Comprehensive molecular profiling of UV-induced metastatic melanoma in Nme1/Nme2-deficient mice reveals novel markers of survival in human patients.

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 and Nme2 genes exert metastasis-suppressor activities in a genetically engineered mouse model of UV-induced 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.

A melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent 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.

Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment.

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.

Non-thermal nanoelectroablation of UV-induced murine melanomas stimulates an immune response.

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.

Shedding light on melanocyte pathobiology in vivo.

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.

Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the 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, identifying a novel candidate therapeutic target for a subset of melanoma patients.

UVB and UVA initiate different pathways to p53-dependent apoptosis in melanocytes.

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.

Economic evaluation of the US Environmental Protection Agency's SunWise program: sun protection education for young children.

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 11,000 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.

Initial studies on an in vivo action spectrum for melanoma induction.

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.

Arctic stratospheric ozone depletion and increased UVB radiation: potential impacts to human health.

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.

Ultraviolet B but not ultraviolet A radiation initiates melanoma.

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.

Immunotoxic effects of cis-urocanic acid exposure in C57BL/6N and C3H/HeN mice.

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.

Animal models of melanoma: an HGF/SF transgenic mouse model may facilitate experimental access to UV initiating events.

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.

Mice with genetically determined high susceptibility to ultraviolet (UV)-induced immunosuppression show enhanced UV carcinogenesis.

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 < 0.0005). Differences between strains were significant for the higher dose (p = 0.03) but not for the lower dose (p = 0.19) of UV, suggesting a dose-strain interaction. Comparing the higher UV dose regimen to the lower UV dose regimen within a strain at three reference points, tumor-free survival was reduced significantly more (p < 0.05) in the CB6F1 mice than in the B6CF1 mice. Histologic assessment of all tumors revealed fibrosarcomas, squamous carcinomas, and mixed tumors. Immunohistochemistry of the mixed tumors for vimentin, keratin, and E-cadherin confirmed the presence of squamous and fibrosarcomatous elements. The enhanced susceptibility to UV carcinogenesis of CB6F1 males treated with the higher UV protocol was attributable to a significantly enhanced proportion (p < 0.005) of mixed tumors. Analysis of the data by comparing the proportion of animals tumor free at three reference time points confirmed a dose-strain interaction only in the development of mixed tumors, putatively the malignantly advanced carcinomas (p < 0.03). A dose-strain interaction was also observed for systemic UV immunosuppression of contact hypersensitivity (p < 0.025). These findings support the concept that genetic differences in susceptibility to UV-induced immunosuppression may be a risk factor for skin cancer.

Ink4a/arf deficiency promotes ultraviolet radiation-induced melanomagenesis.

Cutaneous malignant melanoma (CMM), already known for its highly aggressive behavior and resistance to conventional therapy, has evolved into a health crisis by virtue of a dramatic elevation in incidence. The underlying genetic basis for CMM, as well as the fundamental role for UV radiation in its etiology, is now widely accepted. However, the only bona fide genetic locus to emerge from extensive analysis of CMM suppressor candidates is INK4a/ARF at 9p21, which is lost frequently in familial and occasionally in somatic CMM. The functional relationship between INK4a/ARF and UV radiation in the pathogenesis of CMM is largely unknown. Recently, we reported that hepatocyte growth factor/scatter factor (HGF/SF)-transgenic mice develop melanomas after a single erythemal dose of neonatal UV radiation, supporting epidemiological data implicating childhood sunburn in CMM. Here we show that neonatal UV irradiation induces a full spectrum of melanocyte pathology from early premalignant lesions through distant metastases. Cutaneous melanomas arise with histopathological and molecular pathogenetic features remarkably similar to CMM, including loss of ink4a/arf. A role for ink4a/arf in UV-induced melanomagenesis was directly assessed by placing the HGF/SF transgene on a genetic background devoid of ink4a/arf. Median time to melanoma development induced by UV radiation was only 50 days in HGF/SF ink4a/arf(-/-) mice, compared with 152 and 238 days in HGF/SF ink4a/arf(+/-) and HGF/SF ink4a/arf(+/+) mice, respectively. These studies provide experimental evidence that ink4a/arf plays a critical role in UV-induced melanomagenesis and strongly suggest that sunburn is a highly significant risk factor, particularly in families harboring germ-line mutations in INK4a/ARF.