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High-mobility-group-Box1 (HMGB1) promotes melanoma progression through the recruitment of M2 macrophages
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Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
metastatic melanomas were significantly higher than the percentage of cells in melanocytic nevi. Combined, these results indicate that Espin is not only a metastatic regulator for melanoma but also a potential biomarker of disease progression. http://dx.doi.org/10.1016/j.jdermsci.2016.08.256 P13-12[C02-07] High-mobility-group-Box1 (HMGB1) promotes melanoma progression through the recruitment of M2 macrophages Roman Huber ∗ , Atsushi Otsuka, Barbara Meier, Daniel Widmer, Takashi Satoh, Gabriele Fenini, Johanna Mangana, Tatiana Proust, Reinhard Dummer, Emmanuel Contassot, Lars E. French Department of Dermatology, University Hospital Zurich, Zurich, Switzerland High-mobility-group-Box1 (HMGB1) is a highly conserved nuclear protein regulating gene expression. Upon cell damage, HMGB1 is actively or passively released into the extracellular space where it displays chemokine and cytokine activities. In cancer, both anti-tumoral and pro-tumoral functions of HMGB1 have been reported. HMGB1 can modulate tumorassociated immune responses and deliver important signals to tumor cells. Here, we investigated the role of tumor-derived HMGB1 on melanoma progression. First, we observed that HMGB1 was overexpressed in the serum from patients with metastatic melanoma as compared to healthy individuals. The HMGB1 expression was elevated in hypoxic conditions both in vivo and in vitro. To investigate the role of extracellular HMGB1 in vivo, HMGB1 was knocked-down with shRNA in murine B16 melanoma (shHMGB1-B16). The silencing of HMGB1 led to reduced cutaneous tumor growth in mice as compared to mice bearing B16 tumors transfected with an irrelevant shRNA (shLamin-B16). Consistent with the cytokine activity of HMGB1, treatment of mice receiving wild-type B16 with a soluble HMGB1 inhibitor exhibited reduced tumor growth as compared to mice treated with placebo. The analysis of immune cells infiltrating shHMGB1- and shLamin-tumors revealed that HMGB1 expression in the tumor was associated to a preferential infiltration by M2 macrophages. In vitro exposure of M2 macrophages to HMGB1 led to an upregulation of IL-10 and CXCR4. The presence of IL-10 producing M2 macrophages was also observed in human melanoma metastases. Taken together, our results suggest that HMGB1 secreted by hypoxic tumor cells promotes tumor growth through the CXCR4mediated recruitment of M2 macrophages, the latter secreting IL-10, a cytokine known to suppress anti-tumor immunity. http://dx.doi.org/10.1016/j.jdermsci.2016.08.257 P13-13 Melanoblast maturation is mediated by the aryl hydrocarbon receptor pathway Motoki Nakamura ∗ , Emi Nishida, Akimichi Morita Department of Geriatric and Environmental Dermatology, Nagoya City University, Nagoya, Japan Pigmentation mediated by the aryl hydrocarbon receptor (AhR) is well studied. FICZ-mediated AhR activation is an essential part of the UVB- induced stress response in epidermal keratinocytes,
and AhR mediates UVB-induced skin tanning. We previously reported that AhR-mediated activation of human melanocytes is caused not only by UVB irradiation, but also by smoking tobacco. We hypothesized that melanoblast-to-melanocyte maturation is also mediated by AhR, and investigated AhR expression in melanoblasts stimulated by UVB irradiation and other AhR agonists. We used a cloned cell line, NCCmelb4, derived from mouse neural crest cells. NCCmelb4 cells are immature melanocyte precursors, ‘melanoblasts’, as they are positive for tyrosinase-related protein-1, tyrosinase-related protein-2, and KIT (a stem cell factor receptor), but are negative for tyrosinase and endothelin B receptors. Tyrosinase activation (an indicator of melanoblast-tomelanocyte maturation) was measured by real-time polymerase chain reaction. UVB ranging from 280 to 380 nm in 10-nm increments was irradiated using a multi-wavelength irradiation spectral apparatus. Tyrosinase expression was significantly increased, with bimodal peaks at 310 nm and 360 nm. AhR expression significantly increased at 360 nm, but not at 310 nm. AhR agonist VAF347 and water-soluble tobacco smoke extract induced melanoblast maturation