DNp73-induced degradation of tyrosinase links depigmentation with EMT-driven melanoma progression.

Melanoma is an aggressive cancer with poor prognosis, requiring personalized management of advanced stages and establishment of molecular markers. Melanomas derive from melanocytes, which specifically express tyrosinase, the rate-limiting enzyme of melanin-synthesis. We demonstrate that melanomas with high levels of DNp73, a cancer-specific variant of the p53 family member p73 and driver of melanoma progression show, in contrast to their less-aggressive low-DNp73 counterparts, hypopigmentation in vivo. Mechanistically, reduced melanin-synthesis is mediated by a DNp73-activated IGF1R/PI3K/AKT axis leading to tyrosinase ER-arrest and proteasomal degradation. Tyrosinase loss triggers reactivation of the EMT signaling cascade, a mesenchymal-like cell phenotype and increased invasiveness. DNp73-induced depigmentation, Slug increase and changes in cell motility are recapitulated in neural crest-derived melanophores of Xenopus embryos, underscoring a previously unnoticed physiological role of tyrosinase as EMT inhibitor. This data provides a mechanism of hypopigmentation accompanying cancer progression, which can be exploited in precision diagnosis of patients with melanoma-associated hypopigmentation (MAH), currently seen as a favorable prognostic factor. The DNp73/IGF1R/Slug signature in colorless lesions might aid to clinically discriminate between patients with MAH-associated metastatic disease and those, where MAH is indeed a sign of regression.

Interferon-inducible T-cell alpha chemoattractant (ITAC) induces the melanocytic migration and hypopigmentation through destabilizing p53 via histone deacetylase 5: a possible role of ITAC in pigment-related disorders.

BACKGROUND: Cell migration plays a major role in the immune response and in tumorigenesis. Interferon-inducible T-cell alpha chemoattractant (ITAC) elicits a strong chemotactic response from immune cells. OBJECTIVES: To examine the effect of ITAC on melanocyte migration and pigmentation and its involvement in related disorders, and to investigate potential key players in these processes. METHODS: Human melanocytes or melanoma cells were treated with ITAC and a migration assay was carried out. Global gene expression analysis was performed to find genes regulated by ITAC treatment. The function of key players involved in ITAC-induced cellular processes was addressed using knockdown or overexpression experiments in combination with ITAC treatment. ITAC expression in the inflammation-associated hypopigmentary disorder, vitiligo, was examined. RESULTS: Among CXCR3 ligands, only ITAC induced melanocyte migration. ITAC treatment upregulated the expression of histone deacetylase 5 (HDAC5) and downregulated that of p53, a known target of HDAC5. Through knockdown or overexpression of HDAC5 and p53, we confirmed that HDAC5 mediates ITAC-induced migration by decreasing levels of p53 via deacetylation. In addition, ITAC treatment could decrease pigmentation in a p53- and HDAC5-dependent manner. Finally, the increased migration of human melanoma cells by ITAC treatment and the increased ITAC expression in the epidermis of vitiligo skin were verified. CONCLUSIONS: This study provides in vitro evidence for the migratory and hypopigmentation effects of ITAC on melanocytic cells, gives translational insights into the roles of ITAC in pathological conditions, and suggests that HDAC5 and its substrate p53 are potent targets for regulating ITAC-induced cellular processes.

Biochemical, cytological, and immunological mechanisms of rhododendrol-induced leukoderma.

Recently, an unexpected outbreak of patients with leukoderma occurred in Japan with the use of brightening/lightening cosmetics containing rhododendrol (RD). Patients developed leukoderma mostly on the skin sites repeatedly applied with RD, but some patients also had vitiligo-like lesions on the non-applied sites. RD is a tyrosinase-competitive inhibiting substance, thereby serving as an inhibitor of melanin synthesis. Upon inhibition of tyrosinase, RD is converted to new products such as tyrosinase-catalyzed hydroxyl-metabolite, which damage melanocytes. The melanocyte cell lysates seem to induce T-cell response. The frequencies of CD8+ T cells in both lesional skin and peripheral blood are significantly higher in the RD leukoderma as well as non-segmental vitiligo patients than in normal controls. In HLA-A*02:01 positive cases, circulating Melan-A-specific cytotoxic T cells can be detected at a high frequency. It is thus suggested that RD-induced leukoderma is induced by not only cytolysis of melanocytes but also subsequent immune reactions toward melanocytes.

