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Increasing studies have suggested that dysregulation of RNA‐binding proteins (RBPs) contributes to cancer progression.,Neuro‐oncological ventral antigen 1 (NOVA1) is a novel RBP and plays an important role in tumour development.,However, the expression and role of NOVA1 in melanoma remain unknown.,In this study, we indicated that NOVA1 expression was up‐regulated in melanoma samples and cell lines.,Moreover, we demonstrated that knockdown of NOVA1 suppressed melanoma cell proliferation, migration and invasion in both A375 and A875 cell lines.,In addition, we showed that suppressed expression of NOVA1 enhanced forkhead box O3a (FOXO3a) expression while inhibited AKT expression in melanoma cell.,Furthermore, we demonstrated that inhibited expression of FoxO3A rescued NOVA1‐mediated cell proliferation, migration and invasion in melanoma cell line A375.,These results suggested that NOVA1 acted as an oncogene in the development of melanoma partly through regulating FoxO3A expression.

MITF (microphthalmia-associated transcription factor) represents a melanocytic lineage-specific transcription factor whose role is profoundly extended in malignant melanoma.,Over the last few years, the function of MITF has been tightly connected to plasticity of melanoma cells.,MITF participates in executing diverse melanoma phenotypes defined by distinct gene expression profiles.,Mutation-dependent alterations in MITF expression and activity have been found in a relatively small subset of melanomas.,MITF activity is rather modulated by its upstream activators and suppressors operating on transcriptional, post-transcriptional and post-translational levels.,These regulatory mechanisms also include epigenetic and microenvironmental signals.,Several transcription factors and signaling pathways involved in the regulation of MITF expression and/or activity such as the Wnt/β-catenin pathway are broadly utilized by various types of tumors, whereas others, e.g., BRAFV600E/ERK1/2 are more specific for melanoma.,Furthermore, the MITF activity can be affected by the availability of transcriptional co-partners that are often redirected by MITF from their own canonical signaling pathways.,In this review, we discuss the complexity of a multilevel regulation of MITF expression and activity that underlies distinct context-related phenotypes of melanoma and might explain diverse responses of melanoma patients to currently used therapeutics.

In this Review, Rambow et al. use melanoma as a model to present a series of theoretical arguments coupled to recent experimental evidence.,The authors discuss key roles for nongenetic state switching at various steps of the evolution of disease progression and therapy resistance.,An incomplete view of the mechanisms that drive metastasis, the primary cause of cancer-related death, has been a major barrier to development of effective therapeutics and prognostic diagnostics.,Increasing evidence indicates that the interplay between microenvironment, genetic lesions, and cellular plasticity drives the metastatic cascade and resistance to therapies.,Here, using melanoma as a model, we outline the diversity and trajectories of cell states during metastatic dissemination and therapy exposure, and highlight how understanding the magnitude and dynamics of nongenetic reprogramming in space and time at single-cell resolution can be exploited to develop therapeutic strategies that capitalize on nongenetic tumor evolution.

Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma.,In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy.,Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins.,Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma.,We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status.,Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry.,Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line.,Immunoblot was applied to analyze the IFN-ɣ signaling pathway.,For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed.,Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53.,Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53L22Q,W23S, a transcriptionally-impaired variant, in TP53-wt cells.,Accordingly, expression of p53L22Q,W23S in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression.,The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression.,While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression.,Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment.,Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment.,Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity.,Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1.,Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer.,Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration.,Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies.,Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.,There are many patients who do not respond to immune checkpoint inhibitor (ICI) immunotherapy.,Here, the authors show a significant negative correlation between sphingosine kinase-1 (SK1) expression and survival for ICI-treated melanoma patients, and further show that targeting SK1 improves response to ICI in mouse cancer models.

Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids.,Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells.,Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells.,However, the characteristics and functions of exosomes derived from NK cells remain unknown.,In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo.,Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads.,The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays.,Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation.,NK-92 Exo were characterized by transmission electron microscopy and western blotting.,We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells.,The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays.,To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line.,Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells.,In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo.,We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging.,Tumor mass was monitored after in vivo experiments.,Results: RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells.,B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay.,For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes.,The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX.,We demonstrated by western blot analysis that NK-92 Exo presented two functional NK proteins, namely perforin and FasL.,Moreover, we confirmed the membrane expression of FasL.,The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway.,The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p < 0.001) and CCK-8 assays (p < 0.001).,Furthermore, in normal healthy cells, even after 24 h of co-culture, NK-92 Exo did not exhibit significant side effects.,In the in vivo experiments, tumors in the vehicle control group were significantly increased, compared with those in the NK-92 Exo-treated group (p < 0.05).,Conclusion: The results of the current study suggest that exosomes derived from NK cells exert cytotoxic effects on melanoma cells and thus warrant further development as a potential immunotherapeutic strategy for cancer.

RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61.,Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch.,Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma.,When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis.,RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors.,These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.,•RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes•RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK•RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis•RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes,RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK,RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis,RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S is a common mutation in human cutaneous melanoma.,Lionarons et al. show that RAC1P29S induces a melanocytic to mesenchymal switch via an SRF/MRTF-mediated gene expression program, cooperates with BRAF in melanomagenesis, and drives BRAF inhibitor resistance, which is reversed by SRF/MRTF inhibition.

Somatic mutations of BRAF or NRAS activating the MAP kinase cell signaling pathway are present in 70% of cutaneous melanomas.,The mutant allele frequency of BRAF V600E (M%BRAF) was recently shown to be highly heterogeneous in melanomas.,The present study focuses on the NRAS Q61 mutant allele frequency (M%NRAS).,Retrospective quantitative analyze of 104 NRAS mutated melanomas was performed using pyrosequencing.,Mechanisms of M%NRAS imbalance were studied by fluorescence in situ hybridization (FISH) and microsatellite analysis.,M%NRAS was increased in 27.9% of cases.,FISH revealed that chromosome 1 instability was the predominant mechanism of M%NRAS increase, with chromosome 1 polysomy observed in 28.6% of cases and intra-tumor cellular heterogeneity with copy number variations of chromosome 1/NRAS in 23.8%.,Acquired copy-neutral loss of heterozygosity (LOH) was less frequent (19%).,However, most samples with high M%NRAS had only one copy of NRAS locus surrounding regions suggesting a WT allele loss.,Clinical characteristics and survival of patients with either <60% or ≥60% of M%NRAS were not different.,As recently shown for M%BRAF, M%NRAS is highly heterogeneous.,The clinical impacts of high M%NRAS should be investigated in a larger series of patients.,The online version of this article (doi:10.1186/s12895-017-0061-x) contains supplementary material, which is available to authorized users.

Melanoma is a form of cancer that initiates in melanocytes.,Melanoma has multiple phenotypically distinct subpopulation of cells, some of them have embryonic like plasticity which are involved in self-renewal, tumor initiation, metastasis and progression and provide reservoir of therapeutically resistant cells.,Cancer stem cells (CSCs) can be identified and characterized based on various unique cell surface and intracellular markers.,CSCs exhibit different molecular pattern with respect to non-CSCs.,They maintain their stemness and chemoresistant features through specific signaling cascades.,CSCs are weak in immunogenicity and act as immunosupressor in the host system.,Melanoma treatment becomes difficult and survival is greatly reduced when the patient develop metastasis.,Standard conventional oncology treatments such as chemotherapy, radiotherapy and surgical resection are only responsible for shrinking the bulk of the tumor mass and tumor tends to relapse.,Thus, targeting CSCs and their microenvironment niche addresses the alternative of traditional cancer therapy.,Combined use of CSCs targeted and traditional therapies may kill the bulk tumor and CSCs and offer a promising therapeutic strategy for the management of melanoma.

Nerve endings are often identified within solid tumors, but their impact on the tumor growth and progression remains poorly understood.,Emerging data suggests that the central nervous system may affect cancer development and spreading via the hypothalamic-pituitary-adrenal axis and autonomous nervous system.,However, the role of the afferent sensory neurons in tumor growth is unclear, except some reports on perineural invasion in prostate and pancreatic cancer and cancer-related pain syndrome.,Here, we provide the results of primary testing of the concept that the interaction between melanoma cells and sensory neurons may induce the formation of tumor-supporting microenvironment via attraction of immune regulatory cells by the tumor-activated dorsal root ganglion (DRG) neurons.,We report that despite DRG cells not directly up-regulating proliferation of melanoma cells in vitro, presence of DRG neurons allows tumors to grow significantly faster in vivo.,This effect has been associated with increased production of chemokines by tumor-activated DRG neurons and attraction of myeloid-derived suppressor cells both in vitro and in vivo.,These initial proof-of-concept results justify further investigations of the sensory (afferent) nervous system in the context of tumorigenesis and the local protumorigenic immunoenvironment.

