Normal Skin Melanocytes Research Articles

Detecting normal and cancer skin cells via glycosylation and adhesion signatures: A path to enhanced microfluidic phenotyping

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.

Role of Dermal Factors Involved in Regulating the Melanin and Melanogenesis of Mammalian Melanocytes in Normal and Abnormal Skin.

Mammalian melanin is produced in melanocytes and accumulated in melanosomes. Melanogenesis is supported by many factors derived from the surrounding tissue environment, such as the epidermis, dermis, and subcutaneous tissue, in addition to numerous melanogenesis-related genes. The roles of these genes have been fully investigated and the molecular analysis has been performed. Moreover, the role of paracrine factors derived from epidermis has also been studied. However, the role of dermis has not been fully studied. Thus, in this review, dermis-derived factors including soluble and insoluble components were overviewed and discussed in normal and abnormal circumstances. Dermal factors play an important role in the regulation of melanogenesis in the normal and abnormal mammalian skin.

Elastin is Related to Functions of Melanocytes in Normal and Abnormal Skin

Context: Functions of melanocytes are supported by many growth factors and cytokines derived from epidermal and dermal cells, especially keratinocytes and fibroblasts. In addition to these skin cells, elastin fibers are thought to be important for regulating melanocyte functions in normal and abnormal skin. However, the role of elastin has not been fully investigated. Recently, the research on the role of elastin in melanocyte functions has greatly increased even in human skin, whereas no full review article on this subject has been presented so far. Thus, the author tried to present a complete review article on this subject in animals and humans. Methods: The author searched the literature on elastin and melanocytes in research databases. Results: Elastin fibers can affect the normal functions of melanoblasts/melanocytes in animals and humans. Moreover, elastin fibers influence melanocyte functions in abnormal skin. In vitiliginous skin, where melanin and melanoblasts/melanocytes are completely lost, elastin fibers are dramatically reduced. However, elastin fibers are completely restored or even increased in the dermis of repigmented skin after phototherapy and/or skin transplantation. Moreover, elastin fibers penetrate the basement membrane of repigmented skin, suggesting a direct contact between dermal elastin fibers and epidermal melanoblasts/melanocytes. Conclusions: Elastin/elastin fibers may control the functions of mammalian melanocytes in normal and abnormal skin.

Kaempferol promotes melanogenesis and reduces oxidative stress in PIG1 normal human skin melanocytes

Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO-1 protein level under H2 O2 stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H2 O2 , kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO-1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress-induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H2 O2 -induced oxidative stress damage.

Exosomal miR-22-3p from Mesenchymal Stem Cells Inhibits the Epithelial-Mesenchymal Transition (EMT) of Melanoma Cells by Regulating LGALS1.

The mortality rate from melanoma has been rising and hence new therapeutic approaches for this disease have received extensive attention, especially the search for novel therapeutic targets. The aim of this study was to find new targets for the treatment of melanoma through a bioinformatics and experimental approach. First, we screened for differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) between melanoma and normal tissues using the TCGA-SKCM, GTEX, and GSE24996 datasets. Next, we identified epithelial-mesenchymal transition (EMT)-related DEGs and analyzed their expression levels and association with patient survival. The expression level of DEGs was then confirmed in normal human melanocytes and melanoma cells. Bioinformatics analysis was used to identify miRNAs that targeted the most highly expressed DEG, LGALS1, and their binding confirmed using dual luciferase. Enriched pathways for the LGALS1 target miR-22-3p were also analyzed. miR-22-3p was overexpressed in cells in order to investigate changes in cell activity and in related genes and proteins. Exosomes from human bone marrow mesenchymal stem cells (MSCs) were coated with miR-22-3p to examine its effect on EMT. The expression levels of LGALS1, CPXM1, and APLNR were higher in melanoma than in normal tissues and were associated with worse patient survival. The differential expression of these genes was confirmed using normal human skin melanocytes (PIG1) and human melanoma cells (WM-266-4). LGALS1 was the most differentially expressed gene between WM-266-4 and PIG1 cells, and was also predicted to be a target for miR-22-3p. The results of dual luciferase experiments confirmed that miR-22-3p could bind to LGALS1. Following the overexpression of miR-22-3p in WM-266-4 cells, the cell viability decreased, the expression levels of LGALS1, VIM and SNAI2 decreased, the expression level of CDH1 increased, and cell apoptosis increased. Transfection of miR-22-3p using exosomes resulted in similar effects. We identified three genes (LGALS1, CPXM1, APLNR) that showed a high level of differential expression in melanoma. LGALS1 is a target for miR-22-3p binding and this can inhibit the EMT of melanoma cells, thereby preventing the development of melanoma. Moreover, exosomes secreted by MSCs can be loaded with miR-22-3p, thus regulating the EMT process in melanoma cells.

Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma.

The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.

Use of PRAME immunostaining to distinguish early melanoma in situ from benign pigmented conditions.

PRAME (PReferentially expressed Antigen in MElanoma) is an antigen that shows marked overexpression in melanoma compared to normal skin melanocytes. PRAME immunohistochemistry has proven effective in distinguishing melanocytic nevi from melanoma, but it is unclear if it may be used to distinguish melanoma in situ from other benign pigmented lesions. In particular, differentiating from melanocytic hyperplasia in sun-damaged skin is sometimes clinically and histopathologically challenging. We hypothesized that PRAME staining of solar lentigo, sun-damaged skin, and melanoma in situ would aid in setting a threshold of positivity that could be useful in evaluating such conditions. We collected and stained typical examples of solar lentigo, melanoma in situ, and non-lesional sun-damaged skin by PRAME immunohistochemistry to assess a potential cutoff of PRAME positivity. Solar lentigo and non-lesional sun-damaged skin had 10 or fewer PRAME-positive cells per millimeter (mean 1.2), on the other hand melanoma in situ had at least 16 (mean 75.1). PRAME immunostaining appears sensitive and specific in the current series. This could be clinically useful for distinguishing melanoma in situ from benign melanocytic hyperplasia in sun-damaged skin. However, further studies are required to determine if 10 cells per millimeter is an acceptable threshold of positivity.

Characterization of Primary Cultures of Normal and Neoplastic Canine Melanocytes.

Simple SummaryMelanoma is one of the most aggressive cancers in humans, with high rates of metastasis and a poor prognosis. Because of its environmental, biological and genetic features, numerous studies indicate the dog as a good comparative model for human melanoma. Primary cell cultures of healthy and neoplastic melanocytes derived from skin and oral mucosa of dogs with spontaneous tumors are established in this study. This model could represent a suitable tool to compare biological and molecular features of normal and neoplastic melanocytes from the same patient, to investigate the pathways underlying the oncogenic transformation, and to apply a more personalized therapeutic strategy. The cell cultures also meet international guidelines that encourage the use of alternative models to animal ones for the study of oncological diseases.Although numerous animal models, especially mouse models, have been established for the study of melanoma, they often fail to accurately describe the mechanisms of human disease because of their anatomic, physiological, and immune differences. The dog, as a spontaneous model of melanoma, is nowadays considered one of the most valid alternatives due to the heterogeneity of clinical presentations and of histological and genetic similarities of canine melanoma with the human counterpart. The aim of the study was to optimize a protocol for the isolation and cultivation of healthy and neoplastic canine melanocytes derived from the same animal and obtained from cutaneous and mucosal (oral) sites. We obtained five primary tumor cell cultures (from 2 cutaneous melanoma, 2 mucosal melanoma and 1 lymph node metastasis) and primary normal melanocyte cell cultures (from normal skin and mucosa) from the same dogs. Immunocytochemical characterization with Melan A, PNL2 and S100 antibodies confirmed the melanocytic origin of the cells. This work contributes to expanding the case record of studies on canine melanoma cell cultures as suitable model to study human and canine melanoma. To the authors’ knowledge, this is the first report of isolation of normal skin and mucosal canine melanocytes.

