% at a concentration range of 1 mM to 1 nM. The trans-UCA induction of cAMP can also be downregulated with an H2 histamine receptor antagonist cimetidine. These results support the hypothesis that a cellular target for cis-UCA is the dermal fibroblast and the effects reported here may represent the initial biochemical and cellular event for UVB-induced immune suppression i.e. the immediate step following the isomerization of trans to cis-UCA is the down regulation of cAMP by cis-UCA. Regulation of such an important second messenger such as cAMP could then allow cascading signals to occur, leading to immune suppression.

Irradiation with UV-B, a component of natural sunlight, initiates systemic immunosuppression of delayed-type hypersensitivity responses. This may be a fundamental regulatory mechanism, controlling the interaction between mammals and potentially deleterious environmental UV radiation. Here, Frances Noonan and Edward De Fabo assess the evidence that suppression is initiated by the photoisomerization of trans-urocanic acid (UCA) in the stratum corneum, discuss the significance of this mechanism for skin cancer outgrowth and propose applications for UCA in transplantation.

The incidence of cutaneous malignant melanoma (CMM) has more than doubled in the past 25 years and continues to increase at over 3% per year across all age groups (Linos et al., this issue), and invasive and disseminated melanoma in young women has increased by almost 10% since 1992 (Purdue et al., 2008). Early detection and excision of CMM can result in successful treatment, but disseminated disease is resistant to current therapies and has a very poor prognosis (Garbe and Eigentler, 2007). Sunlight exposure is a major risk factor for melanoma. In this issue, Waster and llinger investigate the effects of UVB and UVA on melanocytes.

Mechanisms of juvenile susceptibility to cancer are not well understood. The immune response in neonates favors nonresponsiveness or TH2-dominant responses, raising the question of a role for neonatal immunity in this susceptibility. We have investigated the postulate that the inflammatory response differs in neonatal and adult skin. We found no inflammatory infiltrate into neonatal mouse skin in response to UV irradiation as a function of time, dose, or wavelength, although UV-induced DNA damage was readily detected. In contrast, UV irradiation of adult mice initiated a dose- and time-dependent influx of inflammatory cells, chiefly CD11b+Ly6G+ neutrophils, into the skin, detected by immunohistochemistry and quantitated by FACS analysis. This inflammatory response was initiated by UVB (290–320 nm) but not by UVA (320–400 nm). Further, in neonates, in contrast to adults, neither topical trinitrochlorobenzene (TNCB) nor i.p. thioglycollate initiated an inflammatory infiltrate. Conversely, topical TNCB applied to neonates was tolerogenic, resulting in a subsequent antigen-specific decrease of the contact-hypersensitivity response in adults. Neonatal blood contained abundant neutrophils, which exhibited impaired chemotaxis to the chemokine growth-related oncogene-α but efficient chemotaxis to the bacterial product fMLP, concomitant with decreased expression of CXCR2 but normal levels of CD11b. We propose this neonatal deficiency in the inflammatory response is a significant, previously unrecognized factor in neonatal immune tolerance and may contribute to neonatal susceptibility to cancer, including melanoma and other UV-induced cancers.

Linan Ha, Frances P. Noonan,w Edward C. De Fabo,w and Glenn Merlino Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, Maryland, USA; wLaboratory of Photobiology and Photoimmunology, Department of Environmental and Occupational Health, School of Public Health and Health Services, The George Washington University Medical Center, Washington, District of Columbia, USA

Contrary to popular belief, stratospheric ozone depletion, and the resultant increase in solar UV-B (280-320 nm), are unlikely to fully recover soon. Notwithstanding the success of the Montreal Protocol in reducing the amount of ozone destroying chemicals into the stratosphere, the life-times of these compounds are such that even with full compliance with the Protocol by all countries, it will be decades before stratospheric ozone could return to pre-1980 levels. This raises the question, therefore, of what will happen to biological processes essential to the maintenance of life on earth which are sensitive to damage by increased UV-B radiation, particularly those involved with human health? The polar regions, because of the vagaries of climate and weather, are the bellwether for stratospheric ozone depletion and will, therefore, be the first to experience impacts due to increases in solar UV-B radiation. The impacts of these are incompletely understood and cannot be predicted with certainty. While some UV-B impacts on human health are recognized, much is unknown, unclear and uncertain. Thus, this paper attempts, as a first approximation, to point out potential impacts to the health and welfare of human inhabitants of the Arctic due to increased solar UV-B radiation associated with stratospheric ozone depletion. As will be seen, much more data is critically needed before adequate risk assessment can occur. (Int J Circumpolar Health 2005; 64(5):509-522) Keywords: Arctic population, ozone depletion, UV-B effects

