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Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Zaidi MR, Davis S, Noonan FP, Graff-Cherry C, Hawley TS, Walker RL, Feigenbaum L, Fuchs E, Lyakh L, Young HA, Hornyak TJ, Arnheiter H, Trinchieri G, Meltzer PS, De Fabo EC, Merlino G - Nature (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
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.

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Melanocyte-specific GFP expression reveals UVB-induced activationa, E11.5 iDct-GFP embryo showing GFP+ cells in neural crest (red arrow) and telencephalon (white arrow). b, In 7-day old pup skin GFP+ cells are located in the bulb (lower arrow) and bulge (upper arrow) regions of hair follicles. Blue = DAPI; E, epidermis; D, dermis. c, Immunohistochemistry with anti-Dct antibody shows co-localization with GFP in iDct-GFP skin. d, UVB-induced activation of melanocytes, characterized by proliferation and migration towards epidermis. Dorsal skins were examined at 1 day (at age P2) and 6 days (P7) post-irradiation. Scale bars = 40 μm. e, Schematic of the regime for isolating GFP+ melanocytes by FACS. Pups are irradiated at P1, and dorsal skins harvested at either P2 (24 h post-UV) or P7 (6 d post-UV). Doxycycline injections are always given after irradiation, 24 h prior to skin harvest.
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Figure 1: Melanocyte-specific GFP expression reveals UVB-induced activationa, E11.5 iDct-GFP embryo showing GFP+ cells in neural crest (red arrow) and telencephalon (white arrow). b, In 7-day old pup skin GFP+ cells are located in the bulb (lower arrow) and bulge (upper arrow) regions of hair follicles. Blue = DAPI; E, epidermis; D, dermis. c, Immunohistochemistry with anti-Dct antibody shows co-localization with GFP in iDct-GFP skin. d, UVB-induced activation of melanocytes, characterized by proliferation and migration towards epidermis. Dorsal skins were examined at 1 day (at age P2) and 6 days (P7) post-irradiation. Scale bars = 40 μm. e, Schematic of the regime for isolating GFP+ melanocytes by FACS. Pups are irradiated at P1, and dorsal skins harvested at either P2 (24 h post-UV) or P7 (6 d post-UV). Doxycycline injections are always given after irradiation, 24 h prior to skin harvest.

Mentions: Mechanisms associated with UV-mediated alterations to melanocytes and their microenvironment have been inscrutable because they cannot be adequately studied in cultured cells. Moreover, melanocytes represent only ~1% of skin cells, and bear few specific cell surface markers permitting efficient isolation. To enable detailed study of melanocyte biology in vivo, we generated a mouse model in which expression of the reverse tetracycline-activated transactivator rtTA2s-M2, characterized by minimal leakiness and background, was regulated by the melanocyte-specific dopachrome tautomerase (Dct) gene promoter (Supplementary Fig. 1a). Dct-rtTA mice bred with transgenic mice bearing a histone H2B-GFP fusion construct controlled by the tetracycline response element (TRE) created Dct-rtTA/TRE-H2BGFP bi-transgenic mice (hereafter iDct-GFP) (Supplementary Fig. 1b). iDct-GFP mice exhibited an inducible GFP profile from embryonic through adult stages consistent with known Dct expression patterns. GFP expression was observed in embryonic neural crest, retinal pigment epithelium and telencephalon, as expected (Fig. 1a; Supplementary Fig. 2). Neonatal and adult skin GFP+ cells were strictly localized to hair follicles, where most GFP+ cells were in bulb regions, with smaller numbers in the outer root sheath and bulge regions, harboring melanocyte precursors5 (Fig. 1b). Co-localization of GFP and anti-Dct antibody by immunohistochemistry (IHC) unequivocally identified GFP+ cells as melanocytes (Fig. 1c). No background GFP expression was detectable without doxycycline. Full GFP induction was achieved within 12–18 hours of a single intraperitoneal injection of a non-toxic doxycycline dose in neonatal or adult mice (Supplementary Fig. 1c).


Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Zaidi MR, Davis S, Noonan FP, Graff-Cherry C, Hawley TS, Walker RL, Feigenbaum L, Fuchs E, Lyakh L, Young HA, Hornyak TJ, Arnheiter H, Trinchieri G, Meltzer PS, De Fabo EC, Merlino G - Nature (2011)

Melanocyte-specific GFP expression reveals UVB-induced activationa, E11.5 iDct-GFP embryo showing GFP+ cells in neural crest (red arrow) and telencephalon (white arrow). b, In 7-day old pup skin GFP+ cells are located in the bulb (lower arrow) and bulge (upper arrow) regions of hair follicles. Blue = DAPI; E, epidermis; D, dermis. c, Immunohistochemistry with anti-Dct antibody shows co-localization with GFP in iDct-GFP skin. d, UVB-induced activation of melanocytes, characterized by proliferation and migration towards epidermis. Dorsal skins were examined at 1 day (at age P2) and 6 days (P7) post-irradiation. Scale bars = 40 μm. e, Schematic of the regime for isolating GFP+ melanocytes by FACS. Pups are irradiated at P1, and dorsal skins harvested at either P2 (24 h post-UV) or P7 (6 d post-UV). Doxycycline injections are always given after irradiation, 24 h prior to skin harvest.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3140101&req=5

Figure 1: Melanocyte-specific GFP expression reveals UVB-induced activationa, E11.5 iDct-GFP embryo showing GFP+ cells in neural crest (red arrow) and telencephalon (white arrow). b, In 7-day old pup skin GFP+ cells are located in the bulb (lower arrow) and bulge (upper arrow) regions of hair follicles. Blue = DAPI; E, epidermis; D, dermis. c, Immunohistochemistry with anti-Dct antibody shows co-localization with GFP in iDct-GFP skin. d, UVB-induced activation of melanocytes, characterized by proliferation and migration towards epidermis. Dorsal skins were examined at 1 day (at age P2) and 6 days (P7) post-irradiation. Scale bars = 40 μm. e, Schematic of the regime for isolating GFP+ melanocytes by FACS. Pups are irradiated at P1, and dorsal skins harvested at either P2 (24 h post-UV) or P7 (6 d post-UV). Doxycycline injections are always given after irradiation, 24 h prior to skin harvest.
Mentions: Mechanisms associated with UV-mediated alterations to melanocytes and their microenvironment have been inscrutable because they cannot be adequately studied in cultured cells. Moreover, melanocytes represent only ~1% of skin cells, and bear few specific cell surface markers permitting efficient isolation. To enable detailed study of melanocyte biology in vivo, we generated a mouse model in which expression of the reverse tetracycline-activated transactivator rtTA2s-M2, characterized by minimal leakiness and background, was regulated by the melanocyte-specific dopachrome tautomerase (Dct) gene promoter (Supplementary Fig. 1a). Dct-rtTA mice bred with transgenic mice bearing a histone H2B-GFP fusion construct controlled by the tetracycline response element (TRE) created Dct-rtTA/TRE-H2BGFP bi-transgenic mice (hereafter iDct-GFP) (Supplementary Fig. 1b). iDct-GFP mice exhibited an inducible GFP profile from embryonic through adult stages consistent with known Dct expression patterns. GFP expression was observed in embryonic neural crest, retinal pigment epithelium and telencephalon, as expected (Fig. 1a; Supplementary Fig. 2). Neonatal and adult skin GFP+ cells were strictly localized to hair follicles, where most GFP+ cells were in bulb regions, with smaller numbers in the outer root sheath and bulge regions, harboring melanocyte precursors5 (Fig. 1b). Co-localization of GFP and anti-Dct antibody by immunohistochemistry (IHC) unequivocally identified GFP+ cells as melanocytes (Fig. 1c). No background GFP expression was detectable without doxycycline. Full GFP induction was achieved within 12–18 hours of a single intraperitoneal injection of a non-toxic doxycycline dose in neonatal or adult mice (Supplementary Fig. 1c).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus