16th Meeting of the European Society for Pigment Cell Research 4–7 September 2010 Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

  • The Wellcome Trust is a charity registered in England, no. 210183. Its sole trustee is The Wellcome Trust Limited, a company registered in England, no. 2711000, whose registered office is at 215 Euston Road, London NW1 2BE, UK.


Dot Bennett, St George’s, University of London, UK

Colin Goding, University of Oxford, UK

Robert Kelsh, University of Bath, UK



Heinz Arnheiter NIH, Washington, USA


Fiona Watt CRUK Cambridge Research Institute, Cambridge, UK

Masashi Narita CRUK Cambridge Research Institute, Cambridge, UK

Greg Barsh Stanford University, USA

Dorothy Bennett St George’s, University of London, UK

Corine Bertolotto University of Nice, France

Markus Böhm University of Münster, Germany

Janice Brissette Massachusetts General Hospital, USA

Lynda Chin Dana-Farber Cancer Institute, USA

Marco d’Ischia University of Naples Federico II, Italy

Jose-Carlos García-Borrón University of Murcia, Spain

Dave Gillespie Beatson Institute for Cancer Research, UK

Colin Goding Ludwig Institute for Cancer Research, UK

Graça Raposo Institut Curie, France

Rudy Happle Philipp University of Marburg, Germany

Keith Hoek University Hospital of Zurich, Switzerland

Stephen Johnson Washington University Medical School, USA

Robert Kelsh University of Bath, UK

Shigeru Kondo Fronteir Bio-Science Osaka University, Japan

Lionel Larue Institut Curie, France

Caroline Le Poole Loyola University, Chicago, USA

Richard Marais Institute for Cancer Research, UK

Alain Mauviel University of Paris, France

Lluis Montoliu Centro Nacional de Biotecnologia, Spain

Nick Mundy University of Cambridge, UK

Julia Newton-Bishop University of Leeds, UK

Liz Patton MRC Human Genetics Unit, UK

Daniel Peeper Netherlands Cancer Institute, The Netherlands

Mauro Picardo San Gallicano Dermatologic Institute - IRCCS

Rome, Italy

Francesca Pignoni SUNY Upstate Medical University, USA

Gloria Ribas Fundacion Investigacion Hospital Clinico Valencia,


José Neptuno Rodríguez-López University of Murcia, Spain

Erik Sahai Cancer Research UK

Vittoria Schiaffino San Raffaele Scientific Institute, Italy

Maria S Soengas Spanish National Cancer Research Centre,


Eirikur Steingrímsson University of Iceland, Iceland

Alain Taieb CHU Bordeaux, France

Conference Programme
Saturday 4 September 2010
10.00–12.00ESPCR Council Meeting (Council Members only)
12.00–18.00Registration with lunch
12.00–18.00Exhibitor set-up
14.00–15.00Fritz Anders Memorial Lecture: Heinz Arnheiter
15.00–17.00Session 1: Developmental biology of pigment cells and MSCs
17.00–18.00Pre Dinner Drinks
19.00–21.00Session 2: Genetics of pigment cell transcription
21.00–00.00Bar open
Sunday 5 September 2010
08.45–10.15Session 3: Pigmentary disorders
10.15–10.45Morning Coffee
10.45–12.45Session 4: Melanocyte cell biology
13.00–14.30Vitiligo European Task Force (Members only)
14.00–16.00Session 5: Melanoma biology and therapeutics
16.00–16.30Afternoon Tea and Exhibits
16.30–18.30Session 6: Melanocytes, migration and melanoma
18.30–19.30Drinks Reception
19.00–21.00IFPCS Council meeting (Council Members Only)
21.00–23.00IFPCS Council dinner (Council Members Only)
20.30–00.00Posters & Bar open
Monday 6 September 2010
08.30–08.45Shigeki Shibahara IFPCS and IPCC; Alain Taieb IPCC 2011
08.45–10.15Session 7: Senescence
10.15–10.45Morning Coffee and Exhibits
10.45–12.45Session 8: Signalling pathways in melanocytes and melanoma cells
13.00–14.00ESPCR General Assembly
14.00–16.00Session 9: Melanocyte and melanoma genomics
16.00–17.45Posters, Tea and Exhibits
17.45–18.45Plenary Lecture 1: Masashi Narita
19.00Buses to Conference Dinner in Cambridge
19.30–00.00Drinks Reception & Conference Dinner at Queens College in Cambridge
Tuesday 7 September 2010
08.45–10.15Session 10: The sun, DNA damage and epidermal biology
10.15–10.45Morning Coffee
10.45–11.45Plenary Lecture 2: Fiona Watt
13.00–16.00Three parallel workshops
Workshop A –Pigment patterns – their formation and evolution
Workshop B –Function and formation of the melanosome
Workshop C –Vitiligo
16.00Conference Closure
16.30Coaches to Cambridge, Stansted and Heathrow Airport depart

Fritz Anders Memorial Lecture

Development and function of retinal pigment epithelium cells

Heinz Arnheiter, Kapil Bharti

Mammalian Development Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States

Compared with neural crest-derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of an entirely different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct signaling pathways, and provide functions that far exceed those of a light screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11-cis retinal by re-isomerizing all-trans retinal that is generated during photoconversion, phagocytose daily a portion of the photoreceptors’ outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of blindness worldwide, and RPE replacement with RPE cells generated from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells provides for a promising cell-based therapy. By focusing on the molecular biology of normal and abnormal RPE development, we hope to gain insights that will ultimately help in the characterization of RPE cells intended for therapeutic purposes. It has been observed, for instance, that cultured ES cell-derived RPE cells can be unstable, switching between RPE-like and neuroectodermal-like phenotypes. This phenotype switching is reminiscent of developmental transdifferentiations that can be found, for example, after tissue injury in adult urodele amphibians, or in rodent embryos with genetic mutations. We have studied such developmental phenotype switching between RPE and retina in the mouse and find it to be controlled by dynamic, cell-autonomous interplays between the transcription factors Mitf and Pax6 and their respective target genes. For instance, Pax6 partially alleviates the RPE-to-retina transdifferentiation due to Mitf mutations by inducing the Mitf paralog Tcfec and genes regulating cell proliferation. We are currently extending these studies to later stages in development and hope to eventually provide a comprehensive analysis of the factors controlling RPE phenotype stability.

Plenary Lecture 1

Stem cells in mammalian epidermis

Fiona M. Watt1,2

1Cancer Research UK Cambridge Research Institute, Cambridge, UK;
2Wellcome Trust Centre for Stem Cell Research, Cambridge, UK

Adult mammalian epidermis is maintained by proliferation of stem cells and differentiation of their progeny. In undamaged epidermis stem cells in a particular location only give rise to the lineages that are appropriate for that location. However, following wounding or genetic manipulation any epidermal stem cell is capable of repopulating all of the epidermal lineages, revealing a remarkable degree of plasticity of the adult tissue. My lab is currently using a range of in vitro and in vivo approaches to define the environmental signals that regulate epidermal stem cell fate. Our studies have uncovered the importance of reciprocal interactions between epidermal cells and cells of the dermis, both in normal epidermal homeostasis and in tumour development.

Plenary Lecture 2

Gene expression in cellular senescence

Masashi Narita

Cancer Research UK, Cambridge Research Institute, Cambridge, UK

Cellular senescence is a stress responsive phenotype that accompanies stable cell cycle arrest. Despite their static appearance, senescent cells are metabolically active. It has been shown that multiple effector mechanisms are involved in this process, depending on the cell type and the particular stress. Such cellular stresses include constitutively active oncogenic stimuli, which paradoxically trigger pro-senescence effectors as a fail-safe mechanism. During oncogene-induced senescence (OIS) in human diploid fibroblasts (HDFs), the gene expression profile dramatically changes. We have previously identified a global and progressive chromatin structure alteration during senescence – senescence associated heterochromatic foci (SAHFs) – which have since been widely used as a marker of senescence. Our current model is that SAHF formation somehow contributes to the altered gene expression profile in senescent cells. More recently, we have also identified that autophagy, a cytoplasmic protein degradation program, is a new effector mechanism of senescence. Autophagy is highly activated during OIS, and facilitates the production of some secretory proteins essential for senescence establishment, at the post-transcription level. Here we propose that global protein turnover might be involved in the regulation of gene expression in parallel with the epigenetic regulation of transcription during senescence.

Session 1 – Developmental biology of pigment cells and MSCs

The chromatoblast concept – evidence from zebrafish genetics

Robert N. Kelsh, Xueyan Yang1, Masataka Nikaido

Department for Biology & Biochemistry, University of Bath, Bath, UK;
1Present address: The State Key Laboratory of Genetic Engineering and The MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, China

It is over 30 yrs since Bagnara proposed the chromatoblast concept – that all neural crest-derived pigment cells derive from a partially-restricted progenitor (Bagnara et al, 1979, Science 203, 410). Surprisingly, although widely accepted, the concept has remained untested. Unlike other major model systems, zebrafish retain multiple pigment cell-types, with adults and larvae showing xanthophores and iridophores in addition to melanocytes. Zebrafish genetic screens have identified multiple mutations affecting specification of individual fates, and in the case of mitfa mutants loss of melanophores is accompanied by gain of iridophores. Leukocyte tyrosine kinase (ltk) mutants fail to specify iridophores, and we proposed that early phase ltk expression might mark chromatoblasts (Lopes et al, 2008, PLoS Genetics 4, e1000026). Here we take advantage of a constitutively active version of Ltk to demonstrate that Ltk signalling in premigratory neural crest can drive other pigment cells to an iridophore fate. Furthermore, by BAC recombineering we show that ltk regulatory elements drive expression of GFP in precursors of not just iridophores, but of all pigment cells. Together our data provide strong evidence that ltk-expression initially marks chromatoblasts, with Ltk-signalling necessary to drive a subset of these cells to an iridophore fate. Furthermore, our data helps to clarify the pathways of progressive fate restriction in the neural crest in vivo.

Regulation of the zebrafish melanocyte stem cell

Stephen L. Johnson

Department of Genetics, Washington University Medical School, St. Louis, MO, USA

The zebrafish pigment pattern provides an ideal substrate for investigating the mechanisms that regulate the establishment and maintenance of, and differentiation from, adult stem cells. I will attempt to discuss several aspects of melanocyte stem cell regulation. This will include: (i) ablation of melanocytes in larval stages and evidence that new melanocytes regenerate from a melancyte stem cell (MSC), (ii) evidence that most embryonic melanocyte develop directly, without a stem cell intermediate, (iii) evidence that the MSC that supports larval regeneration has same genetic requirements as the MSC that supports transition to thte adult pigment pattern, (iv) how many MSCs support larval melanocyte regeneration and probability of individual MSCs to be recruited for regeneration, and (v) number of MSCs, or organ founding stem cells, that colonize the adult fins, and how they contribute to growth and morphogenesis of the fin pigment pattern.

Biological and mathematical modeling of melanocyte development

Lionel Larue1, Flavie Luciani1, Delphine Champeval1, Aurélie Herbette1, Laurence Denat1, Bouchra Aylaj2, Silvia Martinozzi1, Robert Ballotti3, Rolf Kemler4, Colin R. Goding5, Florian De Vuyst2, Véronique Delmas1

1Institut Curie, CNRS UMR3347, INSERM U1021, Orsay, France;
2Laboratoire Mathématiques Appliquées aux systèmes, Ecole Centrale Paris, Grande Voie des Vignes, 94235 Chatenay-Malabry Cedex, France;
3INSERM U895, Nice Cedex, France;
4Department of Molecular Embryology, Max-Planck Institute of Immunobiology, Freiburg, Germany;
5Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion including proliferation and migration from the dermis to epidermis. The model allows the calculation of doubling times of melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function β-catenin mutants in the melanocyte lineage. We found that any alteration of β-catenin activity, whether positive or negative, substantially reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wt and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo, and appropriate feedback control through β-catenin.

Session 2 – Genetics of pigment cell transcription

Regulation of Mitf by acetylation and novel insights from the crystal structure

Eiríkur Steingrímsson1, Alexander Schepsky1, Vivian Pogenberg2, Gunnhildur A. Traustadottir1, Matthias Wilmanns2, Colin Goding3

1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, Vatnsmyrarvegur, Reykjavik, Iceland;
2EMBL, Hamburg, Germany;
3Ludwig Institute for Cancer Research, Oxford, UK

The microphthalmia-associated transcription factor (Mitf) is essential for normal melanocyte development and has also been shown to play an important role in melanoma where it acts downstream of the B-Raf pathway. Mitf plays multiple roles in melanocytes and melanoma cells by regulating the expression of genes involved in various different processes including survival, proliferation, migration and differentiation. However, how it performs these different tasks in the same cell type is not clear, although signaling and DNA-binding specificity have been proposed to play an important role. We have evidence suggesting that acetylation of Mitf (which by itself is regulated by signaling) acts as an on/off switch for the DNA binding and transcription activation ability of the protein. Furthermore, analysis of the structure of the Mitf protein using X-ray crystallography has revealed how Mitf selects binding sites and how this may be affected by acetylation. Our studies reveal the functional importance of post-translational modifications in Mitf function in melanocytes and melanoma cells and provide new insights into the mechanisms of transcription regulation.

MITF controls the DNA damage response and a lineage specific senescence program in melanomas

Corine Bertolotto, Sandy Giuliano, Robert Ballotti

INSERM U895, NICE Cedex, France

Apoptosis and senescence are cellular failsafe programs that counteract excessive mitogenic signaling observed in cancer cells. Melanoma is known for its notoriously resistance to apoptotic processes, therefore, senescence, that remains poorly understood in melanomas, can be viewed as a therapeutic alternative. Microphthalmia associated transcription factor (MITF) which M-transcript is specifically expressed in melanocyte cells plays a critical role in melanoma proliferation and its specific inhibition is associated with G0/G1 growth arrest. Interestingly, decreased MITF expression has been described in senescent melanocytes and we have observed an inhibition of MITF expression in melanoma cells exposed to chemotherapeutic drugs that induce their senescence. All these observations thereby question the role of MITF in controlling senescence in melanoma cells. Here we report that long-term depletion of MITF in melanoma cells triggers a senescence program characterized by typical morphologic and biochemical changes associated with a sustained growth arrest. Further, we demonstrate that MITF-silenced cells engage a DNA damage-response signaling pathway, leading to p53 up-regulation, that is critically required for senescence entry.

This study uncovers the existence of a lineage-restricted DNA damage response/p53 signaling pathway that is inhibited by MITF to prevent senescence and favor melanoma cell proliferation.

Analysis of Mitf in drosophila

Francesca Pignoni1,2, Tianyi Zhang1, Qingxiang Zhou1, Cara Pina1, Donghui Yang-Zhou3, Alyna Dale3

1Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, NY, USA;
2Departments of Neuroscience and Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA;
3Department of Ophthalmology, Harvard Medical School and Massachusetts Eye & Ear Infirmary, Boston, MA, USA

MiT family genes are represented by a single locus, dMitf, in the invertebrate model animal Drosophilamelanogaster. The function of Mitf in the fly shows many similarities to the role of Mitf and other family members in the Retinal Pigmented Epithelium and other tissues. Analyses of protein function by over-expression and dominant negative reveal roles in controlling cell survival, differentiation, proliferation and epithelial integrity. The fly serves as a genetically amenable model for the identification of signaling pathways and other genetic interactors thus providing a list of potentially evolutionarily conserved modifiers or mediators of Mitf function. Current progress will be presented.

Session 3 – Pigmentary disorders

Towards a universal genetic diagnosis of all types of albinism

Lluís Montoliu1,2, Eduardo Moltó1,2, Almudena Fernández1,2, Chris Phillips3, María Torres2,4, Olalla Maronas3, Benoit Arveiler5, Fanny Morice-Picard5, Alain Taïeb6, Robert Aquaron7, Vittoria Schiaffino8, Tamio Suzuki9, Monica Martínez2,10, María José Trujillo2,10, Carmen Ayuso2,10, Ángel Carracedo2,3,4

1Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain;
2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Spain;
3Grupo de Medicina Xenómica, Instituto de Medicina Legal, Universidad de Santiago de Compostela, Spain;
4Grupo de Medicina Xenómica, CEGEN-Universidad de Santiago de Compostela, Spain;
5Laboratoire de Génétique Humaine, Université Victor Segalen Bordeaux, Bordeaux, France;
6Service de Dermatologie et Dermatologie Pédiatrique, Centre de Référence des maladies rares de la peau, Hôpital St André, CHU de Bordeaux, France;
7Faculté de Medecine, Univ. de la Méditerranée, Marseille, France;
8San Raffaele Scientific Institute, Milan, Italy;
9Department of Dermatology, School of Medicine, Yamagata University, Japan;
10Servicio de Genética, Fundación Jiménez Díaz, Madrid, Spain

Albinism includes a heterogeneous group of rare genetic conditions globally characterized by hypopigmentation. If the pigmentation impairment impacts both the skin and the eyes then it is referred to as oculocutaneous albinism (four types termed OCA1 to OCA4), whereas if it primarily impacts the melanin in the eyes it is known as ocular albinism (OA1). These pigment deficiencies can be more severe when associated with other symptoms such as bleeding, breathing and digestive disorders, in the case of Hermansky-Pudlak Syndrome (HPS, eight types termed HPS1 to HPS8), or in combination with immunological deficiencies, leading to infections, in the case of Chediak-Higashi Syndrome (CHS1). There are at least 14 genes with mutations known to be associated with albinism: TYR (OCA1), OCA2 (OCA2), TYRP1 (OCA3), SLC45A2/MATP (OCA4), GPR143 (OA1), LYST (CHS1), HPS1 (HPS1), AP3B1 (HPS2), HPS3 (HPS3), HPS4 (HPS4), HPS5 (HPS5), HPS6 (HPS6), DTNBP1 (HPS7) and BLOC1S3 (HPS8). All these genes encode proteins that are involved in melanin synthesis, melanosome function or lysosome-related organelle biogenesis. Animal models, notably transgenic or knockout mice, have been created for most of these genes. Globally, albinism affects about 1 in 17 000 people and, besides the hypopigmented phenotype, the main handicapping feature is their common severe visual deficiencies, leading to blindness in most cases. More than 500 mutations have been reported in those 14 loci, causing albinism, as obtained from the literature and human gene mutation databases. Very few of these genes are included in human genetic diagnostic tests (i.e. TYR, OCA2, GPR143,…) and are not offered in all reference hospitals, therefore most persons with albinism are not genetically diagnosed. Therefore this knowledge is missing from prenatal test regimes and is unavailable to help devise more accurate support and care recommendations for improving quality of life. In this regard, from the Biomedical Network Research Centre on Rare Diseases (CIBERER) in Spain, we have envisaged a new technological strategy aiming to establish a universal genetic diagnosis of all types of albinism. This approach uses Sequenom iPLEX methodology, which combines automated array processing of DNA samples with mass spectrometry (MALDI-TOF) and can be applied to discriminate, simultaneously, up to 1000 different alleles with known mutations, from a given DNA sample. At first, having obtained the mandatory ethical approval for this project, we collected DNA from persons with and without albinism, and their close relatives, from several places around the world, including a subset of human subjects that had been genetically diagnosed by independent methods, for validation purposes. Members of ALBA (http://www.albinismo.es), the Spanish association in support of persons with albinism, have contributed extensively to this project. The first results of this innovative proposal will be reported and its potential value discussed, in comparison to currently existing methods.

Grant Support: CIBERER INTRA/07/704.1

Pigmentary mosaicism: patterns and mechanisms

Rudolf Happle

Department of Dermatology, Philipp University of Marburg, Germany

The melanocytic system is strikingly versatile in forming different patterns of mosaicism. The best known type is the arrangement following Blaschko's lines in narrow bands. For heuristic purposes we distinguish a more rarely occurring subtype characterized by broad bands as noted in the McCune-Albright syndrome. The checkerboard pattern is defined by alternating flag or block-like areas as noted in naevus spilus maculosus and naevus spilus papulosus. The phylloid pattern is reminiscent of a floral ornament or Jugendstil painting. It is found in two different forms. Phylloid hypomelanosis is a well-defined neurocutaneous syndrome reflecting trisomy 13 mosaicism. More specifically, mosaic trisomy 13q is usually found. By contrast, phylloid hypermelanosis appears to be a heterogeneous mosaic disorder. The patchy pattern without midline separation is noted in cases of giant melanocytic naevus. With regard to the underlying mechanisms, two major categories of pigmentary mosaicism can be distinguished, in the form of genomic versus epigenetic mosaics. Genomic mosaics following Blaschko's lines include pigmentary mosaicism of the Ito type (‘hypomelanosis of Ito’ is not an entity) and linear and whorled naevoid hypermelanosis (which is not an entity either). Recently, naevus lentiginosus linearis has been described as a new disorder following Blaschko's lines. Cutis tricolor is a peculiar genomic mosaic characterized by paired hyper- and hypopigmented macules reflecting allelic didymosis (twin spotting). Different forms of cutis tricolor include a type characterized by large macules (Ruggieri-Happle syndrome) and another one showing small macules (cutis tricolor parvimaculata). In 2010, giant congenital melanocytic naevus has been proposed to represent a superimposed patchy manifestation of a polygenic trait in the form of predisposition to develop disseminated small nevi that have so far been mistaken as being ‘satellite nevi’. Epigenetic mosaics include X-inactivation patterns as observed in incontinentia pigmenti or in women heterozygous for ectodermal dysplasia of Zonana. More recently, autosomal epigenetic mosaicism (‘monoallelic expression’) has been proposed to explain an unusual familial constellation of pigmentary mosaicism following Blaschko's lines (Clin Exp Dermatol 2010). Perhaps, such cases can be taken as a human counterpart of the canine autosomal trait brindle that is caused by epigenetic control of the kbrgene.

Vitiligo: emerging targets to limit the subclinical inflammatory phase

Alain Taïeb

Service de Dermatologie et Dermatologie Pédiatrique, Centre de Référence des Maladies Rares de la Peau, CHU de Bordeauxx and INSERM U876, Bordeaux, France

Skin events, which are easily detected using skin biopsies in most other situations, have not been precisely recorded in vitiligo, with the argument that a clinical diagnosis was sufficient for the management of the patient.

Clinically inflammatory vitiligo is a rare clinical presentation of common vitiligo, described so far in the context of nonsegmental vitiligo (NSV). When associated with other cutaneous diseases it may correspond to a particular mode of onset of depigmentation triggered by local inflammation different from that of Sutton's or even Koebner's phenomena, and so far poorly understood. When associated with HIV infection, it has a clearly distinct clinical presentation for which the outcome of severe cutaneous inflammation leads to a vitiligo-like disorder, difficult to distinguish from common NSV. However, besides such rare observations, consistent histopathological findings in still pigmented progressing borders in NSV and SV suggest that common vitiligo could be considered as a clinically silent chronic microinflammatory skin disorder, clinically visible forms being the emerged tip of an iceberg. Newer animal models suggest that after a phase of lack of tolerance to melanocytic antigens, a chronic inflammation is constant and can be partially blocked by anti CD8 antibodies, and that specific chemokines receptors and cytokines are involved in a time and pattern dependent fashion in generalized vitiligo. Such information is paving the way to targeted therapies in vitiligo.

HSP70I as the sole requirement for activating depigmentation in vitiligo-prone mice

I. C. Le Poole1, J. Mosenson1, A. Zloza2, J. Klarquist1, J. A. Guevara-Patino2

1Department of Pathology, Microbiology and Immunology/Oncology Insitute, Loyola University Chicago, IL, USA;
2Department of Surgery, University of Chicago, IL, USA

Published studies from our lab support the ability of HSP70i, secreted in increased amounts by vitiligo melanocytes, to activate DC mediated cytotoxicity. HSP70i also accelerated TRP-2 DNA vaccine induced, T cell mediated depigmentation in C57BL/6 mice. As several stress proteins are capable of mediating DC activation, experiments were performed in knockout mice lacking constitutive or inducible HSP70 to challenge the redundancy of HSP70i involvement in vitiligo. Also, pmel-1 mice transgenic for a T cell receptor reactive with gp100 were subjected to vaccination with an expression vector encoding HSP70i versus empty vector DNA. Whereas Hsp70-2 knockout mice depigment in response to TRP-2, ruling out involvement of constitutive HSP70 in depigmentation, the response to antigen challenge was severely impaired in Hsp70-1 knockout mice. Reduced depigmentation in mice lacking inducible HSP70 was accompanied by significantly impaired in vivo cytotoxicity towards peptide challenged splenocytes. This supports a crucial and non-redundant role for inducible HSP70 in accelerating vitiligo. To further support the same, it was demonstrated that vaccination with HSP70i was necessary and sufficient to induce depigmentation in pmel-1 transgenic animals. Taken together with in vitro data using a monoclonal antibody to block HSP70i-induced activation of DC, these data offer strong support for a crucial role of stress-induced HSP70 in vitiligo and suggest means of interfering with progressive depigmentation. Supported by NIH/NIAMS grant R01AR054749 to CLP.

Session 4 – Melanocyte cell biology

Skin as a living coloring book: epithelial cell roles in the patterning of pigmentation

Janice L. Brissette, Rong Han, Wenyu Fu, Lorin Weiner

Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA;
Department of Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY, USA

Foxn1, the product of the nude locus, is a transcription factor present in specific epithelial cell populations. Our work to date suggests that Foxn1 confers distinct properties on its host cells, namely, a ‘pigment recipient phenotype’. A cell with this phenotype attracts melanocytes, stimulates pigment transfer, and thereby engineers its own pigmentation. As such, the pigment recipients constitute a specialized, epithelial counterpart to the melanocytes (the pigment donors) and provide a template or blueprint that instructs melanocytes where to place pigment. We have hypothesized that Foxn1 instructs melanocytes via two types of downstream effectors: diffusible and cell-bound signals. Presumably, diffusible signals carry out Foxn1's long-range functions, such as recruiting melanocytes to epithelial cells, while cell-bound signals carry out Foxn1's short-range functions, such as flagging epithelial cells for pigmentation and locking connections with melanocytes into place. We now report the identification of intercellular signals through which Foxn1 induces epithelial pigmentation. These results explain, at least in part, how pigment recipients are created and how these cells ultimately determine the pattern of pigmentation.

Mechanisms of Melanosome transfer in human skin

Miguel C. Seabra1,2,3

1Molecular Medicine, National Heart and Lung Institute, Imperial College London, London, UK;
2Instituto Gulbenkian de Ciência, Oeiras, Portugal;
3CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal

Melanin transfer from melanocytes to keratinocytes and accumulation in the perinuclear area to form the ‘melanin cap’ is a critical process in skin pigmentation and photo-protection. However, the molecular mechanisms underlying these processes remain poorly understood. To study melanosome export from donor melanocytes, and subsequent internalisation by recipient keratinocytes, we performed electron microscopy (EM) on human skin samples. Analysis of serial ultra-thin sections revealed naked melanin in the extra-cellular space between melanocytes and keratinocytes. Within the keratinocyte melanin was always surrounded by a single membrane bilayer, even in peripheral (presumably newly ingested) structures. Cryo-immuno EM, showed that melanin-containing structures within keratinocytes lacked the melanosome membrane protein TYRP1. Taken together these observations indicate that melanin is exocytosed by fusion of the melanosome with the melanocyte plasma membrane and is then phagocytosed by the tightly packed surrounding basal keratinocytes. Melanin-loaded phagosomes within keratinocytes cluster in the perinuclear region and coalesce to form the melanin cap. These structures possess abundant LAMP1, a lysosomal marker, but relatively small amounts of the lysosomal protease, Cathepsin D. The expression of relatively modest amounts of lysosomal proteases by keratinocytes may serve to reduce the rate of melanin degradation in these cells and, thereby, prolong photo-protection.

Session 5 – Melanocytes, migration and melanoma

The signalling and transcription network regulating cell fate switching in melanocytes and melanoma

Colin Goding

Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK

Tumours comprise multiple subpopulations of cells with differing biological properties, some of which are proposed to possess stem cell-like properties, able to seed and maintain tumours, and provide a reservoir of therapeutically-resistant cells. Over recent years we have highlighted the key role of the Microphthalmia-associated transcription factor Mitf in defining different melanoma sub-populations. Importantly, expression of Mitf is repressed by Brn-2, a transcription factor that defines a distinct invasive melanoma sub-population. Understanding how Brn-2 mediates its effects and what regulates its expression may give an opportunity to control Mitf expression and drive melanoma sub-populations towards a therapeutically sensitive phenotype. Progress in defining signalling pathways implicated in regulating Brn-2 expression and activity have revealed an integrated network that balances variable levels of Mitf expression and activity with anti-senescence and pro-invasive pathways.

Imaging melanoma dissemination

Erik Sahai, Cerys Manning, Sophie Pinner

Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, London, UK

The invasion and metastasis of melanoma is a major clinical challenge. Our work uses intravital imaging to directly observe melanoma cell invasion in primary tumours. Interestingly, this approach reveals that only a subset of melanoma cells is motile, even in aggressive melanoma models. We therefore asked what is special about these invasive cells. Strikingly, we find that invasive cells are less pigmented than non-motile cells and have increased activity of the Brn-2 promoter. Circulating melanoma cells isolated from the blood also exhibit a similar phenotype. In contrast, metastases contain large numbers of pigmented cells and cells with low Brn-2 promoter activity. Our data indicates that aspects of differentiation hierarchies remain even in an aggressive melanoma model. Although, both primary tumour and metastases contain differentiated cells only the less differentiated cells disseminate. We have now extended this analysis to investigate heterogeneity in Notch signalling during melanoma dissemination.