Cutaneous malignant melanoma is one of the fastest increasing cancers with an incidence that has more than doubled in the last 25 years. Sunlight exposure is strongly implicated in the etiology of cutaneous malignant melanoma and the UV portion of the sunlight spectrum is considered responsible. Data are, however, conflicting on the roles of ultraviolet B [UVB; 280–320 nanometers (nm)] and ultraviolet A (UVA; 320–400 nm), which differ in their ability to initiate DNA damage, cell signaling pathways and immune alterations. To address this issue, we have used specialized optical sources, emitting isolated or combined UVB or UVA wavebands or solar simulating radiation, together with our hepatocyte growth factor/scatter factor-transgenic mouse model of UV-induced melanoma that uniquely recapitulates human disease. Only UVB-containing sources initiated melanoma. These were the isolated UVB waveband (>96% 280–320 nm), the unfiltered F40 sunlamp (250–800 nm) and the solar simulator (290–800 nm). Kaplan-Meier survival analysis indicated that the isolated UVB waveband was more effective in initiating melanoma than either the F40 sunlamp or the solar simulator (modified log rank P < 0.02). The latter two sources showed similar melanoma effectiveness (P = 0.38). In contrast, transgenic mice irradiated with either the isolated UVA waveband (>99.9% 320–400 nm, 150 kJ/m2), or an F40 sunlamp filtered to remove > 96% of the UVB, responded like unirradiated control animals. We conclude that, within the constraints of this animal model, UVB is responsible for the induction of mammalian cutaneous malignant melanoma whereas UVA is ineffective even at doses considered physiologically relevant. This finding may have major implications with respect both to risk assessment from exposure to solar and artificial UVB, and to development of effective protection strategies against melanoma induction by UVB. Moreover, these differences in wavelength effectiveness can now be exploited to identify UV pathways relevant to melanomagenesis.

Exposure to ultraviolet radiation results in increased levels of intradermal cis-urocanic acid (cUCA) and alters cutaneous immunity by interfering with processing and presentation of antigen by Langerhans cells. Reports on effects of systemic immunotoxicity with 30 day cUCA exposure in laboratory rodents include thymic atrophy, thymic hypocellularity and decreased T-cell-mediated immunity; however, immune effects of single exposure or 5 day cUCA administration, which may better mimic human exposures, are poorly defined. The present study initially evaluated immune effects of single, 5 day, and 4 week cUCA exposure in C57BL/6N mice. Single administration of intradermal cUCA resulted in decreased splenocyte phagocytosis that persisted for 30 days after cUCA exposure. Five day consecutive cUCA exposure decreased numbers of phenotypically mature CD4(+)CD8(-) and CD4(-)CD8(+) (single positive) thymocytes, increased CD4(+)CD8(+) (double positive) immature thymocytes and increased splenocyte proliferation. Prolonged cUCA exposure (4 weeks) caused profound thymic hypocellularity and splenic hypercellularity and increased splenic macrophage chemiluminescence. Because of this apparent sensitivity of C57BL/6N mice to cUCA, thymic hypocellularity was compared between C57BL/6N and C3H/HeN mice dosed with cUCA, and was found to be more pronounced in the C57BL/6N strain. These results are an extension of previous conclusions on immune modulation caused by cUCA in the spleen and thymus. Further, the observed variation in sensitivity between the mouse strains is consistent with known genetic susceptibility of these strains to the immunomodulatory effects of exposure to sunlight.

