Melanocyte Differentiation Research Articles

Human fibroblasts treated with hydrogen peroxide stimulate human melanoblast proliferation and melanocyte differentiation, but inhibit melanocyte proliferation in serum-free co-culture system

BackgroundOxidative stress caused by hydrogen peroxide (H2O2) elicits harmful effects on human melanocytes such as DNA damage and cell death. On the contrary, H2O2 is known to possess beneficial effects on melanocytes. However, mechanisms of the beneficial effects of H2O2 on melanocytes have not been fully understood, especially the indirect effects on melanocyte proliferation and differentiation from cells constituting surrounding tissue environment such as fibroblasts. ObjectiveThe aim of this study was to clarify whether H2O2-treated human fibroblasts affect the proliferation and differentiation of human melanocytes using serum-free co-culture system. MethodsEpidermal melanoblasts and melanocytes were co-cultured with H2O2-treated or control fibroblasts in serum-free culture media. The effects of H2O2-treated fibroblasts were detected by changes in the proliferation and differentiation of melanoblasts/melanocytes. ResultsH2O2-treated fibroblasts stimulated the proliferation of melanoblasts and the differentiation, melanogenesis, and dendritogenesis of melanocytes, but inhibited the proliferation of melanocytes. In the melanocytes co-cultured with H2O2-treated fibroblasts, the expression of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and KIT was increased, whereas TYRP2 and microphthalmia-associated transcription factor showed no change. ConclusionThese results suggest that H2O2-treated fibroblasts can produce and release some mitogenic and melanogenic factors toward melanoblasts in addition to some proliferation-inhibiting factors toward melanocytes. The stimulation of melanocyte differentiation seems to be performed through the upregulation of TYR, TYRP1, and KIT.

A hexIM1 on your melanocytes: transcription elongation – the Achilles’ heel of melanoma?

A hexIM1 on your melanocytes: transcription elongation – the Achilles’ heel of melanoma?

Expression of natural killer cell regulatory microRNA by uveal melanoma cancer stem cells.

Natural killer (NK) cells are implicated in the control of metastasis in uveal melanoma, a process that has been ascribed to its cancer stem cell subpopulation. NK cell activation is regulated by specific microRNA (miR). The NK cell sensitivity and regulatory miR production of uveal melanoma cancer stem cells was examined. Cancer stem cells enriched from aggressively metastatic MUM2B uveal melanoma cells by selecting CD271+ cells or propagating as non-adherent spheres in stem-cell supportive were more resistant to NK cell cytolysis than cancer stem cells enriched from less aggressively metastatic OCM1 uveal melanoma cells. Both MUM2B and OCM1 cells expressed and secreted NK cell regulatory miRs, including miR 146a, 181a, 20a, and 223. MUM2B cells expressed and secreted miR-155; OCM1 cells did not. Transfecting MUM2B cells with anti-miR-155 increased NK cell sensitivity. CD271+ cells were identified in the blood of patients with metastatic uveal melanoma and were characterized by low expression of melanocyte differentiation determinants and by the ability to form non-adherent spheres in stem-cell supportive media. These cells also expressed NK cell regulatory miRs, including miR-155. These results indicate that uveal melanoma cancer stem cells can vary in their sensitivity to NK cell lysis and their expression of NK cell regulatory miRs. Circulating CD271+ cells from patients with metastatic uveal melanoma manifest cancer stem cell features and express miRs associated with NK cell suppression, including miR-155, that may contribute to metastatic progression.