and AhR activation. Culture supernatant derived from the NS47 fibroblast cell line (NS sup) also induced melanoblast maturation and AhR activation. These findings suggest that UVB and environment-stimulated melanoblast-to-melanocyte maturation is enhanced via the AhR pathway. Further, AhR may mediate the interaction between fibroblasts and melanoblasts. http://dx.doi.org/10.1016/j.jdermsci.2016.08.258 P13-14 Anti-B16 melanoma effects of a STAT3 inhibitor (rR9-GRIM19) are strongly enhanced by co-treatment with recombinant interferons Takashi Okamoto ∗ , Naotaka Shibagaki, Shinji Shimada Department of Dermatology, University of Yamanashi, Yamanashi, Japan Induction of cell death and inhibition of cell survival are main principles of cancer therapy. Although rIFN-alpha/beta, or rIFN-gamma is clinically utilized as an optional cancer/melanoma treatment, direct anti-melanoma effects are not satisfactory. STAT3 mediates critical changes in gene expression and molecular events that dysregulate cell growth and apoptosis. Generally, activated STAT3 could inhibit STAT1-mediated IFN signaling transduction. Therefore, inhibiting the STAT3-mediated signaling cascades within cancer microenvironment is a novel and prominent strategy for cancer therapy. Previously, we have demonstrated that rR9GRIM19 (our novel STAT3-targeting inhibitor) elicited enhanced anti-B16 melanoma effects in vivo when combined either with rR9-OVA (as our Th1/Tc1-inducible adjuvant) or CpG-ODN intratumorally, but only the combination of CpG-ODN, rR9-OVA, and rR9-GRIM19 (COG therapy) elicited complete B16 tumor regression with IFN-gamma-producing T cell activation/infiltration. In this study, we investigated whether rR9-GRIM19-mediated direct anti-B16 tumor effects could be enhanced when co-treated with rIFNs (as STAT1 activators). In culture, exposure of rIFN-beta, rIFNgamma, or rR9-GRIM19 alone suppressed marginal B16 cell growth. In contrast, combination with rIFN-beta/rIFN-gamma and rR9GRIM19 significantly suppressed B16-cell growth with apoptosis. In vivo, intratumoral injections of rIFN-beta/IFN- gamma plus rR9GRIM19 also elicited strong anti-B16 tumor effect with complete regression as well as our original COG therapy. Thus, our results indicate that combination therapy with STAT3 inhibitors and rIFNs
Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
metastatic melanomas were significantly higher than the percentage of cells in melanocytic nevi. Combined, these results indicate that Espin is not only a metastatic regulator for melanoma but also a potential biomarker of disease progression. http://dx.doi.org/10.1016/j.jdermsci.2016.08.256 P13-12[C02-07] High-mobility-group-Box1 (HMGB1) promotes melanoma progression through the recruitment of M2 macrophages Roman Huber ∗ , Atsushi Otsuka, Barbara Meier, Daniel Widmer, Takashi Satoh, Gabriele Fenini, Johanna Mangana, Tatiana Proust, Reinhard Dummer, Emmanuel Contassot, Lars E. French Department of Dermatology, University Hospital Zurich, Zurich, Switzerland High-mobility-group-Box1 (HMGB1) is a highly conserved nuclear protein regulating gene expression. Upon cell damage, HMGB1 is actively or passively released into the extracellular space where it displays chemokine and cytokine activities. In cancer, both anti-tumoral and pro-tumoral functions of HMGB1 have been reported. HMGB1 can modulate tumorassociated immune responses and deliver important signals to tumor cells. Here, we investigated the role of tumor-derived HMGB1 on melanoma progression. First, we observed that HMGB1 was overexpressed in the serum from patients with metastatic melanoma as compared to healthy individuals. The HMGB1 expression was elevated in hypoxic conditions both in vivo and in vitro. To investigate the role of extracellular HMGB1 in vivo, HMGB1 was knocked-down with shRNA in murine B16 melanoma (shHMGB1-B16). The silencing of HMGB1 led to reduced cutaneous tumor growth in mice as compared to mice bearing B16 tumors transfected with an irrelevant shRNA (shLamin-B16). Consistent with the cytokine activity of HMGB1, treatment of mice receiving wild-type B16 with a soluble HMGB1 inhibitor exhibited reduced tumor growth as compared to mice treated with placebo. The analysis of immune cells infiltrating shHMGB1- and shLamin-tumors revealed that HMGB1 expression in the tumor was associated to a preferential infiltration by M2 