Cutaneous malignant melanoma, the most lethal of the skin cancers, known for its intractability to current therapies, continues to increase in incidence, providing a significant public health challenge. There is a consensus that skin cancer is initiated by sunlight exposure. For non-melanoma skin cancer there is substantial evidence that chronic exposure to the ultraviolet B radiation (UVB) (280-320 nm) portion of the sunlight spectrum is responsible. Experimentally, UVB is mutagenic and chronic UVB exposure can cause non-melanoma skin cancer in laboratory animals. Non-melanoma tumors in animals and in humans show characteristic UVB signature lesions in the tumor suppressor p53 and/or in the patched (PTCH) gene. An action spectrum or wavelength dependence for squamous cell carcinoma in the mouse shows a major peak of efficacy in the UVB. For malignant melanoma, however, the situation is unclear and the critical direct target(s) of sunlight in initiating melanoma and even the wavelengths responsible are as yet unidentified. This lack of information is in major part a result of a paucity of animal models for melanoma which recapitulate the role of sunlight in initiating this disease. The epidemiology of melanoma differs significantly from non-melanoma skin cancer. Intense sporadic sunlight exposure in childhood, probably exacerbated by additional adult exposure, is associated with elevated melanoma risk. Melanoma is also a disease of gene-environment interactions with underlying genetic factors playing a significant role. These major differences indicate that extrapolation from information for non-melanoma skin cancer to melanoma is unlikely to be useful. We summarize in this review the experimental information available on the role of UV radiation in melanoma and give an overview of animal melanoma models. A new model derived by neonatal UV irradiation of hepatocyte growth factor/scatter factor (HGF/SF) transgenic mice is described which recapitulates the etiology, the histopathology and molecular pathogenesis of human disease. It is anticipated that the HGF/SF transgenic model will provide a means to access the mechanism(s) by which sunlight initiates this lethal disease and provide an appropriate vehicle for derivation of appropriate therapeutic and preventive strategies.

Recurrence and metastasis following resection and therapy are the most critical problems in progressive cancers and the main causes of cancer-related death. This problem is magnified in cutaneous malignant melanoma, which is highly metastatic in advanced stages and generally resistant to chemotherapy. Although new drugs such as Vemurafenib successfully target BRAFV600E and typically demonstrate a significant clinical response, progressive melanomas recur resulting in near uniform patient mortality. Dissection of processes underpinning recurrent and metastatic melanoma is certain to identify new therapeutic targets for more efficacious treatment. However, current preclinical studies do not model progressive recurrent disease, but rather rely on the growth response of human melanoma cell lines subcutaneously xenografted into immunocompromised mice as the efficacy endpoint; these studies have shown poor predictive power for clinical activity. Therefore we have designed new and improved genetically engineered mouse (GEM) models of progressive melanoma. These are being used to test targeted and immune-based therapies singly and in combination, and to study mechanisms associated with the resistance of recurrent/metastatic melanoma to promising clinical drugs. To identify novel targets for advanced disease we hypothesized that metastatic melanomas can exploit hard-wired pathways employed by migratory embryonic melanocytes, but not mature melanocytes, to achieve a more aggressive malignant phenotype. We had previously generated a GEM model (iDCT-GFP) that expresses Green Fluorescent Protein specifically in all melanocytic cells in a doxycycline-regulatable manner. The iDCT-GFP mouse was used in concert with FACS to isolate melanoblasts from key developmental stages, sequence their transcriptomes using RNA-Seq, and identify genes/pathways common to metastatic melanoma cells. We have thus generated a full library of the genes expressed during embryonic development of the melanocyte. A detailed comparative analysis of mouse melanoblast gene expression profiles with human metastatic melanoma cells was then performed, identifying what we believe to be a small number of critical genes common to both. We anticipate that this approach will enhance our understanding of this fatal disease at both mechanistic and prognostic levels, and facilitate the identification of novel therapeutic targets for the treatment of metastatic melanoma. Citation Format: Glenn T. Merlino, Chi-Ping Day, Pravin Mishra, Theresa Guo, Raza Zaida, Sean Davis, Paul Meltzer, Frances Noonan, Edward De Fabo, Zoe Ohler-Weaver, Terry Van Dyke. Modeling recurrent metastatic melanoma in the mouse. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA22.