The capacity of cancer to adapt to treatment and evolve is a major limitation for targeted therapies.,While the role of new acquired mutations is well-established, recent findings indicate that resistance can also arise from subpopulations of tolerant/persister cells that survive in the presence of the treatment.,Different processes contribute to the emergence of these cells, including pathway rebound through the release of negative feedback loops, transcriptional rewiring mediated by chromatin remodeling and autocrine/paracrine communication among tumor cells and within the tumor microenvironment.,In this review, we discuss the non-genetic mechanisms that eventually result in cancer resistance to targeted therapies, with a special focus on those involving changes in gene expression.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family.,So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma.,In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines-keratinocytes (HaCaT) and the primary fibroblast line.,Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed.,Cell viability was studied using an MTT assay.,A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD).,Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B.,This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A.,No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line.,Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line.,The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence.,Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future.,Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer associated with poor survival outcomes in patients with distant metastatic disease (mMCC).,In an initial analysis from JAVELIN Merkel 200, a phase 2, prospective, open-label, single-arm trial in mMCC, avelumab-a human anti-programmed death-ligand 1 (PD-L1) monoclonal antibody-showed promising efficacy and a safety profile that was generally manageable and tolerable.,Here, we report the efficacy of avelumab after ≥1 year of follow-up in patients with distant mMCC that had progressed following prior chemotherapy for metastatic disease.,Patients received avelumab 10 mg/kg by 1-h intravenous infusion every 2 weeks until confirmed disease progression, unacceptable toxicity, or withdrawal.,The primary endpoint was best overall response.,Secondary endpoints included duration of response (DOR), progression-free survival (PFS), and overall survival (OS).,Patients (N = 88) were followed for a minimum of 12 months.,The confirmed objective response rate was 33.0% (95% CI, 23.3%-43.8%; complete response: 11.4%).,An estimated 74% of responses lasted ≥1 year, and 72.4% of responses were ongoing at data cutoff.,Responses were durable, with the median DOR not yet reached (95% CI, 18.0 months-not estimable), and PFS was prolonged; 1-year PFS and OS rates were 30% (95% CI, 21%-41%) and 52% (95% CI, 41%-62%), respectively.,Median OS was 12.9 months (95% CI, 7.5-not estimable).,Subgroup analyses suggested a higher probability of response in patients receiving fewer prior lines of systemic therapy, with a lower baseline disease burden, and with PD-L1-positive tumors; however, durable responses occurred irrespective of baseline factors, including tumor Merkel cell polyomavirus status.,With longer follow-up, avelumab continues to show durable responses and promising survival outcomes in patients with distant mMCC whose disease had progressed after chemotherapy.,Clinicaltrials.gov identifier: NCT02155647.

Merkel cell carcinoma is a primary cutaneous neuroendocrine carcinoma, which once metastatic is difficult to treat.,Recent mutation analyses of Merkel cell carcinoma revealed a low number of mutations in Merkel cell polyomavirus-associated tumors, and a high number of mutations in virus-negative combined squamous cell and neuroendocrine carcinomas of chronically sun-damaged skin.,We speculated that the paucity of mutations in virus-positive Merkel cell carcinoma may reflect a pathomechanism that depends on derangements of chromatin without alterations in the DNA sequence (epigenetic dysregulation).,One central epigenetic regulator is the Polycomb repressive complex 2 (PRC2), which silences genomic regions by trimethylating (me3) lysine (K) 27 of histone H3, and thereby establishes the histone mark H3K27me3.,Recent experimental research data demonstrated that PRC2 loss in mice skin results in the formation of Merkel cells.,Prompted by these findings, we explored a possible contribution of PRC2 loss in human Merkel cell carcinoma.,We examined the immunohistochemical expression of H3K27me3 in 35 Merkel cell carcinomas with pure histological features (22 primary and 13 metastatic lesions) and in 5 combined squamous and neuroendocrine carcinomas of the skin.,We found a strong reduction of H3K27me3 staining in tumors with pure histologic features and virus-positive Merkel cell carcinomas.,Combined neuroendocrine carcinomas had no or only minimal loss of H3K27me3 labeling.,Our findings suggest that a PRC2-mediated epigenetic deregulation may play a role in the pathogenesis of virus-positive Merkel cell carcinomas and in tumors with pure histologic features.

The mechanism of gender disparity in cutaneous melanoma incidence remains unclear.,Steroid hormones including estrogens have long been implicated in the course of melanoma, but the conclusion is controversial.,Estrogen receptors (ERs) and insulin-like growth factor 1 receptor (IGF1R) show extensive crosstalk in cancer development, but how the ER/IGF1R network impacts melanoma is currently unclear.,Here we studied the melanoma associations of selected SNPs from the ER/IGF1R network.,Part of the International Genes, Environment, and Melanoma (GEM) cohort was used as a discovery set, and the Gene Environment Association Studies Initiative (GENEVA) dataset served as a validation set.,Based on the associations with other malignant disease conditions, thirteen single nucleotide polymorphism (SNP) variants in ESR1, ESR2, IGF1, and IGF1R were selected for candidate gene association analyses.,The rs1520220 in IGF1 and rs2229765 in IGF1R variants were significantly associated with melanoma risk in the GEM dataset after Benjamini-Hochberg multiple comparison correction, although they were not validated in the GENEVA set.,The discrepancy may be caused by the multiple melanoma characteristics in the GEM patients.,Further analysis of gender disparity was carried out for IGF1 and IGF1R SNPs in the GEM dataset.,The GG phenotype in IGF1 rs1520220 (recessive model) presented an increased risk of melanoma (OR = 8.11, 95% CI: 2.20, 52.5, p = 0.006) in men but a significant opposite effect in women (OR = 0.15, 95% CI: 0.018, 0.86, p = 0.045).,The AA genotype in IGF1R rs2229765 (recessive model) showed a significant protective effect in men (OR = 0.24, 95% CI: 0.07, 0.64, p = 0.008) and no effect in women.,Results from the current study are warranted for further validation.

The total number of acquired melanocytic nevi on the skin is strongly correlated with melanoma risk.,Here we report a meta-analysis of 11 nevus GWAS from Australia, Netherlands, UK, and USA comprising 52,506 individuals.,We confirm known loci including MTAP, PLA2G6, and IRF4, and detect novel SNPs in KITLG and a region of 9q32.,In a bivariate analysis combining the nevus results with a recent melanoma GWAS meta-analysis (12,874 cases, 23,203 controls), SNPs near GPRC5A, CYP1B1, PPARGC1B, HDAC4, FAM208B, DOCK8, and SYNE2 reached global significance, and other loci, including MIR146A and OBFC1, reached a suggestive level.,Overall, we conclude that most nevus genes affect melanoma risk (KITLG an exception), while many melanoma risk loci do not alter nevus count.,For example, variants in TERC and OBFC1 affect both traits, but other telomere length maintenance genes seem to affect melanoma risk only.,Our findings implicate multiple pathways in nevogenesis.,Melanocytic nevus count is associated with melanoma risk.,In this study, a meta-analysis of 11 nevus GWAS studies identifies novel SNPs in KITLG and 9q32, and bivariate analysis with melanoma GWAS meta-analysis reveals that most nevus genes affect melanoma risk, while melanoma risk loci do not alter the nevus count.

Uveal melanomas are highly metastatic and have high rate of recurrence due to the lack of effective systemic therapy.,The identification of important survival pathways in uveal melanomas provides novel therapeutic targets for effective treatment.,In the present study, we found that the NF-κB signaling pathway was constitutively and highly activated in uveal melanoma cells.,Treatment with the pharmacological NF-κB specific inhibitor BAY11-7082 markedly decreased the nuclear translocation of NF-κB.,In a dose-dependent setting, BAY11-7082 inhibited the proliferation and growth of uveal melanoma cells by inducing apoptosis without effect on cell cycle.,The migration capacity of uveal melanoma cells was also significantly suppressed by BAY11-7082 treatment.,Mechanistically, BAY11-7082 increased the activity of caspase 3 and reduced the expression of anti-apoptotic protein Bcl-2, but did not influence the expression of pro-apoptotic protein Bax.,Furthermore, BAY11-7082 induced uveal melanoma cell apoptosis and inhibited xenograft tumor growth in vivo.,Collectively, the present study identified NF-κB as an important survival signal for uveal melanoma cells and suggested that administration of specific NF-κB inhibitor BAY11-7082 could serve as an effective treatment for patients with uveal melanoma.

Treatment of uveal (intraocular) melanoma is aimed at prolonging life, if possible conserving the eye and useful vision.,About 50% of patients develop fatal metastatic disease despite successful eradication of the primary intraocular tumour.,The effect of ocular treatment on survival is unknown, because the same survival data from case series can be interpreted in different ways.,Treatment is therefore based on intuition and varies greatly between centres.,Randomised trials of treatment vs non-treatment of asymptomatic tumours are desirable but would be controversial, difficult, expensive and possibly inconclusive.,Strategies for coping with uncertainty are needed to avoid unethical care.

Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM).,While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated.,Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM.,UM cells were examined for expression of HIF-1α, VEGF, and ANGPTL4.,Their contribution to the angiogenic potential of UM cells was assessed using the endothelial cell tubule formation and directed in vivo angiogenesis assays.,These results were corroborated in tissue from UM animal models and in tissue from patients with UM.,Inhibition of VEGF partially reduced tubule formation promoted by conditioned medium from UM cells.,Inhibition of ANGPTL4, which was highly expressed in hypoxic UM cells, a UM orthotopic transplant model, a UM tumor array, and vitreous samples from UM patients, inhibited the angiogenic potential of UM cells in vitro and in vivo; this effect was additive to VEGF inhibition.,Targeting both ANGPTL4 and VEGF may be required for the effective inhibition of angiogenesis in UM.

Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these patients develop liver metastases.,Exosomes are small RNA containing nano-vesicles released by most cells, including malignant melanoma cells.,This clinical translational study included patients undergoing isolated hepatic perfusion (IHP) for metastatic uveal melanoma, from whom exosomes were isolated directly from liver perfusates.,The objective was to determine whether exosomes are present in the liver circulation, and to ascertain whether these may originate from melanoma cells.,Exosomes were isolated from the liver perfusate of twelve patients with liver metastases from uveal melanoma undergoing IHP.,Exosomes were visualised by electron microscopy, and characterised by flow cytometry, Western blot and real-time PCR.,Furthermore, the concentration of peripheral blood exosomes were measured and compared to healthy controls.,The liver perfusate contained Melan-A positive and RNA containing exosomes, with similar miRNA profiles among patients, but dissimilar miRNA compared to exosomes isolated from tumor cell cultures.,Patients with metastatic uveal melanoma had a higher concentration of exosomes in their peripheral venous blood compared to healthy controls.,Melanoma exosomes are released into the liver circulation in metastatic uveal melanoma, and is associated with higher concentrations of exosomes in the systemic circulation.,The exosomes isolated directly from liver circulation contain miRNA clusters that are different from exosomes from other cellular sources.,The online version of this article (doi:10.1186/1471-2407-14-962) contains supplementary material, which is available to authorized users.

Recent work has explored a putative role for the E6 protein from some β-human papillomavirus genus (β-HPVs) in the development of non-melanoma skin cancers, specifically β-HPV 5 and 8 E6.,Because these viruses are not required for tumor maintenance, they are hypothesized to act as co-factors that enhance the mutagenic capacity of UV-exposure by disrupting the repair of the resulting DNA damage.,Supporting this proposal, we have previously demonstrated that UV damage signaling is hindered by β-HPV 5 and 8 E6 resulting in an increase in both thymine dimers and UV-induced double strand breaks (DSBs).,Here we show that β-HPV 5 and 8 E6 further disrupt the repair of these DSBs and provide a mechanism for this attenuation.,By binding and destabilizing a histone acetyltransferase, p300, β-HPV 5 and 8 E6 reduce the enrichment of the transcription factor at the promoter of two genes critical to the homology dependent repair of DSBs (BRCA1 and BRCA2).,The resulting diminished BRCA1/2 transcription not only leads to lower protein levels but also curtails the ability of these proteins to form repair foci at DSBs.,Using a GFP-based reporter, we confirm that this reduced foci formation leads to significantly diminished homology dependent repair of DSBs.,By deleting the p300 binding domain of β-HPV 8 E6, we demonstrate that the loss of robust repair is dependent on viral-mediated degradation of p300 and confirm this observation using a combination of p300 mutants that are β-HPV 8 E6 destabilization resistant and p300 knock-out cells.,In conclusion, this work establishes an expanded ability of β-HPV 5 and 8 E6 to attenuate UV damage repair, thus adding further support to the hypothesis that β-HPV infections play a role in skin cancer development by increasing the oncogenic potential of UV exposure.

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role.,We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models.,To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter.,Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates.,Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis.,In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21WAF1 and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls.,Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals.,In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions.,Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.

Treatment with programmed death receptor-1 (PD-1) antibodies is associated with high response rates in patients with advanced melanoma.,Reliable markers for early response and outcome are still sparse.,We evaluated 66 consecutive patients with advanced/metastatic melanoma treated with nivolumab or pembrolizumab between 2013 and 2014.,The main objectives of this study were to investigate whether, first, serum lactate dehydrogenase (LDH) at baseline (normal vs above the upper limit of normal) correlates with overall survival (OS), and, second, whether the change of LDH during treatment predicts response before the first scan and OS in patients with an elevated baseline LDH.,After a median follow-up of 9 months, patients with an elevated baseline LDH (N=34) had a significantly shorter OS compared with patients with normal LDH (N=32; 6-month OS: 60.8% vs 81.6% and 12-month OS: 44.2% vs 71.5% (log-rank P=0.0292).,In those 34 patients with elevated baseline LDH, the relative change during treatment was significantly associated with an objective response on the first scan: the 11 (32%) patients with partial remission had a mean reduction of −27.3% from elevated baseline LDH.,In contrast, patients with progressive disease (N=15) had a mean increase of +39%.,Patients with a relative increase over 10% from elevated baseline LDH had a significantly shorter OS compared with patients with ⩽10% change (4.3 vs 15.7 months, log-rank P<0.00623).,LDH could be a useful marker at baseline and during treatment to predict early response or progression in patients with advanced melanoma who receive anti-PD-1 therapy.

