Paola Zanna

Melanogenesis in visceral tissues of Salmo salar. A link between immunity and pigment production

Melanogenesis is mostly studied in melanocytes and melanoma cells, but much less is known about other pigment cell systems. Liver, spleen, kidney, and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes. In teleosts, melanin-containing cells occur in the reticulo-endothelial system and are mainly in the kidney and spleen. The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature adversely affects the quality of fish products and is a problem for the aquaculture industry. With the aim to reveal novel functions and behaviour of the salmonid extracutaneous pigment system, we investigated aspects of the melanogenic systems in the tissues of Atlantic salmon, as well as in SHK-1 cells, which is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney. We demonstrate that a melanogenic system is present in SHK-1 cells, head-kidney, and spleen tissues. As teleosts lack lymph nodes and Peyers patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs. The detection of tyrosinase activity in lymphoid organs indicates that a link exists between the extracutaneous pigmentary system and the immune system in salmon.

Sporadic melanoma in South-Eastern Italy: the impact of melanocortin 1 receptor (MC1R) polymorphism analysis in low-risk people and report of three novel variants.

Environmental and genetic risk factors are involved in the development of melanoma. The role of the melanocortin 1 receptor (MC1R) gene has been investigated and differences according to geographic areas have been described. To evaluate the role of some clinical and genetic risk factors in melanoma development, we performed a case–control study involving 101 melanoma patients and 103 controls coming from South-Eastern Italy (Puglia), after achieving informed consent. We confirmed the role of known clinical risk factors for melanoma. Furthermore, 42 MC1R polymorphisms were observed. Three of these variants (L26V, H232L, D294Y) were not previously reported in the literature. Their predicted impact on receptor function was evaluated using bioinformatic tools. We report an overall frequency of MC1R variants in our population higher than in Northern or Central Italy. The most common polymorphism found was V60L, that has been recently reported to spread among South Mediterranean population. This variant influenced phenotypic characteristics of our population while it did not impinge on melanoma risk. An increased risk of melanoma was associated with two or more MC1R variants, when at least one was RHC, compared to people carrying the MC1R consensus sequence or a single MC1R polymorphism. Interestingly, we observed an increased risk of melanoma in subjects with darker skin and lower nevus count, usually considered at low risk, when carrying MC1R polymorphisms.

Oral malignant melanoma: immunopathological analysis of a multiphasic case

Mucosal melanoma is a rare malignant neoplasm, more aggressive than the cutaneous counterpart. The first sign is usually a mass lesion that may display uniform pigmentation (black or brown) or a variegated discoloration (from black, brown to grey, purple and red), or may be depigmented. We report a case of oral malignant melanoma in a white man characterized by an unusual clinical appearance. A 65-year-old white man presented with an unusual lesion in the mouth. On physical examination, a heterogeneously pigmented lesion involving the soft and hard palates and the maxillary alveolar crest was seen (Fig. 1). The lesion had a nodular, nonpigmented, ulcerated central region (AM), measuring about 20 mm on its maximum diameter, surrounded by a nodular, ulcerated, pigmented area (UPM) and by other flat and not ulcerated pigmented lesions (PM), with an area resembling dysplastic naevi (DN). The patient did not smoke and had no history of trauma or chronic inflammation. He underwent hemimaxillectomy. The surgical sample was fixed in 10% buffered formalin, embedded in paraffin wax, cut into slices 5 lm thick, and stained with haematoxylin and eosin. The histology results confirmed the diagnosis of melanoma. Immunohistochemical studies were performed using an avidin–streptavidin method with anti-N-cadherin, E-cadherin and b-catenin antibodies, and using diaminobenzidine (DAB) as the chromogen. Distinct multiphasic melanoma areas showing differential expression of b-catenins, E-cadherins and N-cadherins were found (Fig. 2). Intracytoplasmic b-catenin immunoreactivity was detected in the PM, whereas the UPM and AM were negative and weak membranous positivity was found in the surrounding normal tissue. DN-like areas of melanoma were positive for b-catenins in the melanoma cells located in the deep papillary dermis. The DN-like areas also had very weak staining for E-cadherin immunoreactivity in scattered melanoma cells. Both the PM and UPM ⁄ AM areas also had weak staining for E-cadherin positivity, despite consistently strong staining of the surrounding epithelial cells. The PM areas had intense and membranous E-cadherin immunoreactivity. N-cadherin immunoreactivity was strong in the melanoma cells of the UPM ⁄ AM areas, while it was less intense in the PM areas and absent in the DN areas. In the UPM ⁄ AM areas, the surrounding epithelial cells did not have N-cadherin immunoreactivity. The patient experienced recurrence of the distant lymphonodal tumour 2 years later; at that time, the lesion was of the nonpigmented type both clinically and histologically. E-cadherins are the main adhesion molecules responsible for adhesion between melanocytes and keratinocytes in the normal skin. N-cadherins are expressed on fibroblasts, endothelial cells and neurones, and they provide for the homotypic anchorage of these cells. The high metastasizing capacity of melanoma cells is linked to the shift from E-cadherin to N-cadherin. This change might facilitate the interaction of melanoma cells with stromal fibroblasts and endothelial cells, increasing the invasive capacity of these cells. b-catenins are involved in the cell–cell adhesion because they link the cytoplasmic domain of E-cadherins to the b-actin cytoskeleton. Several studies have reported that altered expression and subcellular compartmentalization of cell adhesion markers are important in the development and progression of melanocytic tumours. In particular, it has been reported that the switch of cadherin subtypes from the E-type to the N-type during melanoma development frees melanocytes from keratinocyte control and enhances their Figure 1 Non-homogeneously pigmented lesion involving the soft and hard palate and the maxillary alveolar crest. Viewpoints in dermatology • Correspondence