Wnt/β-catenin signaling inhibitor ICG-001 enhances pigmentation of cultured melanoma cells

BackgroundWnt/β-catenin signaling is important in development and differentiation of melanocytes. ObjectiveThe object of this study was to evaluate the effects of several Wnt/β-catenin signaling inhibitors on pigmentation using melanoma cells. MethodsMelanoma cells were treated with Wnt/β-catenin signaling inhibitors, and then melanin content and tyrosinase activity were checked. ResultsAlthough some inhibitors showed slight inhibition of pigmentation, we failed to observe potential inhibitory effect of those chemicals on pigmentation of HM3KO melanoma cells. Rather, one of powerful Wnt/β-catenin signaling inhibitors, ICG-001, increased the pigmentation of HM3KO melanoma cells. Pigmentation-enhancing effect of ICG-001 was reproducible in other melanoma cell line MNT-1. Consistent with these results. ICG-001 increased the expression of pigmentation-related genes, such as MITF, tyrosinase and TRP1. When ICG-001 was treated, the phosphorylation of CREB was significantly increased. In addition, ICG-001 treatment led to quick increase of intracellular cAMP level, suggesting that ICG-001 activated PKA signaling. The blockage of PKA signaling with pharmaceutical inhibitor H89 inhibited the ICG-001-induced pigmentation significantly. ConclusionsThese results suggest that PKA signaling is pivotal in pigmentation process itself, while the importance of Wnt/β-catenin signaling should be emphasized in the context of development and differentiation.

Abstract 296: Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma

Abstract Background: The duration of survival benefit from MAPKi in B-raf mutant melanoma (MEL) is limited by the near universal development of resistance (RES). Recently, MiTF, a transcription factor and master regulator of melanocyte differentiation - was identified as a hub of MAPKi RES. Given its dual role as both a tumor promoter and inducer of MEL senescence, MiTF is a problematic therapeutic target. Pigmentation is a MiTF-dependent feature of MELs correlates with poor survival. We have interrogated the MAPKi/MiTF-dependent regulation of a panel of MITF-regulated melanosomal differentiation antigens (MDAs) for their potential as therapeutic targets to overcome MAPKi RES. Methods: The TCGA MEL gene expression data set was interrogated. B-raf mutant MEL cells were exposed to dabrafenib (D), or trametinib (T). qRT-PCR, Immunoblot or FACS were used to assess MiTF and MDA expression. Serial biopsy tissue and serum samples from MEL patients taken before treatment, during treatment, and at progression with MAPKi therapy were obtained, and analyzed by IHC and qRT-PCR, RNAseq, and ELISA. Transient siRNA-mediated knockdown was used to assess the requirement for MiTF, in MAPKi-mediated induction of MDAs, including GPNMB. To assess the interaction between MAPKi and CDX011 (C), a GPNMB- targeted antibody drug conjugate, mice bearing A375 and WM2664 xenografts were treated with T or DT + C in combination. Results: MiTF is strongly correlated with MDAs, including GPNMB in gene-expression datasets derived from human MEL tumors. A MiTF driven MDA-gene-signature correlates with poor survival among 291 melanoma patients. MAPKi treatment increased MiTF and MDAs in MEL cells, and induced pigmentation in vivo. This phenotype was reversible as MEL acquired RES to MAPKi. Knockdown of MiTF leads to abrogation of MAPKi-mediated GPNMB induction. GPNMB promotes MEL invasion in vitro. MiTF and MDA were elevated in tumors and serum from patients receiving MAPKi treatment and returned to baseline in tumors that acquired RES. The addition of C to either T or DT led to, decreased pigmentation, prolonged tumor regression and inhibited the development RES to T or DT treatment in vivo. Tumors that acquired RES to DT displayed increased c-Met expression and MAPK activation; tumors treated with DT+C failed to develop this phenotype. Conclusions: MAPKi induce MDA in a MiTF-dependent manner in MEL. This process leads to melanocytic differentiation and increased pigmentation, which is subsequently reversed as a tumor acquires RES to MAPKi. This demonstrates the plasticity of MEL in response to MAPKi. The pigmented phenotype is less proliferative but more invasive which is in part mediated by GPNMB. This transient phenotype may allow tumors to acquire characteristics (ie. c-Met upregulation and MAPK re-activation) which contribute to MAPKi RES. Targeting a pro-invasive MDA, GPNMB with CDX-011 subverts this plasticity and impairs the development of MAPKi RES. Citation Format: April A.N. Rose, Matthew G. Annis, Dennie T. Frederick, Zhifeng Dong, Lawrence Kwong, Tibor Keller, Thomas Hawthorne, Ian R. Watson, Keith T. Flaherty, Peter M. Siegel. Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 296.

Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.

Lineage-specific transcription factors determine the cell fate during development. Direct conversion of several cell types into other lineages has been achieved by the overexpression of specific transcription factors. Even cancer cells have been demonstrated to be amenable to transdifferentiation. Here, we identified a distinct set of transcription factors, which are sufficient to transform cells of the keratinocytic lineage to melanocyte-like cells. Melanocyte marker expression was induced and melanosome formation was observed in non-tumorigenic keratinocytes (HaCaT) and tumorigenic squamous cell carcinoma (MET-4) cells. Moreover, reduced proliferation, cell metabolism, invasion and migration were measured in vitro in transdifferentiated MT-MET-4 cells. A loss of tumorigenic potential of squamous cell carcinoma cells could be due to the upregulation of the melanocyte differentiation associated gene IL-24. Our data show that cells from the keratinocytic lineage can be transdifferented into the melanocytic lineage and provide a proof of principle for a potential new therapeutic strategy.

Frizzled with age: an opportunity for ‘gerontological medicine’

Frizzled with age: an opportunity for ‘gerontological medicine’

The role of microphthalmia transcription factor (Mitf) in prediction of distant metastases in cutaneous melanoma.

9564Background: Microphthalmia Transcription Factor (Mitf) plays a central role in melanocyte maturation and differentiation, as well as tumorigenesis. We previously proved that Mitf expression of ...

Platelet-derived growth factor regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner

BackgroundAlthough many kinds of keratinocyte-derived factors are known to regulate the proliferation and differentiation of human melanocytes, it is not well defined whether dermis-derived factors work in a similar way. ObjectiveThe aim of this study is to clarify whether dermal factors are involved in regulating the proliferation and differentiation of human melanocytes. MethodsHuman epidermal melanoblasts were cultured serially in a serum-free growth medium. Platelet-derived growth factor-BB (PDGF-BB) was supplemented to the medium, and the effects on the proliferation of melanoblasts/melanocytes and the differentiation of melanocytes were studied. ResultsPDGF-BB stimulated the proliferation of melanoblasts cultured in melanoblast-proliferation medium, but inhibited the proliferation of melanocytes cultured in melanocyte-proliferation medium. By contrast, PDGF-BB stimulated the differentiation, dendritogenesis, and melanogenesis of melanocytes through the stimulation of tyrosinase activity and the expressions of tyrosinase and tyrosinase-related protein-1. ConclusionThese results suggest that PDGF-BB regulates the proliferation and differentiation of human melanocytes in a differentiation-stage-specific manner. PDGF-BB seems to be one of the dermal factors that regulate the proliferation and differentiation of human melanocytes.

Adoptive transfer of induced pluripotent stem cell-derived regenerated T cells mediates effective regression of large tumors and establishes immunological memory

Abstract Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. However, therapeutic efficacy of iPSC-derived regenerated T cells remains to be elucidated. To this end, we established iPSCs from Pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen as a preclinical model of evaluating antitumor reactivity of regenerated antigen-specific T cells from iPSCs (Pmel-1 iPSCs). Pmel-1 iPSCs retained the same rearranged configuration as the original Pmel-1 CD8+ T cells, and differentiated into CD4+CD8+ double positive (DP) T cells that expressed CD3 and the desired TCR in vitro. Stimulation of iPSC-derived DP T cells with the cognate antigen in the presence of IL-7 and IL-15 followed by expansion with IL-2, IL-7 and IL-15 generated large numbers of less-differentiated CD8+ single positive T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8+ T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. These findings support the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional and expandable autologous antigen-specific T cells harboring characteristics necessary for in vivo effectiveness.