The aim of this study was to investigate the feasibility of using serum miR-221 as a noninvasive prognostic biomarker for cutaneous malignant melanoma (CMM).,We measured the expression levels of miR-221 in serum samples from 72 CMM patients and 54 healthy controls by real-time quantitative polymerase chain reaction (RT-PCR).,The overall survival (OS) and disease-free survival (DFS) were calculated using the Kaplan-Meier method.,The differences between the survival curves were tested by using the log-rank test.,The COX proportional hazards regression model was used to determine the joint effects of several variables on survival.,The serum miR-221 levels were significantly higher in patients with CMM than in healthy controls (p<0.0001).,Patients with high serum miR-221 levels had a significantly lower 5-year OS rate (22.1% vs.,54.6%; P=0.018) and RFS rate (12.5% vs.,45.2%; P=0.008) than those with low serum miR-221 level.,In a multivariate Cox model, we found that miR-221 expression was an independent predictor of poor 5-year OS (hazards ratio [HR]=3.189, 95% confidence interval [CI]=1.782-6.777, P=0.007) and 5-year DFS (HR=2.119, CI=1.962-8.552, P=0.01) in CMM patients.,Our data indicate that serum miR-221expression level has prognostic value in patients with CMM.

In less than 10 years, melanoma treatment has been revolutionized with the approval of tyrosine kinase inhibitors and immune checkpoint inhibitors, which have been shown to have a significant impact on the prognosis of patients with melanoma.,The early steps of this transformation have taken place in research laboratories.,The mitogen-activated protein kinase (MAPK) pathway, phosphoinositol-3-kinase (PI3K) pathway promote the development of melanoma through numerous genomic alterations on different components of these pathways.,Moreover, melanoma cells deeply interact with the tumor microenvironment and the immune system.,This knowledge has led to the identification of novel therapeutic targets and treatment strategies.,In this review, the epidemiological features of cutaneous melanoma along with the biological mechanisms involved in its development and progression are summarized.,The current state-of-the-art of advanced stage melanoma treatment strategies and the currently available evidence of the use of predictive and prognostic biomarkers are also discussed.

Epidermal melanocytes are particularly vulnerable to oxidative stress due to the pro-oxidant state generated during melanin synthesis, and to intrinsic antioxidant defences that are compromised in pathologic conditions.,Melanoma is thought to be oxidative stress-driven, and melanocyte death in vitiligo is thought to be instigated by a highly pro-oxidant state in the epidermis.,We review the current knowledge about melanin and the redox state of melanocytes, how paracrine factors help counteract oxidative stress, the role of oxidative stress in melanoma initiation and progression and in melanocyte death in vitiligo, and how this knowledge can be harnessed for melanoma and vitiligo treatment.

In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition.,Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric analysis, Ion Torrent technology, immunofluorescence and biochemistry.,All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition.,However, melanoma cells with TGF-β1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF-β1-induced EMT melanoma cells, suggesting that TGF-β1-induced EMT was not associated with dabrafenib resistance.,Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected.,These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure.,Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression.,In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.

Loss of expression of surface antigens represents a significant problem for cancer immunotherapy.,Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes.,We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels.,Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines.,We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR.,Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma.,Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation.,This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy‐resistant melanomas.,Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX‐derived models.,We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy.,Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines.,Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment.,The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof.,Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF‐MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy.,Ganetespib is a potent ATP competitive inhibitor of HSP90.,Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations.,Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM.,Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition.,Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase.,Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21Cip1 and p27Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4.,HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition.

Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role.,In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors.,Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.,We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy.,We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples.,In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples.,No NRAS mutations were detected in pre-resistant samples.,By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells.,The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.,We conclude that BRAF and NRAS co-mutations are not mutually exclusive.,However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy.,In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells.,This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion.,We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells.,These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro.,Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells.,Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells.,Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels.,We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a deficient background, two frequent driver mutations in human melanoma.,Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain and liver metastases.,Whole genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis.,Notably, loss or knockdown of STAT3 in mouse or human cells resulted in up-regulation of MITF and induction of cell proliferation.,Mechanistically we show that STAT3-induced CEBPa/b expression was sufficient to suppress MITF transcription.,Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus.,Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression.,We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.

Integrins are a large family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions.,Among the 24 integrin isoforms, many have been found to be associated with tumor angiogenesis, tumor cell migration and proliferation, and metastasis.,Integrins, especially αvβ3, αvβ5 and α5β1, participate in mediating tumor angiogenesis by interacting with the vascular endothelial growth factor and angiopoietin-Tie signaling pathways.,Melanoma patients have a poor prognosis when the primary tumor has generated distant metastases, and the melanoma metastatic site is an independent predictor of the survival of these patients.,Different integrins on the melanoma cell surface preferentially direct circulating melanoma cells to different organs and promote the development of metastases at specific organ sites.,For instance, melanoma cells expressing integrin β3 tend to metastasize to the lungs, whereas those expressing integrin β1 preferentially generate lymph node metastases.,Moreover, tumor cell-derived exosomes which contain different integrins may prepare a pre-metastatic niche in specific organs and promote organ-specific metastases.,Because of the important role that integrins play in tumor angiogenesis and metastasis, they have become promising targets for the treatment of advanced cancer.,In this paper, we review the integrin isoforms responsible for angiogenesis and organ-specific metastasis in malignant melanoma and the inhibitors that have been considered for the future treatment of metastatic disease.

Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma.,Five-year outcomes in all patients and treatment-naive patients are reported herein.,Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed.,Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw.,Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated.,Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017).,KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months.,Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively.,Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively.,Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months.,Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab.,One patient each achieved CR and partial response (after data cut-off).,Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE.,This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma.,ClinicalTrials.gov, NCT01295827.

Metastatic melanoma (mM) and renal cell carcinoma (mRCC) are often treated with anti-PD-1 based therapy, however not all patients respond and further therapies are needed.,High dose interleukin-2 (HD IL-2) can lead to durable responses in a subset of mM and mRCC patients.,The efficacy and toxicity of HD IL-2 therapy following anti-PD-1 or anti-PD-L1 therapy have not yet been explored.,Reports on mM and mRCC patients who had received HD IL-2 after PD-1 or PD-L1 inhibition were queried from the PROCLAIMSM database.,Patient characteristics, toxicity and efficacy were analyzed.,A total of 57 patients (40 mM, 17 mRCC) were treated with high dose IL-2 after PD-1 or PD-L1 inhibition and had data recorded in the PROCLAIM database.,The best overall response rate to HD IL-2 was 22.5% for mM (4 complete response (CR), 5 partial responses (PRs)) and 24% for mRCC (2 CRs, 2 PRs).,The toxicity related to HD IL-2 observed in these patients was similar to that observed in patients treated with HD IL-2 without prior checkpoint blockade.,One patient who had received prior PD-L1 blockade developed drug induced pneumonitis with HD IL-2 requiring steroid therapy.,In this retrospective analysis, HD IL-2 therapy displayed durable antitumor activity in mM and mRCC patients who progressed following treatment with PD-1 and PD-L1 inhibition.,The toxicities were generally manageable and consistent with expectations from HD IL-2 but physicians should watch for immune related toxicities such as pneumonitis.,This analysis supports the development of randomized prospective trials to assess the proper sequencing and combination of immune checkpoint blockade and cytokine therapy.,The online version of this article (10.1186/s40425-019-0522-3) contains supplementary material, which is available to authorized users.

A vast array of tumor-derived genetic, proteomic and cellular components are constantly released into the circulation of cancer patients.,These molecules including circulating tumor DNA and RNA, proteins, tumor and immune cells are emerging as convenient and accurate liquid biomarkers of cancer.,Circulating cancer biomarkers provide invaluable information on cancer detection and diagnosis, prognosticate patient outcomes, and predict treatment response.,In this era of effective molecular targeted treatments and immunotherapies, there is now an urgent need to implement use of these circulating biomarkers in the clinic to facilitate personalized therapy.,In this review, we present recent findings in circulating melanoma biomarkers, examine the challenges and promise of evolving technologies used for liquid biomarker discovery, and discuss future directions and perspectives in melanoma biomarker research.

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs.,Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway.,We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs.,These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma.,They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors.,Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.,•pan-RAF inhibitors also inhibit SRC family kinases•The compounds do not induce paradoxical activation of ERK in RAS mutant cells•The compounds are active in BRAF and NRAS mutant melanomas•The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors,pan-RAF inhibitors also inhibit SRC family kinases,The compounds do not induce paradoxical activation of ERK in RAS mutant cells,The compounds are active in BRAF and NRAS mutant melanomas,The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors,Girotti et al. describe two pan-RAF inhibitors that also inhibit SRC-family kinases.,These compounds do not drive paradoxical MEK/ERK activation and can inhibit MEK in NRAS mutant cells.,Moreover, the agents can overcome resistance to clinical BRAF or combination BRAF/MEK inhibitors in patient-derived xenografts.