Molecular cloning and biochemical characterization of the skin tyrosinase from Rana esculenta L.

Amphibian tyrosinases display unique and poorly understood properties such as seasonal activity variations, different activities in dorsal and ventral skin and the occurrence as inactive forms requiring proteolytic activation. For the first time we have sequenced and characterized Rana esculenta L. tyrosinase by functional expression of the cloned cDNA, and compared it with frog skin extracts. R. esculenta tyrosinase ORF is well conserved compared with tyrosinases of various sources. The amino acid similarities between the tyrosinases from R. esculenta and other amphibia range from 85% to 98%. Homology remains high with mammalian tyrosinases (65% identity with Homo sapiens, and 63% with Mus musculus) and with bird orthologues (66% identity with Gallus gallus). Tyrosinase was expressed in HEK293T cells as an active enzyme. Activity staining on non reducing SDS-PAGE revealed two bands around 63 and 68 kDa. R. esculenta skin extracts were mildly active and reached maximal activity upon protease treatment, revealing a high molecular weight dopa-positive band in the 200 kDa range and one of higher MW, after nagarse treatment, in activity stainings. The different behaviour of recombinant tyrosinase compared to skin extracts suggests formation in vivo of a multimeric complex.

The mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines: correlations with BRAF mutational status

Background Metabolic reprogramming is commonly found in cancer but it is poorly understood in melanoma. Recent works [1,2] provided new insights concerning molecular mechanisms involved in mitochondrial biogenesis of melanoma. This work aims to find possible correlations between pathways involved in the onset and progression of the disease in order to provide supporting information in this field. In particular we studied the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines and its relations with BRAF mutational status.

New insight into the role of metabolic reprogramming in melanoma cells harboring BRAF mutations.

This study explores the V600BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastatic V600BRAF melanoma cell lines. V600BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/β, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario, V600BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in V600BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma. Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.

Molecular and Ultrastructural Analysis of a Multiphasic Oral Malignant Melanoma

Melanomas of the oral cavity are extremely rare. Their rarity and their independence on exposure to UV radiation make them particularly interesting. The authors analyzed an oral multiphasic melanoma composed by a nodular nonpigmented ulcerated central region, a nodular ulcerated pigmented area, a pigmented nonulcerated region, and an area similar to a dysplastic nevus. They determined the expression of some genes involved in the differentiation and cellular transformation in morphologically different regions of melanoma. All these areas were also analyzed by electron microscopy. The various regions composing the melanoma expressed genes involved in melanogenesis and melanoma progression in a different manner. Electron microscopy observation of ultrathin sections of each region evidenced ultrastructural differences, being the cellular architecture more compromised in the most aggressive parts of the neoplasm. This pilot study identified morphological, molecular, and ultrastructural differences that characterize each region of the multiphasic melanoma.