Interacting Genetic Lesions of Melanoma in the Tumor Microenvironment: Defining a Viable Therapy.

Melanoma is the most aggressive form of skin cancer with an estimated 106,110 newly diagnosed cases in the United States of America in 2021 leading to an approximated 7180 melanoma-induced deaths. Cancer typically arises from an accumulation of somatic mutations and can be associated with mutagenic or carcinogenic exposure. A key characteristic of melanoma is the extensive somatic mutation rate of 16.8 mutations/Mb, which is largely attributed to UV exposure. Bearing the highest mutational load, many of them occur in key driver pathways, most commonly the BRAFV600E in the mitogen-activated protein kinase (MAPK) pathway. This driver mutation is targeted clinically with FDA-approved therapies using small molecule inhibitors of oncogenic BRAFV600E and MEK, which has greatly expanded therapeutic intervention following a melanoma diagnosis. Up until 2011, therapeutic options for metastatic melanoma were limited, and treatment typically fell under the spectrum of surgery, radiotherapy, and chemotherapy.Attributed to the extensive mutation rate, as well as having the highest number of neoepitopes, melanoma is deemed to be extremely immunogenic. However, despite this highly immunogenic nature, melanoma is notorious for inducing an immunosuppressive microenvironment which can be relieved by checkpoint inhibitor therapy. The two molecules currently approved clinically are ipilimumab and nivolumab, which target the molecules CTLA-4 and PD-1, respectively.A plethora of immunomodulatory molecules exist, many with redundant functions. Additionally, these molecules are expressed not only by immune cells but also by tumor cells within the tumor microenvironment. Tumor profiling of these cell surface checkpoint molecules is necessary to optimize a clinical response. The presence of immunomodulatory molecules in melanoma, using data from The Cancer Genome Atlas and validation of expression in two model systems, human melanoma tissues and patient-derived melanoma cells, revealed that the expression levels of B and T lymphocyte attenuator (BTLA), TIM1, and CD226, concurrently with the BRAFV600E mutation status, significantly dictated overall survival in melanoma patients. These molecules, along with herpesvirus entry mediator (HVEM) and CD160, two molecules that are a part of the HVEM/BTLA/CD160 axis, had a higher expression in human melanoma tissues when compared to normal skin melanocytes and have unique roles to play in T cell activation. New links are being uncovered between the expression of immunomodulatory molecules and the BRAFV600E genetic lesion in melanoma. Small molecule inhibitors of the MAPK pathway regulate the surface expression of this multifaceted molecule, making BTLA a promising target for immuno-oncology to be targeted in combination with small molecule inhibitors, potentially alleviating T regulatory cell activation and improving patient prognosis.

Surgical interventions for vitiligo

Since nonsurgical treatment of vitiligo is not always successful, surgical interventions are viable options for patients with refractory vitiligo. Surgical treatment is a method in which melanocytes of normal skin are transplanted into vitiligo lesions and provided as a repigmentation source. Such treatments are primarily divided into tissue grafting and cellular grafting, depending on the nature of the graft. Tissue grafting includes split-thickness skin grafting, suction blister grafting, punch grafting, hair follicle transplantation, and smashed-skin grafting. Cellular grafting includes non-cultured epidermal cell suspension transplantation, non-cultured follicular cell suspension transplantation, and cultured epidermal cell suspension transplantation. Among these, suction blister grafting and micro-punch grafting have been widely performed for localized refractory vitiligo, and non-cultured epidermal cell suspension transplantation is adopted as the standard treatment for extensive vitiligo. Research on cultured cellular grafting to treat larger vitiligo areas is also ongoing. Selecting patients with stable vitiligo that has not spread for over 12 months is the most critical factor in the surgical outcome. It is also important to choose an appropriate surgical modality for each patient, and a combination of various procedures often improves the overall outcome. In conclusion, surgical intervention can be an effective and safe option for patients with vitiligo refractory to conventional treatments. Non-cultured epidermal cell suspension transplantation, which was denied by New Health Technology Assessment in Korea, should be approved to benefit patients with refractory vitiligo.