265 Pilot trial to evaluate the effect of vitamin D on melanocyte biomarkers

264 Topical treatments improved periorbital aging J Namkoong, B Cook, D Kern and HE Knaggs Global R&D, Nu Skin Enterprises, Inc., Provo, UT Facial aging is manifested by different symptoms. One area people pay more attention to and use additional cosmetic products to keep healthier is the eye area e the periorbital region. There are multiple reasons why the periorbital region manifests an unhealthy and aged look. The skin in the eye area is thinner than other parts of face, and repetitive muscle movements and UV exposures lead to skin aging. Periorbital aging includes eyelid drooping, dark circles, under eye bags, or fine lines/wrinkles. Topical and mechanical treatments to improve periorbital aging is challenging due to the proximity to eyes. Hypersensitivity to treatment options are common occurrence in the eyes. In order to improve the skin in the eye area, topical ingredients were evaluated for their potential to modulate different skin aging target genes, such as improving skin structures, hydration, anti-inflammation, antioxidant capacity and cell turnover, using normal human skin equivalent cultures. Some interesting finding includes modulation of Prostaglandin E2 (PGE2). Induction of cellular stress with 5mM or 10mM hydrogen peroxide increased PGE2 expression, which would increase inflammation. In addition, PGE2 increase has been suggested in the skin of elderly and inhibition of PGE2 resulted in collagen production in skin organ culture. Experimental ingredients were able to suppress that H2O2-induced PGE2 expression, similar to ascorbic acid. After evaluating ingredients using in vitro models, a specific formula was developed to target clinical signs of periorbital skin aging. Prior to running a clinical study, the formula was evaluated for safety, due to potential hypersensitivity. This eye formulation did not trigger any safety concerns during in vitro evaluations and non-ocular evaluations. The formula was further assessed for safety during the efficacy testing by both dermatological and ophthalmologic evaluations. This formula improved periorbital aging in 50% of subjects with sagging skin, eyelid firmness and puffiness after 12 weeks of application by dermatological evaluations. 265 Pilot trial to evaluate the effect of vitamin D on melanocyte biomarkers E Anderson, M Rezaee, I Bailey-Healy, K Sarin and J Tang 1 School of Medicine, Stanford University, Stanford, CA and 2 Dermatology, Stanford University, Stanford, CA The role of vitamin D for cancer prevention is an expanding area of interest. In clinical and epidemiologic studies, vitamin D has been shown to predict incidence and severity of melanoma skin cancer. Although the role of vitamin D in melanocyte differentiation and melanoma carcinogenesis has been described, a clear mechanism is yet to be established. Previous studies have suggested that vitamin D supplementation and increased serum vitamin D levels are associated with decreased melanoma incidence. We sought to determine the effect of oral vitamin D supplementation on gene expression within melanocytic nevi of women previously diagnosed with non-melanoma skin cancer. A total of 24 healthy women were included in this randomized placebo-controlled study. Half of the cohort received 4,000 IU oral vitamin D3 daily for 8 weeks. Skin checks were performed at baseline and 8 week follow-up, and nevi were biopsied at both time points. RNAwas collected from these nevi for gene expression profiling to identify activity in signaling pathways associated with vitamin D treatment. Women who received vitamin D supplementation experienced a significant increase in vitamin D levels at follow-up (p 1⁄4 0.0001) while women who received placebo did not (p 1⁄4 0.50). Gene expression analysis of melanocytic nevi revealed 1048 genes that were significantly differentially expressed between vitamin D-treated and placebo-controlled women. Ingenuity Pathway Analysis indicated that the most significantly differentiated molecular signaling pathways were related to immune system signaling, including primary immunodeficiency signaling (p 1⁄4 3.53 x 10), T cell receptor signaling (p 1⁄4 4.31 x 10), iCOS-iCOSL signaling in T helper cells (p 1⁄4 1.24 x 10), calcium-induced T lymphocyte apoptosis (p 1⁄4 7.52 x 10), and crosstalk between dendritic cells and natural killer cells (p 1⁄4 1.68 x 10). This finding suggests that increased immune surveillance could underlie the previously described association between vitamin D supplementation and decreased melanoma incidence.