Menadione (Vitamin K3) decreases melanin synthesis through ERK activation in Mel-Ab cells.

Menadione is a synthetic vitamin K3 derivative. Here, we examined the effects of menadione on melanogenesis and its related signaling pathways. Our results showed that melanin content was significantly reduced after menadione treatment in a dose-dependent manner. However, menadione treatment did not reduce tyrosinase activity directly. Wnt signaling is known to play a major role in the control of melanin synthesis. Thus, we tested the effects of menadione treatment on GSK3ß and ß-catenin signaling, but found that menadione did not influence either of these signaling pathways. We also investigated changes in the phosphorylation of ERK, which is related to melanin regulation. These results indicated that menadione treatment led to the phosphorylation of ERK. Additionally, menadione treatment reduced both MITF and tyrosinase protein levels. Treatment with PD98059, a specific ERK pathway inhibitor, restored menadione-induced melanin reduction and also prevented MITF and tyrosinase downregulation by menadione. These results suggest that the hypopigmentary action of menadione is due to MITF and tyrosinase downregulation by ERK activation.

PP2A and DUSP6 are involved in sphingosylphosphorylcholine-induced hypopigmentation.

Activation of extracellular signal-related kinase (ERK) is involved in decreased melanogenesis by sphingosylphosphorylcholine (SPC). In the present study, we confirmed that SPC activated ERK and that a specific inhibitor of the ERK pathway (PD98059) recovered SPC-induced hypopigmentation. Moreover, we found that SPC significantly reduces protein phosphatase 2A (PP2A) activity in Mel-Ab cells, and that PP2A activator treatment abrogated SPC-induced hypopigmentation. We determined that α-melanocyte-stimulating hormone (α-MSH) increased the expression of dual-specificity phosphatase 6 (DUSP6), an ERK phosphatase, in a time-dependent manner. In contrast, SPC decreased the level of DUSP6 in Mel-Ab cells. Furthermore, inhibiting DUSP6 increased ERK activation and subsequently augmented the SPC-induced hypopigmenting effects. Taken together, our data suggest that SPC-induced phosphatase inhibition is also responsible for the hypopigmentary effects.

Hereditary leukonychia, or porcelain nails, resulting from mutations in PLCD1.

Hereditary leukonychia (porcelain nails or white nails) is a rare nail disorder with an unknown genetic basis. To identify variants in a gene underlying this phenotype, we identified four families of Pakistani origin showing features of hereditary leukonychia. All 20 nails of each affected individual were chalky and white in appearance, consistent with total leukonychia, with no other cutaneous, appendageal, or systemic findings. By using Affymetrix 10K chip, we established linkage to chromosome 3p21.3-p22 with a LOD score (Z) of 5.1. We identified pathogenic mutations in PLCD1 in all four families, which encodes phosphoinositide-specific phospholipase C delta 1 subunit, a key enzyme in phosphoinositide metabolism. We then identified localization of PLCD1 in the nail matrix. It was recently shown that PLCD1 is a component of the human nail plate by proteomic analysis and is localized in the matrix of human nails. Furthermore, mutations detected in PLCD1 resulted in reduced enzymatic activity in vitro. Our data show that mutations in PLCD1 underlie hereditary leukonychia, revealing a gene involved in molecular control of nail growth.

Mechanisms of spatial and temporal development of autoimmune vitiligo in tyrosinase-specific TCR transgenic mice.