Abstract 1271: UV irradiation and epigenetic change in melanocyte 5-hydroxymethylcytosine

Abstract Background Melanoma is the main cause of death from skin cancer. Excessive sun exposure with resultant UV irradiation, particularly during childhood, is established as its major environmental risk factor. Epigenetics are becoming increasingly recognized as potential reversible regulators of genomic anomalies responsible for cancerous growth. Loss of DNA hydroxymethylation marks 5-hydroxymethylcytosine (5-hmC) a functionally significant epigenetic event in melanomagenesis, but whether decreased 5-hmC is driven by UV irradiation remains unknown. The aim of this study is to characterize 5-hmC levels in potential melanoma precursor lesions and cultured samples by UV irradiation in vitro. Methods 70 human skin excision cases of chronic UV exposed skin and matched controls were evaluated. In addition, discarded human foreskins were exposed to biologically relevant doses of UVB (302 nm) and UVA (365 nm) irradiation. Tissue samples were then incubated in DMEM medium and after 24 hours fixed in 10% formalin overnight and stored in 70% ethanol prior to sectioning and staining. A dual anti-5-hmC and anti-MART-1 immunohistochemical (IHC) staining protocol was performed, and resultant semiquantitative immunoreactivity scores were analyzed statistically by one-way ANOVA analysis and Bonferroni's Multiple comparisons test or unpaired T-test using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA). All p-values were two-tailed, with a p<0.05 considered statistically significant. Results When comparing sun-protected versus chronically sun-exposed sites of the excision specimens, lower levels of 5-hmC immunoreactivity were noted in melanocytes of sun-protected specimens than sun-exposed samples (p = 0.017). Moreover, a more significant difference (p = 0.0053) was detected between 5-hmC staining in normal skin melanocytes versus 5-hmC loss detected in so-called “atypical melanocytic hyperplasia of sun-damaged skin” that surrounds melanomas. Finally, we sought to determine whether we could induce in vitro a UV effect on 5-hmC similar to that seen with chronic sun-damage. Significant decreased immunoreactivity for 5-hmC was observed in foreskin melanocytes after UV irradiation- a finding that replicated what we observed in patient samples containing chronically UV-altered melanocytes. Conclusions Our results suggest that UVB irradiation's effect on the epigenome may play a crucial role in the development of melanoma. Implementation of the dual staining for anti-5-hmC and anti-MART-1 may be helpful in defining melanocytes altered in the direction of melanomagenesis, and thus may serve as a practical assay in determining potential melanoma precursor fields amenable to surveillance and prophylactic therapies. Citation Format: Kevin Y. Wang, Tanja Zdravkovic, Christine Lian. UV irradiation and epigenetic change in melanocyte 5-hydroxymethylcytosine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1271.

Long non-coding RNA MIAT promotes the growth of melanoma via targeting miR-150.

Melanoma is a common skin cancer and it can lead to high mortality probably by earlyinvasion and metastasis. LncRNA MIAT is involved in tumor proliferation, invasion and epithelial-to-mesenchymal transition (EMT). However, the roles of MIAT in melanoma still require further investigation. Thus, the aim of the study is to investigate the roles of MIAT in melanoma, especially the effects of MIAT on EMT of melanoma cancer cells. The results showed that the expression of MIAT was significantly upregulated in melanoma tissue and cells compared with the normal skin and normal melanocytes; moreover, miR-150 was confirmed as a target of MIAT. Furthermore, knockdown of MIAT inhibited cell proliferation and invasion in melanoma cancer cells and transfection of miR-150 inhibitors partial abrogated the anti-tumor effects of MIAT siRNA. In addition, MIAT siRNA also inhibited the EMT of melanoma cells, while miR-150 inhibitors can reverse the effects of MIAT siRNA. Finally, knockdown of MIAT also inhibited the carcinogenic effects of melanoma in vivo by targeting miR-150. In conclusion, we reported that MIAT promotes the proliferation, invasion and EMT of melanoma cells via targeting miR-150, which suggested that MIAT might be a therapeutic target for the treatment of melanoma.