345 Cell culture media that are less proliferative and more homeostatic improve differentiation, responsiveness and co-culture of keratinocytes, fibroblasts and melanocytes in 2D and 3D culture

Combinations of highly proliferative cell culture additives such as serum (FCS), pituitary extract (BPE), insulin and EGF/FGF-family members are routinely used to generate rapid in vitro proliferation of a variety of skin cell types. Highly concentrated proliferative signals such as these are ideal for amplifying cells, however they are poorly suited to many other situations where very different cell behaviours are required. We have investigated a range of less proliferative, more homeostatic media for their suitability when inducing differentiation and co-culturing keratinocytes, fibroblasts and melanocytes. Differentiation was improved in all three cell types, with 2x better barrier (3D keratinocytes), 2.5x more melanin (2D melanocytes), and 5x more ECM (2D fibroblasts). Homeostatic media were also found to increase responsiveness of melanocytes and fibroblasts to external stimuli such as tyrosinase inhibitors and vitamin C treatment respectively. Variants of standard proliferation media were also found to deliver balanced 2D co-culture of keratinocytes and fibroblasts, with each cell type delivering similar proliferation rates when seeded at equal density on standard tissue culture plastic. Finally, a fibroblast medium tailored for ECM production was also found to be ideally suited to the fast and simple establishment of full-thickness skin models with fully natural dermal matrix, thereby avoiding the handling challenges and variability associated with traditional collagen scaffolds. In conclusion, less proliferative, more homeostatic culture media were found to deliver a range of improved cell behaviours that complement the single rapid-growth behaviour created by standard proliferation media.

A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen

Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.

Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2

Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100.

Induction of MITF expression in human cholangiocarcinoma cells and hepatocellular carcinoma cells by cyclopamine, an inhibitor of the Hedgehog signaling

Microphthalmia-associated transcription factor (MITF) is a key regulator of differentiation of melanocytes and retinal pigment epithelial cells, but it also has functions in non-pigment cells. MITF consists of multiple isoforms, including widely expressed MITF-A and MITF-H. In the present study, we explored the potential role played by the Hedgehog signaling on MITF expression in two common types of primary liver cancer, using human cholangiocarcinoma cell lines, the KKU-100 and HuCCT1, along with the HepG2 human hepatocellular carcinoma cell line. Importantly, cholangiocarcinoma is characterized by the activated Hedgehog signaling. Here we show that MITF-A mRNA is predominantly expressed in all three human liver cancer cell lines examined. Moreover, cyclopamine, an inhibitor of the Hedgehog signalling, increased the expression levels of MITF proteins in HuCCT1 and HepG2 cells, but not in KKU-100 cells, suggesting that MITF expression may be down-regulated in some liver cancer cases.

Abstract B035: Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells

Abstract Background: Naturally occurring regulatory T (Treg) cells play an essential role for immunological unresponsiveness to self-tumor-antigens. Yet, how they stably suppress self-tumor-antigen-specific T cells remains unclear. In particular, it is unknown whether Treg cell-mediated suppression for a limited period has a critical long-lasting effect on cell fate and antigen reactivity of self-tumor-antigen-specific T cells. Experimental procedures: To demonstrate Treg-cell effect on proliferation, cytokine production, and cell fate of self-tumor-antigen-specific CD8+ T cells, CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of healthy individuals were stimulated with a melanocyte differentiation antigen Melan-A/ MART-1, expressed by normal melanocytes and some melanoma cells, with/without FoxP3+CD25+CD4+ Treg cells. Melan-A/ MART-1-specific CD8+ T-cell induction and their phenotypes were investigated. Results: Melan-A/ MART-1-specific CD8+ T-cell induction was reduced by the presence of Treg cells. Self-tumor-antigen-specific CD8+ T cells co-cultured with Treg cells underwent one cell division and stopped further proliferation due to the lack of co-stimulation from antigen-presenting cells through Treg-cell suppression. They harbored low-affinity T-cell receptors and an anergic phenotype (i.e., hypo-proliferative and cytokine hypo-producing upon antigen re-stimulation) defined by expression of CCR7 and co-inhibitory molecules such as CTLA-4. Melan-A/ MART-1-specific CD8+ T cells with this anergic phenotype were detected ex vivo in the periphery and contributed to the tolerance of self-tumor-antigens. Conclusions: Treg-cell-mediated induction of anergy in self- antigen tumor-specific CD8+ T cells inhibits their activation in cancer patients after immunotherapy, and neo-antigens associated with tumor-specific gene mutations may become a main target of CD8+ T cells inducing actual tumor regression. Citation Format: Yuka Maeda, Hiroyoshi Nishikawa, Shimon Sakaguchi. Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B035.