Complex regulation of the MC1R underscores its central role in controlling human pigmentation and the UV response

Zalfa A. Abdel-Malek

Department of Dermatology, University of Cincinnati, Cincinnati, Ohio, USA

The MC1R gene an important determinant of the diversity of human pigmentation and a melanoma susceptibility gene. We demonstrated that expression of two MC1R variants that are strongly associated with red hair phenotype and increased melanoma risk results in loss of function MC1R in human melanocytes (HM), and an aberrant response to UV, exemplified by compromised nucleotide excision repair capacity and increased induction of oxidative DNA damage. We reported that α-MSH enables melanocytes to overcome UV-induced DNA damage by enhancing nucleotide excision repair and reducing the generation of oxidative DNA damage, and that these effects are dependent on expression of functional MC1R, and were inhibited by treatment of HM with the MC1R antagonist agouti signaling protein (ASIP). Here we report that in addition to the MC1R genotype, the expression and activity of the MC1R are regulated transcriptionally, and post translationally by desensitization. We found that human β-defensin 3 (HBD3), similar to ASIP, acts an antagonist of the human MC1R, blocking the stimulatory effects of α-MSH on cAMP generation and tyrosinase activity. Treatment of HM with the MC1R agonists α-MSH or ACTH up regulated, while treatment with the antagonists ASIP or HBD3 had no effect on MC1R gene expression. Treatment of HM with either forskolin or TPA up regulated MC1R gene expression, suggesting the involvement of cAMP and PKC in this effect. Exposure to UV markedly down regulated MC1R expression. Treatment with the paracrine factors endothelin 1 or basic FGF increased MC1R mRNA levels. The MC1R is a Gs-protein coupled receptor (GPCR). We investigated whether or not MC1R, like most other GPCRs, undergoes desensitization. Using a panel of 10 different HM strains derived from 10 different donors, we compared the expression of the G protein coupled receptor kinases (GRKs) 2, 3, 5 and 6, as well as β-arrestin 1 that is recruited once the receptor is phosphorylated by GRKs. Our results showed differences in the protein levels of GRKs and β-arrestin 1 among the different HM strains, with maximal differences in GRK5 and GRK6 expression. Following brief treatment with 1 nM α-MSH, we found that different HM strains had different levels of desensitization (none, partial, or complete desensitization). In most HM strains, there was a residual activity of the MC1R following removal of the agonist, suggesting the presence of spare receptors. The desensitization profiles of the different HM strains were independent of the MC1R genotype and did not correlate with the expression of the GRKs. Interestingly, brief prior treatment with either ASIP or HBD3 blocked the response of HM to α-MSH. Whether or not this is due to MC1R desensitization is being further investigated. These results point out the complexity of regulation of the MC1R, and explain the wide variation in the responses of individuals to its physiological agonists and UV.

Session 6 – Signalling pathways in melanocytes and melanoma cells

Multiplicity and complexity of the OA1 signaling pathway(s)

M. Vittoria Schiaffino, Ilaria Palmisano, Tiziana Daniele, Angela Palmigiano, Rosa Lucia D’Ambrosio

San Raffaele Scientific Institute, Milan, Italy

The protein product of the ocular albinism gene, named OA1, is a pigment cell-specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). However, in contrast to canonical GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely melanosomes and lysosomes, by specific sorting determinants. Skin melanocytes and retinal pigment epithelium (RPE) of patients or mice with ocular albinism show not only a significant reduction in melanosome number and the presence of giant melanosomes, but also an abnormal distribution of the organelles. These findings indicate that OA1 plays critical role in both melanosome biogenesis and transport, most likely by means of a G-alpha-i-mediated pathway. Furthermore, a recent report by Lopez et al., 2008, provides evidence for a direct role of OA1 in retinal development, mediated by its activation by L-DOPA and resulting in the triggering of a G-alpha-q-dependent downstream pathway at the plasma membrane of RPE cells. Results addressing these novel aspects of OA1 signaling and their implications for the function of the receptor will be discussed.

Non-canonical from wild type and mutant human melanocortin 1 receptor (MC1R)

Jose C. Garcia-Borron, Celia Jimenez-Cervantes, Herraiz Serrano Cecilia

Department of Biochemistry and Molecular Biology, University of Murcia, Spain

MC1R, a G protein-coupled receptor (GPCR) expressed in melanocytes, is a major determinant of skin pigmentation and phototype. Upon stimulation by ( melanocyte-stimulating hormone ((MSH) or related melanocortin (MC) peptides, MC1R triggers two major signal transduction pathways. Functional coupling to the Gs protein activates cAMP production, ultimately leading to transcriptional activation of cAMP-regulated genes such as Microphthalmia. MC1R also activates the module linking RAS to the MAP kinases ERK1 and ERK2, a pathway crucial to melanocyte proliferation and very frequently hyperfunctional in melanoma. It has been assumed that in MC-stimulated melanocytic cells cAMP is responsible for ERK activation, so that the two pathways are activated sequentially. However, the major R151C, R160W and D294H MC1R alleles associated with red hair, fair skin (the RHC phenotype) and increased skin cancer risk encode for forms with decreased through cAMP, yet they activate the ERKs at least as effectively as wild-type MC1R. In keeping with this dramatically different impact of the RHC mutations on cAMP and ERK signaling, MC1R-mediated ERK activation in melanocytes and melanoma cells is independent on cAMP, and displays different dose-response curves and conformational requirements. Pharmacological interference, siRNA and functional reconstitution studies showed that (MSH-induced ERK activation resulted from transactivation of the cKIT receptor tyrosine kinase (RTK), thus pointing to an unexpected link between MC1R and KIT signaling in melanocytes and melanoma. Treatment of HBL human melanoma cells (wild type for MC1R, NRAS and BRAF) with NDP-MSH but not with the cKIT ligand SCF activated the Src non-RTK. Moreover specific pharmacological inhibition of Src kinase activity blocked MC1R-dependent activation of the ERKs. This suggests that Src is located upstream of cKIT in the pathway mediating MC-dependent ERK activation in melanocytic cells. Thus, the cAMP and ERK pathways are not activated sequentially by the MC1R, but rather they are triggered independently. Nevertheless, the two pathways appear to establish an asymmetric crosstalk, whereby ERK activity has little effect on MC-induced cAMP production, whereas increased cAMP levels might antagonize ERK activation.

GLI2, not just a Hedgehog mediator – its role in melanoma metastasis

Alain Mauviel

Curie Insititute, INSERM U1021-CNRS UMR3347, Orsay, France

GLI2, a mediator of Hedgehog signaling, is implicated in the progression of a number of human malignancies. We demonstrated that its expression is transcriptionally upregulated by TGF-β in a variety of cell types (Dennler et al., Cancer Res., 2007). Characterization of the 5′-flanking sequence of human GLI2 mRNA led to the identification of a transcription start site, the cloning of ~1600 bp of the regulatory promoter region and the identification and functional mapping of a TGF-β-responsive region to a 91 bp sequence between nucleotides -119 and -29 of the promoter. This region harbors putative SMAD and LEF/TCF binding sites that allow a functional cooperation between SMAD3 and β-catenin, recruited to the promoter in response to TGF-β, to drive GLI2 gene transcription (Dennler et al., J Biol. Chem., 2009).

The TGF-β pathway is often constitutively active in melanoma and a marker of poor prognosis. We found that GLI2 is upregulated in a TGF β-dependent fashion in the most aggressive melanoma cell lines. GLI2 promotes melanoma migration and invasion by controlling a mesenchymal transition characterized by loss of expression of E-cadherin and increased MMP secretion. GLI2 knockdown in melanoma cells that express high levels of GLI2 significantly inhibits their invasive capacity and the development of experimental bone metastases in mice (Alexaki, Javelaud et al., J. Natl. Cancer Inst., 2010). GLI2 expression is heterogeneous within human melanoma lesions, associates with tumor regions in which E-cadherin is lost, and is increased in distant metastases as compared to primary lesions. Our data thus provide functional evidence for a direct role for GLI2 in driving melanoma invasion and metastasis.

Session 7 – Senescence

Treating the untreatable? Cell-based screening for senescence-based cancer drug discovery

Dorothy C. Bennett1, Rebecca S. Collinson1, Mahito Sadaie2, Masashi Narita2, W. Nicol Keith3, Christian Dillon4

1BMS, St George's University of London, Cranmer Terrace, London, UK;
2CR-UK Cambridge Research Institute, Cambridge, UK;
3University of Glasgow and CR-UK Beatson Laboratories, Glasgow, UK;
4Cancer Research Technology Discovery Laboratories, London, UK

Cell senescence, a natural form of irreversible cellular arrest, has emerged as a powerful tumour-suppressor mechanism, disabled when advanced cancers develop. This raises the possibility of artificial restoration of senescence as therapy, especially for cancers resistant to known therapies. CR-UK has established a consortium of teams with relevant expertise, aiming to develop high-throughput cell-based screens to identify senescence-inducing agents. There has been an initial focus on melanoma – a particularly intractable disease with a genetic signature strongly reflecting bypass of senescence. Using normal human IMR90 fibroblasts with inducible oncogenic HRAS as a well-characterized first model, an automated screen was developed with a Cellomics ArrayScan Screening Reader. This screen detects morphological changes associated with senescence, including SAHF (senescence-associated heterochromatic foci). A parallel cancer model was developed by exposing A375P metastatic melanoma cells to etoposide, then culturing without drug for a period. This could yield arrested cells with senescence markers, including β-galactosidase and persistent nuclear foci of 53BP1/γH2AX. These foci could be quantitated using the ArrayScan, as could nuclear size and number following DAPI-staining, giving a potential melanoma-based screen. By screening fibroblasts followed by secondary molecular senescence marker-based screens, several active compounds were identified from a small-molecule library. Three of the compounds were also positive in the melanoma screen, again confirmed by senescence markers. These compounds could likewise arrest other metastatic melanoma lines and other therapy-resistant cancer cell types. The system thus shows promise for identification of candidate novel anticancer drugs.

Oncogene-induced cell senescence and its relevance for melanoma

Daniel Peeper

Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands

The BRAF protein kinase is frequently mutated (commonly V600E) in melanoma and a number of other tumors. Remarkably, BRAFE600 is also found in the vast majority of melanocytic nevi (moles). Nevi typically show little proliferative activity and are the benign counterparts of malignant melanoma. Recently, we reported that BRAFE600 is associated with activation of tumor suppressors (including p16INK4A) and induction of long-term, senescence-like cell cycle arrest of nevi (1, 2). Similar results have now been reported across a wide variety of in vivo model systems and in the context of several oncogenes and tumor suppressor genes.

In spite of the arrested state of nevus cells, we observed that nevi often display a mosaic immunopositivity for p16INK4A. This might suggest that in addition to p16INK4A other factors contribute to the senescent state of BRAFE600-expressing cells. As our recent findings suggest that oncogene-induced senescence serves to suppress tumorigenesis, such factors may include novel tumor suppressors. In addition to taking a candidate gene approach, we are combining gene expression analysis, RNAi and unbiased functional ‘barcode’ screens. This integrative genomics approach may identify novel signaling networks involved in (BRAFE600-associated) cancers (3, 4).

1 Michaloglou, C., Vredeveld, L.C., Soengas, M.S., Denoyelle, C., Kuilman, T., van der Horst, C.M., Majoor, D.M., Shay, J.W., Mooi, W.J., and Peeper, D.S. (2005). BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724.

2 Mooi, W.J., and Peeper, D.S. (2006). Oncogene-induced cell senescence – halting on the road to cancer. N. Engl. J. Med. 355, 1037–1046.

3 Kuilman, T., Michaloglou, C., Vredeveld, L.C., Douma, S., van Doorn, R., Desmet, C.J., Aarden, L.A., Mooi, W.J., and Peeper, D.S. (2008). Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133, 1019–1031.

4 Kuilman, T., and Peeper, D.S. (2009). Senescence-messaging secretome: SMS-ing cellular stress. Nat. Rev. Cancer 9, 81–94, Epub 2009 Jan 9.

Session 8 – Melanoma biology and therapeutics

BRAF and RAS signalling in human melanoma

Richard Marais

Signal Transduction Team, The Institute of Cancer Research, London, UK

The small G-protein NRAS is mutated in about 20% of human melanomas and its downstream effector BRAF is mutated in a further 45% of cases. We have developed mouse models of melanoma driven by these oncogenes expressed at physiological levels and show that oncogenic BRAF can induce melanoma, whereas oncogenic RAS cannot. Curiously however, kinase-dead BRAF potentiates the oncogenic potential of oncogenic RAS, or oncogenic RAS can drive tumorigenesis when over-expressed. Kinase-dead BRAF potentiates tumorigenesis by oncogenic RAS through a mechanism involving RAS-dependent direct binding of BRAF to CRAF, leading to CRAF and pathway hyper-activation. We are currently using these models to dissect the genetics of melanoma and to develop novel understanding of the biology to assist development of therapeutic approaches to this disease.

β-catenin LEF/TCF co-factors are expressed according to the rhythm of melanoma progression

Keith Hoek, Ossia Eichhoff, Daniel Widmer, Phil Cheng, Marie Zipser, Reinhard Dummer

Department of Dermatology, University of Zurich, Zurich, Switzerland

We present data which shows that while β-catenin activity is critical to phenotype-specific gene expression in melanoma cells, it is regulated by the phenotypic-specific exchange of Lef/Tcf transcription co-factors. With the melanoma cell phenotype switching model for disease progression as our starting point, in which melanoma cells are thought to transition back-and-forth between states of proliferation and invasion to drive metastatic spread, we first explored an early hypothesis that melanoma cell phenotype was in part determined by the regulation of β-catenin levels. We found that while there were phenotypically distinct characteristics of β-catenin at the membrane and in the cytosol, nuclear levels and activity showed no such differential and the hypothesized relationship was discarded. It was then determined that β-catenin nuclear activity was dominated by one Lef/Tcf co-factor in proliferative phenotype cells, and by a different Lef/Tcf co-factor in invasive phenotype cells. Knock-down of proliferative phenotype-specific Lef/Tcf co-factor expression in proliferative phenotype cells resulted in down-regulation of proliferative phenotype-specific genes. Likewise, knock-down of invasive phenotype-specific Lef/Tcf co-factor in invasive phenotype cells down-regulated the expression of invasive phenotype-specific genes. Interestingly, knock-down of proliferative phenotype-specific Lef/Tcf co-factor in proliferative phenotype cells up-regulated invasive phenotype-specific Lef/Tcf expression and these cells became less proliferative and more invasive. However, while invasive phenotype-specific Lef/Tcf knock-down in invasive phenotype cells reduced invasiveness, it did not affect proliferative phenotype-specific Lef/Tcf co-factor expression or increase proliferation rates. These data show that phenotype-specific gene expression is in large part determined by which Lef/Tcf co-factor is being expressed, and not by regulation of β-catenin levels as previously thought.

Session 9 – Melanocyte and melanoma genomics

Human pigmentation genes and novel genetic susceptibilities to melanoma: a candidate gene approach using illumina platform

Gloria Ribas1,2, Lara P. Fernandez1, Maider Ibarrola-Villava1,2, Santos Alonso3, María D. Boyano4, María C. Peña-Chilet2, Guillermo Pita5, Enrique Boldó6, Rafael Lozoya6, Matias Mayor7, Marta Feito7, Neskuts Izagirre3, Concepción. De la Rúa3, Aintzane Asumendi4, Gorka Perez-Yarza4, Yoana Arroyo-Berdugo4, Jose Antonio Avilés8, Jesús Jesús Gardeazabal9, M. Careaga10, Iñigo Martínez-de Lizarduy10, Ana Sánchez-Díez10, Carlos Valdés10, Angel Pizarro7, Mariano Casado7, Gregorio Carretero11, Manuel Martin-Gonzalez12, Eduardo Nagore11, Pablo Lázaro8, Javier Benítez1,5, Conrado Martínez-Cadenas6

1Human Genetics Group, CNIO, Madrid, Spain;
2Department of Hematology and Medical Oncology, Fundación Hospital Clínico Universitario, Valencia, Spain;
3Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Spain;
4Department of Cell Biology and Histology, University of the Basque Country, Spain;
5CeGen, CNIO, Madrid, Spain;
6Surgical Oncology Unit, Castellon Province Hospital, Madrid, Spain;
7Department of Dermatology, La Paz Hospital, Madrid, Spain;
8Department of Dermatology, Gregorio Marañon Hospital, Madrid, Spain;
9Department of Dermatology, Cruces H, Madrid, Spain;
10Department of Dermatology, Basurto Hospital, Madrid, Spain;
11Dr. Negrín Hospital, Madrid, Spain;
12Department of Dermatology, Ramon y Cajal Hospital, Madrid, Spain;
13Instituto Valenciano de Oncología, Valencia, Spain

The aetiology of malignant melanoma (MM) remains unclear but it is known that both genetic and environmental factors influence the development of sporadic disease. The main reason for the increasing incidence of MM is greater sun exposure. Human pigmentation and consequently sun sensitivity are complex characteristics. In humans there is a long list of genes known to be involved in rare pigmentary disorders. These genes contribute most of the variation in pigmentation phenotypes seen in human populations.

Three hundred and eighty four SNPs in 68 genes belonging to the pigmentation pathway were genotyped using an Illumina array (Golden GateTM). Tag SNPs with MAF >0.05 were selected using HapMap database. Phase I_Discovery was done with a case-control study including a total of 590 Spanish MM patients and 507 control subjects. Phenotypic information was collected using a standardised questionnaire. Twenty-nine SNPs had a P-value <0.05 of which 10 had a P-value <0.01 and specially two SNPs were statistically significant after bonferroni correction (P = 0.03). Of these top 10 associated SNPs we selected the best six hits corresponding to SCL45A2, SILV, MYO7A, ADAMTS20, TYR and KIT genes and we validated them in an independent series of 775 Spanish MM patients and 789 control subjects (PhaseII_Validation). We confirmed the novel association of rs35414 located at the SLC45A2 gene with MM. Other associations with melanoma and pigmentary characteristics such as hair, skin and eye colour will be discussed. These results confirm the contribution of pigmentation genes to genetic predisposition to MM.

Session 10 – The Sun, DNA damage and epidermal biology

Truncated alpha-MSH tripeptides and derivatives – novel tools for the prevention of UVB-induced stress and inflammation

Markus Böhm, Agatha Kokot, Thomas A. Luger

Department of Dermatology, University of Münster, Münster, Germany

Although alpha-melanocyte-stimulating hormone (alpha-MSH) has potent melanotropic, anti-inflammatory and anti-oxidative effects, its topical usefulness for dermatology remains a problem due the physiochemical nature of this tridecapeptide. At present, truncated tripeptides, either those containing three amino acids of the central pharmacophor, or those related to the C-terminal domain of alpha-MSH, are emerging as a new class of ‘biologics’ in medicine. While the first class of alpha-MSH tripeptides have retained melanotropic and preserved cytoprotective effects against UVB-induced DNA damage due to high-affinity binding to the melanocortin-1 receptor (MC-1R), the latter class, among the tripeptide KdPT, does not bind to MC-1R as shown by ligand binding studies and melanin assays using normal melanocytes and B16 melanoma cells. However, KdPT (and KPV) share with alpha-MSH a number of remarkable anti-inflammatory in vitro and in vivo effects as well as the induction of transcription factor Nrf2 and the upregulation of Nrf-dependent anti-oxidative enzyme heme oxygenase-1 (HO-1) in normal human melanocytes. While in models of inflammation and in presence of IL-1beta, KdPT attenuates IL-1beta-mediated proinflammatory effects presumably by interfering with IL-1beta binding to the IL-1 receptor type I, the molecular mechanisms how KdPT or KPV per se induce Nrf2 and HO-1 in melanocytes, remains unclear. Possible mediators of the biological effects of these small peptides are distinct oligopeptide transporters, i.e. PHT1 and 2, which were detected in normal human melanocytes. It will be fascinating to explore the expression, regulation and function of these oligopeptide transporters in pigment cells as well as in pigmentary disorders.

Chk1 is essential for skin tumorigenesis

David A. Gillespie1,2,4, Lye Mun Tho1,2, Silvana Libertini1, Owen Sansom1,2, Steve Elledge3, Roy Rampling2

1Beatson Institute for Cancer Research, Glasgow, UK;
2Faculty of Medicine, University of Glasgow, Glasgow, UK;
3Department of Genetics, Division of Genetics, Howard Hughes Medical Institute, Brigham and Women's Hospital, Harvard University Medical School, Boston, MA, USA

Chk1 is a key regulator of DNA damage responses and genome stability in eukaryotes. To better understand how checkpoint proficiency relates to cancer development, we investigated the effect of genetic ablation of Chk1 in mouse skin on tumors induced by chemical carcinogens. We found that homozygous deletion of Chk1 immediately prior to carcinogen exposure strongly suppressed benign tumor (papilloma) formation, and that the few, small lesions that formed in ablated skin always retained Chk1 expression. Remarkably, Chk1 deletion rapidly triggered spontaneous cell proliferation, DNA damage, and apoptosis within the hair follicle, a principal site of origin for carcinogen-induced tumors. At later times, ablated skin was progressively repopulated by non-recombined Chk1-expressing cells, most likely via proliferation of label-retaining stem cells within the bulge region of the follicle, and ultimately normal sensitivity to tumour induction was restored when carcinogen treatment was delayed. In marked contrast, papillomas formed normally in Chk1 hemizygous skin but showed an increased propensity to progress to carcinoma. Thus, complete loss of Chk1 is incompatible with epithelial tumorigenesis whereas partial loss of function (haploisufficiency) fosters benign-malignant tumor progression.

The sun, vitamin D and melanoma

Julia Newton-Bishop

University of Leeds, Cancer Genetics Building, UK

Melanoma is predominantly a cancer of fair skinned peoples with incidence rates highest at low latitudes and therefore is undoubtedly related aetiologically to sun exposure. Meta-analyses of case-control studies (1) and more recently pooled data analyses (2) have provided strong evidence that sunburn and intermittent sun exposure are risk factors. Sun-sensitive phenotypes are therefore not surprisingly risk factors, which is supported by genetic association between risk and the inheritance of variants in the melanocortin 1 receptor gene (MC1R) seen both in early candidate gene studies (3) and genome wide association studies (GWAS) (4). The lack of evidence linking risk with large cumulative exposures to the sun in temperate climates at least, and indeed some evidence for a protective effect of occupational sun exposures (1) has puzzled researchers for some time. We report evidence from a new case-control study that suggested that higher levels of regular weekend sun exposure was associated with reduced risk of melanoma in the UK and discuss the hypothesis that vitamin D resulting from that sun exposure might be protective for melanoma. Thus the hypothesis is that sun exposure may be both causal and protective for melanoma depending on phenotype and pattern of sun exposure. The presence of large numbers of melanocytic naevi is the most potent phenotypic risk factor and GWAS have recently identified some so-called naevus genes (4, 5).

1 Gandini, S., Sera, F., Cattaruzza, M.S., Pasquini, P., Picconi, O., Boyle, P., et al. (2005). Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure. Eur. J. Cancer 41, 45–60.

2 Chang, Y.M., Barrett, J.H., Bishop, D.T., Armstrong, B.K., Bataille, V., Bergman, W., et al. (2009). Sun exposure and melanoma risk at different latitudes: a pooled analysis of 5700 cases and 7216 controls. Int. J. Epidemiol. 38, 814–830.

3 Valverde, P., Healy, E., Sikkink, S., Haldane, F., Thody, A.J., Carothers, A., et al. (1996). The Asp84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma. Human Molec. Genet. 5, 1663–1666.

4 Bishop, D.T., Demenais, F., Iles, M.M., Harland, M., Taylor, J.C., Corda, E., et al. (2009). Genome-wide association study identifies three loci associated with melanoma risk. Nat. Genet. 41, 920–925.

5 Falchi, M., Bataille, V., Hayward, N.K., Duffy, D.L., Bishop, J.A., Pastinen, T., et al. (2009). Genome-wide association study identifies variants at 9p21 and 22q13 associated with development of cutaneous nevi. Nat. Genet. 41, 915–919.

Workshop 1: Pigment Patterns – their formation and evolution

Interactions between zebrafish pigment cells responsible for the generation of turing patterns

Shigeru Kondo

Graduate School of Frontier Biosciences, Osaka University, Yamadaoka, Suita, Osaka, Japan

The reaction-diffusion system is one of the most studied nonlinear mechanisms that generate spatially periodic structures autonomous. On the basis of many mathematical studies using computer simulations, it is assumed that animal skin patterns are the most typical examples of the Turing pattern (stationary periodic pattern produced by the reaction–diffusion system). However, the mechanism underlying pattern formation remains unknown as the molecular or cellular basis of the phenomenon has yet to be identified. We have been investigating the mechanism of skin pigment pattern formation using the zebrafish system. In this talk, we present our resent data about the inter-cellular interactions among the pigment cells and about the molecules those transfer the signals.

When the pigment cells in a restricted region were killed with laser treatment, new pigment cells developed to regenerate the striped pattern. We also found that the development and survival of the cells were influenced by the positioning of the surrounding cells. When melanophores and xanthophores were located at adjacent positions, these cells excluded one another. However, melanophores required a mass of xanthophores distributed in a more distant region for both differentiation and survival. Interestingly, the local effect of these cells is opposite to that of their effects long range. This relationship satisfies the necessary conditions required for stable pattern formation in the reaction-diffusion model. Simulation calculations for the deduced network generated wild type pigment patterns, as well as other mutant patterns.

Recently, we found three different molecular systems may involve in the process of pigment pattern formation. The detailed data about the relationship between the molecules and skin patterns will be discussed. Our findings allow further investigation of Turing pattern formation within the context of cell biology.

Workshop 2 – Function and formation of melanosome

The dark side of pigment cells: from DHICA to eumelanin, key issues and clues from chemistry

Marco d’Ischia, Alessandra Napolitano, Alessandro Pezzella, Lucia Panzella

Department of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Naples, Italy

The protective role of epidermal melanocytes against ultraviolet (UV) radiation and oxidative stress is believed to depend, among other factors, on functionally active MC1R receptors, competence for eumelanin synthesis and efficient melanosome transfer to keratinocytes. Several lines of evidence that accumulated during the past decades indicated, however, that melanocyte roles and function are not simply related to the provision of melanin pigments, but involve the entire melanogenic pathway and its diffusible intermediates. The detailed mechanisms by which the eumelanin pathway contributes to skin homeostasis and (photo)protection are however still not entirely understood. Relevant to this issue is the intriguing function of dopachrome tautomerase (Dct, or Tyrp2), the enzyme that diverts the rearrangement of dopachrome toward the formation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) rather than 5,6-dihydroxyindole (DHI). Early studies suggested that DHICA is an effective inhibitor of lipid peroxidation (1) and Fenton (2) processes in vitro and enhances LPS-induced stimulation of macrophage activity (3). Very recently, new data showed that Dct inactivation in knockout mice elevates the level of ROS, increases the numbers of sunburn cells and apoptotic cells, and decreases the amount of eumelanin in the epidermis upon exposure to chronic UVA radiation (4). Moreover, it was found that DHICA-melanin exhibits a potent hydroxyl radical-scavenging activity, whereas DHI melanin does not, suggesting the important role of Dct in the regulation of eumelanin antioxidant properties. Aim of the present communication is to provide a detailed analysis of the chemical properties of DHICA as a basis to interpret at molecular level its possible role in melanogenesis. In this frame, new chemical data are reported supporting a role of DHICA as an effective endogenous antioxidant. Moreover we show for the first time that the main circulating DHICA metabolite, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), shares with the parent indole a potent and comparable antioxidant activity, suggesting a possible action distal from the main site of eumelanin synthesis. It is proposed that the actual significance of Dct is related to the dual role of DHICA as important determinant of eumelanin protective action and as diffusible antioxidant and chemical messenger, partly in the form of 6H5MICA.

1 Memoli, S., Napolitano, A., d’Ischia, M., Misuraca, G., Palumbo, A., and Prota, G. (1997). Lipids and lipid metabolism. Biochim. Biophys. Acta 1346, 61–68.

2 Novellino, L., Napolitano, A., and Prota, G. (1999). Chem. Res. Toxicol. 12, 985–992.

3 D’Acquisto, F., Carnuccio, R., d’Ischia, M., and Misuraca, G. (1995). Life Sciences 57, PL401-PL406.

4 Jiang, S., Liu, X.-M., Dai, X., Zhou, Q., Lei, T.-C., Beermann, F., Wakamatsu, K., and Xu, S.-Z. (2010). Free Rad. Biol. Med. 48, 1144–1151.

Activation of 3-o-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin in melanoma to a potent irreversible inhibitor of dihydrofolate reductase

José Neptuno Rodríguez-López1, Luís Sánchez-del-Campo1, María F. Montenegro1, Juan Cabezas-Herrera2

1Department of Biochemistry and Molecular Biology A, School of Biology, University of Murcia, Murcia, Spain;
2Research Unit of Clinical Analysis Service, University Hospital Virgen de la Arrixaca, Murcia, Spain

Human melanoma is a significant clinical problem because it is resistant to treatment by most chemotherapeutic agents, including antifolates. It is therefore a desirable goal to develop a second generation of low-toxicity antifolate drugs to overcome acquired resistance to the prevention and treatment of this skin pathology. In our efforts to improve the stability and bioavailability of green tea polyphenols for cancer therapy, we synthesized a trimethoxy derivative of epicatechin-3-gallate, which showed high antiproliferative and pro-apoptotic activity against melanoma. This derivative, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), is a prodrug that is selectively activated by the specific melanocyte enzyme tyrosinase. Upon activation, TMECG generates a stable quinone methide product that strongly inhibits dihydrofolate reductase in an irreversible manner. The treatment of melanoma cells with TMECG also affected cellular folate transport and the gene expression of DHFR, which supported the antifolate nature of this compound. In addition, its pharmacological efficacy has been confirmed in a mouse melanoma model, in which tumor growth and metastasis were inhibited, significantly enhancing the mean survival of the treated groups. TMECG, therefore, shows a potential for clinical use in melanoma therapy.

Grant sponsors: Fundación Séneca, Región de Murcia; Grant number: 08595/PI/08 and Ministerio de Ciencia e Innovación, Gobierno de España; Grant number: SAF2009-12043-C02-01.

Workshop 3 – Vitiligo

Lipid modification in vitiligo melanocytes: a possible role in the alteration of signal transduction pathways

M. Picardo, B. Bellei, M. Ottaviani, D. Kovacs, M. L. Dell’Anna

San Gallicano Dermatologic Institute, IRCCS, Rome, Italy

The mechanism of melanocytes detachment in vitiligo has not been completely clarified. An enhanced sensitivity to external stimuli associated with an increased intracellular generation of reactive oxygen species (ROS) and antioxidant alteration has been suggested as a possible intrinsic defect. We previously demonstrated in vitiligo melanocytes in culture increased ROS production and membrane lipid peroxidation. The pattern of membrane lipids is crucial for the activation of the intracellular pathway involved in cell proliferation and differentiation. Moreover, we detected increased level of membrane cholesterol associated with an altered response to αMSH treatment. Now we have evaluated the modification of intracellular kinases phosphorilation in non stimulated vitiligo melanocytes and the possible biological consequences. Vitiligo melanocytes presented hyper-phosphorilation of the PKA target CREB. Consequently, some CREB target genes, such as COX-2 and NRF2, which control the expression of several antioxidant enzymes, showed a significant upregulation. In addition, a reduced αMSH-dependent modulation of NRF2 was observed in vitiligo melanocytes. Our results suggest that an alteration of the lipid component of the cell membrane in vitiligo melanocytes can be associated with an altered activation of the intracellular pathway controlling the antioxidant system.

Submitted Abstracts

c-Myc in melanocytes and melanoma

Marzia Armaro1, Irina Pshenichnaya1, Andreas Trumpp2, Lionel Larue3, Stuart Gallagher3, Friedrich Beermann1

1EPFL SV ISREC, Lausanne, Switzerland;
2DKFZ, Heidelberg, Germany;
3UMR146 CNRS, Institut Curie, Orsay, France

c-Myc is an oncogenic transcription factor that is frequently upregulated in human malignancies including melanoma. We have recently addressed the role of c-Myc in the melanocyte lineage using mice lacking c-Myc specifically in pigment cells (Tyr::Cre, c-Mycflox/flox). Such mice display hair graying which is directly caused by a reduction in melanocyte/melanoblast number during embryonic development, without any obvious effect on melanocyte function or differentiation. We have recently begun to address the role of c-Myc in melanoma formation, using lentiviral transduction of mouse melanoma cells, and conditional c-Myc knock-out mice developing melanoma (Tyr::NrasQ61K, INK4a-/-). Preliminary in vitro results suggest that depletion of c-Myc in melanoma cells blocks cell differentiation and induces differentiation.

Overexpression of the Strawberry Notch homolog 2 gene in the melanocyte lineage triggers a fate switch in melanocyte stem cells

Geneviève Aubin-Houzelstein1,2, Stéphanie Gadin1, Florence Bernex1, Jean-Jacques Panthier1,2

1INRA, Maisons-Alfort, France;
2Institut Pasteur, Paris, France

Conditional mutations in three members of the Notch pathway lead to a failure in melanocyte stem cell maintenance and to hair greying. Strawberry Notch homolog 2 (Sbno2) is the mouse homologue of the sno gene, a regulator of the Notch pathway in Drosophila. If this gene also acts as a Notch modifier in the mouse, mutations in Sbno2 should alter melanocyte stem cell homeostasis. To test this hypothesis, we produced mice overexpressing Sbno2 in the melanocyte lineage (Tg(Dct::Sbno2) mice) and studied their coat colour phenotype. At birth, the transgenics had a wild-type coat except for their white tail tip and feet. Their coat started whitening from 3 months of age. The whitening phenotype was not total as the transgenics never turned completely white. We compared the distribution of melanocytes and their precursors in hair follicles isolated from Tg(Dct::Sbno2); Tg(Dct::lacZ) mutants and Tg(Dct::lacZ) control mice. In Tg(Dct::Sbno2); Tg(Dct::lacZ) hair follicles, there was a drastic reduction in the number of melanocyte progenitors from the first hair cycle onward and the number of β-galactosidase positive cells in the bulge region decreased in the successive hair cycles, suggesting an incomplete maintenance of the melanocyte stem cells. Strikingly, a ring of β-galactosidase positive cells was systematically found encircling several hair follicles at the bulge level. These cells had the same localization and shape as the nerve fibers of the deep cutaneous nerve plexus and hair follicle neural network, consisting of neurons and Schwann cells. We are characterising the cell type of the β-galactosidase positive cells by immunofluorescence. Our results suggest that the overexpression of Sbno2 in melanocyte stem cells or their progeny triggers a cell fate switch responsible for the reduced number of melanocyte progenitors and differentiated melanocytes in the hair follicles and the ring of β -galactosidase positive cells around the hair follicles in the Tg(Dct::Sbno2); Tg(Dct::lacZ) mice.

MITF is the key molecular switch between melanoma initiating cells and their differentiated progeny

Yann Cheli1,2, Sandy Guiliano1,2, Thomas Botton1,2, Stéphane Rocchi1,2, Véronique Hofman3, Paul Hofman2,3, Philippe Bahadoran1,2,4, Corine Bertolotto1,2,4, Robert Ballotti1,2,4

1INSERM, U895, Centre Méditerranéen de Médecine Moléculaire (C3M), Equipe 1, Biology and pathologies of melanocytes, Equipe Labellisée par la Ligue Contre le Cancer, Nice, Cedex, France;
2Université de Nice-Sophia Antipolis, UFR médecine, Nice, France;
3ERI21/EA 4319 and Human Biobank, Cedex, Nice, France;
4CHU NICE, Department of dermatology, Cedex, NICE, France

We have identified MITF, the master regulator of melanocyte differentiation, and p27, a CDK inhibitor, as the key molecular switches that control the transition between melanoma-initiating cells and their differentiated progeny. MITF silencing is sufficient to increase the tumorigenic and metastatic potential of B16 melanoma cells. MITF silencing also upregulates the stem cell markers Oct4 and Nanog and induces mesenchymal transition of melanoma cells that might explain their increased invasiveness in vitro and in vivo. Notably, the cell cycle inhibitor p27 is increased in MITF-depleted cells and is required for the induction of the ‘stemness’ and exacerbation of the metastatic properties of melanoma cells. These data were confirmed using human melanoma cell lines and freshly isolated human melanoma cells. Further, a low-MITF population that has high tumorigenic potential exists spontaneously in melanoma cell lines. Ablation of this population by cell sorting, forskolin-induced differentiation or MITF forced expression dramatically decreases tumor formation, indicating that eradication of low-MITF cells is an appealing strategy to cure melanoma.

Rab GTPases, the recycling endosome and regulation of melanosome trafficking and release

Kimberley A. Beaumont1, Nicholas A. Hamilton1, Matthew T. Moores1, Oliver Cairncross1, Anthony L. Cook1, Aaron G. Smith1, Ryo Misaki1, Mitsunori Fukuda2, Tomohiko Taguchi1, Richard A. Sturm1, Jennifer L. Stow1

1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia;
2Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi, Japan

Specific Rab GTPases have been shown to contribute to pigmentation. A mouse coat color mutation in Rab27a revealed its role in capturing melanosomes onto actin filaments for subsequent transfer to keratinocytes. Additionally, Rab32 and Rab38 regulate melanosome protein trafficking. However, the role of Rab GTPases in other steps in melanosome maturation, movement and transfer are less well defined. Rab17 – previously only studied in epithelial cells – is on recycling endosomes and, in melanocytic cells, we find it also localized to melanosomes. Rab17 mRNA expression is regulated by Microphthalmia Transcription Factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in B16 melanoma cells quantitatively increased melanosome concentration at the cell periphery, contrasting starkly with Rab27a knockdown, which caused perinuclear accumulation as expected. Rab17 knockdown also increased the length, but not number, of cell dendrites, an effect attributed to recycling endosomes. Rab17 knockdown did not alter melanosome maturation or movement, but melanosome release was inhibited, resulting in abnormally high levels of intracellular melanin. The effects of double knockdown of Rab17/Rab27a suggest that Rab17 acts on melanosomes downstream of Rab27a. These results show a novel role for MITF-regulated Rab17 in pigmentation where it defines a new distal step in the process of melanosome release. Preliminary data indicates that other recycling endosome Rabs may be involved in melanosome trafficking and release.

β-catenin critically involved in α-MSH-induced regulation of melanogenesis

Barbara Bellei1, Angela Pitisci1, Lionel Larue2, Mauro Picardo1

1Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS, Rome, Italy;
2Developmental Genetics of Melanocytes, UMR 3347, CNRS, U1021 INSERM, Institute Curie, Orsay, Cedex, France

Extensive examination in different organism and cell lineages over the past decade has illustrated the complexity of the Wnt signaling pathway and versatile role of β-catenin as a transcription factor. Wnt/β-catenin signaling plays important roles in many developmental processes, including neural crest-derived melanocyte development, and migration. However, the effective contribution of the canonical Wnt/β-catenin pathway in melanogenesis in adult human melanocyte has not been fully elucidated. Wide advances have been made over the past few years in understanding the complexity of Wnt signaling and the cross-talk between Wnt and other signaling pathways. Certain peptide hormones may stimulate β-cat/Tcf-Lef1-mediated gene transcription via activation of their corresponding G-protein coupled receptors. Here we report that in B16 melanoma cells and in NHM treatment with α-MSH induces phosphorylation of β-catenin at Ser675. Such phosphorylation that is mediated by protein kinase A (PKA) cells appears to stabilize β-catenin at protein levels and to increase β-catenin-mediated gene transcription. Glycogen synthase kinase-3β (GSK3β), which regulates ubiquitin-dependent degradation of β-catenin, is also a physiological target of PKA. Activation of adenylate cyclase by α-MSH or forskolin attenuates GSK3β activity by phosphorylation of Ser9 suggesting a coordinated mechanism of β-catenin activity stimulation. Consistent with nuclear β-catenin increase, several Wnt target genes (Axin2, Lef1, Mitf, Sox9 and Wisp1) were coordinately upregulated in B16 cells and the observed stimulation was significantly reduced in β-cat-siRNA-treated cells. Moreover, as a consequence of reduced expression of β-catenin, we observed a significant reduction of melanin synthesis induced by α-MSH treatment and a more pronounced inhibition on unstimulated cells. In addition, chromatin immunoprecipitation assays demonstrated an increased association of β-catenin with the proximal promoter of microphthalmia-associated transcription factor (M-Mitf), the master regulator of pigmentation. These results demonstrate the existence of a cross talk between the cAMP and Wnt/β-catenin pathways in melanocytes demonstrating that β-catenin could play a key role in physiological regulation of epidermal melanogenesis.

Anti-melanoma effects of hsp an inhibitor of the mitotic driver class

Caroline Bonet, Sandy Giuliano, Robert Ballotti, Corine Bertolotto

INSERM U895, Team 1, Biology and pathology of melanocytes: from cutaneous pigmentation to melanoma, route Saint Antoine de Ginestière, NICE Cedex, France

Melanoma originates from melanocytes and is the most deadly form of skin cancer mainly due to absence of efficient treatment for late stage of the disease. Therefore there is an urgent need to find new or improve existing therapeutic strategies.

AURORA B is a serine/threonine kinase that belongs to the chromosomal passenger proteins that are critically required for mitosis, a highly coordinated process that assures the fidelity of chromosome segregation. More particularly, AURORA B has been involved in chromosome condensation, organization and stabilization of the bipolar mitotic spindle, chromosome congression at the metaphase plate and cytokinesis. Errors in this process result in aneuploidy and oncogenesis but accumulation of excessive errors that can't be repaired by the cell machinery leads to cell cycle arrest or cell death.

AURORA B has been implicated in cancer being overexpressed in several tumors notably in melanoma cells. Recently, a new class of pharmacological inhibitors termed ‘mitotic drivers’, as opposed to the well-known classic anti-mitotics, has been developed. The aim of the current study is to investigate the potential anti-melanoma effect of this new class of inhibitors.

First we confirmed an over-representation of AURORA B in melanoma cells at different stage of progression relative to normal melanocytes suggesting that mitotic drivers will act in melanoma cells but not in melanocytes. We confirmed this hypothesis and showed a decreased cell proliferation and colony formation by treatment with HSP, a selective pharmacological inhibitor of AURORA B. Further, we found that AURORA B inhibition by HSP or RNA interference promoted severe mitotic defects altering cytokinesis and resulting in massive polyploidy. Consistently, HSP induced caspase activation and cleavage of PARP, indicating that HSP induces apoptosis of melanoma cells. All of these results were validated in several human melanoma cell lines.

Our results suggest an important role of AURORA B in melanoma tumorigenesis and a novel potential target in its treatment.

MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma

Simone Braig1*, Daniel W. Mueller1*, Tanja Rothhammer1, Anja-Katrin Bosserhoff1

1Institute of Pathology, University of Regensburg Medical School, Franz-Josef-Strauss-Allee, Regensburg, Germany
*Contributed equally

It its known, that bone morphogenetic proteins (BMPs) play an important role in melanoma progression. We, therefore, aimed to determine the molecular mechanisms leading to overexpression of BMP4 in melanoma cells compared to normal melanocytes.

By our experimental approach we revealed that loss of expression of a microRNA represents the starting point for a signaling cascade finally resulting in overexpression of BMP4 in melanoma cells. In detail, strongly reduced expression of the microRNA miR-196a in melanoma cells compared to healthy melanocytes leads to enhanced HOX-B7 mRNA and protein levels, which subsequently raise Ets-1 activity by inducing basic fibroblast growth factor (bFGF). Ets-1 finally accounts for induction of BMP4 expression.

Functional assays revealed strongly reduced migratory potential of melanoma cell clones stably transfected with miR-196a expression plasmids compared to mock transfected cells. Vice versa, treatment of normal human melanocytes with anti-miR-196a increases the expression of the miR-196a target genes HOX-B7, bFGF and BMP4 and enhances the migratory potential of anti-miR-196a transfected cells compared to control cells.

RACK1 labelling in horse skin melanocytes marks genetic predisposition to melanoma

Campagne Cécile1, Julé Sophia1, Chaumien Matthieu1, Golovko Anna2, Beermann Friedrich3, Bernex Florence1,4, Towanou Narcisse4, Robert Céline5, Seltenhammer Monika6, Andersson Leif2, Aubin-Houzelstein Geneviève1,7, Panthier Jean-Jacques7, Egidy Giorgia1,7

1INRA-ENVA UMR955, Ecole Nationale de Vétérinaire d’Alfort (ENVA), Maisons-Alfort, France;
2Uppsala University, Uppsala, Sweden;
3EPFL, Lausanne, Switzerland;
4Anatomie pathologique ENVA, Maisons-Alfort, France;
5Service d’Anatomie ENVA, Maisons-Alfort, France;
6Max Perutz Laboratories, University of Vienna, Austria;
7Génétique fonctionnelle de la souris, Institut Pasteur, Paris, France

Melanoma occurs in horses regardless of age, sex or coat colour. Non-gray horses may also develop melanocytic malignant tumours. RACK1 protein was shown to be a marker of malignancy for human melanoma (Egidy et al, Molecular Cancer 7:34, 2008). Using immunofluorescent labelling for RACK1, we identified specific patterns in healthy skin melanocytes, benign melanocytic tumours and melanomas from non-gray horses.

Gray colour in horses shows an autosomal dominant inheritance. Gray horses (G/G or G/g) are born coloured and after some years their hair gradually whitens while their skin pigmentation is maintained. Aged gray horses are known to have a high incidence of melanocytic tumours, yet a high survival rate. In gray horses distinguishing between benign and malignant melanocytic tumours is difficult. Although most melanomas in gray horses have benign features initially, as many as 66% of these tumours eventually become malignant.

We studied RACK1 labelling in normal skin and melanomas from gray horses. In healthy skin from gray horses, we found a RACK1 pattern different from that previously identified in non-gray horse skin. This RACK1 pattern was also found in skin melanocytes of transgenic Tyr::NRasQ61K; Ink4a-/- mice which spontaneously develop cutaneous metastasizing melanomas beyond 6 months of age. These data suggest a correlation between the RACK1 status in healthy skin melanocytes and predisposition to melanoma.

Melanocytes modulate the alteration of epidermal homeostasis induced by exposure to electromagnetic radiation

M. Cario-André1, D. Simon1,2, A. Daubos1, C. Pain1, R. Fitoussi2, R. Anane3, R. Ennamany3, K. Vié2, A. Taieb1, L. de Benetti2

1Universiy V Segalen, INSERM, Bordeaux, France;
2Laboratoires Clarins, Pontoise, France;
3Eurotest, Cestas, France

In this study we have looked at the effects of a strong acute electromagnetic radiation exposure (GSM basic, 900 MHz, SAR 2 mW/g, 6 h) on a model of epidermis reconstructed with or without melanocytes. We have analyzed the variation in expression and localisation of various epidermal markers (stress, differentiation, proliferation) and melanocyte behaviour markers 6 and 24 h after exposure.

Analysis after Fontana-Masson or anti-melanA staining revealed that exposure to electromagnetic radiation did not induce change in epidermal architecture whatever the type of reconstructs. In pigmented reconstructs, no change in the location and dendricity of melanocytes and in transfer of melanin to neighbouring keratinocytes was detected after exposure. Apoptotic cells were not detected in reconstructs at 24 h. Similarly, by immunohistochemistry, no changes were found in the localisation of epidermal markers of differentiation/proliferation, presence of apoptotic cells and induction of p53 and Hsp70 in both types of epidermis. If no effect of melanocytes could be detected by immunohistochemistry, probably due to a lowest sensibility, we observed some variations of protein expression by western blot analysis. A decrease at 6 h was found in the levels of filaggrin, loricrin, cytokeratin 14 and cytokeratin 10 for both types of reconstructs. Cytokeratins 5 was decreased at 6 h in pigmented reconstructs whereas it was increased in non pigmented reconstructs. The level of filaggrin was not fully recovered at 24 h contrary to cytokeratins 5 and 10 in pigmented reconstructs. In non pigmented reconstructs cytokeratin 5 and filaggrin were markedly increased at 24 h whereas cytokeratin 10 was partially recovered. In both types of reconstructs, the 20S proteasome activity was decreased at 24 h. At 24 h we observed an increase in oxidized protein only in non pigmented reconstucts. Hsp70 was significantly decreased at 6 h in both types of reconstructs. Caspase 3 was markedly but transiently activated in non pigmented reconstructs at 6 h whereas it was slightly increased at 24 h in pigmented ones. No effect of electromagnetic radiation was noted on MITF expression and phosphorylation.

In conclusion, our data indicate that exposure to 900 MHz frequency induce an alteration of the epidermal barrier which may alter the protective capacity of the skin against external factors. If the behaviour of melanocytes themselves does not seem to be affected by exposure to 900 MHz frequency, their presence seems to minimize oxidative stress and variation of expression of structural proteins.

Chemico-genetic analysis of the parade mutant; insights into embryonic pigment pattern formation in zebrafish

Sarah Colanesi, Jeanette Muller, Robert N. Kelsh

Centre for Regenerative Medicine, University of Bath, UK

Pigment pattern formation is a classic model system in which to identify general mechanisms of pattern formation. The complex and beautiful body patterns seen in vertebrates remain unexplained. Although they can be readily modelled using reaction-diffusion systems, direct dissection of the genetic basis of these patterns is required to assess the biological mechanisms underlying these patterns. Zebrafish as an ideal vertebrate model organism for this problem, due to the availability of multiple mutants affecting pigment pattern formation, accessibility for live imaging in the transparent embryos, and a broad range of genetic tools. Zebrafish embryos have three types of pigment cells which form a highly reproducible pattern, of four horizontal stripes of melanophores (melanocytes), some of which are associated with iridophores (shiny), and a more homogeneous distribution of xanthophores (yellow) in-between the other pigment cells. Parade mutants show a unique pigment pattern defect of ectopic melanophores and iridophores on the distal medial migration pathway, although the rest of the pigment pattern is normal. Here we will present the positional cloning of the parade gene. Although this gene has been previously associated with cranial cartilage development, there is no precedent for a role in pigment cell patterning. In situ hybridization reveals the expression to be prominent in the midline blood vessels, suggesting that it acts non-autonomously to the pigment cells. Interestingly, numbers of pigment cells in the ventral stripe are substantially increased. We hypothesised that the ectopic cells may result from over-filling of the ventral stripe. The earliest visible phenotype is an increase in iridophore marker expression from the very early stage of 30 hpf, consistent with our idea.Using a chemical biology approach, we screened 1300 drug compounds for those that would specifically alter the ectopic pigment phenotype in parade, and were able to identify 31 (rescreened and confirmed) drugs that do so. Further work is required to identify the link between midline blood vessels and excess pigment cell numbers. Our data suggests that pigment pattern formation depends in part upon favoured target locations, with other sites occupied only when the former are fully occupied.

Co-Regulation of TYR expression by bHLH-LZ transcription factor in response to UV

Sebastien Corre1, Marco Pontecorvi2, Amine Bouafia1, Marie-Dominique Galibert1, Colin Goding2

1CNRS UMR 6061 Genetic and Development Institute of Rennes, RTO Team, Université de Rennes-1, Faculté de Médecine, IFR140 GFAS, 2 av du Pr Léon Bernard, CS, Rennes cedex, France;
2Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Old Road Campus, Headington, Oxford, UK

The skin is the body's first barrier that is exposed to various physical, chemical and biological hazards that can alter DNA structure. To maintain the integrity of the genome, cells are equipped with specific defense machinery, which targets different types of mechanisms such as melanin synthesis, DNA repair and cell cycle progression. In recent years, the ubiquitous USF1 transcription factor has been implicated in the regulation of the stress response in the skin by controlling the expression of a wide number of genes in response to UV. Indeed, USF1 has been shown to induce pigmentation gene expression in different type of skin models. More recently we have implicated USF1 in regulation of the NER machinery, one of the most versatile DNA repair systems that eliminates a wide variety of DNA lesions. In addition, we have shown that USF1 is a key sensor of stress signaling; while phosphorylation of USF1 after low dose of stress is associated with the active form, phospho-acetylation of USF1 after high stress is associated with loss of transcriptional activation properties. Activation of transcription by bHLH-LZ transcription factors is also linked to the occupancy of target genes by one or several dedicated activators. By analyzing the regulation of the pigmentation gene Tyrosinase in response to UV, we described for the first time a specific kinetic of binding for the USF and Mitf factors to the consensus elements of the Tyr promoter and demonstrate that USF and Mitf cooperation is crucial for significant Tyr induction. Furthermore, we identified a Tyr antisense transcripts (lnRNA), which is required for full transcriptional activation of Tyr by UV and that is dependent on USF1 regulation. Our findings depict how promoter occupancy and the recruitment of the transcriptional machinery by transcription factors are essential to drive gene expression and molecular processes.

Molecular characterization of melanin uptake and processing by keratinocytes

Maria S. Correia1,5, Giulia Bolasco2,3, Alistair N. Hume2, Clare E. Futter4, Miguel C. Seabra1,2,5

1Instituto Gulbenkian de Ciência, Oeiras, Portugal;
2Molecular Medicine, National Heart and Lung Institute, Imperial College London, London, UK;
3EMBL, Mouse Biology Unit, Monterotondo, Italy;
4Institute of Ophthalmology, University College London, London, UK;
5CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal

Melanosome transfer from melanocytes to keratinocytes and later redistribution in keratinocytes supra-nuclear area is a critical process in skin pigmentation and UV-protection. However, the uptake and ultimate fate of melanin within keratinocytes has been poorly characterized. Here we attempt to validate in vitro systems to study the mechanisms of entry and the traffic of melanin within keratinocytes. In vitro studies were carried out on keratinocytes fed with 500 nm fluorescent latex beads, and melanocyte-keratinocytes co-cultures. These studies revealed that, for both systems, the internalisation process of the microspheres and melanin granules by keratinocytes was similar to our in vivo observations. However, subsequent to the uptake, the beads appeared to be processed by keratinocytes through a degradative pathway and to accumulate in lysosome-like structures that appeared morphologically different from the melano-phagosomes observed in vivo. Conversely, melanin present in keratinocytes co-cultured with melanocytes was trafficked to a degradative compartment that highly resembled the physiological supranuclear cap observed in human skin. Taking together, our observations confirmed the use of latex beads as a valid approach to identify factors involved in the uptake of pigment by keratinocytes. Currently we are performing knock-down experiments of candidate proteins involved in phago/endocytosis to assess their relevance in this process.

Loss of E-cadherin increases metastasic potential of melanoma

V. Delmas1, F. Luciani1, F. Beermann2, R. Kemler3, L. Larue1

1Developmental Genetics of Melanocytes, UMR 146 CNRS, Institut Curie, Orsay, France;
2EPFL SV ISREC GR-BEERMANN, SV 2539 (Bâtiment SV) Station, Lausanne, Switzerland;
3Max-Planck Institut für Immunbiologie, Stübeweg, Freiburg, Germany

E-cadherin is a calcium-dependent cell-cell adhesion molecule expressed by melanocytes and responsible for their adhesion to the surrounding keratinocytes in the epidermis. Disruption of E-cadherin-mediated cell-cell adhesion has been implicated in tumour progression and metastasis. In order to investigate its role in melanomagenesis in vivo, we have generated a mouse model in which E-cadherin is specifically deleted in melanocytes (Tyr::Cre/°, E-cadherinfloxed/floxed = Ecad mice). At the molecular level, E-cadherin is neither replaced by N- nor P-cadherin in defloxed melanocytes. Furthermore the amount of β-catenin, normally bound to E-cadherin, is less important at the membrane but accumulates in the cytoplasm and nucleus, which has been linked to 30% of human melanoma. However Ecad mice do not develop melanoma, suggesting a minor role of E-cadherin in the initiation steps of melanomagenesis including proliferation and immortalization. We crossed Ecad mice with Tyr::NrasQ61K and Ink4a mice to induce proliferation and immortalization in vivo. As expected the latency period and the penetrance of melanoma production were not altered in the absence of E-cadherin. However, the lack of E-cadherin in melanocytes promotes dramatically lymph node invasion and lung macrometastasis.

Gene expression profiles of human melanoma cells with different invasive potential reveal tetraspanin 8 as a novel mediator of invasion

Manale El Kharbili1, Jérôme Lamartine1, Arnaud de la Fouchardière2, Thomas Pointecouteau1, Patrick Verrando3, François Le Naour4, Odile Berthier-Vergnes1

1Centre de Génétique Moléculaire et Cellulaire, UMR-CNRS 5534, Université C. Bernard Lyon, France;
2Département d’Anatomie et Cytologie Pathologiques, Centre Léon Bérard, Lyon, France;
3INSERM UMR 911 – CRO2, Faculté de Médecine Timone, Université de la Méditerranée, Marseille, France;
4INSERM U602, Université Paris-XI, Villejuif, France

The events occurring during the penetration of melanoma cells through the dermal-epidermal junction, the first step in the process of metastasis, remain ill defined. Previously, we successfully selected a pair of isogenic melanoma clones from the same parental cell line that differ in their ability to invade dermis in human skin reconstructs. To gain insight into the mechanisms of dermal invasion and in search of early prognostic markers, gene expression profiles of these two clones were compared by oligonucleotide microarray. Our data spotlighted TSPAN8, a member of the tetraspanin family not yet described in cutaneous melanoma. Tspan8 is exclusively expressed by invasive melanoma cells, but not by normal melanocytes or non-invasive melanoma cells. Its cell surface expression correlated with the invasive phenotype of a panel of well-defined melanoma cell lines, as assessed by flow cytometry. Consistent with the in vitro findings, an immunohistochemical study on paraffin-embedded melanocytic lesions revealed that Tspan8 is expressed in primary tumors and lymph node metastasis but not in healthy skin. More importantly, transient knockdown of endogenous Tspan8 by RNA interference reduced the invasive outgrowth of melanoma tumor spheroids embedded in matrigel, without impact on the cell proliferation and survival. Collectively, our data implicate Tspan8 in the acquisition of an invasive behavior of melanoma cells and may thus be a promising novel target for anti-invasive therapy.