Generalized vitiligo is thought to have an autoimmune etiology and has been correlated with the presence of CD8 T cells specific for melanocyte differentiation Ag. However, limited animal models for the disease have hampered its understanding. Thus, we generated TCR transgenic mice that recognize an epitope of the melanocyte protein, tyrosinase. These animals develop vitiligo with strikingly similar characteristics to the human disease. Vitiligo develops temporally and spatially, with juvenile lesions forming bilaterally in head and facial areas, and only arising later in the body of adult animals. Vitiligo is entirely dependent on CD8 T cells, whereas CD4 T cells exert a negative regulatory effect. Importantly, CD8 T cells can be pervasively present in the skin in the steady state without inducing vitiligo in most areas. This points to developmental differences in melanocyte susceptibility and/or immunological effector mechanisms over time, or in different body locations. Disease is strongly dependent on both IFN-gamma and CXCR3, whereas dependence on CCR5 is more limited, and both CCR4 and perforin are dispensable. Genetic ablation of CXCR3 or IFN-gamma also resulted in scarce CD8 T cell infiltration into the skin. Our results identify unexpected complexity in vitiligo development and point toward possible therapeutic interventions.

Provision of granulocyte-macrophage colony-stimulating factor converts an autoimmune response to a self-antigen into an antitumor response.

Many tumor Ags recognized by T cells are self-Ags. Because high avidity, self-reactive T cells are deleted in the thymus, any residual self-reactive T cells existing in the periphery are likely to be low avidity and nonresponsive due to peripheral tolerance mechanisms. Activation of these residual T cells is critical for targeting tumors for immunotherapy. In this study, we studied immune responses against the murine B16 melanoma using a tyrosinase-related protein 2 (TRP-2) peptide as a model tumor/self-Ag. Our results showed that TRP-2 peptide vaccination alone elicited a weak T cell response and modestly decreased B16 lung tumor nodules. The combination of peptide vaccination and treatment with an Ab directed against the inhibitory receptor CTLA-4 enhanced the immune response against TRP-2 peptide, inducing autoimmune depigmentation and further decreasing lung tumor nodules. However, both vaccination methods failed to protect against orthotopic (s.c.) B16 tumor challenge. The addition of an irradiated GM-CSF-expressing, amelanotic tumor cell vaccine significantly delayed s.c. B16 tumor growth. Subsequent studies revealed that provision of GM-CSF increased dendritic cell numbers in lymph nodes and spleen. Furthermore, addition of CTLA-4 blockade increased the frequency of TRP-2-specific, IFN-secreting T cells in spleen and lymph nodes. Overall, our results indicate that combining enhancement of Ag presentation with removal of CTLA-4-mediated inhibition can convert a "weaker" autoimmune response into a more potent antitumor immune response.

An established light ear mutant (C57BL/6J-Pdeb(rd1) le) mouse cell line exhibits a block to secretion of lysosomal enzymes.

The hypopigment mutant mice, light ear, pallid, and beige, possess defects in melanosomes, lysosomes, and platelet dense granules, suggesting that these organelles share a common biogenesis and processing. Light ear and pallid mutants are animal models for Hermansky Pudlak syndrome, whereas the beige mouse is an animal model for Chediak Higashi syndrome. An established skin cell line from the light ear mouse was tested along with pallid and beige cell lines for mutant effects on secretion of lysosomal hydrolase activities of six different lysosomal glycosidases and the trafficking of N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythrosphingosine (C(5)-DMB-ceramide). There were no consistently significant differences between the pallid and the beige mutant cell lines or between these two mutant lines and the control cell line in the percentage secretion of lysosomal hydrolase activities. The light ear mutant cell line, however, displayed a significantly lower percentage secretion of lysosomal hydrolase activities than all other cell lines tested. The light ear mutant cells processed C(5)-DMB-ceramide completely, as seen in the control cell line, whereas pallid and beige cell lines retained fluorescent material and exhibited a block in the complete processing of C(5)-DMB-ceramide 20 h after labeling. The block to secretion of lyososomal hydrolase activities in the light ear mutant cell line will be useful for further studies on this mutant's lysosomal defect.