IFN-γ Promotes Apoptosis of Melanocytes in Vitiligo

Vitiligo is an immune-mediated inflammatory condition characterized by selective destruction of epidermal and or follicular melanocytes. It is well established that cell-mediated immunity has been implicated in the pathogenesis of this common skin disorder. Although abnormalities in different subpopulations of T cells may play a role in induction and development of vitiligo, great attention has been paid to CD4+ Th1 cells and cytotoxic CD8+ T lymphocytes (CTLs) (1, 2). The significance of both subgroups in vitiligo development is supported by different observations. For instance, CD8+ T cell-mediated melanocyte destruction has been demonstrated in vitiligo. In addition, an increased accumulation of CD8+ T cells in peripheral blood mononuclear cells (PBMCs) and perilesional skin of patients with progressive vitiligo has been reported (1, 3). On the other hand, elevated expression of Th1 cell specific transcription factor (T-bet) and cytokine (IFNγ) was indicated in PBMCs of patients with vitiligo (2). The exact mechanism by which these cells exert their cytotoxic effects on melanocytes is not well understood. Nonetheless, the results of different laboratory and histological examinations support the hypothesis that apoptosis is an important mechanism for melanocyte disappearance. Apoptosis is a complex process and can be initiated through intrinsic and extrinsic pathways. These pathways are activated by a variety of factors such as cytokines, certain chemicals and other molecular mechanisms. Amongst these factors, cytokines appear to play a pivotal role in promoting apoptotic melanocyte cell death. Cytokines are small secreted proteins that participate in cellular communication and many immunologic functions. Abundant evidence indicated cytokine imbalance (such as IFN-γ, IL-10, IL-17) in the skin and circulation of patients with vitiligo. Therefore, disturbed expression of these molecules may be involved in disease pathogenesis. IFN-γ is a proinflammatory cytokine mainly produced by Th1 lymphocytes, NK cells and CD8+ cytotoxic T cells. This cytokine plays a major role in both innate and adaptive immune response. It can also affect the action of other cytokines in a synergistic or antagonistic manner. The exact function(s) of IFN-γ in the pathology of vitiligo is not known and it may act via different mechanisms. For instance, IFN-γ may participate in the homing of CD8+ T cells to the skin through local induction of chemokines and expression of adhesion molecules on endothelial cells (4). Cytotoxicity via induction of apoptosis has been considered as a major effector mechanism of autoreactive CTLs. Therefore, recruited CD8+ T cells may specifically induce autologous melanocyte apoptosis resulting in disappearance of melanocytes (3). On the other hand, CD4+ Th1 cells (one of the main sources of IFN-γ production) are required for optimal induction of antigen-specific CD8+ T cell effector responses. CD8+ T cells can be considered as a main effector cell of Th1 immunity (5). Therefore, cooperation between CD4+ and CD8+ T cells plays an important role in complete melanocyte clearance. In addition, IFN-γ may influence melanin production. For instance, treatment of melanocytes with IFNγ resulted in significant suppression of melanin production and reduction of mRNA expression levels of tyrosinase and microphthalmia-associated transcription factor (MITF), which regulates differentiation and development of melanocytes and retinal pigment epithelium (6). Moreover, Natarajan et al. indicated a regulatory role of IFN-γ in skin pigmentation. They showed that IFN-γ signaling impedes maturation of the melanosome, which is a major site for synthesis and storage of melanine (7). Altogether, the actual mechanism of melanocytes killing in the skin of patients with vitiligo has not been definitively determined. Current studies indicate a potential role of Th1 and CTL subpopulations in vitiligo pathology as well as a link between IFN-γ and melanocyte abnormalities in this disease. However, further studies are needed to unravel the exact mode of IFN-γ action. A deeper understanding of these mecha-

A novel synthetic Piper amide derivative NED-180 inhibits hyperpigmentation by activating the PI3K and ERK pathways and by regulating Ca2+ influx via TRPM1 channels.