What are the priorities for clinical research in vitiligo?

Viktoria Eleftheriadou1, Kim Thomas1, Maxine Whitton2, David J. Gawkrodger3, Jeff Corne4, Bernard C. Lamb5, Jonathan Batchelor6, Jane Ravenscroft7

1Centre of Evidence Based Dermatology, University of Nottingham, Nottingham, England;
2NHS Library, Skin Cochrane Group, London, England;
3Royal Hallamshire Hospital, Sheffield, England;
4Vitiligo Society, London, England;
5Vitiligo Society, London, England;
6Addenbrooke's Hospital, Cambridge, UK;
7Queen's Medical Centre, University of Nottingham, Nottingham, England

Background: Vitiligo is the most common depigmentation disorder of the skin. It affects around 0.5% of the population worldwide, and can be cosmetically and psychologically devastating. However, vitiligo has been historically neglected, and the evidence base for clinical practice is poor. A recent Cochrane systematic review showed that the majority of trials to date have small number of participants; are methodologically poor; and involve multiple treatment comparisons. This makes it difficult to make firm statements regarding recommendations for clinical practice.

In order to address these issues, and to identify the most important treatment uncertainties in the field of vitiligo, we conducted a prioritisation exercise involving patients and health professionals.

Methods: This study was conducted in collaboration with the Centre of Evidence Based Dermatology, the James Lind Alliance, the Vitiligo Society, the British Association of Dermatologists, the British Dermatological Nursing group, the Primary care Dermatology Society, and cosmetic camouflage groups, in order to bring clinicians and patients together in a working partnership. The process had three stages:

1 Postal and on-line survey – to collect treatment uncertainties.

2 Ranking exercise – to prioritise the Top 20–30 treatment uncertainties.

3 Final Prioritisation Workshop – to agree upon the Top 10 treatment uncertainties.

Results: The survey resulted in 660 treatments uncertainties submitted by 460 participants. Two hundred and thirty people voted during the ranking exercise for their favourite topics. The resulting Top 23 uncertainties were then discussed at a Final Prioritisation Workshop, and the list of Top 10 was created. These included interventions such as phototherapy, topical preparations (tacrolimus, steroids, pseudocatalase, piperine), the impact of psychological interventions and camouflage on quality of life, and the role of gene therapy.

Conclusions: The Top 10 uncertainties will provide reference and guidance for future researchers and funding bodies, to ensure that future research is important to both patients and healthcare professionals. We next plan to conduct a feasibility study in collaboration with UK Dermatology Clinical Trials Network on one of the identified priorities. This will inform the development of a protocol for a large multicentre trial.

Autoantibodies against tyrosine hydroxylase in patients with non-segmental (generalised) vitiligo

Sherif Emhemad1, Samia Akhtar1, Philip F. Watson1, David J. Gawkrodger2, Anthony P. Weetman1, E. Helen Kemp1

1Department of Human Metabolism, School of Medicine, University of Sheffield, Sheffield, UK;
2Department of Dermatology, Royal Hallamshire Hospital, Sheffield, UK

Vitiligo is an acquired idiopathic hypomelanotic skin disorder characterised by depigmented macules due to loss of cutaneous melanocytes. Although the exact cause of vitiligo remains obscure, evidence suggests that autoimmunity plays a role in the pathogenesis of the disease. Previously, tyrosine hydroxylase (TH) was identified as a putative autoantigen in vitiligo using phage-display technology. In the present study, the prevalence of TH antibodies in vitiligo patients was further investigated.

A radioimmunoassay was used to detect TH antibodies in sera from patients with either non-segmental vitiligo (n = 79), segmental vitiligo (n = 8) or other autoimmune diseases without concomitant vitiligo (n = 91). Sera from healthy individuals (n = 28) were also tested.

Of 79 non-segmental vitiligo patients, 18 (23%) were positive for TH antibodies. TH antibody reactivity was not demonstrated in segmental vitiligo patients, healthy controls or patients with other autoimmune diseases without concomitant vitiligo. A significant increase in the prevalence of TH antibodies in non-segmental vitiligo patients was evident when compared with controls (P = 0.003). TH antibody prevalence was also significantly increased in patients with active vitiligo (4/6 = 67%) compared to those with stable disease (14/81 = 17%) (P = 0.015).

Overall, the results indicate that TH is an antibody target in non-segmental but not in segmental vitiligo. TH antibodies also appear to be more frequent in patients with active vitiligo, although this will need to be confirmed in a larger patient cohort.

Analysis of coat colour loci in Vicugna pacos (Alpaca) and association with coat colour variation

Natasha L. Feeley1, Kylie A. Munyard1

1School of Biomedical Science, Curtin University of Technology, Perth, WA, Australia

Alpaca fibre is highly valued in the textile industry due to its exceptional strength, softness and lustre. It is increasingly being used in high end fashion and is a growing industry in Australia and around the world. Colour is one of the important fibre characteristics because it has substantial impact on the applications and value of the final product. Unfortunately, little is known about the way fibre colour is inherited in alpacas. Therefore there is a need for research into the genetic mechanisms, which control colour inheritance in alpacas. There are several genes which are known to be the key regulators of pigmentation in mammals. These include the Melanocortin-1 receptor, Agouti and Tyrosinase Related Protein-1 genes and these have been the basis of our research into colour genetics in alpacas. These genes control the relative amounts of dark and light pigment present in the fibre. Our research has identified several mutations occurring within these genes, some of which appear to be functionally significant. These results may be valuable to the alpaca industry in allowing them to selectively breed animals for desired colours, enabling the industry to tailor fibre production to the current demands of the market.

2,4,6-octatrienoic acid promotes melanogenesis in normal human melanocytes

E. Flori1, A. Mastrofrancesco1, V. Maresca1, B. Bellei1, G. Giuliani2, S. Briganti1, Mauro Picardo1

1Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute (IRCCS) Rome, Italy;
2Giuliani SpA, Milan, Italy

Melanin synthesis is a complex process controlled by several regulators affecting intracellular signal transduction pathways. Peroxisome-activated receptors (PPARs) belong to a family of ligand-activated transcription factors that include receptors for retinoids and regulate important functions in the skin including proliferation and differentiation. Evidence in literature shows that activation of PPARγ, one of the three PPARs isoforms, can induce melanocytes differentiation and melanogenesis. We have observed that Parrodienes, a class of compounds sharing some structural features with carotenoids, natural precursors of retinoids, possess antioxidant activities and counteract senescence-like phenotype in fibroblasts. Moreover we found that the same compounds are able to promote PPARγ activation. Considering that PPARγ agonists can stimulate melanogenesis, we selected 2,4,6-octatrienoic acid (Octa), a derivative of parrodienes, to study its ability to promote melanogenesis in normal human melanocytes (NHM). Exposure of NHM to Octa resulted in a significant induction of tyrosinase expression and activity. The melanin content was also significantly increased by Octa treatment. Moreover we examined the expression level of PPARγ and microphthalmia-associated transcription factor (MITF), the master transcription factor regulating melanocyte differentiation, proliferation and survival. The observed increase in pigmentation was associated with a stimulation of both PPARγ and MITF expression. These results suggest a regulatory role of Octa in melanogenesis of NHM, offering future perspectives of its employment to enhance skin photoprotection and possibly for the treatment of pigmentary disorders.

Signals involved in oncogenic activities of metabotropic glutamate receptor 1

Yoko Funasaka1, Abdel-Daim Mohamed2, Takeshi Harada3, Atsu Aiba3, Seiji Kawana, Chikako Nishigori2

1Department of Dermatology, Nippon Medical School, Tokyo, Japan;
2Division of Dermatology, Kobe University Graduate School of Medicine, Kobe, Japan;
3Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

Metabotropic glutamate receptor subtype 1 (mGluR1) is a G protein coupled receptor activated by glutamate, and is functionally expressed in the central nervous system. We have previously shown that ectopic expression of mGluR1 in melanocytes is essential for both development and in vivo growth of melanoma in transgenic mice. Upon stimulation by glutamate, mGluR1 can couple to multiple signaling pathways through different G proteins, such as activation of phospholipase C (PLC) β which leads to trigger IP3-mediated Ca2+ release and activation of PKC, increase of cytoplasmic cAMP, and activation of ERK1/2 MAP kinase. Our aim is to elucidate the signals involved in development and growth of melanoma using inhibitors and stimulators of these signals. Inhibitors to phospholipase CPKC, MEK1/2, and cAMP phosphodiesterase, antagonists to Ca2+ and calmodulin, and activator of adenylyl cyclase were administered to transgenic mice. Except cAMP inducers, each signal inhibitor to PLC, PKC, Ca2+ release, calmodulin, and MEK1/2 inhibited melanoma development. Although administration of a glutamate release inhibitor suppressed melanoma growth, these signal inhibitors only suppressed melanoma growth partially. These results indicate that for development of melanoma, activation of every signal is required, however, for the growth of melanoma, lack of several signals can be compensated by these downstream signals of mGluR1.

TYRP1 function in melanoma progression

A. Mogha1,2, A. Primot1,2, J. Debbache1,2, D. Bennett3, G. Ghanem4, B. Dreno5, M. D. Galibert1,2,6

1CNRS UMR 6061 Institut de Génétique et Développement de Rennes, Equipe RTO, France;
2Université de Rennes 1, IFR140 GFAS, Faculté de Médecine, 2 avenue du Pr Léon Bernard, CS, Rennes, France;
3St. Georges Hospital Medical School, Department of Basic Medical Sciences, London, UK;
4Institut Jules Bordet, Université Libre de Bruxelles Brussels, Belgium;
5Department of Dermato-Oncology Nantes University Hospital, INSERM, France;
6CHU Rennes, Departement de Génomique Médicale, Rennes, France

Melanoma is one of the most aggressive skin cancers arising from the malignant transformation of melanocytes or precursors. The incidence of this cancer has increased over the last 40 years in most European and US populations. While early stages of melanoma have an excellent prognosis following surgery, metastatic melanoma has a 5-year survival that does not exceed 15%. The pressure to understand the development of melanoma on a molecular level is thus increasing, as this is important for early diagnosis and for the identification of effective drug treatments.

In an attempt to identify biological markers and new therapeutical targets, we have screen gene expression level of about 60 metastatic melanoma tumour samples (lymph nodes and sub-cutaneous metastasis), focusing on Mitf 6a/b splicing forms, Tyr, TRYP1, Dct mRNA levels and others. Our interest for TYRP1 started with its intriguing expression profile.

TYRP1 implication in pigmentation has been proposed according to its high sequence homology with Tyrosinase and Dct, however its precise function remains unclear. We thus investigated TYRP1 role by inhibiting TYRP1 expression in melanoma (VGP, metastatic) derived cell lines. Using a gene silencing approach (shRNA) we could show that the absence of TYRP1 impacts cell proliferation rate and drives a G1/S cell cycle arrest. Level of TYRP1 affects also cell morphology modulating cell dendricity and the capacity of cells to migrate.

Taken together our findings highlight new functions for TYRP1 and suggest that the level of TYRP1 may participate in tumour progression. In this context, TYRP1 could possibly be considered as a promising drug-target.

The topical improvement of the marginal repigmentation pattern of vitiligo

Yvon Gauthier1, Laila Benzekri2

1Pigmentary Disorders outpatient Clinic, Department of Dermatology, Bordeaux, France;
2Mohamed V University Souissi Rabat, Morocco

Melanocytes in skin are present both in the basal layer of the epidermis and in hair follicles. For many authors, the perifollicular repigmentation pattern is the only mode of repigmentation often partial in hairy skin and absent in glabrous skin .Nevertheless it has been clinically observed on vitiligo patches a slight marginal repigmentation.It has been hypothesized that a defective melanocyte migration is responsible at least in part of this abortive marginal repigmentation. But the datas reported from experimental study (organotypic culture of vitiligo skin Le Poole 1994) and from clinical studies(Anbar 2008, Ahmad TJ 2008)indicate that there is no inherent migration defect responsible for impaired repigmentation from the edges In all these cases, under the influence of inflammatory agents, a significative epidermal melanocyte migration could occur from the edges. We hypothesized that this process requires changes in the microenvironment such as the widening of intercellular spaces. Unlike epidermal melanocytes, amelanotic hair follicles melanocytes can move easily in wide spaces with only few desmosomes to repopulate likely growing anagen hair follicles. In a preliminary study we have tried to study the influence of changes in microenvironment on the epidermal melanocytes migration from the edges both in guinea pig skin and on vitiligo skin submitted to local NB UVB irradiation. We have assessed the effects of EDTA and proteases on dissolving cellular attachment. The enlarging by many ways of the intercellular spaces seemed to facilitate the horizontal melanocyte migration from the edges. We suggested that the control of melanocytes movement into and along the basal layer of the epidermis is essential to improve repigmentation of vitiligo.

Flexible nanosomes enable siRNA activity in cultured primary melanocytes and siRNA penetration in the viable epidermis of ex vivo human skin after topical application

Barbara Geusens1, Mireille Van Gele1, Sien Braat1, Stefaan C. De Smedt2, Marc C. A. Stuart3, Tarl Prow4, Washington Sanchez4, Michael S. Roberts4,5, Niek N. Sanders6, Jo Lambert1

1Department of Dermatology, Ghent University Hospital, De Pintelaan, Ghent, Belgium;
2Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat, Ghent, Belgium;
3Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh, AG Groningen, The Netherlands;
4Therapeutics Research Centre, Southern Clinical Division, Department of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Australia;
5School of Pharmacy & Medical Sciences, University of South Australia, North Terrace, Adelaide, Australia;
6Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat, Merelbeke, Belgium

The extent to which nano-engineered systems cross intact human skin and can exert pharmacological effects in viable epidermis is controversial. The present research sought to develop a new lipid-based nanosome that enabled the effective delivery of siRNA into primary human melanocytes. Effective delivery was demonstrated by using a siRNA that can modulate pigment transport by specifically blocking the expression of a Myosin Va exon F containing isoform that is physiologically involved in melanosome transport in human melanocytes. The overall goal was to use topical nanotechnology delivery in developing a new therapeutic strategy for the treatment of (hyper) pigmentation. Our major finding is that an ultraflexible siRNA containing nanosome, prepared using DOTAP, cholesterol, sodium cholate and 30% of ethanol, did penetrate into the epidermis of freshly excised intact human skin and was able to enter into keratinocytes. The nanosomes, called Surfactant-Ethanol-Cholesterol-osomes (SECosomes) showed excellent size, surface charge, morphology, deformability, transfection efficiency, stability and skin penetration capacity after complexation with siRNA. Importantly, these nanosomes had ideal characteristics for siRNA encapsulation, in that the siRNA was stable for at least 4 weeks, they enabled transfection of in vitro cultured cells and were shown to transportsiRNA delivery through intact human skin where changes in keratinocytes were demonstrated. It is concluded that increasing flexibility in nanosomes greatly enhances their ability to cross the intact human epidermal membrane and to unload their payload into targeted epidermal cells.

Redox potential and expression of catalase and in grey and pigmented hair follicles

Melanie Giesen, Guido Fuhrmann

Henkel AG & Co. KGaA, Duesseldorf, Germany

Excessive exposure of melanocytes to oxidative stress accompanied with decreased expression of catalase has been proposed to be one reason for greying in human hair follicles. Belonging to the detoxifying system which is mandatory for cells with an aerobe metabolism, catalase neutralizes hydrogen peroxide by producing oxygen and water. Thus, catalase is one of the melanocyte's foremost lines of defense against the impact of reactive oxygen species. Additionally there is also evidence that decreased expression of catalase might be associated with an imbalanced anti-oxidant status. To evaluate the relation between gene expression of catalase and the anti-oxidant status of grey versus pigmented hair follicles we analyzed the cellular redox potential as well as the catalase gene expression of both hair follicle types in 10 volunteers. Therefore, electron spin resonance spectroscopy (ESR) has been used to evaluate the degradation of semi steady test radicals in plucked grey and pigmented hair roots. As the potential to decompose the introduced test radicals correlates with the redox potential of the challenged hair follicles the experiments should allow a conclusion regarding the ability of grey hair follicles to cope with reactive oxygen species. Our results demonstrate that in grey hair follicles of some volunteers decreased gene expression of catalase is actually accompanied by impaired redox potential in grey hair follicles. However, this could not be proven for all volunteers. Some of them showed no difference between grey and pigmented follicles, neither in the gene expression of catalase nor in the redox potential. Strong inter-individual differences between volunteers could be aetiological for these findings. Furthermore, as human hair follicles themselves act as individual, not synchronized mini-organs also intra-individual differences between several hair follicles of one individual imply a certain impact. To further elucidate the correlation between catalase gene expression and anti-oxidant status of grey versus pigmented hair follicles and their contribution to greying processes the additional evaluation of catalase activity is of interest.

SLC24A5 and cholesterol homeostasis – a route to human skin colour variation?

Rebecca S. Ginger, Tony Dadd, Martin R. Green, David Gunn, Fei-Ling Lim, Magda Sawicka, Stephen Wilson

Unilever Discover, Colworth Science Park, Sharnbrook, Bedfordshire, UK

Human skin colour is determined both by environmental exposure to UV light and through inherited genetic variation in just a handful of genes. Variation of a non-synonymous single nucleotide polymorphism (SNP; rs1426654) in one of these genes (SLC24A5) is associated with differences in constitutive skin colour in South Asians (1). The alternate alleles of this SNP are near fixation in African and Caucasian populations (2), with the SNP encoding the substitution of Alanine for Threonine at residue 111. The SLC24A5 gene product (NCKX5) is located at the trans-Golgi-network (TGN) of melanocytes and functions as a potassium-dependent sodium-calcium exchanger. When heterologously expressed, the Thr111 variant of NCKX5 confers significantly lower exchange activity than the Ala111 variant and this reduced exchanger function is postulated to mediate the lower melanogenic activity of lighter skinned individuals (3). We have used gene expression microarrays to assess the impact of siRNA-mediated knockdown of SLC24A5 on the transcriptome of cultured normal human melanocytes (NHM). Surprisingly, very few genes previously associated with melanogenesis were altered at the transcript level but did include MC1R, suggesting that SLC24A5 interacts with at least one well characterized melanogenic signaling pathway. Furthermore α-MSH treatment increased both SLC24A5 transcript and NCKX5 protein expression, whilst MITF knockdown decreased their expression. More surprisingly the expression of a number of cholesterol homeostatic genes was altered after SLC24A5 knockdown and the total cholesterol content of NHM was increased. Cholesterol has previously been identified as a potential melanogenic regulator (4,5) and our data imply that SLC24A5 could control natural variation in skin pigmentation by modulating intracellular sterol levels from its location in the melanocyte TGN.

1 Stokowski, R.P., Pant, P.V.K., Dadd, T., Fereday, A., Hinds, D.A., Jarman, C., Filsell, W., Ginger, R.S., Green, M.R., van der Ouderaa, F.J., and Cox, D.R. (2007). Am. J. Hum. Genet. 81, 1119–32.

2 International HapMap Consortium (2005). Nature 437, 1299–1320.

3 Ginger, R.S., Askew, S.E., Ogborne, R.M., Wilson, S., Ferdinando, D., Dadd, T., Smith, A.M., Kazi, S., Szerencsei, R.T., Winkfein, R.J., Schnetkamp, P.P., and Green, M.R. (2008). J. Biol. Chem. 283, 5486–5495.

4 Schallreuter, K.U., Hasse, S., Rokos, H., Chavan, B., Shalbaf, M., Spencer, J.D., Wood, J.M. (2009). Exp. Dermatol. 18(8), 680–688.

5 Hall, A.M, Krishnamoorthy, L., Orlow, S.J. (2004) Pigment Cell Res. 17(4), 396–406.

Intracellular sorting of the melanosomal G Protein-Coupled Receptor OA1 involves ubiquitination and ESCRT function

Francesca Giordano1,2, Sabrina Simoes1,2, Ciro Bonetti3, Enrico M. Surace3, Valeria Marigo4, Graça Raposo1,2

1Institut Curie, Centre de Recherche, Paris, France;
2Structure and Membrane Compartments CNRS, UMR, Paris, France;
3Telethon Institute of Genetics and Medicine, TIGEM, Naples, Italy;
4Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

OA1 (GPCR143), the protein product of the Ocular albinism type 1 gene, is a pigment-cell specific membrane glycoprotein that displays structural and functional features of G Protein-Coupled Receptors. On the basis of its sequence it defines a unique subfamily of GPCRs. The peculiarity of OA1 as GPCR is its localization to intracellular organelles namely lysosomes and melanosomes. We have previously shown that OA1 functions at early stages of melanosome biogenesis to maintain melanosome identity and composition.(1)OA1 function in melanogenesis is regulated by a tight balance between its targeting to melanosomes and its downregulation. One important mechanism controlling intracellular trafficking events within the endo-lysosomal system is protein modification by ubiquitination. Such ubiquitin modified proteins can be recognized by components of the Endosomal Sorting Complex Responsible for Transport (ESCRT) machinery for targeting to different compartments. Such components are highly expressed in melanocytic cells and have already been shown to control sorting of melanosomal proteins from endosomes to melanosomes.(2)To shed light on the mechanisms regulating the intracellular sorting of OA1 and in particular the involvement of ESCRT machinery and/or its ubiquitination we used a combination of morphological and biochemical methods together with inactivation approaches. Our ongoing studies show for the first time that OA1 can be ubiquitinated and that its sorting and degradation requires functional ESCRT components. We are currently investigating the nature of such ubiquitination and how this modification impacts on OA1 trafficking to melanosomes or lysosomes. Our study has important implications in the physiopathology of Ocular Albinism type 1. Moreover given the unique features of this GPCR our studies will open new avenues to understand the trafficking of these signaling receptors within the endomembrane system.

1 Giordano, F., Bonetti, C., Surace, E.M., Marigo, V., and Raposo, G. (2009). Hum Mol Genet. Dec 1;18(23), 4530–45.

2 Truschel, S.T., Simoes, S., Setty, S.R.G., Harper, D.C., Tenza, D., Thomas, P.C., Herman, K.E. Sackett, S.D., Cowan, D.C., Theos, A.C., Raposo, G., and Marks, M.S. (2009). Traffic. 10(9), 1318–1336.

Functional characterization of STX17 in Gray horse melanoma

Golovko Anna1, Hu Guo-Zhen1, Hjälm Göran1, Jiang Lin1, Seltenhammer Monika4, Sundström Elisabeth2, Ronne Hans1,2, Andersson Leif1,3

1Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden;
2Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden;
3Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden;
4Max Perutz Laboratories, University of Vienna, Vienna, Austria

Gray is a dominantly inherited coat colour found in a number of horse breeds. Gray horses are born coloured but with age gradually lose the hair pigmentation, while the skin pigmentation is usually maintained. This phenotype is strongly associated with a high incidence of melanoma and vitiligo. We have previously established that the Gray phenotype is caused by a duplication in intron 6 of Syntaxin 17 (STX17), that represents a cis-acting regulatory mutation. The mutation upregulates expression of both STX17 and the neighboring NR4A3 in Gray horse melanomas, implicating these genes in the establishment of the Gray phenotype. We found that STX17, a distinct member of the syntaxin family of trafficking proteins, encodes a previously uncharacterized truncated isoform, in addition to the full-length protein. This truncated protein lacks the N-terminal portion including a well-conserved SNARE domain, but retains the two-pass membrane region and carboxyterminal tail, the two characteristic features of STX17.

Here, we studied STX17 expression in melanocytes and melanoma from Gray and non-Gray horses. We found that both STX17 isoforms are overexpressed in Gray horse melanomas, with the full-length being the predominant form. Immunofluorescence studies showed a complete co-localization of the isoforms and their partial co-localization with the ER.

We have previously shown that STX17 long mediates the ERK signaling in Gray horse melanoma (Jiang et al, submitted). In order to identify the protein partners we conducted a yeast two-hybrid screen using the N- and C-terminal portions of STX17 and a cDNA library from a Gray horse melanoma cell line. We identified several interesting candidate interactors, one of which has a firm link to human melanoma development. We are currently investigating the role of this interaction in the ERK signaling and proliferation in Gray horse melanoma cells.

Metabolic parameters of B16 F10 murine melanoma cell lines with different Akt status treated with fenofibrate

Maja Grabacka

Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture, Krakow, Poland

Metabolic alterations are frequently observed in cancer cells and are regarded as a one of the hallmarks of neoplastic transformation. Cancer cells avidly consume glucose and glutamine in glycolysis and glutaminolysis, respectively. Enhanced aerobic glycolysis and reduced oxidative metabolism observed in cancer cells are known as the ‘Warburg effect’. Peroxisome Proliferator activated Receptors (PPARs) are the nuclear receptors that are known to control cellular metabolism and promote fatty acid oxidation and down regulate glucose and aminoacid consumption. Oncogenic proteins that are responsible for the transformation exert a strong impact on cellular metabolism. Akt (protein kinase B, PKB), which is frequently overactive in melanoma, is responsible for induction of glucose uptake and glycolysis as well as enhanced lipidogenesis. PPAR alpha counteracts Akt activity and promotes the metabolic switch from glycolysis to fatty acid oxidation and blocks fatty acid synthesis.

Parental B16 F10 murine melanoma cell line was used to develop the B16-Myr Akt cell line with overexpression of constitutively active (myristoylated) Akt, and B16-DN Akt cell line overexpressing a dominant negative mutant of Akt (K179M Akt). The metabolic characteristics and sensitivity to PPAR alpha ligand fenofibrate was studied in all these three cell lines. It was revealed that they show different glucose consumption and fatty acid oxidation intensity, as is demonstrated in the experiments assessing the glucose levels in cell culture medium and the activities of mitochondrial acyl-CoA dehydrogenase and glutaminase. Fatty acid oxidation is not a preferred source of energy for the proliferating cells such as cancer cells, as it is much slower process than glycolysis. Moreover, it requires the presence of functional mitochondria, which are often abnormal in cancer cells. Fenofibrate is a candidate drug to induce a metabolic crisis in melanoma cells by as it might be able to influence the glucose consumption and glutaminolysis with simultaneous enhancement of fatty acid oxidation, which is not a convenient energetic pathway for rapidly proliferating cells. A beneficial action of fenofibrate as an anti-cancer agent supplementary to the classical chemotherapeutic drugs is postulated.

Melanin content does not correlate with ultraviolet radiation induced erythema or cyclobutane pyrimidine dimer formation in untanned Caucasian skin.

Eugene Healy1, Samantha Robinson1, Kazumasa Wakamatsu2, Sandra Dixon1, Carol Gough3, Shosuke Ito2, Brian Diffey4, Peter Friedmann1

1Dermatopharmacology, University of Southampton, UK;
2Department of Chemistry, Fujita Health University School of Health Sciences, Japan;
3Wellcome Trust Clinical Research Facility, University of Southampton, UK;
4Department of Medical Physics, University of Newcastle upon Tyne, UK

The role of cutaneous melanin in photoprotection is well established, as documented by less ultraviolet radiation (UVR)-induced damage in Asian and Negroid skin and in animal models with significantly elevated levels of epidermal eumelanin. The incidence of skin cancer is higher in fair skinned individuals than in Caucasians who tan well, and based on the trend that Caucasians who tan well experience less sunburn, it may well be assumed that less UVR-induced damage occurs in those Caucasians with the greatest amounts of melanin in their skin. However, it is not clear whether an inverse relationship between levels of epidermal melanin and UVR damage exists in untanned Caucasian skin, something which is relevant to initial summertime UVR exposures before skin has acclimatised to repetitive UVR exposure.

In this study, we have investigated whether the levels of total melanin, eumelanin and phaeomelanin are inversely associated with the levels of erythema and cyclobutane pyrimidine dimers (cpd) in untanned Caucasian skin. Forty six individuals with skin types I–IV were irradiated on normal untanned buttock skin with incremental doses of UVB. At 24 h, erythema was measured and biopsies taken from unirradiated skin (46 cases) and from skin which had received 200 mJ/cm2 UVR (40 cases). Total melanin, eumelanin and phaeomelanin levels in unirradiated skin were quantified by HPLC for pyrrole-2,3,5-tricarboxylic acid (PTCA) and 4-amino-3-hydroxyphenylalanine (4-AHP) respectively. Erythema was quantified by an erythema index meter and the number of epidermal nuclei positive for cpd was determined by immunohistochemistry and image analysis.

There was no significant association between total melanin content, eumelanin or phaeomelanin levels and UV-induced erythema or cpds in untanned skin (r = 0.064, r = 0.075 and r = -0.141 for erythema (200 mJ/cm2) versus total melanin, eumelanin and phaeomelanin respectively and r = 0.029, r = 0.046 and r = -0.145 for cpd versus total melanin, eumelanin and phaeomelanin respectively). These results suggest that the inverse relationship between epidermal melanin and UVR-induced damage noted in darker pigmentary groups is not present in untanned Caucasian skin. These observations have implications for public health messages on protection against sun exposure for Caucasians and suggest that Caucasians who tan well should not be complacent about sun exposure, especially during the earlier stages of summertime sun exposure.