Seasonal stratospheric ozone depletion in the Arctic has raised the question of whether the associated increases in ultraviolet-B (290-320 nm) constitute a significant health risk in Arctic populations. Increases in skin cancer in Europe and the USA from excess UV-B resulting from ozone depletion have been predicted. Skin cancer is, however, rare in Inuit populations. UV-B also causes a selective down regulation of the immune system which may be a natural regulatory mechanism evolved to prevent autoimmune attack on sunlight-altered skin. The action spectrum for UV-B immunosuppression implicated a unique skin photoreceptor molecule, urocanic acid (UCA), which isomerizes from the trans to the cis isomer on exposure to UV-B, the cis isomer being immunosuppressive. This form of immunosuppression is important in skin cancer and possibly in infectious diseases. The epidemiology of non-Hodgkin's lymphoma shows a relationship with UV exposure, postulated to be via the immunosuppressive effects of UV-B. Cancers which show an excess in Inuit populations include nasopharyngeal and salivary gland cancer. Genetic factors appear to be involved, but these are thought to be virally related cancers possibly associated with the high viral load in these populations. In several studies on non-Arctic populations, salivary gland cancer has been linked to ultraviolet exposure. A potential role for UV-B exposure in these cancers in the Arctic needs to be explored. In view of the high levels of POPS in some Arctic regions, potential interactions between the immunosuppression caused by some of these pollutants and the effects of UV-B need to be investigated.

First of all I would like to thank the organizers, and in particular Environment Canada and the United Nations Environment Programme (UNEP), for the invitation to speak to you today on the topic I have been associated with for the past twenty years, the effects of increased solar ultraviolet B radiation (UV-B), due to stratospheric ozone depletion, on biological and health systems.

In mammals, ultraviolet-B radiation impacts on the skin and on the eyes (Zigman, Chapter 6 this volume). Because of effective absorption by skin components, very little of the incident UV-B penetrates beyond the epidermis. In spite of this, UV-B radiation is intimately linked with the mammalian immune system. In this chapter, we will describe a specific immunosuppression initiated by UV-B and discuss the mechanism of its generation. UV-B-induced immunosuppression may be a fundamental biological regulatory mechanism, designed to protect against autoimmune attack on sunlight-altered skin, the adverse effect of which is to permit the outgrowth of UV-induced skin cancers. In addition to this specific immunosuppression, irradiation with UV-B also releases a number of cytokines and induces the expression on keratinocytes of cell-surface immunologic receptors, which may be of significance in UV-related dermatologie disorders.

Retrospective epidemiological data have indicated that cutaneous malignant melanoma may arise as a consequence of intense, intermittent exposure of the skin to ultraviolet radiation, particularly in children, rather than from the cumulative lifetime exposure that is associated with other forms of skin cancer. Here we use a genetically engineered mouse model to show that a single dose of burning ultraviolet radiation to neonates, but not adults, is necessary and sufficient to induce tumours with high penetrance which are reminiscent of human melanoma. Our results provide experimental support for epidemiological evidence that childhood sunburn poses a significant risk of developing this potentially fatal disease.

Using information on solar irradiance at different latitudes derived from a radiative transfer model and a detailed in vivo action spectrum for immune suppression in a murine system, we report here calculations of the "biologically effective" irradiance of sunlight for immune suppression. From 40 degrees N to 40 degrees S in summer, under normal stratospheric ozone concentrations this value ranged from 0.27 W/m2 (40 degrees N or S) to a peak of 0.33 W/m2 (20 degrees N or S) predicting that 50% immune suppression in the Balb/c mouse would occur after 21-26 min of sunlight exposure within this latitude range. We also found that the most effective wavelengths for immune suppression shift from a peak of 270 nm in the laboratory to near 315 nm in sunlight. Furthermore, using ozone depletion scenarios of 5 to 20%, at latitudes 20 degrees S and 40 degrees N, a 0.6% increase in biologically effective irradiance levels of solar UVB for immune suppression was predicted for each 1% decrease of ozone. This value rose to a nearly 1% increase for each 1% decrease in ozone at 60 degrees N latitude in wintertime. These data indicate that activation of immune suppression, in a murine model, requires relatively low levels of sunlight and that these levels are easily obtainable over most of the populated regions of the world. Since a UVB-activated photoreceptor, urocanic acid, regulates immune suppression in mice and since this same compound exists on other mammalian skin, including human skin, suppression of the mammalian immune system is predicted to increase if substantial stratospheric ozone depletion takes place.