macrophages. In vitro exposure of M2 macrophages to HMGB1 led to an upregulation of IL-10 and CXCR4. The presence of IL-10 producing M2 macrophages was also observed in human melanoma metastases. Taken together, our results suggest that HMGB1 secreted by hypoxic tumor cells promotes tumor growth through the CXCR4mediated recruitment of M2 macrophages, the latter secreting IL-10, a cytokine known to suppress anti-tumor immunity. http://dx.doi.org/10.1016/j.jdermsci.2016.08.257 P13-13 Melanoblast maturation is mediated by the aryl hydrocarbon receptor pathway Motoki Nakamura ∗ , Emi Nishida, Akimichi Morita Department of Geriatric and Environmental Dermatology, Nagoya City University, Nagoya, Japan Pigmentation mediated by the aryl hydrocarbon receptor (AhR) is well studied. FICZ-mediated AhR activation is an essential part of the UVB- induced stress response in epidermal keratinocytes,
and AhR mediates UVB-induced skin tanning. We previously reported that AhR-mediated activation of human melanocytes is caused not only by UVB irradiation, but also by smoking tobacco. We hypothesized that melanoblast-to-melanocyte maturation is also mediated by AhR, and investigated AhR expression in melanoblasts stimulated by UVB irradiation and other AhR agonists. We used a cloned cell line, NCCmelb4, derived from mouse neural crest cells. NCCmelb4 cells are immature melanocyte precursors, ‘melanoblasts’, as they are positive for tyrosinase-related protein-1, tyrosinase-related protein-2, and KIT (a stem cell factor receptor), but are negative for tyrosinase and endothelin B receptors. Tyrosinase activation (an indicator of melanoblast-tomelanocyte maturation) was measured by real-time polymerase chain reaction. UVB ranging from 280 to 380 nm in 10-nm increments was irradiated using a multi-wavelength irradiation spectral apparatus. Tyrosinase expression was significantly increased, with bimodal peaks at 310 nm and 360 nm. AhR expression significantly increased at 360 nm, but not at 310 nm. AhR agonist VAF347 and water-soluble tobacco smoke extract induced melanoblast maturation and AhR activation. Culture supernatant derived from the NS47 fibroblast cell line (NS sup) also induced melanoblast maturation and AhR activation. These findings suggest that UVB and environment-stimulated melanoblast-to-melanocyte maturation is enhanced via the AhR pathway. Further, AhR may mediate the interaction between fibroblasts and melanoblasts. http://dx.doi.org/10.1016/j.jdermsci.2016.08.258 P13-14 Anti-B16 melanoma effects of a STAT3 inhibitor (rR9-GRIM19) are strongly enhanced by co-treatment with recombinant interferons Takashi Okamoto ∗ , Naotaka Shibagaki, Shinji Shimada Department of Dermatology, University of Yamanashi, Yamanashi, Japan Induction of cell death and inhibition of cell survival are main principles of cancer therapy. Although rIFN-alpha/beta, or rIFN-gamma is clinically utilized as an optional cancer/melanoma treatment, direct anti-melanoma effects are not satisfactory. STAT3 mediates critical changes in gene expression and molecular events that dysregulate cell growth and apoptosis. Generally, activated STAT3 could inhibit STAT1-mediated IFN signaling transduction. Therefore, inhibiting the STAT3-mediated signaling cascades within cancer microenvironment is a novel and prominent strategy for cancer therapy. Previously, we have demonstrated that rR9GRIM19 (our novel STAT3-targeting inhibitor) elicited enhanced anti-B16 melanoma effects in vivo when combined either with rR9-OVA (as our Th1/Tc1-inducible adjuvant) or CpG-ODN intratumorally, but only the combination of CpG-ODN, rR9-OVA, and rR9-GRIM19 (COG therapy) elicited complete B16 tumor regression with IFN-gamma-producing T cell activation/infiltration. In this study, we investigated whether rR9-GRIM19-mediated direct anti-B16 tumor effects could be enhanced when co-treated with rIFNs (as STAT1 activators). In culture, exposure of rIFN-beta, rIFNgamma, or rR9-GRIM19 alone suppressed marginal B16 cell growth. In contrast, combination with rIFN-beta/rIFN-gamma and rR9GRIM19 significantly suppressed B16-cell growth with apoptosis. In vivo, intratumoral injections of rIFN-beta/IFN- gamma plus rR9GRIM19 also elicited strong anti-B16 tumor effect with complete regression as well as our original COG therapy. Thus, our results indicate that combination therapy with STAT3 inhibitors and rIFNs