The vascular phenotype, vessel stabilization and maturation affect the resistance to anti-angiogenic therapy in malignant melanoma

Iris Helfrich1,2, Inka Scheffrahn3,Sönke Bartling4, Joachim Weis5, Verena von Felbert5, Mark Middleton6, Masahi Kato7, Süleyman Ergün3, Dirk Schadendorf2

1Joint Research Division of Vascular Biology, Medical Faculty Mannheim, University of Heidelberg, and German Cancer Research Center, Heidelberg, Germany;
2Department of Dermatology, University Hospital Essen, Germany;
3Institute of Anatomy, University Hospital Essen, Germany;
4Medical Physics in Radiology, German Cancer Research Center Heidelberg, Germany;
5Institute for Neuropathology, Medical Faculty, RWTH Aachen University, Germany;
6University of Oxford, Department of Medical Oncology, Churchill Hospital Oxford, UK;
7Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Matsumoto-cho, Kasugai-shi, Aichi, Japan

Clinical efficacy of angiogenesis inhibitors targeting the vascular endothelial growth factor signaling pathway is still limited to date. Not all cancer patients benefit from such anti-angiogenic therapies and some, who benefit initially, might develop resistance during the treatment or show some adverse effects. However, angiogenesis is not only dependent on endothelial cell invasion and proliferation, it also requires pericyte coverage of vascular sprouts for stabilization of vascular walls. We hypothesized that the level of vessel maturation is critically involved in the response to anti-angiogenic therapies. To test this hypothesis we evaluated the vascular network in spontaneously developing melanomas of MT/ret transgenic mice after using PTK787/ZK222584 for anti-VEGF therapy but also analyzed human melanoma metastases taken at clinical relapse in patients undergoing adjuvant treatment using bevacizumab. In summary, we were able to show in both experimental settings, MT/ret-transgenic melanoma and human melanoma metastases, taken after adjuvant treatment using bevacizumab, that tumor vessels, resistant to anti-VEGF therapy, are characterized by enhanced vessel diameter (P ≤ 0.001) and normalization of the vascular bed by coverage of mature pericytes, immunoreactivity for desmin, NG-2, PDGFR-β and the late stage maturity marker α-SMA in contrast to partly reduced pericyte coverage or lack of α-SMA-expressing pericytes on intratumoral microvessels vessels, sensitive to anti-VEGF therapy (P ≤ 0.001). Our findings emphasize that the level of mural cell differentiation and stabilization of the vascular wall significantly contribute to the response towards anti-angiogenic therapy in melanoma. This study may be useful to pave the way towards a more rational development of second generation anti-angiogenic combination therapies and provide for the first time a murine model to study this.

Human stress oxidative genes and genetic susceptibility to melanoma: a candidate gene approach using Sequenom platform

Maider Ibarrola-Villava1, María Peña-Chilet1, Cristina Gómez2, Beatriz Casado2, Jose A. Avilés3, Manuel Martin-Gonzalez4, Pablo Lázaro3, Rafael Lozoya5, Enrique Boldó5, Conrado Martinez-Cadenas5, Gloria Ribas1

1Fundación Investigación Clínico de Valencia, Valencia, Spain;
2La Paz Hospital, Madrid, Spain;
3Gregorio Marañón Hospital, Madrid, Spain;
4Ramon y Cajal Hospital, Madrid, Spain;
5Castellon Province Hospital, Castellon, Spain

Malignant melanoma (MM) incidence is currently increasing faster than that of any other malignancy in almost all Western countries. The key environmental risk factor is exposure to the ultraviolet (UV) component in sunlight, which causes various kinds of DNA damage, including bulky lesions and oxidative damage. UV radiation can indirectly cause oxidative damage via reactive oxygen species generated after the absorption of light energy by cellular chromophores. Furthermore, inflammatory response to UV exposure may further contribute to skin carcinogenesis by oxidative stress mechanisms. In addition, UV radiation can alter antioxidant and other related enzymes, and impair the antioxidant defense in the skin. Therefore, polymorphisms in oxidative stress genes might modulate cancer predisposition.

We present a case-control study including 640 Spanish MM patients and 340 control subjects. Phenotypic information was collected using a standardised questionnaire. All studied subjects gave informed consent. Functional (from coding and regulatory regions) SNPs with MAF < 0.5 were selected using HapMap database. Seventeen SNPs in 11 genes belonging to the oxidative stress pathway were finally selected. Thirteen have been successfully genotyped using the Sequenom platform (in two multiplexes of seven and six SNPs respectively). The four remaining assays failed and were designed by Taqman. Associations with melanoma and pigmentary characteristics such as hair, skin and eye colour will be discussed. These results will confirm the contribution of oxidative stress genes to genetic predisposition to MM in Spain.

Melanocyte response to gravitational stress: an update with a focus on guanylyl cyclase-cGMP signaling

Krassimira Ivanova, Peter Eiermann, Wasiliki Tsiockas, Ingrid Block, Jens Hauslage, Ruth Hemmersbach, Rupert Gerzer

German Aerospace Center, Institute of Aerospace Medicine, Cologne, Germany

Guanylyl cyclase (GC) catalyzes the synthesis of 3’,5’-cyclic guanosine monophosphate (cGMP), a second messenger with multiple functions. While the membrane-bound GC isoforms GC-A and GC-B serve as receptors for natriuretic peptides, which have been implicated in cancers, the soluble isoform of GC (sGC) is a key enzyme in nitric oxide (NO) signaling.

In our previous studies we have reported differential expression of GC isoforms in human melanocytic cells. Normal human melanocytes and low-metastatic melanoma cell lines predominantly express the NO-sensitive sGC isoform, which is associated with melanogenesis, whereas absence of soluble guanylyl cyclase, but the presence of membrane-bound guanylyl cyclase appears to correlate with the metastatic behaviour of melanoma cells. We have further shown, that human melanocytic cells differentially respond to gravitational stress. Hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured human melanocytes and low-metastatic melanoma cell lines, but not in metastatic phenotypes, which is related to an increase in the expression of the selective cyclic nucleotide exporter, the multidrug resistance proteins 4 and 5 (MRP4/5), but not MRP8 on mRNA and protein levels. Here we report that alterations of the MRP4/5 expressions in hypergravity are dependent on the presence of functional sGC, a heterodimeric protein consisting of α and β subunits, since no changes in the expression of mRNA for MRP4/5 were found in low-metastatic melanoma 1F6 cells transfected with siRNA for sGC-β1. In contrast, exposure of 1F6 cells to simulated microgravity (up to 1.21 × 10-2 g for 24 h) on a fast-rotating clinostat with one rotation axis resulted in reduced mRNA levels for MRP5 of approximately 35% in comparison to 1-g controls. More importantly, exposure to simulated microgravity for 6 h reduced the mRNA levels for GC-A of highly metastatic BLM melanoma cells by approximately 30% and of low metastatic 1F6 cells by about 18%, whereas exposure for 24 h attenuated the mRNA levels of both GC-A and GC-B at about 50%. The respective RNA levels of sGC α and β subunits were down-regulated by about 32 and 22% during 24-h exposure. Results of a life microscopy analysis indicate that cellular morphology was not altered at reduced gravity conditions.

Taken together, these results indicate that hypergravity and simulated microgravity inversely regulate the expression of components of the GC-cGMP pathway in melanocytic cells. Moreover, the down-regulation of the GC and of MRP4/5 expression in reduced gravity in comparison to terrestrial conditions may indicate attenuation of the metastatic potential of melanoma cells as well as of the drug resistance in weightlessness.

Identification of novel mutations affecting MC1R signalling

Ian J. Jackson, Peter Budd, Margaret Keighren, Eve Anderson, Sally Cross

MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, UK

Signaling through the G-protein coupled receptor MC1R results in increased intracellular cAMP which leads to upregulation of the synthesis of eumelanin. Absence of MC1R signaling, whether through mutation or antagonism of the receptor, leads to synthesis of phaeomelanin. In collaboration with MRC Mammalian Genetics Unit we have identified new, chemically induced, mouse mutations that affect the agouti pigmentation pattern of the coat. The phenotypes suggest that the balance of synthesis of eumelanin and phaeomelanin has been altered, and that the signaling through MC1R is changed.

The ginger mutation has a homozygous phenotype in which the phaeomelanic band on the dorsal hair is expanded, resulting in a more yellow appearance. We mapped ginger to chromosome 5, in the region of the Corin gene. Corin was initially identified as an atrial natriuteric peptide-converting enzyme highly expressed in the heart. Recently, however, it has been found to be expressed in dermal papilla and Corin knockout mice have an expanded phaeomelanic band. Using RT-PCR of Corin mRNA we find that in ginger mice there is a duplication of exon 4 which will result in frameshift of translation and premature termination. Corin appears to act upstream of MC1R, and is co-expressed with agouti protein, the endogenous antagonist of MC1R. We suggest that corin normally acts to limit the period of action of agouti and in its absence agouti function persists, resulting in an expanded yellow band.

Goth is a mutation with a dominant phenotype very similar to activating mutations of Mc1r. Heterozygotes are much darker than wild type littermates and have almost black hair on the back and dark-agouti flanks, suggesting enhanced MC1R signaling, which lowers the effectiveness of agouti antagonism. Mice homozygous for Goth die perinatally. We mapped the mutation to chromosome 2, and have identified a missense mutation in Gnas, which encodes the G-alphaS subunit, and which couples GPCRs to adenylate cyclase to increase cAMP synthesis. We propose that the mutation enhances the coupling of MC1R and so increases signaling. We have been unable to find evidence of increased signaling through other GPCRs.

Differential regulation of human melanocortin 1 receptor (MC1R) signalling through the cAMP and the mitogen-activated protein kinases ERK1 and ERK2

Cecilia Herraiz Serrano, Marta Abrisqueta, Jose C. Garcia-Borron, Celia Jimenez-Cervantes

Department of Biochemistry and Molecular Biology, University of Murcia, Spain

MC1R, a major determinant of skin phototype, is a G protein-coupled receptor (GPCR) that regulates pigment production in melanocytes. MC1R stimulation by α melanocyte stimulating hormone or related peptides termed melanocortins (MCs) triggers cAMP synthesis leading to activation of the rate-limiting melanogenic enzyme tyrosinase. MC signalling also activates the MAP kinase module leading to ERK1 and ERK2 phosphorylation. This pathway controls key cellular decisions such as proliferation or differentiation. We showed that in human melanocytic cells ERK activation by MCs is achieved by the cAMP-independent transactivation of the cKIT receptor, suggesting that coupling to the ERK and cAMP pathways involves different effectors and, possibly, different regulatory mechanisms. To prove this hypothesis, we considered several layers of MC1R regulation, including: (i) a genotypic level through the expression of variant alleles, (ii) protein-protein interactions modulating the association of MC1R with signalling partners and (iii) crosstalk between different pathways. We analyzed the impact of natural and artificial MC1R mutations on activation of the ERKs or cAMP production. Several mutants that failed to activate cAMP production were able to achieve efficient ERK activation, but we did not find natural mutants preserving functional coupling to cAMP but unable to activate the ERKs. This suggests less stringent conformational requirements for functional coupling to the ERK pathway and imbalanced signalling in mutant melanocytes. Overexpression of members of the GPCR desensitization machinery responsible for uncoupling MC1R from the cAMP pathway had no effect on MC-dependent ERK activation. Moreover, ERK activation was also resistant to mahogunin ring finger 1, a protein that inhibits MC1R signalling to the cAMP pathway by interfering with the coupling to Gs. Finally, we analyzed the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (WT for MC1R, NRAS and BRAF). ERK activation by constitutively active NRAS or BRAF mutants, and ERK inhibition with PD98059 or following ablation of cKIT expression with siRNA had little effect on MC-stimulated cAMP synthesis. Accordingly, MC1R signalling to the cAMP pathway is largely unaffected by ERK activity. Conversely, high cAMP levels efficiently blocked MC1R-dependent activation of the ERKs. Therefore, cAMP is a negative regulator of ERK signalling in human melanocytic cells rather than an activator of this pathway, as suggested by analogy with mouse melanocytes.

Impact of V600EBRAF mutation on melanoma cell proliferation and response to oxidative stress

Fabrice Journe, Mohammad Krayem, Murielle Wiedig, Renato Morandini, Ghanem Ghanem

LOCE, Institut Jules Bordet, ULB, Brussels, Belgium

The mutations of BRAF are described in about 50% of melanoma tumors with 90% of these mutations occurring at a single site, leading to the V600E substitution. However, the impact of V600EBRAF mutation on melanoma progression is unclear. In this study, we compared three cell lines without BRAF mutations (wild-type cells) (HBL, LND1, MM79) and three cell lines with the V600EBRAF mutation (MM32, MM43, MM74) with respect to cell proliferation and MAPK pathway activation. The V600E substitution in BRAF mimics phosphorylation within the activation segment of BRAF and results in the constitutive activation of the protein, leading to the subsequent constitutive activation of the MAPK pathway, which is a critical signaling pathway for cancer cell survival. Then, as expected, we observed that ERK1/2 phosphorylation level was significantly higher in the group of mutated cells. Surprisingly, the proliferation index (D3/D1 ratio) was significantly lower in the group of mutated cells as compared to the group of wild-type cells (1.6 versus 5.0, P = 0.01). In addition, in spite of MITF may be activated through a crosstalk with the MAPK pathway, leading to increased transcription of MITF-dependent genes, such as TYR, TYRP1 and DCT, we did not detect TYRP1 (mRNA/protein) in BRAF mutated cells, indicating a rather limited melanocyte differentiation. To explain these contradictory observations, a series of experiments was conducted: (i) we measured similar levels of apoptotic cells (annexin-positive cells) in both groups, indicating that the lower growth rate of mutated cells was rather due to a reduced proliferation and not to an increase in cell death; (ii) we observed that the dual specificity phosphatase DUSP6, a specific phosphatase of ERK, was higher expressed in mutated cells, suggesting a blockade of the activated MAPK signaling, and therefore an inhibition of cell proliferation; (iii) we induced phosphatase inhibition by oxidative stress using 10−4 M paraquat (superoxide anion generator) and found a 2.5-fold increase in pERK level after 30 min in wild-type cells, with a 5-fold increase in DUSP6 expression after 15 min, while it only weakly affected pERK and DUSP6 in mutated cells; (iv) we showed that paraquat stimulation did not impact cell proliferation in both cell types, indicating that DUSP6 could dramatically inhibited the transduction of the ERK signaling. In conclusion, in spite of an activating V600EBRAF mutation associated with a hyperphosphorylation of ERK, mutated melanoma cells have a lower proliferation index and no TYRP1 expression. Overexpression of DUSP6 could explain the unexpected consequences of V600EBRAF mutation on cell proliferation/differentiation, since the increase in ERK phosphorylation in wild-type cells following an oxidative stress had no effect on cell growth and was associated with DUSP6 upregulation. We hypothesize, therefore, that the overexpression of this phosphatase could be critical in the control of cell proliferation.

GENE expression profiling and RTqPCR validation in 133 melanoma metastases identify TYRP1 mRNA level as a new prognosis marker in high risk melanoma

Fabrice Journe1, Hichame Id Boufker1, Léon Van Kempen2, Marie-Dominique Galibert3, Murielle Wiedig1, Anne Theunis1, François Salès1, Denis Nonclercq4, Annica Frau4, Guy Laurent4, Ahmad Awada1, Ghanem Ghanem1

1Institut Jules Bordet, ULB, Brussels, Belgium;
2Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;
3University of Rennes 1, Rennes, France;
4Université de Mons, Mons, Belgium

Using microarrays, we generated a pigmentation signature comprising six genes overexpressed in melanoma metastases (skin and lymph node metastases, N = 32) of patients with poor survival [TYRP1 (34x), SILV (16x), DCT (13x), OCA2 (8x), TYR (6x) and MITF (3x)]. We calculated scores for the pigmentation signature (mean expression of genes, corrected to baseline) and for TYRP1 (log corrected values), and we found significant correlations between the signature and TYRP1 (rho = 0.801, P < 0.001), indicating that TYRP1 alone may reliably reflect the information brought forth by the signature. We also confirmed that TYRP1 score was significantly higher in the group of patients with poorer survival (P < 0.001), especially in skin metastases. Then, we evaluated TYRP1 mRNA expression in the skin metastasis biopsy subgroup by quantitative RTqPCR and confirmed the microarray data (rho = 0.780, P = 0.002). To validate the data, we evaluated TYRP1 mRNA expression (RTqPCR) in an independent group of skin metastases (N = 101). We used the 25th percentile as a cut off to divide the population into two groups with low and high TYRP1 mRNA levels, and found that high expression was significantly associated with a shorter DMFS (P = 0.01, HR = 0.49, 95% CI = 0.28–0.84, Kaplan-Meier analysis and Cox regression) as well as with a shorter OS (P = 0.007, HR = 0.48, 95% CI = 0.29–0.82). Moreover, we found that high TYRP1 mRNA expression had a positive predictive value of 94% associated with DMFS at 7.5 yr and with OS at 15 yr. We also evaluated the expression of TRP1 protein in a panel of skin metastasis paraffin-embedded biopsies (n = 52) by IHC and observed that, in many cases (56%), the protein was not detected while mRNA was expressed at high levels, suggesting possible posttranscriptional events. However, when only considering samples with positive immunological staining for TRP1, we found a significant correlation between protein (IHC data) and mRNA (RTqPCR data) expressions (rho = 0.488, P = 0.034). In addition, we found that TYRP1 mRNA expression significantly correlated with Breslow thickness and Clark level, confirming that TYRP1 mRNA expression in skin metastases, regardless the lag time to their occurrence, was associated with the main prognosis parameters defined in the corresponding primary lesions. Furthermore, we observed that TYRP1 mRNA levels persisted in successive metastasis biopsies obtained during the course of melanoma progression. Finally, we validated TYRP1 mRNA detection by ISH (n = 40) and found significant correlation between ISH data and RTqPCR data (rho = 0.679, P < 0.001). TYRP1/TRP1 mRNA/protein expression (ISH/IHC) is currently assessed in primaries with particular attention being paid to radial versus vertical invasion phases. To conclude, we consider that TYRP1 mRNA can be regarded as a prognosis marker, at least in skin metastases, and may also be helpful where information on the primary are lacking. Its expression is most probably conserved during tumor progression, suggesting that it could be a potential target for melanoma chemotherapy.

Tendencies of malignant melanoma in Japanese patients younger than 50 yr

Masako Kasami1, Teruki Kataoka2, Yoshio Kiyohara2, Shusuke Yoshikawa2, Dai Ogata2

1Pathology Department, Shizuoka Cancer Center Hospital and Research Institute, Nagaizumi, Shizuoka, Japan;
2Dermatology department, Shizuoka Cancer Center Hospital and Research Institute, Nagaizumi, Shizuoka, Japan

In Japan, the incidence rate of advanced phase of malignant melanoma is still high, and strategies for early detection of melanoma are necessary. We studied the clinical and pathological aspects of 225 patients treated by surgery, chemotherapy, immunotherapy and proton beam therapy in our hospital from 2002 to 2010. Malignant melanoma occurred on the skin;144 cases (range:6–93 years (y), average:59 y, rate of patients younger than 50 y (<50 y) :22%, female rate:49%), in nasal cavity; 27 cases (range: 49–85 y, average:70 y, <50 y:4%, female rate:63%), in digestive site; 16 cases (range: 8–79 y, average:62 y, <50 y:13%, female rate:44%), in ophthalmic site;14 cases (range: 37–85 y, average:68 y, <50 y:14%, female rate:64%), in oral cavity;13 cases(range: 35–89 y, average:66 y, <50 y:15%, female rate:23%), and in genital site;11 cases (range: 39–88 y, average:68 y, <50 y:9%, female rate:91%). In younger patients, cutaneous melanoma occurred most commonly. Mucosal melanoma occurred less commonly. As far details of the cutaneous melanoma, in younger patients, it had a higher rate in female patient (63% for younger patients: 48% for older patients), a higher rate of localization in trunk, arm and leg (75:34%), a lower rate of histologic type of acral lentiginous melanoma (9:46%), no differences in the 5-yr survival rate (68:64%) but a higher frequency of late recurrence (13:2%).More than 60% of patients (74% for younger:61% for older) had been aware of black pigment for more than 1 yr at the diagnosis of melanoma. More than 25% of patients had been aware of symptoms for more than 10 yr. We concluded that public information programs about malignant melanoma in Japan should include education for the predominant localizations. Trunk, arm and legs for younger people, especially for female, and hands and feet for older people should be examined in Japan.

A new spectrophotometric method for simple quantitation of melanosomal transfer from melanocytes to keratinocytes: the trans-membrane melanosomal transfer method

B. Kasraee, M. Patakey, D. Nicolick,D. Salomon, O. Sorg, J.-H. Saurat

Department of Dermatology, Geneva University Hospital, Geneva, Switzerland

Three major difficulties should be overcome for the reliable measurement of melanosomal transfer in vitro: (i) development of a three-dimentional melanocyte-keratinocyte co-culture reassuring a ‘direct’ cell to cell melanosomal transfer, (ii) reliable melanocyte keratinocyte separation after co-culture and (iii) sensitive and specific measurement of melanosomes in each cell population.

Methods: Murine B16 melanocytes and BDVII keratinocytes are cultured on the opposite sides of a porous membrane (Transwell membrane, pore diameter: 1 μm, pore density: 2.6 milion/cm2, membrane thickness: 11 μm). Cells remain in co-culture for three consecutive days. Simple trypsinization is sufficient to separate the two cell popultions. Melanosome quantity is measured in each cell population through an ELISA-like method using Pmel-17 as the detectable antigen.

Results: Electron microscopy, immunocytochemistry and flow cytometric analyses confirmed the direct melanocyte-keratinocyte contact through the pores and the transfer of melanosomes to keratinocytes without any passage of the cells to the opposite side of the membrane (>99% cell purity after trypsinization). The ELISA-like method proved to be very sensitive and specific for the evaluation of known melanosomal transfer inhibitors and inducers.

Conclusion: The trans-membrane melanosomal transfer method is a very simple spectrophotometric method for the accurate quantitation of melanosomal transfer.

The plumage colour DarkBrown in chicken is caused by an 8 kb deletion upstream of SOX10

Susanne Kerje1, Ulrika Gunnarsson1, Bertrand Hed’hom2, Anna-Stina Sahlqvist3, Olov Ekwall3, Michele Tixier-Boichard2, Olle Kämpe3, Leif Andersson1

1Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden;
2UMR Genetique et Diversite Animales, INRA/INA P-G, Centre de Recherhes de Jouy, Jouy-en-Jousas, France;
3Department of Medical Sciences, Uppsala University, Uppsala, Sweden

Dark brown is a colour mutant in chickens that reduces the expression of black eumelanin and enhances the expression of red pheomelanin in certain parts of the plumage. The Dark brown phenotype is segregating in a large intercross between the red junglefowl and the Obese Strain of White Leghorn and a genome scan using SNP markers was carried out to map the Dark brown locus to chicken chromosome 1 in agreement with a previous study. Identical-by-descent mapping including four different strains of chicken, all carrying the Dark brown allele, refined the localization to a 12.8 kb fragment upstream of the SOX10 transcription factor. SOX10 has a well-established role in melanocyte biology and is absolutely essential for melanocyte migration and survival.

The Dark brown haplotype was shown to involve an 8 kb deletion including a highly conserved non-coding sequence. Previous studies in mice indicate that this conserved element is a SOX10 enhancer. Re-sequencing of both Dark brown and wild-type chromosomes provided conclusive evidence that this deletion must be the causative mutation for the Dark brown phenotype. The mechanism of action of this mutation is being investigated by further experimental work and a plausible scenario is that loss of the SOX10 enhancer leads to reduced SOX10 expression in feather follicle melanocytes which in turn leads to down-regulation of the expression of key enzymes in pigment synthesis such as tyrosinase. Lower tyrosinase activity leads to a shift towards a more pheomelanistic (reddish) plumage colour, which is the characteristic feature of the Dark brown phenotype.

Alpha-MSH attenuates TNF-alpha-mediated NF-κB signaling and suppression of melanogenesis key enzymes in human melanocytes possibly by suppressors of cytokine signaling 1/3

Agatha Kokot, Markus Böhm, Thomas A. Luger

Department of Dermatology, University of Münster, Münster, Germany

Suppressors of cytokine signaling (SOCS) are a family of proteins which negatively regulate cytokine signaling. Expression of SOCS1/3 is induced by several cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta. These cytokines are typically produced by UVB irradiation of epidermal keratinocytes and suppress melanocyte melanogenesis and proliferation. Since UVB also induces alpha-melanocyte-stimulating hormone (alpha-MSH), which increases proliferation and melanogenesis we hypothesized that this peptide may act as a coordinator of the UVB-response of melanocytes to the above cytokines. As a first step towards this aim, we examined if alpha-MSH induces expression of SOCS1/3 expression in human melanocytes (NHM). α-MSH time- and dose-dependently increased mRNA expression of SOCS1/3 in NHM as well as of SOCS1 in murine B16 melanoma cells. This inductive effect of alpha-MSH on SOCS1 expression in NHM was confirmed by Western immunoblotting. Preincubation of the cells with actinomycin D revealed that the inductive effect of alpha-MSH on SOCS1/3 expression is mediated transcriptionally. Pathway inhibition studies with the PKA-inhibitor H89, the MEK-inhibitor PD98059, and the adenylate cyclase-blocker SQ22356 further indicated a cAMP-PKA and MAPK-MEK-dependent mechanism of the alpha-MSH-mediated induction of SOCS1/3 in NHM. In accordance with these findings, in silico promoter analysis disclosed several putative CREB binding sites within the promoters of both SOCS1 and SOCS3 genes. Interestingly, preincubation of NHM with alpha-MSH attenuated TNF-alpha-induced nuclear translocation of NF-κB/p65 in line with known molecular interference of SOCS1/3 with p65 and as well suppressed TNF-alpha-induced IL-6 and IL-8 mRNA expression. Moreover, preincubation with alpha-MSH antagonized the TNF-alpha-mediated suppression of melanogenesis key enzymes, such as tyrosinase and dopachrome tautomerase in NHM. Our findings show for the first time that alpha-MSH induces SOCS1/3 in melanocytes. Via induction of SOCS1/3, alpha-MSH may direct the behaviour of melanocytes exposed to cytokines after UVB exposure into a coordinated, unidirectional but protective response.

Solute Carrier Family 45 Member 2 (SLC45A2) of the guppy: molecular cloning, polymorphisms and fine mapping of genomic region

Verena A. Kottler, Margarete Hoffmann, Eva-Maria Willing, Detlef Weigel, Christine Dreyer

Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany

The pigment patterns of male guppies (Poecilia reticulata) are highly polymorphic and an outstanding example for ornamental variation within a species. In natural guppy populations, male pigmentation is primarily shaped by the delicate interplay between sexual and natural selection. Previously, sex-linked inheritance of an array of guppy colour traits could be shown, but the molecular and developmental processes underlying colouration in the guppy have only scarcely been explored. Here we investigate the gene structure and polymorphisms of slc45a2 (aim1), a pigmentation gene that we recently mapped to the sex chromosomes of the guppy. Mutations of this single copy gene are known to cause pigmentation defects in several vertebrate species. We first obtained a full-length slc45a2 cDNA sequence of the Cumaná guppy, encoding a predicted 12 transmembrane protein with a length of 579 aa. A putative truncated splice variant of slc45a2 was detected in the eyes of the guppy. Second, we compared the slc45a2 cDNA sequences of 10 different guppy laboratory strains, including three with significant pigmentation aberrations. Thereby, we identified 27 polymorphic sites in the open reading frame, including nine non-synonymous substitutions. However, none of these correlated with the observed ornamental aberrations. Third, three genomic BACs containing slc45a2 were subjected to Illumina paired-end sequencing. Short read assembly with Velvet revealed the slc45a2 exon-intron structure and a part of the presumptive promoter region. Although no sequenced genome of the guppy is available as reference, we identified nine adjacent genes of slc45a2. Comparison of the slc45a2 region between fish genomes revealed a syntenic block, which is inverted in medaka as compared to stickleback, with the guppy slc45a2 region being most similar to that of the stickleback. These results show that new DNA sequencing technologies allow ab initio assembly of genomic sequence in non-reference species and greatly facilitate the study of pigmentation genes in the guppy.

DHICA induces differentiation and cell protecting mechanisms in melanocytes and keratinocytes

D. Kovacs1, V. Maresca1, E. Flori1, N. Aspite1, L. Panzella2, A. Napolitano2, M. Picardo1, M. d’Ischia2

1Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute (IRCCS), Rome, Italy;
2Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Naples, Italy

Interest in colourless intermediates of melanocyte metabolism has traditionally been related to their role as pigment precursors, however several lines of evidence scattered in the literature suggest that these compounds may exert an antioxidant and protective function per se independent of melanin synthesis. As an extension of early studies showing that 5,6-dihydroxyndole (DHICA) exerts an inhibitory effect against lipid peroxidation and stimulates LPS-induced macrophage activation, we report herein the effects of this diffusible eumelanin intermediate on primary cultures of human melanocytes and keratinocytes. Data showed that DHICA inhibits cell proliferation and stimulates the expression and activities of superoxide dismutase and catalase. In melanocytes, DHICA caused a marked increase in cell dendricity, and melanin content, whereas in keratinocytes it induced the up-modulation of early and late differentiation markers. Notably, DHICA proved able to protect the cells against UV-induced death. In conclusion, these preliminary results demonstrate for the first time that DHICA inhibits cell proliferation, induces differentiation and promotes mechanisms of antioxidant defence both in melanocytes and keratinocytes. On this basis, we propose that DHICA is a diffusible endogenous inducer of differentiation for melanocytes themselves and the other cell populations of the skin, contributing to skin homeostasis and acting as protecting agent against UV-induced cell death.