Piper amides have a characteristic, unsaturated amide group and exhibit diverse biological activities, including proliferation and differentiation of melanocytes, although the molecular mechanisms underlying its antimelanogenesis effect remain unknown. We screened a selected chemical library of newly synthesized Piper amide derivatives and identified (E)-3-(4-(tert-butyl)phenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (NED-180) as one of the most potent compounds in suppressing melanogenesis. In murine melan-a melanocytes, NED-180 downregulated the expression of melanogenic regulatory proteins including tyrosinase, Tyrp1, Dct, and MITF. PI3K/Akt-dependent phosphorylation of GSK3β by NED-180 decreases MITF phosphorylation and inhibits melanogenesis without any effects on cytotoxicity and proliferation. Furthermore, topical application of NED-180 significantly ameliorated UVB-induced skin hyperpigmentation in guinea pigs. Interestingly, data obtained using calcium imaging techniques suggested that NED-180 reduced the TPA-induced activation of TRPM1 (melastatin), which could explain the NED-180-induced inhibition of melanogenesis. All things taken together, NED-180 triggers activation of multiple pathways, such as PI3K and ERK, and inhibits TRPM1/TRPV1, leading to inhibition of melanogenesis.

Transcriptional Analysis of Vitiligo Skin Reveals the Alteration of WNT Pathway: A Promising Target for Repigmenting Vitiligo Patients

Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

Microphthalmia transcription factor in malignant melanoma predicts occult sentinel lymph node metastases and survival.

Microphthalmia transcription factor (Mitf) is involved in melanocyte development and differentiation. We previously reported that Mitf expression, as detected by immunohistochemical analysis, is an independent prognostic marker in patients with intermediate-thickness melanoma. However, the clinical significance of Mitf expression in melanoma is not well delineated. In this prospective study, we attempted to demonstrate the correlation between Mitf expression in primary melanoma and the sentinel lymph node status and prognosis. We prospectively examined primary cutaneous melanomas from 94 patients undergoing nodal staging by sentinel lymph node biopsy. We quantified the percentage of tumor cells whose nuclei stained with the Mitf antibody visually. Survival curves were generated using the Kaplan-Meier method. The correlation between Mitf expression and nodal status was evaluated using the Mann-Whitney U-test. Here we demonstrate that Mitf expression is directly correlated with both disease-free survival (DFS) and overall survival (OS) over a median follow-up of 28.5 months. The mean DFS and OS in the eight patients whose melanomas did not stain positive for Mitf were 15.75±3.36 months (median, 12 months) and 38.17±5.18 months (median, 29 months), respectively. These results are significantly lower than those for patients who showed evidence of Mitf expression, in whom the mean DFS and OS were 66.1±4.03 months (median, not reached, P=0.0001) and 66.75±38.17 months (median, not reached, P=0.0001), respectively. The mean DFS and OS with greater than 25% (67 patients) of the melanoma cells staining positive for Mitf expression were 78.37±2.78 and 82.38±1.6 months, respectively, compared with 26.37±3.2 months (P=0.0001) and 44.53±4.5 months (P=0.0001), respectively, with up to 25% (27 patients) of cells stained positive for Mitf expression. In addition, there was a significant relationship between Mitf expression and nodal status, as evaluated by sentinel node biopsy. For example, none of the melanomas with greater than 50% Mitf expression had a positive sentinel node biopsy. Our study shows that expression of the molecular marker Mitf in primary cutaneous melanomas is a useful tool in assessing lymph node status. Mitf immunostaining in the primary tumor serves as a reliable predictor of occult lymph node metastases, as well as a favorable prognosticator of DFS and OS in melanoma patients.