On\u2013off switching of cell cycle and melanogenesis regulation of melanocytes by non-thermal atmospheric pressure plasma-activated medium

Non-thermal atmospheric pressure (NAP) plasma has demonstrated potential in biomedical applications, such as cancer treatment, bactericidal sterilization, and cell growth promotion or inhibition. In this study, for the first time, we demonstrated on–off switching of cell cycle progression and regulated melanogenesis in normal human skin melanocytes by NAP plasma-activated medium (PAM). The melanocytes were exposed to NAP plasma at durations varying from 0 to 20 min, and the effects of PAM on cell proliferation, cell cycle progression, and melanogenesis were investigated. Although PAM showed no cytotoxicity, the proliferation of melanocytes was inhibited. The melanocyte cell cycle was arrested by PAM for a relatively short period (48 h), after which it recovered slowly. PAM promoted melanogenesis through the activation of the enzymes tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. These effects seem to be related to reactive oxygen species induced by PAM. Our finding that PAM modulates the cell cycle may provide insight into the recurrence of cancer. The regulation of the melanogenesis of melanocytes may facilitate the control of skin tone without incurring negative side effects.

Occurrence of non-toxic bioemulsifiers during polyhydroxyalkanoate production by Pseudomonas strains valorizing crude glycerol by-product

While screening for polyhydroxyalkanoate (PHA) producing strains, using glycerol rich by-product as carbon source, it was observed that extracellular polymers were also secreted into the culture broth. The scope of this study was to characterize both intracellular and extracellular polymers, produced by Pseudomonas putida NRRL B-14875 and Pseudomonas chlororaphis DSM 50083, mostly focusing on those novel extracellular polymers. It was found that they fall into the class of bioemulsifiers (BE), as they showed excellent emulsion stability against different hydrocarbons/oils at various pH conditions, temperature and salinity concentrations. Cytotoxicity tests revealed that BE produced by P. chlororaphis inhibited the growth of highly pigmented human melanoma cells (MNT-1) by 50% at concentrations between 150 and 200 μg/mL, while no effect was observed on normal skin primary keratinocytes and melanocytes. This is the first study reporting mcl-PHA production by P. putida NRRL B-14785 and bioemulsifier production from both P. putida and P. chlororaphis strains.

MYSM1/2A-DUB is an epigenetic regulator in human melanoma and contributes to tumor cell growth.

Histone modifying enzymes, such as histone deacetylases (HDACs) and polycomb repressive complex (PRC) components, have been implicated in regulating tumor growth, epithelial-mesenchymal transition, tumor stem cell maintenance, or repression of tumor suppressor genes - and may be promising targets for combination therapies of melanoma and other cancers. According to recent findings, the histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue differentiation in mice, in part by modulating DNA-damage responses in stem cell and progenitor compartments. Based on the identification of alterations in skin pigmentation and melanocyte specification in Mysm1-deficient mice, we hypothesized that MYSM1 may be involved in melanoma formation. In human melanoma samples, expression of MYSM1 was increased compared with normal skin melanocytes and nevi and co-localized with melanocyte markers such as Melan-A and c-KIT. Similarly, in melanoma cell lines A375 and SK-MEL-28 and in murine skin, expression of the deubiquitinase was detectable at the mRNA and protein level that was inducible by growth factor signals and UVB exposure, respectively. Upon stable silencing of MYSM1 in A375 and SK-MEL-28 melanoma cells by lentivirally-mediated shRNA expression, survival and proliferation were significantly reduced in five MYSM1 shRNA cell lines analyzed compared with control cells. In addition, MYSM1-silenced melanoma cells proliferated less well in softagar assays. In context with our finding that MYSM1 bound to the c-MET promoter region in close vicinity to PAX3 in melanoma cells, our data indicate that MYSM1 is an epigenetic regulator of melanoma growth and potentially promising new target for tumor therapy.