Thyroid dysfunction in 585 Dutch patients with non-segmental vitiligo: a cohort study

Marije W. Kroon1,2, Bas S. Wind1,2, Ivo K. C. W. Joore1, Mariska A. C. Leloup1, Ludmila Nieuweboer-Krobotová1,2,3, Rosalie M. Luiten1,2, Jan D. Bos2, Albert Wolkerstorfer1,2, J. P. Wietze van der Veen1,2,3

1Netherlands Institute for Pigment Disorders (SNIP), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
2Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
3The Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands

Background and Objectives: Previous studies reported an association of non-segmental vitiligo with autoimmune thyroid disease (particularly hypothyroidism) and an increased prevalence of thyroid antibodies. Screening of thyroid function and for the presence of auto antibodies to thyroid antigens has been recommended. However, reports involving a large group of patients are scarce. Aim of this study was to investigate the prevalence of autoimmune thyroid disease and thyroid peroxidase (TPO) antibodies, in a large cohort of Dutch patients with non-segmental vitiligo.

Materials and Methods: We included patients with non-segmental vitiligo who were referred to our institute from January 2008 until September 2009. Thyroid function (TSH and Free T4) and TPO antibody titers were assessed in those patients who had no history of thyroid disease or recent thyroid screening (last 3 months). Furthermore, type, extent, duration, activity, family history and age of onset of vitiligo were determined.

Results: e included 585 patients. A total of 80 patients (13.7%) was screened before, of whom 46 (7.9%) had thyroid disease and 34 (5.8%) had no thyroid disease. In these patients, the assessment of thyroid function was not repeated. Hypothyroidism was detected in 44 patients (7.5%) (women: 5.9%; men: 1.5%) There was a clear female preponderance. Of these 44 patients, 40 of them were already diagnosed with thyroid dysfunction while four of them were newly diagnosed. High TPO antibody titers (>60 kU/l) were detected in 69 of 505 vitiligo patients (11.8%). In a regression model for abnormal TSH significant values were found for age (P < 0.01), surface of vitiligo lesions (P < 0.01) and positive family history of thyroid disease (P < 0.05).

Conclusions: The prevalence of clinical hypothyroidism is 7.5%. However, in our population, prevalence of thyroid disease in subjects never screened before is not higher than in the general population. Screening of all vitiligo patients for thyroid function and TPO antibodies may be unnecessary. Further analysis of different subgroups may justify screening in specific patient groups.

Autochthonous primary and metastatic melanomas in Hgf-Cdk4R24C mice evade T cell-mediated immune surveillance

Jennifer Landsberg, Evelyn Gaffal, Mira Cron, Judith Kohlmeyer, Thomas Tüting

Laboratory of Experimental Dermatology, Department of Dermatology and Allergology, University of Bonn, Germany

Genetically engineered mouse models offer new opportunities to investigate the role of cell-mediated immunity in the natural progression of melanoma in an immunocompetent host. Here we report that Hgf-Cdk4R24C mice spontaneously develop a spectrum of primary melanomas with high penetrance during their first year of life. Malignant transformation proceeds in a stepwise manner from multiple melanocytic nevi to single nodular melanomas and disseminated metastases in most mice. Migrating melanoma cells invade the draining lymph nodes without activating the immune system. Autochthonous primary tumors are destroyed following experimental introduction of immune surveillance using an adoptive lymphocyte transfer approach. However, some tumor cells are able to survive, evade immune cell control and recur both locally and systemically. Immune tolerance in recurring tumors may be supported by immunosuppressive Gr1+ myeloid cells. Taken together, our results demonstrate that primary and metastatic melanomas developing spontaneously in Hgf-Cdk4R24C mice effectively evade cellular immune surveillance.

Copy number variation in pigmentation loci TYR, TYRP1, DCT, MC1R AND SLC24A5

Saioa López1, Concepción de la Rúa1, Neskuts Izagirre1, Montserrat Hervella1, Lara Fontecha1, Isabel Smith2, Santos Alonso1

1UPV/EHU, Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Science and Technology, Barrio Sarriena, Leioa, Biscay, Spain;
2UPV/EHU, Department of Zoology and Animal Cell Biology, Faculty of Science and Technology, Barrio Sarriena, Leioa, Biscay, Spain

Besides SNPs, Copy Number Variants or CNVs contribute too to a large fraction of genetic diversity. Therefore, we hypothesized that CNVs in pigmentary loci could contribute to normal variation in skin pigmentation.Thus, in this study we pursued the detection of CNVs in every exon of a subset of genes related to human skin pigmentation: TYR, TYRP1, DCT, MC1R and SLC24A5, using the Multiplex Amplifiable Probe Hybridization technique (MAPH).

We analyzed a total of 99 DNA samples of unrelated individuals from five population groups (including North-Saharan Africans, Sub-Saharan Africans, Asians and Caucasians). Validation of the results was performed by qPCR, which confirmed the existence of a CNV in exon 1 of SLC24A5. At this point, we analyzed 100 new samples, the 50 most pigmented and the 50 less pigmented, from a total of 650 DNA samples of unrelated individuals living in the Basque Country whose skin pigmentation had been analyzed by reflectance spectrometry.

We found that four of the individuals analyzed shared this novel CNV. All variants detected were the result of a gain of one or more copies, but there was no association between the CNV and the degree of pigmentation. Thus, this rare structural variant (MAF <5%) has not an evident role in normal variation of skin pigmentation, contrary to other polymorphism previously identified in this gene (SNP rs1426654). Thus, CNV at the loci examined does not seem to be responsible for phenotypic differences in skin pigmentation.

Adhesion molecules in primary oral mucosal melanoma: study of integrins and immunoglobulins in a series of 35 cases

Silvia Vanessa Lourenço1,2, Ricardo Hsieh1,2, Sheyla Batista Bologna2, Marcilei E. Buim3, Fernando A. Soares1,3, Martin Sangueza4, Marcello Menta Simonsen Nico2

1Department of General Pathology, Dental School, University of São Paulo, Brazil;
2Department of Dermatology, Medical School, University of São Paulo, Brazil;
3Department of Pathology, A.C. Camargo Hospital, São Paulo, Brazil;
4Department of Pathology, Hospital Obrero, La Paz Bolivia

Primary melanomas of the oral mucosa are rare (with an incidence of 1.2 cases per 10 million per year) and reported to be more aggressive than their cutaneous counterpart. There are well-established factors participating in the pathogenesis of cutaneous melanomas, such as familial and environmental factors. However, the aetiology and pathogenesis of primary oral mucosal melanomas are poorly understood and no intraoral risk has been recognized to date. Studies have shown that cell adhesion molecules belonging to the integrin and immunoglobulin superfamilies have been implicated in tumour progression in cutaneous melanomas. Additionally, these adhesion molecules have been associated with metastatic phenotyes of melanomas, being regarded as a markers of poor prognosis. To investigate the status of adhesion molecules in primary oral mucosal melanomas, and their relationship with clinical-pathological parameters, we examined components of integrin and immunoglobulin superfamilies in a series of 35 primary oral melanomas organised in a tissue microarray (TMA). Patients′ ages ranged from 9 to 91 yr and the average age at time of diagnosis was 61 yr old. There were 18 males and 17 females and the main prevalence of melanomas were amongst whites (21 patients). The majority of the patients (71.42%) had palate commitment and invasive histopathological aspect (grade III) was observed in 80% of the specimens. Long distance metastasis was found in 60% of the cases. Increased expression of integrin beta 3 and immunoglobulin CD166 (ALCAM) was statistically associated with cases with extensive vascular invasion (P < 0.05). Lower expression of the immunoglobulin CD54 (ICAM) was associated with cases with extensive necrosis. Most cases with metastasis were negative for the immunoglobulin CD66 (CEACAM). The present study shows that, at least in part, adhesion molecules are implicated in the pathogenesis and progression of primary oral mucosal melanomas. This participation includes mechanisms of neoplastic cell invasion and metastasis.

Monobenzone induces autoimmune skin depigmentation by specific immunogenic melanocyte modulation

Rosalie M. Luiten1, Jasper G. van den Boorn1, Daisy I. Picavet1, Paul F. van Swieten3, Henk A. van Veen2, Debby Konijnenberg1, Peter A. van Veelen4, Toni van Capel2, Esther C. de Jong2, Eric A. Reits2, Jan Wouter Drijfhout4, Cornelis J. Melief4, Jan D. Bos1

1Department of Dermatology and The Netherlands Institute for Pigment Disorders, Academic Medical Center, University of Amsterdam, The Netherlands;
2Department of Cell Biology & Histology, Academic Medical Center, University of Amsterdam, The Netherlands;
3Bio-Organic Synthesis, Leiden Institute of Chemistry, University of Leiden, The Netherlands;
4Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, The Netherlands

The phenolic compound monobenzone induces progressive skin depigmentation that is clinically and histologically indistinguishable from vitiligo vulgaris. The exact mode of action by which monobenzone induces vitiligo has remained unknown. Monobenzone-induced depigmentation spreads to non-exposed sites, indicating a systemic response rather than only direct cytotoxicity. Here, we present a comprehensive study of how monobenzone increases the immunogenicity of melanocytes and melanoma cells, leading to autoimmunity and vitiligo. We showed that monobenzone inactivates tyrosinase enzyme, leading to decreased cellular melanin synthesis, as well as the formation of quinone metabolites that bind to cysteine residues in proteins, thereby forming quinone haptens to melanosomal proteins. Moreover, monobenzone-tyrosinase interaction induced increased levels of reactive oxygen species, and the release of tyrosinase- and MART-1-containing CD63+ exosomes from monobenzone-exposed pigmented cells. We also found that monobenzone induces melanosome autophagy, as evidenced by the generation of melanin-containing autophagosomes and autolysosomes upon monobenzone exposure. This autophagic degradation of the melanosome induced by monobenzone is a lysosomal degradation process leading to the targeting of tyrosinase to MHC class-II compartments. In addition, monobenzone also induces the ubiquitination of tyrosinase in exposed melanoma cells. Monobenzone-exposed pigmented cells activated dendritic cells (DC), thereby generating a signal for immune activation. Using autologous healthy donor DC-T cell stimulations, we demonstrated that monobenzone-exposed melanoma cells induced the activation of melanoma-reactive human CD8+ T cells, in contrast to unexposed melanoma cells. Importantly, 78% of the melanoma cell-reactive CD8+ T cell clones generated with monobenzone were found to recognize monobenzone-treated and –untreated melanoma cells equally well. Together, these data show that monobenzone, by its specific effects on the enzyme tyrosinase, induces potent CD8+ T cell immunity against auto-antigens expressed by melanocytes and melanoma cells. Monobenzone thereby presents a powerful new drug in the field of melanoma immunotherapy.

Effective melanoma immunotherapy in mice by the skin-depigmenting agent monobenzone and the adjuvants imiquimod and CpG

Rosalie M. Luiten1, Jasper G. van den Boorn1, Debby Konijnenberg1, Esther P. M. Tjin1, Daisy I. Picavet1, Nico J. Meeuwenoord2, Dmitri V. Filippov2, J. P. Wietze van der Veen1,4, Cornelis J. M. Melief3, Jan D. Bos1

1Department of Dermatology and The Netherlands Institute for Pigment Disorders, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
2Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands;
3Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands;
4Department of Dermatology, Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands

Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity.

We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16.F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation.

MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B- and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clinic.

Translational control mechanisms in the progression of cutaneous malignant melanoma: the role of eIF2alpha

Immacolata Maida1, Paola Zanna1, Marcella Arciuli1, Claudia Greco1, Stefania Guida2, Domenica Inverardi2, Nicoletta Cassano2, Raffaele Filotico2, Gino A. Vena2, Rosa Cicero1, Gabriella Guida1

1Dipartimento di Biochimica Medica, Biologia Medica e Fisica Medica Facoltà di Medicina e Chirurgia, Università degli Studi di Bari, Italy;
2Clinica Dermatologica II, Università degli Studi di Bari, Italy

Malignant transformation and tumorigenesis include a complex series of cellular and biomecular events which are not yet completely known. Emerging evidence supports the fundamental role of translational control mechanisms in the modulation of cell functions. Cell growth and proliferation depend on protein synthesis that is regulated, in part, by translational initiation factors. These factors transiently increase in normal cells in response to growth factors and are constitutively elevated in transformed cells. In particular, the eIF2 translation factor seems to be involved in the abnormal regulation of protein synthesis that leads to a tumorigenic phenotype. Recent works suggest the translation factor eIF2 to be involved in the pathogenesis and/or tumour progression in some kind of tumours such as bronchioloaveolar carcinoma, non-Hodgkin lymphomas, breast tumour, brain tumour, gastrointestinal carcinomas, melanoma. Moreover other data indicate that the pERK levels could influence the RAS/RAF/MAPK pathway modifying the cell proliferation parameters.

In this study we have analysed the phosphorylated eIF2-alpha and ERK levels in different melanoma cell lines derived from metastatic or primary cutaneous melanoma lesions, as well as in a line from the primary skin lesion of a patient with metastatic melanoma.

Our results show higher levels of phosphorylated eIF2alpha and ERK in the metastatic cell lines (including the line from the primary lesion in the patient with distant metastases) as compared to the primary melanoma cell lines. In such cell lines, the gene expression of proteins involved in cell cycle regulation (i.e. cyclin D1 and p53) was also examined.

The results of our study suggest the relationship between p-eIF2 alpha and tumoral progression in the malignant melanoma. Further analyses could allow to identify phosphorylated eIF2 as a possible prognostic marker.

Imaging heterogeneity in melanoma

Cerys Manning1, Sophie Pinner1,2, Erik Sahai1

1Cancer Research UK London Research Institute, Lincoln's Inn Fields, London, UK;
2Current address, Cancer Immunology and AIDS department, Dana-Farber Cancer Institute, Boston, USA

Melanoma is a highly aggressive and often metastatic disease. Metastasis is a multi-step process that begins with the acquisition of motility in the primary tumour. Intra-vital imaging of xenograft tumours can be used to explore the dynamics of cell motility and has revealed that cells move in two main ways in vivo; as single cells using either amoeboid or mesenchymal motility, or collectively as groups or chains of cells. Motility can be influenced by many factors including signals in the microenvironment, mutations in the cell and differentiation status of the cell. Here we show that in a genetically homogeneous xenograft melanoma model there is heterogeneity in cell behaviour. Less than 10% of melanoma cells are motile and only certain regions of the tumour contain motile cells. We have developed a method of imaging pigment of melanoma cells in vivo and have found that motile single cells have lower pigment levels. In addition Brn2 promoter activity is increased in single motile cells suggesting motile melanoma cells may be less differentiated. We are further extending this to look at differences in signalling between motile and non-motile cells using clonal melanoma reporter cell-lines. We will present data relating to Notch and TGF-β signalling in melanoma in vivo.

α-MSH-mediated MC1R stimulation induces PPAR-γ through a Phospholipase-C mediated pathway

V. Maresca, E. Flori, B. Bellei, N. Aspite, E. Camera, G. Cardinali, M. Picardo

Laboratory of Cutaneous Physiophathology and Integrated Center of Metabolomics Research, San Gallicano Dermatologic Institute, IRCCS Rome, Italy

The knowledge about the possible function of Peroxisome Proliferator Activated receptors (PPARs) in human skin is just beginning to emerge. In melanocytes PPAR-γ is expressed at detectable levels and is able to promote melanogenesis and differentiation in response to pharmacological agonists. These effects are also promoted by α-MSH treatment. On this basis we investigated whether PPAR-γ could act as an effector component of melanocyte differentiation, downstream MC1R activation. We evaluated whether or not PPAR-γ is activated, in melanocytes, in response to α-MSH-mediated stimulation. This treatment up-regulated PPAR-γ mRNA and promoted its transcriptional activity. In order to define the mechanism responsible for PPAR-γ activation in response to α-MSH exposure, we focused on the cAMP/PKA pathway, as a pivotal way, in mediating melanogenic effects downstream activated MC1R. Surprisingly, Forskolin up-regulated PPAR-γ at mRNA levels, but failed to promote its transcriptional activity. Since MC1R belongs to the family of seven-transmembrane G-protein-coupled receptors, we have investigated whether or not it could mediate its effects even through Phophoinositide-Phosphoplipase-C (PLC)-dependent pathway. Cells exposure to α-MSH, in combination with a PLC inhibitor, significantly abrogated the activation of PPAR-γ and the up-regulation of its mRNA levels promoted by α-MSH alone. Moreover, the involvement of PLC in the α-MSH mediated PPAR-γ activation was confirmed by stimulating cells with a specific PLC inductor. We obtained a direct evidence of phosphoinositide-PLC-dependent pathway activation, in response to α-MSH, by assessing a rapid generation of Inosytol triphosphate (IP3) and Diacylglycerol (DAG) as hydrolysis products of PLC activity. We also investigated the possible influence of PPAR-γ downstream α-MSH-dependent MC1R stimulation on melanogenesis, as a typical aspect of melanocyte differentiation. Stimulation of cells with α-MSH, after silencing PPARγ expression, significantly counteracted melanogenic response induced by α-MSH alone. This study delineates a new signalling axis from the α-MSH to its G-protein coupled receptor MC1R, through PLC, down to PPAR-γ. This signal axis, which is independent from the well characterised cAMP/PKA pathway, is also involved in the control of melanocyte differentiation. This study places the activation of PPAR-γ downstream a PLC-dependent lipid transduction pathway, which so far has not been investigated under MC1R.

Drug screening to overcome chemoresistance in metastatic melanoma

Ewan McNeil, Elizabeth Patton, David Melton

Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit, University of Edinburgh, Edinburgh, UK

Cutaneous melanoma is the sixth most commonly diagnosed cancer in the UK and the most common in young people aged 15–34 (data from 2006 excluding non-melanoma skin cancer). Furthermore, while less common than NMSC, malignant melanoma accounts for 4% of skin cancer cases and 74% of skin cancer related deaths. Early surgical removal of primary tumours is an effective treatment, but patients that go on to develop metastatic disease have a very poor prognosis (5 yr survival rate is only 5%) and there is still no effective therapy for metastatic melanoma.

Elevated expression of a number of DNA repair genes has been reported in primary melanomas that subsequently metastasised when compared to non-recurrent primary tumours. This increased expression could contribute to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic that is effective against a range of other cancers is cisplatin. Elevated levels of the DNA repair enzyme ERCC1, which is needed to remove cisplatin-induced DNA damage has been found to be an indicator of poor prognosis in ovarian and lung cancer.

To test our hypothesis that elevated ERCC1 levels account for an increased resistance to cisplatin in melanoma, a genetic deletion in mouse melanocytes was performed. As expected, the derivative cell line harbouring the Ercc1 deletion was 50 fold more sensitive to cisplatin than the Ercc1 proficient parent cell line. We therefore performed a drug screen to identify small molecule inhibitors that would overcome cisplatin resistance in A375 human melanoma cells mirroring that observed by genetic deletion in mouse melanocytes. Our findings demonstrate a novel approach and class of compounds to overcome chemoresistance in malignant melanoma.

A novel in vivo model for Braf function in the medaka fish (Oryzias latipes)

Luciana Menescal, Daniel Liedtke, Manfred Schartl

Physiological Chemistry I, Biocenter, University of Wuerzburg, Germany

In the medaka fish (Oryzias latipes) melanoma model the activity of the Xiphophorus melanoma receptor kinase (Xmrk), a homologue of the human Egfrb receptor altered by a constitutively activating mutation, is expressed in pigment cells and leads to pigment cell tumor formation. As Xmrk signals are transmitted through known cancer pathways of human melanomas, malignant transformation in medaka cells can be compared to the human situation. One active pathway downstream of Xmrk is the MAP kinase signaling pathway, including Braf. Braf is mutated in 80% of nevi and 60% of human melanoma. In melanoma, mutated Braf leads to increased cell proliferation and spread of tumor cells. Our work aims to reveal how tumor progression is influenced in vivo by Braf function. For that purpose medaka braf was identified and cloning of full length gene constructs was performed. Also, a constitutively activated version of braf was produced by site directed mutagenesis leading to substitution of a valine in position 614 to a glutamic acid. This mutation corresponds to the human ‘gain-of-function’ mutation braf V600E, which is the most common mutation found in melanomas. In order to test protein functionality in vitro, we transfected mouse Melan A cells with medaka wildtype braf variant or the constitutively activated medaka braf V614E. We observed that the medaka Braf V614E mutant is able to phosphorylate MAP kinase even in starving medium, thereby confirming its constitutive activity. Subsequently, both the wildtype and mutant coding sequences were cloned downstream of the medaka mitfa promoter which drives expression exclusively in pigment cells. These construct were injected into one cell stage medaka embryos in order to establish stable fish lines. Remarkably, in a large number of transiently expressing mitfa:braf V614E G0 fishes, abnormal eye development was observed and investigated in detail. These data provide insights into physiological and oncogenic Braf function in vivo.

Primary oral mucosal melanoma: study of the P16-CYCLIN D1-CDK4-pRB pathway

Marcello Menta Simonsen Nico1, Ricardo Hsieh1,2, Sheyla Batista Bologna1, Marcilei E. Buim3, Fernando A. Soares2,3, Martin Sangueza4, Silvia Vanessa Lourenço1,2

1Department of Dermatology, Medical School, University of São Paulo, Brazil;
2Department of General Pathology, Dental School, University of São Paulo, Brazil;
3Department of Pathology, A.C. Camargo Hospital, São Paulo, Brazil;
4Department of Pathology, Hospital Obrero, La Paz Bolivia

Primary melanomas of the oral mucosa are rare, accounting for only 0.5% of all oral malignancies and with an incidence of 1.2 cases per 10 million per year. According to literature, there are well-established factors participating in the development and evolution of cutaneous melanoma, such as familial and environmental factors. However, the aetiology and pathogenesis of oral mucosal melanomas are yet poorly understood. Recently, studies have postulated that DNA repair capacity may modify the risk of melanoma in the presence of other strong risk factors. A reduced efficiency if the cell-repairing mechanism is capable of explaining the increasing trends of melanoma incidence that is noticed since the mid-20th century. In this scenario, the Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A), is the major gene involved in melanoma pathogenesis and predisposition. It is located on chromosome 9p21 and encodes two proteins, p16CDKN2A (including exons 1α, and 3) and p14CDKN2A. Its alteration in cutaneous melanomas is regarded to UV-exposition. To assess the status of the p16CDKN2A pathway in primary oral mucosal melanomas pathogenesis, our study examined the key components of this G1 checkpoint mechanism by immunohistochemistry in a series of 35 primary oral melanomas organized in tissue microarray (TMA). Patients’ ages ranged from 9 to 91 yr and the average age at time of diagnosis was 61 yr old. There were 18 males (51.43%) and 17 (48.57%) females and the main prevalence of melanomas were amongst whites (21 patients or 75%) against non-whites (25%) (five Black/Afro-Brazilian, two Asian). The majority of the patients (71.42%) had palate commitment and invasive histopathological aspect (grade III) was observed in 80% of the specimens. Long distance metastasis was found in 60% of the cases. Our study showed downregulation of many proteins of this pathway (p16, CDK4 and pRB). Upregulation of cyclin D1 was associated with cases of extensive perineural infiltration. These results point to important alterations of the cell cycle mechanisms in primary oral mucosal melanomas, independent of the UV-exposition.

Progression of cutaneous human melanoma. No stress mediated by neural no synthase (nNOS), a potential accelerator

Frank L. Meyskens Jr, Sun Yang

Chao Family Comprehensive Cancer Center, University of California, Irvine, Orange, CA, USA

Our laboratory has been extensively involved in the study of abnormal redox status and redox-sensitive signalings such as APE/Ref-1, AP-1 and NF-κB in the past decade as a basis for the experimental therapeutics of melanoma. It has been well-documented that UV radiation exposure, especially sunburn at a young age, is particularly linked to melanoma incidence. As an important environmental carcinogen, UV radiation not only leads to ROS, but also activates iNOS in keratinocytes resulting in a large amount of nitric oxide (NO) in human skin. Utilizing a NO-donor DETA to mimic NO stress, long-term exposure of primary normal human melanocytes resulted in cellular over-growth with formation of foci in in vitro culture, indicating gain of vertical growth potential.

We also demonstrated that melanoma proliferation and invasion potential were significantly stimulated by DETA treatment, associated with induction of many proteins involved in cell growth (c-Jun, JunD), anti-apoptosis (Bcl-2, APE/Ref-1) and metastasis signalings (MMP-1, Snail). Both our in vitro cell culture (immunoblotting) and in vivo human biopsy (immunohistochemistry) studies demonstrated marked elevation of nNOS expression in melanoma. Induction of nNOS was evident with UVB radiation and bFGF incubation. Knockdown of nNOS with siRNA efficiently reduced the DETA-induced melanoma proliferation and invasion potential, with reduction of c-Jun, Bcl-2 and MMP-1. A novel synthesized nNOS inhibitor JI-11 was utilized to inhibit nNOS activity; our data showed that co-treatment of JI-11 (1 μM) significantly attenuated the alterations induced by UVB radiation and DETA treatments. Based on our studies, we propose that targeting nNOS with application of specific synthetic inhibitors to diminish NO stress represents an innovative and promising strategy for prevention of progression of human melanoma.

Long-term follow up of scenesse® (afamelanotide, an alpha-MSH analogue) in patients with erythropoietic protoporphyria (EPP)

Elisabeth I. Minder, Stephan Lautenschlager

Stadtspital Triemli, Central Laboratory and Dermatological Department, Zurich

Aims: Phototoxicity or intolerable pain of the skin following sunlight exposure is the main symptom of EPP, an inborn error of porphyrin metabolism. Afamelanotide, a first in class melanocortin-1-receptor agonist and analogue of alpha-MSH, induced skin melanization and showed beneficial effects on EPP symptoms in a preliminary study (Harms 2009). We administered afamelanotide long-term within a compassionate use program treating EPP patients, collected clinical and laboratory data and evaluated adverse effects. In addition, data on the patient's daily sun exposure, EPP-related phototoxic pain as well as melanin density were collected.

Methods: Fifteen EPP patients requested after completion of the placebo-controlled randomized trial continuous therapy with afamelanotide 16 mg dosing. During 2 yr, a controlled release implant was administered every 60 day. Patients were asked to record daily any adverse event, the time spent in sunlight and EPP-related pain level (11-point Lickert scale, 0 equals no pain, 10 intolerable pain). Melanin density measurements and laboratory safety controls were performed every second month and – at beginning and after 1.5 yr –a whole-body dermatological check was accomplished as well.

Results: Two hundred and sixty-six treatment months were covered by our observations. The mean duration of survey per patient was 532 days (median 634, range 91–711 days). An average of 8.9 doses per patient (median 9.5, range 3–12) was implanted. The longest duration of sunlight exposure within one day per patient ranged between 5 and 12.5 h (mean 8.3, median 7.25). 88% of the recorded days (mean, median 93, range 46–100) were pain-free. 10/15 patients never reported a pain score above 6 and none of the 15 ever had pain intensity above 8. Maximal melanin density (according to Dwyer) per patient ranged between 3.69 and 5.18. Slight nausea for 1–2 days after implant application was reported by seven patients, disappearing in 4/7 during prolonged treatment. Neither laboratory nor dermatological examinations revealed any clinically significant adverse effects.

Conclusion: During afamelanotide treatment patients tolerated sunlight for several hours a day, a phenomenon not reported as possible in the lifetime of these patients before starting treatment. Yet, they remained pain-free for most of the time and severe pain episodes were uncommon in them. Prolonged afamelanotide administration well tolerated by all 15 EPP patients.

Acknowledgement: SCENESSE®, afamelanotide, implant formulations were sponsored by Clinuvel Pharmaceuticals Limited, Melbourne Australia.

Early onset and highly penetrant melanoma in zebrafish derives from transformation of larval melanocytes

Marina Mione1, Cristina Santoriello1, Elisa Gennaro1, Viviana Anelli1, Martin Distel2, Amanda Kelly3, Adam Hurlstone3, Reinhard Köster2

1IFOM Foundation – FIRC (Italian Foundation for Cancer Research) Institute of Molecular Oncology, Milan, Italy;
2Helmholtz Zentrum München, Institute of Developmental Genetics, Ingolstädter Landstrasse, Neuherberg, Germany;
3University of Manchester, Faculty of Life Sciences, Oxford Road, Manchester, UK

The cells that are able to initiate and maintain melanoma development are still unknown. On one hand melanoblast stem cells could provide a source of renewable and drug-resistant tumor cells similar to what has been shown for many cancers of epithelial origin. On the other hand studies in a variety of animal models have shown that mature melanocytes undergoing oncogenic transformation are able to sustain melanoma development.