Urocanic acid (UCA, deaminated histidine) is a major ultraviolet-absorbing component of the stratum corneum. On UV irradiation, the naturally occurring trans form converts to the cis isomer. We have previously postulated that UV-induced systemic suppression is initiated by cis-UCA by way of an antigen-presenting cell defect. To test this hypothesis further, we have investigated the antigen-presenting cell (APC) function of splenic dendritic cells (DC). Splenic DC were prepared from mice 7 days after 1 h UV irradiation (27 kJ/m2) or i.v. administration of 50-200 micrograms/mouse of cis- or trans-UCA. Dendritic cells from UV-irradiated or cis-UCA-treated mice had a significantly impaired (APC) ability, assessed by the proliferative response of purified T cells from mice immune to DNP6 OVA to DC pulsed with this antigen. Dendritic cells from mice given trans-UCA had normal APC ability. The number of FcR+ cells was the same in DCs from all four treatment groups, and the number of IAd+ cells and the intensity of IAd expression were not decreased in DCs from UV-irradiated or cis-UCA-treated mice. Mixture of DCs from UV- or cis-UCA-treated mice with DCs from normal mice did not suppress APC activity. Dendritic cells taken 3 days after UV or cis-UCA treatment, in contrast to DC taken 7 days after treatment, had normal APC ability, indicating a time delay in the generation of the APC defect. In contrast, addition of cis-UCA or trans-UCA (66 micrograms/ml) directly to an in vitro proliferation assay had no effect, suggesting that cis-UCA may be activated in vivo. These results support our original hypothesis that cis-UCA has a natural role as a modulator of immune function.

The leishmaniases remain serious public health problems in the tropics and semitropics. These regions receive two to ten times as much solar ultraviolet radiation (UV) as do the temperate or arctic zones.

Ultraviolet radiation has a suppressive effect on the immune system in vivo which appears to be a critical step in ultraviolet carcinogenesis, preventing the immunologic rejection of the highly antigenic UV-induced tumours.

Studies on the immunologic factors involved in skin cancer induction in mice by ultraviolet (UV) radiation have suggested that several immunologic processes can be altered by UV exposure. Following the observation that many UV-induced murine skin cancers are rejected immunologically upon transplantation into normal syngeneic animals [12], studies were initiated to investigate how these highly antigenic squamous carcinomas and fibrosarcomas escaped immunologic destruction in the primary host [15]. These studies led to the discovery that UV irradiation of the shaved dorsum of mice produced systemic changes in the animals that interfered with the normal immunologic rejection of these skin cancers [13,15]. One aspect of this systemic change has been elucidated in recent experiments, which produced the rather startling finding that a subcarcinogenic exposure of mice to UV radiation resulted in the induction of suppressor T lymphocytes. These cells, present in spleens and lymph nodes of UV-irradiated mice, prevent the development of an immune response against UV-induced tumors [2, 5, 6, 24]. The specificity of this immunologic alteration in UV-irradiated mice was investigated by testing their immune responses to a variety of antigens [15–17,22,25]. Although many immune responses proceeded normally in UV-irradiated animals, a depressed response to the contact sensitizer, dinitrochlorobenzene (DNCB), was observed [16]. Studies on the mechanism of this systemic suppression of immunity to DNCB demonstrated that there was a second immunologic alteration in UV-irradiated mice and suggested that this alteration occurred at the level of the cells involved in antigen uptake, processing, and presentation, i.e., macrophages or Langerhans cells [11].

The association between skin cancer and ultraviolet radiation has been known for a long time. In 1934, it was demonstrated that by filtering sunlight with ordinary window glass and then exposing white rats to this light the carcinogenic activity associated with unfiltered sunlight was eliminated.1 This suggested that the carcinogenic wavelengths had to be between 290 nm, the approximate lower wavelength limit of sunlight penetration through the atmosphere2 and 320 nm radiation, the approximate lower wavelength limit of penetration through window glass. This waveband is commonly defined as UVB. Since that time a large amount of circumstantial data supporting the role of sunlight (UVB) in at least three types of human skin cancer has accrued.2 These three types are basal cell epitheliomas, squanous cell carcinomas and melanoma.