Distinct Role of Sesn2 in Response to UVB-Induced DNA Damage and UVA-Induced Oxidative Stress in Melanocytes.

Ultraviolet (UV) radiation, including both UVB and UVA irradiation, is the major risk factor for causing skin cancer including melanoma. Recently, we have shown that Sesn2, a member of the evolutionarily conserved stress-inducible protein family Sestrins (Sesn), is upregulated in human melanomas as compared to melanocytes in normal human skin, suggesting an oncogenic role of Sesn2. However, the role of Sesn2 in UVB and UVA response is unknown. Here, we demonstrated that both UVB and UVA induce Sesn2 upregulation in melanocytes and melanoma cells. UVB induces Sesn2 expression through the p53 and AKT3 pathways. Sesn2 negatively regulates UVB-induced DNA damage repair. In comparison, UVA induces Sesn2 upregulation through mitochondria but not Nrf2. Sesn2 ablation increased UVA-induced Nrf2 induction and inhibits UVA-induced ROS production, indicating that Sesn2 acts as an upstream regulator of Nrf2. These findings suggest previously unrecognized mechanisms in melanocyte response to UVB and UVA irradiation and potentially in melanoma formation.

Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.

BackgroundConstitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses.MethodsGrey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting.ResultsWe found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses.ConclusionsThese findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-857) contains supplementary material, which is available to authorized users.

SIRT1 deacetylase is overexpressed in human melanoma and its small molecule inhibition imparts anti-proliferative response via p53 activation

Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.

Reduced MiR-675 in Exosome in H19 RNA-Related Melanogenesis via MITF as a Direct Target

H19 non-coding RNA downregulation stimulates melanogenesis in melasma patients. However, its mechanism is unclear. In this study, the potential role of a H19 microRNA, miR-675, in melanogenesis was examined. Real-time PCR using cultured normal human skin keratinocytes, melanocytes, and fibroblasts with or without H19 knockdown showed accompanying changes between expression levels of H19 and those of miR-675 in keratinocytes. MiR-675 was also detected in concentrated culture supernatants and showed expression levels parallel with those of cell lysates. In addition to RNase resistance, FACS analysis showed anti-CD63-positive exosomes in culture supernatants, suggesting miR-675 could be released extracellularly and delivered to neighboring cells without degradation. In western blot analysis, the miR-675 mimic reduced the expression of microphthalmia-associated transcription factor (MITF) and phosphorylation of cAMP-responsive element-binding protein, extracellular signal-regulated kinase and apoptosis signal-regulating kinase, whereas these expressions were increased by the miR-675 inhibitor. Although H19 was not a miR-675 target, luciferase reporter assay showed a direct binding of miR-675 to 3'-untranslated region of MITF. In addition, localized in vivo miR-675 overexpression in mouse using a cationic polymer transfection reagent showed reduced mRNA expression levels of MITF, tyrosinase, tyrosine-related protein-1 (Trp-1), and Trp-2. Collectively, the results suggest that miR-675 derived from keratinocytes could be involved in H19-stimulated melanogenesis using MITF as a target of miR-675.

Phenotyping of Human Melanoma Cells Reveals a Unique Composition of Receptor Targets and a Subpopulation Co-Expressing ErbB4, EPO-R and NGF-R

Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45−/CD31− cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4–40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R− subpopulations produced melanoma lesions in NOD/SCID IL-2Rgammanull (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.