Here we use the transparent zebrafish to investigate this question. The zebrafish has the advantage of two temporally distinct populations of melanocytes and melanoblast stem cells for one of these populations have been identified. Moreover, genetic and chemical tools for precise ablation of different melanocyte populations are available. Using the combinatorial Gal4 –UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. kita-GFP-RAS larvae show a hyper-pigmentation phenotype at 3 days and start to develop melanoma at 3 weeks of life. The tumors reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show dependence on mitfa expression and do not require additional mutations in tumor suppressors, such as p53 or PTEN.

This indicates that the kita driver line is a robust tool for melanoma studies in fish and prompted us to investigate whether this was due to oncogene expression in melanocyte stem cells. By chemically ablating a population of ErbB3-dependent melanoblast stem cells that give rise to regenerating and adult melanocytes (Hultman et al., 2009) we found that hyperpigmentation and melanoma develop from ras transformed larval melanocytes, rather than from ErbB3+ stem cells.

These results suggest that transformation of melanoblast stem cells is not required for the development of a highly aggressive form of melanoma.

Presence versus absence of autoimmunity in non segmental vitiligo patients

Silvia Moretti, Meena Arunachalam, Roberta Colucci, Samantha Berti, Torello Lotti

Division of Dermatology, Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy

Nonsegmental vitiligo is commonly considered an autoimmune phenomenon, however the possible differences between autoimmune vitiligo versus non autoimmune vitiligo have not been clearly established. The objective of our study was to perform a comparison of non segmental vitiligo patients with autoimmune phenomena versus those without autoimmune aspects in regards to clinical characteristics and toxic/drug exposure.

One hundred and fifty non segmental vitiligo patients have been examined at a university based dermatology outpatient hospital (2nd dermatology unit). Medical assessment was performed by dermatologists using the modified Vitiligo European Task Force form and signs of autoimmunity were evaluated. Significant differences were found in disease duration, growth, signs of inflammation/pruritus, history of thyroid disease, family history of psoriasis, the quantity of cigarettes consumed in the past, the use of thyroid hormone, a long-term consummation of drugs, consummation of goitrogenic foods, which were all significantly increased in the autoimmune group. On the other hand, koebner phenomenon was the sole parameter significantly correlated with the non autoimmune group. In conclusion, the evaluation of non segmental vitiligo patients according to the presence versus the absence of autoimmunity allows us to make a new correlation of autoimmune patients with signs of inflammation/pruritus and moderate past smoking, and of non autoimmune patients with koebner phenomenon. In doing so, we can provide a more effective diagnostic evaluation of vitiligo patients.

An integrated approach to melanoblast behavior during embryonic development

Richard L. Mort1, Leonard Hay1, Kevin J. Painter2, Ian J. Jackson1

1MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, UK;
2Department of Mathematics, School of Mathematical and Computer Sciences, Heriot-Watt University, Edinburgh, UK

Melanoblasts, the embryonic precursors of melanocytes, are derived from the neural crest and migrate along the dorsolateral pathway. They populate the epidermis around embryonic day 12 (E12) and localise to embryonic hair follicles between E13.5 and E16.5. The partial failure of this process leads to white spotting phenotypes. Many factors may contribute to the complete colonisation of the embryonic skin including tissue growth, melanoblast motility, melanoblast proliferation and extracellular signaling factors. Integrating these factors into a testable hypothesis requires mathematical modeling. We labeled the melanoblast lineage by crossing transgenic mice that express Cre recombinase under the control of the tyrosinase promoter with ROSA26-EYFP reporter mice. We cultured embryonic skin in an ex vivo system and were able to perform live imaging of melanoblast migration in embryonic skin using confocal microscopy. Analysis of time lapse sequences reveals that melanoblasts are highly mobile and allows measurement of the dynamics of their migration. We have begun to construct mathematical models from this data to allow us to explain how individual cellular properties and tissue expansion can contribute to the net dispersal of the melanoblast population.

In vivo identification of solar radiation-responsive gene network: role of the p38 stress-dependent kinase

Nicolas Mouchet1,2,3, Henri Adamski4, Régis Bouvet5, Sébastien Corre1,2, Yann Courbebaisse3, Eric Watier6, Jean Mosser5, Christophe Chesné3, Marie-Dominique Galibert1,2,5

1CNRS UMR 6061 Institut de Génétique et Développement de Rennes, Equipe RTO, Rennes, France;
2Université de Rennes 1, IFR140 GFAS, Faculté de Médecine, Rennes, France;
3PROCLAIM, Saint Grégoire, France;
4CHU Rennes, Service de Dermatologie, Rennes, France;
5CHU Rennes, Laboratoire de Génomique Médicale, Plateforme Transcriptomique GenOuest, Rennes, France;
6CHU Rennes, Service de Chirurgie Plastique, Rennes, France

Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm2), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out 5 h after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects.

MicroRNA miR-196a controls melanoma-associated genes by regulating HOX-C8 expression

Daniel W. Mueller, Anja-Katrin Bosserhoff

Institute of Pathology, University of Regensburg Medical School, Franz-Josef-Strauss-Allee 11, Regensburg, Germany

Resulting from a screening for miRNAs differentially regulated in melanocytes and melanoma cells we found expression of miR-196a, beyond others, to be significantly down-regulated in malignant melanoma cell lines and tissue samples. As it was stated before that miR-196a might negatively regulate expression of the transcription factor HOX-C8, we analyzed HOX-C8 levels in NHEMs and melanoma cells and found a strong up-regulation of HOX-C8 expression in malignant melanoma cell lines and tissue samples compared to melanocytes. Several HOX-C8 target genes are known to be involved in processes like oncogenesis, cell adhesion, proliferation, and apoptosis. We, therefore, aimed to further investigate a potential ‘miR-196a → HOX-C8 → HOX-C8 target gene’ relationship.

Stabile transfection with a miR-196a expression plasmid led to strong down-regulation of HOX-C8 expression in melanoma cells. Luciferase assays using a reporter plasmid containing a fragment of the HOX-C8 3’UTR confirmed that regulation of HOX-C8 expression is mediated by direct interaction of miR-196a with a target-sequence in the HOX-C8 mRNA. Focusing on target genes of HOX-C8 which might play an important role in melanomagenesis, we identified three genes (cadherin-11, calponin-1, and osteopontin) which are up- or down-regulated, respectively, by the altered HOX-C8 expression in miR-196a expressing stabile cell clones and are thus indirectly regulated by microRNA miR-196a. As those target genes are closely related to important cellular mechanisms like cell adhesion, cytoskeleton remodeling, tumor formation, and invasive behavior of tumor cells, altered miR-196a expression exerts strong effects contributing to tumor cell transformation and formation and progression of malignant melanoma.

The Insall chamber: a novel assay for direct visualisation of cancer cell chemotaxis

Andrew Muinonen-Martin, Rob Jones, Robert Insall

Beatson Institute for Cancer Research, Glasgow, UK

Chemotaxis is the process by which the movement of cells is biased towards a concentration gradient of extracellular signals. Due to its involvement in fundamental biological processes it is conserved across species from amoebas to eukaryotic cells (1). Recently, it has emerged as an important mechanism in the field of cancer migration, invasion and metastasis (2). Many of the proteins that regulate cell motility are involved in chemotaxis and are also markers for metastasis and poor patient outcomes (3). Drugs targeting cancer cell invasion and metastasis have proven difficult to develop, so understanding chemotaxis may enable the prioritisation of drug targets, better drugs, appropriate patient selection and smarter early clinical study design (4).

Melanoma motility continues to be extensively researched and harnessing new imaging techniques in vivo is producing exciting new insights into the metastatic process (5). Research into the field of cancer chemotaxis is still in its infancy and investigative tools have limitations, having been developed for fast moving Dictyostelium cells. Here we describe an improved direct visualisation chamber and its use for measuring melanoma chemotaxis. The Insall chamber is a refined derivative of the Dunn chemotaxis chamber. Particular advantages include easy handling, gradients with defined directions and two different steepnesses in the same assay, and above all the ability to use normal thin cover slips, allowing the use of high numerical aperture oil immersion lenses. As with the Dunn chamber, gradients are maintained for at least 24–48 h, allowing slowly-moving cells to be tracked.

Chemotaxis assays can be divided into direct and indirect visualisation assays for which there are several pros and cons. The fundamental problem with indirect methods is their inability to distinguish chemotaxis from orthokinesis (6), whilst direct visualisation assays are often limited by low magnification, inability to perform immunofluorescence and a short assay period. We describe in detail the fabrication and methodology for the Insall chamber and the results of tests to characterise the gradient and validate our claims using a melanoma cell line.

1 Insall, R., and Andrew, N. (2007). Curr. Opin. Microbiol. 10, 578–581.

2 Condeelis, J., Singer, R.H., and Segall, J.E. (2005). Annu. Rev. Cell. Dev. Biol. 21, 695–718.

3 Stathopoulos, G.T. et al. (2008). Mol. Cancer Res. 6, 364–371.

4 Kedrin, D. et al. (2007). J. Mammary Gland Biol. Neoplasia 12, 143–152.

5 Pinner, S. et al. (2009). Cancer Res. 69, 7969–7977.

6 Rhodes, J.M. (1982). J. Immunol. Methods 49, 235–236.

Isoquinoline building blocks in red hair pheomelanin uncovered

Alessandra Napolitano, Giorgia Greco, Lucia Panzella, Marco d’Ischia

Department of Organic Chemistry and Biochemistry, University of Naples ‘Federico II’, Naples Italy

Investigation of the main structural units of pheomelanins is of critical importance for an understanding of the basic photophysical and photochemical properties of these pigments. At pH 7.4, natural and synthetic pheomelanins show absorption features that are not compatible with the exclusive involvement of benzothiazine structural units, as recently pointed out (1), but would be more consistent with a contribution from other components. One possible option is provided by 3-oxo-3,4-dihydrobenzothiazine and benzothiazole motifs, which are generated in the later stages of pheomelanogenesis in vitro (2). Whether other precursor units contribute to pheomelanin structural features and absorption properties remains an open issue. A clue comes from early work (3) on gallopheomelanin from red chicken feathers, reporting the identification by oxidative degradation of a series of pyridinecarboxylic acids, including 2-[2’-(4’,5’-dicarboxythiazolyl)]-3,4,6-pyridine-tricarboxylic acid (TPCA) suggesting the occurrence of isoquinoline units in the natural pigment, but this hypothesis has never been demonstrated, not was it known what structural components account for TPCA formation (4). Herein we report biomimetic studies showing that oxidation of 5-S-cysteinyldopa in the presence of Zn2+ ions leads to two unprecedented isomeric dimers featuring a thiazinedihydroisoquinoline linked to a benzothiazole unit. A plausible formation pathway for the dimers from 2H-1,4-benzothiazine-3-carboxylic acid, precursor also of 2,2’-bibenzothiazine dimers and trichochromes, was postulated based on mechanistic experiments. The dihydroisoquinoline compounds exhibit absorption characteristics similar to those of red hair pheomelanin, including a main band around 400 nm. On chemical degradation the dimers afforded all typical pheomelanin markers, including TPCA. The latter is also obtained in significant yields by degradation of natural and synthetic pheomelanins. This finding would support the long postulated existence of isoquinoline-containing structural motifs in pheomelanins and provides novel insights into the mechanisms of UVA-induced phototoxicity and photocarcinogenesis in red haired individuals.

1 Napolitano, A., De Lucia, M., Panzella, L., and d’Ischia, M. (2008). Photochem. Photobiol. 84, 593–599.

2 Wakamatsu, K., Ohtara, K., and Ito, S. (2009). Pigment Cell Melanoma Res.22, 474–486.

3 For a review see Thomson, R.H. (1974). Angew Chem Intl Ed.13, 305–312.

4 Simon, J.D., Peles, D., Wakamatsu, K., and Ito, S. (2009). Pigment Cell Melanoma Res.22, 563–579.

Role of oxidative stress in the pathogenesis of vitiligo and its relationship to PUVA therapy

Naglaa Nabil1, Randa Youssef1, Olfat Shaker2, Nahla Hunter1, Yasser El Asuoty3, Isabel Caroline le Poole4

1Department of Dermatology, Cairo University, Egypt;
2Department of Medical Biochemistry& Molecular Biology, Cairo University, Egypt;
3Department of Dermatology,BanySweif University, Cairo, Egypt;
4Department of Microbiology and Immunology, Loyola University Chicago, USA

Background: HO-1 is a marker of oxidative stress, it is cited as an etiological factor in progressive vitiligo and its up regulation is required to protect melanocytes from apoptosis in response to UVA. The aim of this work was to investigate the role of HO-1 and HO-2 mRNA gene expression in pathogenesis of vitiligo, and in response of vitiligo to PUVA therapy.

Subjects and Methods: Five groups were involved in this study: Twenty Egyptian vitiligo patients (group I), ten vitiligo patients of group I before starting PUVA (group II), ten healthy volunteers (group III), ten american vitiligo patients (group IV), and ten american neonatal foreskin (group V). Biopsies were taken from lesional skin in group I before and after PUVA therapy, from non lesional skin in groups II and IV and from normal skin in groups III and V. HO-1 mRNA was evaluated by RT-PCR technique. Immunostained cryosections and confocal microscopy were examined for HO-1 and HO-2 localization by melanocytes. Western blot was done to detect HO-1 and HO-2 expression in non lesional vitiligo melanocytes and control melanocytes foreskin.

Results: HO-1 mRNA was induced in 19 patients after PUVA treatment. Indirect immunoperoxidase, immunodouble-staining and confocal microscopy confirmed the results which revealed colocalization of HO-1 expression by melanocytes. Western blot also revealed upregulation of HO-1 after UVA.

Conclusion: Successful UV treatment of vitiligo patients requires high inducibility of HO-1 to protect the melanocytes from apoptosis, and it is the inducible HO-1 which provided photoimmune protection and not the constitutive HO-2.

Keywords: heme oxygenase-1; heme oxygenase -2; vitiligo; PUVA; melanocyt.

Mahogunin Ring Finger 1 (MGRN1) targeting to the cell nucleus

Ana B. Pérez Oliva, Celia Jimenez-Cervantes, Jose C. Garcia-Borron, Conchi Olivares

Department of Biochemistry and Molecular Biology, University of Murcia, Spain

The ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) interacts physically with the MC1R and inhibits melanocortin signalling. MGRN1 is mutated in mahoganoid, a mouse mutation causing coat colour darkening, heart defects and spongiform neurodegeneration. Melanocytes express 4 MGRN1 isoforms differing in the length of the C-terminal exon and in the usage of exon 12. All isoforms are located in the cytosol when expressed in heterologous cells, but isoforms containing exon 12 are translocated to the nucleus when co-transfected with wild type (WT) MC1R, suggesting that MGRN1 might interact with nuclear partners. We analyzed the specificity and mechanisms of MGRN1 translocation to the nucleus. Co-expression of MGRN1 with MCR family members, but not distantly related GPCRs, triggered cAMP-independent translocation, suggesting that nuclear targeting of MGRN1 might be specific to the MCRs. The subcellular location of several truncated constructs and point mutants demonstrated that the trafficking information is conveyed by a NLS located in exon 12. Residue Ser370 is centrally located in this NLS and has a high phosphorylation probability. We compared the behaviour of WT MGRN1 and two mutants: S370A, unable to undergo phosphorylation, and the phosphorylation-mimicking S370D. S370D was found in the cytoplasm, whereas S370A was located in the nucleus, in the presence or absence of MC1R. Moreover, the phosphatase inhibitor okadaic acid inhibited nuclear accumulation of WT MGRN1, whereas the PKC inhibitor Ro31-8425 promoted movement to the nucleus. We next compared the efficacy of several MC1R variants to determine nuclear localization of MGRN1. The major R151C and R160W RHC mutants were as effective as WT, but D294H was markedly impaired. D294H accumulates on the plasma membrane due to inefficient internalization, suggesting a potential relationship of internalization and MGRN1 translocation to the nucleus. In agreement with this, the ability of several other artificial MC1R mutants to promote MGRN1 translocation correlated with their internalization rates. Moreover, MGRN1 was targeted to the nucleus by β-arrestin 2, which promotes MC1R internalization, but not by β-arrestin 1, which does not. Finally, inhibition of internalization with sucrose abolished MGRN1 translocation. Overall, these data support a model of regulation of the subcellular location of MGRN1, whereby the critical parameter would be a phosphorylation status of residues in exon 12 NLS. This, in turn, would be modulated by MC1R-dependent interactions with the internalization machinery.

Effects of terrein and dihydropyranocoumarin D2 via different degradation mechanism

Kyoung-Chan Park1, Hye-Ryung Choi1, Dong-Seok Kim2

1Department of Dermatology, Seoul National University College of Medicine, Seoul, Korea;
2Department of Biochemistry, Chung-Ang University College of Medicine, Seoul, Korea

Tyrosinase is a critical rate-limiting enzyme in melanin synthesis. Tyrosinase is degraded endogenously, at least in part, by the ubiquitin protease system (UPS). It is known that a variety of intrinsic factors have the potency to increase tyrosinase degradation, e.g. TGF-β1, TNF-α and linoleic acid. Microphthalmia-associated transcription factor (MITF) is a transcription factor that regulates melanocyte pigmentation, proliferation and survival. It is well known that MITF is a major transcription factor that modulates tyrosinase transcription. Previosuly, we reported that a fungal metabolite, terrein, decreases melanin synthesis via down-regulation of MITF. Recently, we also found that terrein decreases melanogenesis through ubiquitination-dependent proteasomal degradation as well as via decreased production of tyrosinase.

Interestingly, we observed hypopigmenting effects of dihydropyranocoumarin D2, a decursin derivative, as a MITF-degrading agent compared to terrein, which act as a tyrosinase degrading agent. Furthermore, dihydropyranocoumarin D2 down-regulate MITF via both lysosomal and proteosomal degradation. Hypopigmenting effects of dihydropyranocoumarin D2 and terrein will be discussed in terms of differential mechanism on MITF and tyrosinase degradation.

Role of apoptosis and melanocytorrhagy: a comparative study of melanocytes adhesion in stable and unstable vitiligo

D. Parsad, R. Kumar, A. J. Kanwar

Department of Dermatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Apoptosis and melanocytorrhagy have been proposed as mechanisms of melanocyte disappearance although there are not many controlled studies.

We undertook this project to study melanocyte morphology and adhesion defects in patients with stable and unstable disease and to compare it with control. In this comparative study we included seven patients with stable disease and seven patients with unstable vitiligo. We cultured perilesional skin melanocyte from these patients which revealed some significant morphological changes along with adhesion defects in the unstable vitiligo melanocytes. Perinuclear region of unstable vitiligo patients melanocytes was bigger in size whereas in stable and control melanocytes perinuclear region was normal in size. More importantly the dendrites of unstable vitiligo patients melanocytes were small with clubbed ends. Unstable vitiligo melanocytes show significantly low adhesion to collagen type IV as compared to control and stable vitiligo melanocytes. After the treatment with okadiac acid the melanocytes from unstable vitiligo patients showed significantly more Annexin V staining as compared to the control and stable melanocytes. Our results revealed that caspase3 staining was significantly more in the unstable vitiligo melanocytes as compared to the control, whereas expression of caspase3 was almost same in stable and control melanocytes. In this study we demonstrated that melanocytes in the unstable vitiligo patients were in their detachment phase which ultimately leads to apoptosis of these cells whereas melanocytes cultured from stable vitiligo patients and control were morphologically normal without any adhesion defects. These morphological and adhesion findings support theory of melanocytorrhagy as the primary defect underlying melanocyte loss in unstable vitiligo.

Treatment of facial melasma with fractional CO2 laser. About three cases

Michel Pascal, F. Albala-Fiszenson, P. Tsianakas

Centre Laser Espace, St-Honoré, PARIS, France

Objective: Melasma is a pigmentary disorder which is resistant to many therapies. A lot of lasers (Qs ruby, Qs alexandrite, Qs Nd-Yag, Erbium or continous CO2) have been used for treating melasma.But most of them require significant downtime and cause side effects like postinflammatory hyperpigmentation. In 2005, FDA gave an agreement for using Fraxel to treat melasma. For this reason, we decided to study the effect of a new fractional CO2 on refractory melasma.

Methods: Three female patients (Fitzpatrick skin types IV) have been included in this open study. They were unresponsive to previous treatment (peeling, topical steroids and hydroquinone). For this protocol, we worked with the DERMATRIX (La Compagnie des Lasers) which is a fractional CO2 laser. Only one session has been performed and response was evaluated according to the percentage of lightening of original pigmentation. Tolerance, down time and side effects have been detailed 4 days after the session then each month. Two physicians evaluated the photographs and each patient was invited to fill a personal evaluation form. The duration of the follow up was 6 months.

Results: We obtained a great improvement of the three patients(60 à 70%) without postinflammatory pigmentation or recurrence in a delay of 6 months after the treatment.

Conclusion: Fractional CO2 laser provides advantages like short down time and real efficacy for the treatment of resistant melasmas but we need larger scale study to confirm these encouraging results.

Synthesis and biological activity of 3-o-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin on melanoma

Luís Sánchez-del-Campo1, Magali Saez-Ayala1, Juan Cabezas-Herrera2, Soledad Chazarra1, José Neptuno Rodríguez-López1

1Department of Biochemistry and Molecular Biology A, School of Biology, University of Murcia, Murcia, Spain;
2Research Unit of Clinical Analysis Service, University Hospital Virgen de la Arrixaca, Murcia, Spain

Despite presenting bioavailability problems, tea catechins have emerged as promising chemopreventive agents due to their observed efficacy in various animal models. To improve the stability and cellular absorption of tea polyphenols, we developed a new catechin-derived compound, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), which has shown significant antiproliferative activity against several cancer cell lines, especially melanoma. The presence of methoxy groups in its ester-bond gallyl moiety drastically decreased its antioxidant and prooxidant properties without affecting its cell-antiproliferative effects, and the data indicated that the 3-gallyl moiety was essential for its biological activity. As regards its action mechanism, we demonstrated that TMECG binds efficiently to human dihydrofolate reductase and downregulates folate cycle gene expression in melanoma cells. Disruption of the folate cycle by TMECG is a plausible explanation for its observed biological effects and suggests that, like other antifolate compounds, TMECG could be of clinical value in cancer therapy.

Grant Sponsors: Fundación Séneca, Región de Murcia; Grant number: 08595/PI/08 and Ministerio de Ciencia e Innovación, Gobierno de España; Grant number: SAF2009-12043-C02-01.

Notch targets in melanocytes

Bhushan Sarode1, Karine Schouwey1, Lionel Larue2, Véronique Delmas2, Freddy Radtke1, Friedrich Beermann1

1Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland;
2Developmental Genetics of Melanocytes, UMR CNRS, Institute Curie, Orsay, France

Cellular signaling mediated by Notch receptors results in coordinated regulation of cell growth, survival, and differentiation. It has been shown that both Notch1 and Notch2 receptors contribute to the maintenance of melanoblasts and melanocyte stem cells, and are essential for proper hair pigmentation. Deletion of Notch1 and Notch2 receptors results in dose dependent hair graying, due to an elimination of melanocytes and melanocyte stem cells. The expression of a NotchIC transgene rescued this deficiency, however, not the deletion of RbpJK, thus proving further evidence that Notch signaling acts via RbpJK transcription factor in pigment cells. The mechanism responsible for defective stem cell maintenance caused by Notch deletion is not known. The identification of targets in melanocytes thus might indicate why melanocyte stem cells disappear in the absence of Notch. For that purpose we will carry out Microarray experiments and isolate Notch deficient melanoblasts by FACS from E16.5 embryos, which will be used for Microarray. In addition, we have established primary mouse melanocytes from mice carrying conditional alleles of Notch1 and Notch2.These will be used for deletion of Notch receptors in vitro.

Melanoma inhibitory activity (MIA) acts as a mediator of oncogene-induced senescence in human melanocytes

Susanne Schiffner, Anja K Bosserhoff

University of Regensburg, Institute of Pathology, Regensburg, Germany

Melanoma inhibitory protein (MIA) is highly expressed and secreted by human melanoma cells and is associated with tumor progression and metastasis in vivo. Elevated MIA levels were also detected in most benign nevus cells but, in contrast, not in normal human epidermal melanocytes (NHEM). Oncogene-induced senescence (OIS) has been found as a mechanism to prevent the genesis of melanomas out of benign nevi which carry mutated BRaf(V600E). We observed that cultured primary NHEM show increasing MIA levels at higher passages of cell culture correlating with an increase in senescent cells but they do not carry the oncogenic BRaf(V600E) mutation. Therefore, we assumed that MIA as an oncogene is involved in the induction of OIS in melanocytes.

In human primary melanocytes cultured at low passages, MIA overexpression led to an increase of senescence-associated β-Galactosidase (SA-βGal) activity and to the expression of the cell cycle inhibitor p21/CIP. On the other hand, siRNA-mediated MIA knockdown in primary NHEM at high passages reduced the proportion of SA-βGal positive cells as well as p21/CIP mRNA levels. These cells further showed higher proliferation rates compared to the controls. We recently described a MIA deficient melanoma cell line, HMB2-MIA. We observed SA-βGal positive cells in the parental HMB2 cell line whereas no stained cells could be detected in the MIA negative clones. We further generated MIA deficient mice on the background of the Tg(Grm1)EPv transgenic melanoma mouse strain which was described to develop spontaneous melanomas with a short latency. All mice developed melanocytic lesions primarily at the tail, ear and perianal region which progressed to distinctive melanomas with 100% penetrance. Interestingly, EPv/MIA-/- animals showed an earlier onset of melanoma initiation compared to EPv/MIA wildtype mice. These data suggest a role of MIA in oncogene-induced senescence and in the early stage of melanocytic transformation.

NR4A nuclear receptors mediate normal and oncogenic signalling in melanocytic cells

Aaron G. Smith, Nicole Luk, Wen Lim, Kasturee Jagidar, Richard A. Newton, George E. O. Muscat, Richard A. Sturm

Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia
Email: a.smith@imb.uq.edu.au

NR4A genes encode a sub-family of the nuclear receptor family of transcriptional regulators. We have recently reported the rapid and transient induction of NR4A expression by Melanocortin-1 Receptor (MCIR) signalling in melanocytes. MCIR is a G-protein coupled receptor known to play a pivotal role in the regulation of pigmentation in mammals and recent studies have demonstrated its involvement in DNA repair responses in melanocytes. In the European population, MC1R has been found to be highly polymorphic with several functional variants associated with the phenotype of Red Hair Colour and fair skin (RHC), cutaneous UV sensitivity and increased risk of developing melanoma and non-melanoma skin cancer. Our data identify the NR4A receptor family as potential mediators of a MC1R co-ordinated DNA damage response to UV exposure in melanocytic cells. Notably, the induction of these genes is impaired in melanocytes that are homozygous for MC1R loss of function alleles, which may contribute to the susceptibility of these individuals to melanoma. While NR4A expression is tightly regulated in normal physiological contexts, examination of NR4A expression in melanoma cell lines reveal high levels of constitutive expression as a consequence of oncogenic MAPK signalling. Gene expression profiling of melanoma cells in which the NR4A1 and NR4A2 genes have been knocked-down using siRNA reveal targets involved in angiogenesis, invasion, metastasis and signalling. Current investigations aim to further understand the potential cyto-protective role of the NR4A genes in the context of melanocytic signalling and explore the implications of aberrant NR4A receptor expression in malignancy.

The Role of Chk1 in melanocytes in vivo

Joanne L. Smith, David A. Gillespie

CR-UK Beatson Laboratories, Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow

Checkpoint kinase 1 (Chk1) is an essential effector kinase in the cell cycle checkpoints of the DNA damage response pathway. These checkpoints are important in ensuring cells do not pass through mitosis in the presence of damaged and/or incompletely replicated DNA. This is important in allowing cells to maintain genome integrity.

We are investigating the role of Chk1 in melanocyte development in vivo. Chk1 deletion in mammals has been shown to be embryonic lethal at day E6.5 due to a peri-implantation defect. We have therefore utilised a conditional knock-out of the Chk1 allele in combination with a melanocyte specific Cre-recombinase in order to delete Chk1 specifically within the melanocyte lineage. Pigmented mice on a C57/Bl6 background which have been deleted for Chk1 do not show evidence for pigmentation in their skin, however still maintain pigmented eyes. In addition we have investigated Chk1 deletion in utero and have observed that in embryos harvested at day E13.5 melanocytes are absent. These observations suggest that Chk1 is essential for melanocyte development.

The effects of narrow band ultraviolet B at therapy dose on melanocytes proliferation and apoptosis and MC-1R expression

Song xiuzu, Xiang wenzhong, Lu liangjun, Xu aie

Department of Dermatology, The Third Hospital of Hangzhou, Hangzhou

Objective: To explore the mechanism of narrow band ultraviolet B(NB-UVB) on vitiligo repigmentation through investigating the effects of NB-UVB on melanocytes proliferation and apoptosis and melanin synthesis.

Methods: The immortal B10BR melanocytes were irradiated with NB-UVB at varying doses. The proliferation and apoptosis of melanocytes were detected by MTT method and flow cytometry. The content of melanin was determined by NaOH method. The expression of BCL-2mRNA was determined by RT-PCR method and the MC-1R expression of melanocytes was detected by immunocytochemistry method and western blot method.

Results: The proliferation and apoptosis of melanocytes had no obvious change after irradiation with NB-UVB at the dose of 400, 800, 1200 mJ/cm2. The melanin content was increased in a dose dependent manner after NB-UVB irradiation, the value of NB-UVB groups were 1.42, 1.78, 2.0 times of the control group. The mRNA expression of BCL-2 was increased after NB-UVB irradiation, the value of NB-UVB groups were 1.75, 2.32, 3.28 times of the control group. The MC-1R expression was also increased after NB-UVB irradiation, the value of NB-UVB groups were 1.68, 2.35, 3.01 times of the control group.

Conclusion:  NB-UVB irradiation at therapy doses have no effects on melanocytes apoptosis, moreover they can strengthen the anti-oxidant capability by up-regulating the BCL-2 and MC-1R expression and increase the melanin synthesis. This may help to elucidate the therapy mechanism of NB-UVB on vitiligo repigmentation.

Keywords: melanocyte; narrow band ultraviolet B; apoptosis.

Hsp70: on the track of tumour cell migration and invasion

Andrea Spachinger, Kata Juhasz, Alois Sonnleitner, Zsolt Balogi

Center for Advanced Bioanalysis GmbH, Linz, Austria

Cancer cells have been found to exhibit elevated cytoplasmic levels of Hsp70, a member of the heat shock protein family, which is known to exert cytoprotective as well as anti-apoptotic effects. Recent investigations propose a link between high Hsp70 expression and tumour invasion and metastasis. However, it still remains elusive how Hsp70 takes its effect in terms of cancer cell migration and invasion.

Our studies aim at understanding the contribution of Hsp70 to melanoma cell migration and invasion. A B16 tumour cell system which showed very low endogenous Hsp70 expression when unstressed (referred to as control cells) was the basis to test the effects of elevated Hsp70 production. In these cells upregulation could be mimicked by use of the inducible TET on/off system which introduced exogenous Hsp70 (‘Hsp70 off’) and further excess Hsp70 upon induction (‘Hsp70 on’).

Regarding tumour cell migration our work focused on tracking alterations due to varying Hsp70 levels and addition of chemical attractants. The impact of Hsp70 levels in resting cells was tracked by comparing the invasive potential of control cells, ‘Hsp70 off’ and ‘Hsp70 on’ cells. To reveal the influence of elevated Hsp70 levels under stress tumour cell invasion was monitored under heat shock. To better understand the invasive behaviour for different Hsp70 levels, we are tracking subcellular localization of Hsp70 and explore its potential role in signalling pathways.

New potencies of immortal postnatal neural crest-like stem cells

Elena V. Sviderskaya, Shabbir A. Kamali, Dorothy C. Bennett

Division of Basic Medical Sciences, St George's, University of London, UK

We have established a novel type of pluripotent neural crest-like stem cells (NCLSCs) from neonatal mouse epidermis (Sviderskaya et al. 2009, FASEB J). These cells were isolated as three independent immortal lines. These cells resemble neural crest stem cells in their capacity to differentiate into several cell types normally derived from the neural crest. Using alternative regulatory factors, they could be converted to large numbers of either melanoblasts and melanocytes, Schwann precursor cells, or functional sensory neurons showing voltage-gated sodium channels. We also showed that when the cells were treated with bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 2 (FGF 2) and ascorbic acid they produced chondrocytes. This was confirmed by Alcian blue staining. Here we investigated whether neural crest-like stem cells can generate smooth muscle cells, another mesenchymal cell type and product of normal neural crest cells in the head. We examined the effect of BMP-2, FGF2 and transforming growth factor β1 (TGFβ1) on these cells growing in DMEM with foetal calf serum (FCS). BMP-2, FGF2 and TGFβ1 significantly increased cell proliferation in 7 days. We identified smooth muscle cells in cultures of NCLSCs grown with or without gelatine, by a marked change in morphology, cell size and production of filaments immunoreactive for α-smooth muscle actin. About 6% of all cells showed positive staining. Thus our immortal NCLSCs can produce cells of two mesenchymal cell types, chondrocytes and smooth muscle cells, and can differentiate into at least five cell lineages. These lines provide a new and valuable model for large-scale studies of the biology and gene expression of neural crest stem cells and the regulation of lineage determination. Supported by the Wellcome Trust.

Irradiation-induced leukoderma, systemic anti-melanoma immunity and tumor-free survival in a patient with end stage melanoma

Hansje-Eva Teulings1, Esther Tjin1, Willemijn Dingemans2, Walter Vervenne3, E. Helen Kemp4, Jan D. Bos1, Wietze van der Veen1,5, Rosalie Luiten1

1Department of Dermatology, Academic Medical Center and the Netherlands Institute for Pigment Disorders, University of Amsterdam, The Netherlands;
2Department of Pathology, Academic Medical Center, University of Amsterdam, The Netherlands;
3Department of Internal Medicine, Deventer Ziekenhuis, Deventer, The Netherlands;
4Department of Human Metabolism, School of Medicine, University of Sheffield, UK;
5Department of Dermatology, Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands

We present a patient with end stage melanoma including brain metastasis with an exceptionally favorable disease course. The patient had been treated with chemo- and radiation therapy and never received immunotherapy. After radiotherapy, he developed vitiligo in the irradiated skin areas (face and axilla) and subsequently demonstrated disease free survival until now, a period of 18 months. Auto-immunity, including vitiligo is associated with prolonged survival in metastatic melanoma patients. We investigated whether a systemic anti-melanoma immune response could be found in this patient. We therefore analysed T-cells derived from the peripheral blood and T-cells cultured from perilesional vitiligo skin lesions for their melanocyte specificity. We found elevated levels of MART-1 specific T-cells in the blood. We also found increased levels of MART-1 and gp100 specific T-cells in the perilesional vitiligo skin. Additionally, we performed immunochemistry on the primary melanoma tissue and on a lymph node metastasis. Both tumor tissues were infiltrated with CD8+ cytotoxic T-cells and expressed the antigens Melan A (MART-1), HBM45 (gp100) and S100. An antibody response against both MART-1 and gp100 antigens was also found in this patient.

These data show the presence of anti-melanoma immunity in this patient which may have been involved in vitiligo development, protection against melanoma relapse and brain metastasis clearance. Considering that radiotherapy does generally not induce total regression of melanoma brain metastasis, it may have enhanced anti-melanoma/melanocyte immunity, after local destruction of melanocytes which was clinically observed by skin depigmentation. As far as we know, this is the first case described of a histologically proven melanoma brain metastasis regressing with subsequent tumor-free survival coinciding with an active anti-melanoma immune response.

Raf signalling is dispensable for early melanocyte lineage development but required for melanocyte stem cell self-maintenance

Agathe Valluet1, Sabine Druillennec1, Magalie Larcher1, Manuela Baccarini2, Lionel Larue1, Alain Eychène1

1Institut CURIE-Recherche, INSERM U1021-CNRS UMR3347, Centre Universitaire, Orsay, France;
2Center for Molecular Biology, University of Vienna, Max F. Perutz Laboratories, Vienna, Austria

BRAF and CRAF protein kinases have recently emerged as critical players in human cancers, especially in cutaneous melanoma. Although in vitro studies have shown that RAF proteins are regulated by several extracellular signals in normal melanocytes in culture, nothing is known about their putative role in the development and homeostasis of the melanocyte lineage in vivo. To address this question, we generated knockout (KO) mice in which ablation of both B-raf and C-raf genes is restricted to this lineage, upon tyrosinase promoter-driven Cre expression. Surprisingly, the double knockout animals displayed normal pigmentation at birth and did not show significant defect in survival, proliferation, migration and homing of melanoblasts in the developing hair follicles. Since these processes were previously demonstrated to require Kit and FGFR signalling, these results suggest that Raf signalling is dispensable downstream of these receptors during early melanocyte lineage development. However, following the first hair molting, 100% of the B-raf;C-raf KO animals unveiled a coat colour phenotype, characterized by a progressive hair graying, due to the replacement of black hair by unpigmented hair during successive hair cycles. Hair graying was proposed to be caused by defective self-maintenance of melanocyte stem cells (MSC) in the bulge of the hair follicle. Accordingly, analysis of B-raf;C-raf KO animals that have undergone several hair cycles revealed a complete disappearance of MSC in the bulge. Single B-raf or C-raf KO animals did not show this hair graying phenotype, indicating redundant functions for Raf proteins in this process. Finally, we successfully derived melanocytes cultures from wild-type animals, whereas melanoblasts/melanocytes from B-raf;C-raf KO mice did not grow in the presence of TPA, a potent mitogen requiring the ERK pathway.

Taken together, our results show that Raf signalling is required for proper MSC maintenance, but dispensable for early melanocyte lineage development. Interestingly, the emergence of the melanocytes that produce pigment in the first hair requires Kit signalling, whereas the presence of MSC in the bulge has been previously demonstrated to become independent of it. These observations reveal an unexpected uncoupling between Kit and Raf signalling during melanocyte lineage development.

Prognostic value and clinical significance of halo nevi with respect to vitiligo

Nanja van Geel, Sarah Vandenhaute, Ilse Mollet, Reinhart Speeckaert, Lieve Brochez, Jo Lambert

Department of Dermatology, Ghent University Hospital, Ghent, Belgium

Background: Vitiligo and halo nevi can be present together or separately from each other. Whether it concerns different entities remains unclear.

Objectives: To assess the prognostic value of halo nevi, both with respect to the future development of vitiligo, and to the clinical profile and course of disease in patients with vitiligo.

Methods: In total 291 patients were examined and questioned in this study, including patients with only halo nevi (group 1; n = 40), generalized vitiligo without halo nevi (group 2; n = 173) and generalized vitiligo with halo nevi (group 3; n = 78). The distribution and extent of the lesions were evaluated using clinical photographs of each patient.

Results: Between patients with only halo nevi (group 1) and generalized vitiligo patients (group 2 + 3), a significant difference in associated auto-immune diseases (P = 0.004), family history of vitiligo (P = 0.013) and presence of Koebner phenomenon (P < 0.001) was observed. Multiple halo nevi (>3) were significantly more observed (P = 0.002) in patients with halo nevi alone (group 1) compared to generalized vitiligo patients with halo nevi (group 3). In group 3, halo nevi were reported prior to the development of vitiligo in 52%, with a mean time interval between both of 33.7 ± 5.17 months. In patients with vitiligo, no significant correlation could be observed between the presence of halo nevi and the extent of lesions, activity status or subtype of vitiligo. However, the presence of halo nevi in patients with vitiligo reduces significantly the risk to have associated auto-immune diseases (P = 0.02), and the age of onset of vitiligo was significantly lower as compared to vitiligo patients without halo nevi (P < 0.001).

Conclusion: Our results support the hypothesis that halo nevi can exist as a separate entity. The risk to develop vitiligo in a patient with halo nevi is lower if halo nevi (in the absence of vitiligo) are present for more than 3 yrs, the total amount of lesions is more than three, the onset occurred during adolescence and in the absence of associated auto-immune diseases, a family history of vitiligo or a Koebner phenomenon. Studying the patterns above gives a guideline in answering patients’ questions with regard to prognosis. The occurrence of halo nevi in a subset of vitiligo patients points out to a possible different underlying etiopathological pathway.

Development of a 3D pigmented skin model to evaluate RNAI-induced pigmentation

Mireille Van Gele, Barbara Geusens, Peter Dynoodt, Reinhart Speeckaert, Jo Lambert

Department of Dermatology, Ghent University Hospital, Medical Research Building, De Pintelaan, Ghent, Belgium

As current skin whitening products often work insufficient or are known to cause side-effects, novel approaches for depigmentation are warranted. Silencing of genes by means of RNA interference (RNAi) has become a powerful tool for gene function analyses in vitro as well as in vivo. The skin represents an easily accessible tissue and holds great promise for topical application of novel RNAi-based therapies. In view of the development of novel treatments to reduce pigmentation we first established a human pigmented full-thickness skin model. Briefly, melanocytes were seeded together with keratinocytes on top of a fibroblast containing collagen matrix and exposed to air-liquid interface after 4 days of submersion into serum-free medium. The reconstructs were cultured further for up to 14 days in vitro. Histological examination of our models showed an accurate histological structure and a correct deposition of melanocytes into the basal layer was confirmed by performing immunocytochemical stainings with the melanocyte specific marker Melan A. In a second part of our study we used this model to specifically silence the expression of tyrosinase, the rate-limiting enzyme in melanogenesis. The transfection efficiency of Stealth™ siRNAs targeting tyrosinase and the duration of silencing was first validated in in vitro cultured primary human melanocytes. Thereafter, melanocytes transfected with the most efficient siRNA directed against tyrosinase were introduced into the model and compared to reconstructed skins containing siScrambled transfected melanocytes (controls). The degree of skin pigmentation was monitored by visual inspection and colorimetry. A significant reduction in pigmentation was observed at D14 with 50 nM siTyrosinase as compared to the controls. These results prove that siRNA-mediated gene silencing works successfully in our self-made 3D pigmented skin model. Other targets, involved in the transfer of melanin from melanocytes to keratinocytes, are currently knocked-down and their effect on pigmentation is tested in the model.

In conclusion, we established a novel reproducible human full-thickness skin model closely resembling the structure of native skin. The presented results show that these pigmented skin models are suitable models for functional testing of RNAi-induced depigmentation.Additionally, this stresses the importance of self-made human skin models as research tools in order to obtain RNAi-based ‘proof-of-principles’, which is a necessary step before evaluating the biological effect of topical applied siRNA-carriers onto skin in vitro or in vivo.

Reversion of cisplatin-induced apoptosis conferred by the function of organic anion transporting polypeptides (OATPs) in human melanoma cells

Patrick Verrando, Hayate Khalladi, Françoise Silvy

INSERM UMR911, Melanoma Research team University of the Méditerranée, Bd Jean Moulin MARSEILLE – France

Melanoma is very resistant to chemotherapeutic agents, a feature that may involve the efflux transporters of the ABC family. However, influx transporters such as those of the OATP/SLCO superfamily have not been considered in melanoma cells (MMC). We have previously reported for the first time that human MMC exhibit a differential expression of several OATPs according to cell aggressiveness. We report here that the transport of various OATP substrates in aggressive MMC lines triggers a common cell signal that leads to a reversion of the apoptosis induced by cisplatin (CisPt). A minimum time of presence of the substrates is required (15–24 h) to reverse by ~30–95% the cytotoxicity induced by 25 μM (EC50) CisPt during 24 h. Investigations aimed at decipher the molecular events that associate the OATP function transport and reversion of CisPt-induced apoptosis (monitored by cell viability, nucleus fragmentation, PARP and caspase cleavages), showed that OATP substrates cause a leak of the intracellular reduced gluthatione (GSHi) by 2–4 h. CisPt alone lowered GSHi only by 15 h, suggesting it was a consequence of apoptosis sequence. The OATP-dependent decrease of GSHi coincided with a modulation of activity of the ERK MAP-kinase. While our investigations are ongoing, they suggest that the transporter function of OATP causes a rapid, substrate-dependent, depletion of GSHi in MMC that is responsible for an ERK-mediated apoptotic resistance to CisPt. Conclusively, OATPs may play important roles in the resistance of MMC to chemotherapy and may open new perspectives for drug design against melanoma.

Testing in vivo the gene regulatory network underlying melanocyte differentiation

Laura Vibert, Masataka Nikkaido, Robert N. Kelsh

University of Bath, Centre for Regenerative Medicine, Bath

Multipotent cells of the neural crest have the potential to differentiate into an extensive range of derivative cell types. Understanding the factors driving fate specification of multipotent cells and maintaining the differentiated phenotype are key challenges in developmental biology. My project focuses on melanocyte differentiation in Zebrafish. Two main transcription factors, Sox10 and Mifta (microphthalmia-associated transcription factor), are already known to be involved in this process (Dutton et al. 2001; Elworthy et al. 2003; Lister et al. 1999). Fate specification of melanocytes depends upon Sox10, and on Wnt signalling, to mediate regulation of mitfa transcription. In contrast, the precise mechanism resulting in stable melanocyte differentiation remains unclear. In order to better define the genetic regulatory network (GRN) underlying melanocyte differentiation, I am testing different aspects of an initial GRN derived from a combination of our recent experimental data and mathematical modelling. Our in vivo data suggests that Sox10 forms part of a Feed-Forward Loop repressing melanocyte differentiation that is relieved by Mitfa-dependent repression of sox10. Our modelling predicts that a Factor Y is then required for maintenance of Mitfa expression. Here I describe our progress in refining our model and in identifying Factor Y. We are exploring the role of Wnt signalling in melanocyte differentiation, as a candidate Factor Y. Wnt signalling has been shown to regulate mitfa expression during fate specification in both mouse and zebrafish (e.g. Dorsky et al. 2000). That Wnt regulation might also contribute to maintenance of mitfa expression in differentiated cells is indicated by studies in mouse (Bellei 2008). To test the hypothesis that the Wnt signalling pathway might be a limiting factor regulating mitfa expression in differentiated melanocytes, we have assessed the effects of GSK3b inhibitor treatment of zebrafish embryos. Our data to date suggests that Wnt signalling is not limiting for melanisation, but that it may well affect cell shape. We will report our attempts to develop further assays to confirm and extend these initial results. These data will then be used to refine our understanding of zebrafish melanocyte differentiation in vivo.

Differential roles of the pRb and p53 pathways in naevo and melanoma genesis

Graeme Walker1, Blake Ferguson1, Herlina Handoko1, H. Konrad Muller2, H. Peter Soyer3

1Skin Carcinogenesis Laboratory, Queensland Institute of Medical Research, Herston, Qld, Australia;
2School of Medicine, University of Tasmania, Hobart, Tasmania, Australia;
3University of Queensland Clinical Cancer Centre, Herston, Qld, Australia

Using transgenic mouse models of melanoma we have performed a systematic analysis of genotype-specific melanocyte responses to UVR, along with the corresponding studies of melanoma age-of-onset and the comparative histopathology of tumours. We found that deregulation of either the pRb (by Cdk4 activation) or p53 (by Arf or p53 inactivation) pathway cooperates with NrasQ61K to destabilize and transform melanocytes. Melanomas were overtly similar in appearance but showed marked differences in histopathology between genotypes. Arf/p53 loss significantly decreased the migratory response to melanocytes to neonatal UVR although the animals developed highly proliferative melanomas with very early age-of-onset. Thus p53 is important in the proliferative ‘stress’ response of normal melanocytes, but in melanoma cells it acts to limit proliferation. Interestingly, mice carrying activated Cdk4 along with NrasQ61K developed dermal melanocyte proliferations reminiscent of naevi. Many melanomas in these mice emanated from these benign precursors. However animals carrying the Arf or p53 defects did not develop ‘naevi’, with all lesions skipping the benign precursor stage in progression. We did not observe differences in UVR-induced DNA damage repair that would help explain the potentially different tumorigenic mechanisms. Instead, differences in cell cycle dysregulation appear to dictate whether lesions develop de novo or via a naevus precursor.

Our data suggest that a functioning p53 stress response pathway is important for proliferation of melanocytes after UVR, and for naevogenesis. Notably, in these animal models the degree to which melanocytes proliferate in response to UVR-induced damage does not in itself correlate with age-of-onset of melanoma. Furthermore, without a functional p53 checkpoint melanocytes skip the naevus phase and progress rapidly to overt malignancy. A similar effect of p53 loss in driving tumour progression without a benign precursor stage has been observed in other cancer types. In contrast, activated Cdk4 drives the formation of naevus-like lesions in mice. These results underline the critical role of cell cycle regulation in controlling naevo and melanoma genesis. How lesions emanate either from naevi, or apparently de novo, is an important clinical question in dermatology, and our murine models may help shed light on these processes.

Ultrastructural and MITF-transcriptional alterations of epidermal melanocytes in lesional, perilesional, and normal vitiligo skin

Ping Wang, Weisong Hong, Li Zhang, Junhui Zheng, Aie Xu

Department of Dermatology, the Third Hospital of Hangzhou, Hangzhou, Zhejiang, China

Objective: To analyze the microphthalmia-associated transcription factor (MITF) transcriptional alterations of epidermal melanocytes in lesional, perilesional, and normal vitiligo skin.

Methods: Suction epidermal sheets were taken from lesional, perilesional, and normal skin respectively from patients of 12 vitiligo vulgaris (VV) and eight segmental vitiligo (SV) between 3–300 months duration. Transmission electron microscopic studies were performed on 10 vitiligo patients (six VV and four SV). MITF and its transcriptional molecules tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), and tyrosinase-related protein-2 (TYRP1) of cultured melanocytes from normal vitiligo skin were detected by western blotting.

Results: In Ultrastructural study, epidermal melanocytes in lesional skin were not found in seven of 10 vitiligo patients, whereas melanocytes with decreased or absent melanin (melanosome) could accidently be seen in other three VV patients with short and long standing lesions; in perilesional skin, abnormal ultrastructure of melanocytes could be found in three of six VV with duration <15 months, and only in one of four SV; all epidermal melanocytes of 10 patients were normal in healthy skin. In VV patients, the depigmentation may be associated with MITF and its transcriptional molecules TYR, TYRP1, and TYRP2. In SV patients, MITF was significant decreased whereas TYR, TYRP1, and TYRP2 were almost normal expression.

Conclusions: There may exist different pathogenesis between VV and SV.

A melanoma specific function of beta-catenin

Claudia Wellbrock, Imanol Arozarena, Daniel Gilby, Helen Bischof

Manchester Cancer Research Centre, Wellcome Trust Centre for Cell Matrix Research, FLS, University of Manchester, Manchester, UK

With the increasing amount of data on cancer genomes and transcriptomes it is becoming more and more evident that different cancer types are genetically distinct entities. Therefore it is crucial for targeted cancer therapies not only to identify but also to functionally dissect tumour-specific genetic alterations. Today several genes are know to be deregulated with high preference in particular cancer types such BRAF in melanoma. Another example is nuclear beta-catenin as a result of mutation or deregulated WNT signalling in colorectal cancer, where this is correlated with reduced overall survival, and where nuclear beta-catenin serves as prognostic marker. However, in melanoma the role of nuclear beta-catenin is still debated with data suggesting that it contributes to malignant transformation contradicting other findings of loss of nuclear beta-catenin during tumour progression. Moreover, more recent studies using automated quantitative analysis (AQUA) found that nuclear beta-catenin is a marker for good prognosis with reduced expression in metastases and that increased total beta-catenin can be assigned to a low risk patient group.

In order to study the function of beta-catenin in human melanoma we performed a comparative analysis of a set of melanoma cell lines expressing high levels of nuclear beta-catenin with melanoma cells expressing low and predominantly membrane and cytoplasmic beta-catenin. Our findings reveal that in contrast to other cancers such as colon cancer, in melanoma high expression of nuclear beta-catenin is indicative for a non-invasive phenotype, thus explaining its correlation with good prognosis in this disease. Moreover, a comparison between melanoma and colon cancer reveals that a cancer-type specific gene expression and regulation determines the tumorigenic potential of beta-catenin. Thus our data also demonstrate the importance of considering the epigenetic background of a particular cancer type in order to correctly interpret prognostic tumour markers.

Whitton pigmentary disorders (poster presentation)

Interventions for vitiligo, Maxine Whitton1, Mariona Pinart2, Jonathan Batchelor3, Claire Lushey4, Jo Leonardi-Bee5, Urbá González2

1Cochrane Skin Group, University of Nottingham, Nottingham, UK;
2Department of Dermatology, Research Unit for Evidence-based Dermatology, Hospital Plató, Barcelona, Spain;
3Department of Dermatology, Addenbrooke's Hospital, Cambridge, UK;
4Centre of Evidence Based Dermatology, University of Nottingham, Nottingham, UK;
5Division of Epidemiology and Public Health, University of Nottingham, Nottingham, UK

Around 1% of the world's population has vitiligo, a disease which causes white patches on the skin. Although many treatments are available, most of them are unsatisfactory.

This review updates the one published in 2006. The objective of the review was to assess all interventions used to manage vitiligo reported in randomised controlled trials (RCTs).

We updated searches of the Cochrane Skin Group Specialised Register, the Cochrane Central Register of Controlled Trials in The Cochrane Library (Issue 4, 2009), MEDLINE, EMBASE, AMED, PsycINFO, LILACS and ongoing trials databases. At least two review authors independently assessed study eligibility and methodological quality, and carried out data extraction. Because of heterogeneity only two of the included studies could be combined for meta-analysis.

We assessed 57 trials, including 19 from the original review, with 3139 participants. We analysed data from 28 studies which met our outcome criteria – >75% repigmentation, and effect on quality of life. Most of the RCTs, which covered a wide range of interventions, had fewer than 50 participants. All of the studies assessed repigmentation, six measured cessation of spread, and five investigated the effect of treatment on quality of life. Most of the studies assessed combination therapies which generally reported better results. Topical preparations, in particular corticosteroids, reported most adverse effects. None of the studies demonstrated long-term benefits. We found one study of psychological interventions.

This review has found some evidence from individual studies to support existing therapies for vitiligo, but different designs and outcome measurements and a lack of quality of life measures limit the usefulness of the findings. There is a need for follow-up studies to assess permanence of repigmentation as well as high quality randomised trials using standardised measures and which also address quality of life.

The role of hypoxia in melanoma phenotype switching

Daniel S. Widmer, Ossia M. Eichhoff, Marie C. Zipser, Phil Cheng, Reinhard Dummer, Keith S. Hoek

Department of Dermatology University Hospital of Zürich, Switzerland

Our group reported a model for melanoma progression which involves melanoma cell switching between phenotypes of invasion and proliferation. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by effecting the shift from a proliferative to an invasive phenotype. In Initial experiments microarray analyses were performed on proliferative phenotype melanoma cells that were exposed to hypoxic conditions in vitro. These experiments showed an up-regulation of invasive phenotype-specific genes as well as a down-regulation of proliferative phenotype-specific gene expression including important melanocytic markers. In vitro invasion assays were performed to show that a hypoxic environment increased invasiveness in proliferative melanoma cell cultures. Importantly, invasiveness was retained after these cells were returned to normoxia for 48 h. Furthermore, extended periods of hypoxia increase the invasiveness proliferative melanoma cells in a ‘dose dependent’ fashion. In contrast, invasive phenotype melanoma cells show no increase in invasive potential upon exposure to hypoxia. Additionally, we stained Clark's level IV primary human cutaneous melanoma biopsies for key markers concerning melanocytic function, hypoxia, proliferation and vascularization. Hypoxic regions revealed a loss of melanocytic markers, supporting our in vitro data. These results suggest that exposure to a hypoxic microenvironment leads to the reprogramming of proliferative phenotype melanoma cells, down-regulating melanocytic marker expression and increasing their invasive potential.

Punch graft testing in vitiligo; effects of uva, nb-uvb and 632.8 nm hene laser on the outcome

Bas S. Wind1,2, Marije W. Kroon1,2, Johan F. Beek1,2,3, J. P. Wietze van der Veen1,2,4, Ludmila Nieuweboer-Krobotová1,2,4, Jan D. Bos2, Albert Wolkerstorfer1,2

1Netherlands Institute for Pigment Disorders (SNIP), University of Amsterdam, The Netherlands;
2Department of Dermatology, University of Amsterdam, The Netherlands;
3Laser Centre, Academic Medical Centre, University of Amsterdam, The Netherlands;
4The Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands

Background and Objectives: In therapy-resistant non-progressive vitiligo, autologous punch grafting is widely used, as it is relatively inexpensive, easy to perform, and successful. Post-operative irradiation (sunlight, UVA, NB-UVB) has been suggested to improve pigment outgrowth after punch grafting, but is unpractical and probably promotes photoageing and photocarcinogenesis. Recently, the Helium-Neon (He-Ne) laser (632.8 nm) was introduced as a safe potential inductor of repigmentation in vitiligo. Aim of this study was to evaluate pigment outgrowth of punch grafts after irradiation with UVA, NB-UVB and He-Ne laser as compared to no phototherapy in patients with non-segmental vitiligo.

Materials and Methods: In a randomized controlled observer-blinded study, six patients were included with stable non-segmental vitiligo. In each of four 2 × 2 cm areas located on the torso or upper extremities, four 1.5 mm (1.8 mm2) punch grafts were placed. These four areas were randomly allocated to NB-UVB, UVA, HeNe laser and no phototherapy. Phototherapy was given twice a week during 3 months. Directly after the last treatment and at 3 and 6 months, pigment spread was assessed by a blinded physician.

Results: At the start of the phototherapy each patient had a total of 16 pigmented grafts (1.8 mm2) in four depigmented test regions. After 24 phototherapy sessions we noticed that in two patients the majority of punch grafts survived, whereas in the other four patients the majority of punch grafts depigmented, irrespective of phototherapy. Moreover, all six patients had stable vitiligo for at least 1 yr before entering the study and during follow-up. There was no apparent difference in pigment outgrowth between UVA, NB-UVB, He-Ne laser and no phototherapy.

Conclusions: These data suggest that intrinsic patient-related factors in the grafted area seem to determine outgrowth of pigment, while the phototherapeutic modalities have minor to no effect. Anamnestically stable vitiligo does not predict successful transplantation and a punch graft-test is probably essential to predict successful outcome of punch grafting in non-segmental vitiligo.