Autophagy is involved in cancer initiation and progression but its role in uveal melanoma (UM) was rarely investigated.,Herein, we built an autophagy-related gene (ARG) risk model of UM patients by univariate Cox regression and least absolute shrinkage and selection operator (Lasso) regression model and filtrated out nine prognostic ARGs in The Cancer Genome Atlas (TCGA) cohort.,Survival and Receiver Operating Characteristic (ROC) Curve analysis in the TCGA and other four independent UM cohorts (GSE22138, GSE27831, GSE44295 and GSE84976) proved that the ARG-signature possessed robust and steady prognosis predictive ability.,We calculated risk scores for patients included in our study and patients with higher risk scores showed worse clinical outcomes.,We found the expressions of the nine ARGs were significantly associated with clinical and molecular features (including risk score) and overall survival (OS) of UM patients.,Furthermore, we utilized univariate and multivariate Cox regression analyses to determine the independent prognostic ability of the ARG-signature.,Functional enrichment analysis showed the ARG-signature was correlated with several immune-related processes and pathways like T-cell activation and T-cell receptor signaling pathway.,Gene set enrichment analysis (GSEA) found tumor hallmarks including angiogenesis, IL6-JAK-STAT3-signaling, reactive oxygen species pathway and oxidative phosphorylation were enriched in high-risk UM patients.,Finally, infiltrations of several immune cells and immune-related scores were found significantly associated with the ARG-signature.,In conclusion, the ARG-signature might be a strong predictor for evaluating the prognosis and immune infiltration of UM patients.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults.,Many previous studies have demonstrated that the infiltrating of immune and stromal cells in the tumor microenvironment contributes significantly to prognosis.,Dataset TCGA-UVM, download from TCGA portal, was taken as the training cohort, and GSE22138, obtained from GEO database, was set as the validation cohort.,ESTIMATE algorithm was applied to find intersection differentially expressed genes (DEGs) among tumor microenvironment.,Kaplan-Meier analysis and univariate Cox regression model were performed on intersection DEGs to initial screen for potential prognostic genes.,Then these genes entered into the validation cohort for validation using the same methods as that in the training cohort.,Moreover, we conducted correlation analyses between the genes obtained in the validation cohort and the status of chromosome 3, chromosome 8q, and tumor metastasis to get prognosis genes.,At last, the immune infiltration analysis was performed between the prognostic genes and 6 main kinds of tumor-infiltrating immune cells (TICs) for understanding the role of the genes in the tumor microenvironment.,959 intersection DEGs were found in the UM microenvironment.,Kaplan-Meier and Cox analysis was then performed in the training and validation cohorts on these DEGs, and 52 genes were identified with potential prognostic value.,After comparing the 52 genes to chromosome 3, chromosome 8q, and metastasis, we obtained 21 genes as the prognostic genes.,The immune infiltration analysis showed that Neutrophil had the potential prognostic ability, and almost every prognostic gene we had identified was correlated with abundances of Neutrophil and CD8+ T Cell.,Identifying 21 prognosis genes (SERPINB9, EDNRB, RAPGEF3, HFE, RNF43, ZNF415, IL12RB2, MTUS1, NEDD9, ZNF667, AZGP1, WARS, GEM, RAB31, CALHM2, CA12, MYEOV, CELF2, SLCO5A1, ISM1, and PAPSS2) could accurately identify patients' prognosis and had close interactions with Neutrophil in the tumor environment, which may provide UM patients with personalized prognosis prediction and new treatment insights.

Circulating tumour cells (CTCs) can be assessed through a minimally invasive blood sample with potential utility as a predictive, prognostic and pharmacodynamic biomarker.,The large heterogeneity of melanoma CTCs has hindered their detection and clinical application.,Here we compared two microfluidic devices for the recovery of circulating melanoma cells.,The presence of CTCs in 43 blood samples from patients with metastatic melanoma was evaluated using a combination of immunocytochemistry and transcript analyses of five genes by RT-PCR and 19 genes by droplet digital PCR (ddPCR), whereby a CTC score was calculated.,Circulating tumour DNA (ctDNA) from the same patient blood sample, was assessed by ddPCR targeting tumour-specific mutations.,Our analysis revealed an extraordinary heterogeneity amongst melanoma CTCs, with multiple non-overlapping subpopulations.,CTC detection using our multimarker approach was associated with shorter overall and progression-free survival.,Finally, we found that CTC scores correlated with plasma ctDNA concentrations and had similar pharmacodynamic changes upon treatment initiation.,Despite the high phenotypic and molecular heterogeneity of melanoma CTCs, multimarker derived CTC scores could serve as viable tools for prognostication and treatment response monitoring in patients with metastatic melanoma.

Approximately 25% of melanoma patients with locoregional metastases are nonresponsive to new molecular target therapy and immunotherapy.,When metastases are located in the pelvis, melphalan hypoxic perfusion can be an optional treatment.,Because methylation of MGMT promoter increases the efficacy of alkylating agents, studies on melanoma outcome of patients treated with melphalan regional chemotherapy should consider this epigenetic change.,This study aims to evaluate whether the survival of stage III melanoma patients treated with melphalan regional chemotherapy may be correlated with MGMT methylation status.,The metastatic tissues of 27 stage III melanoma patients with locoregional metastases located in the pelvis subjected to melphalan hypoxic pelvic perfusion were examined.,The methylation status of the MGMT promoter was investigated by MS-MLPA probes analysis and the presence of the BRAF V600E mutation was analyzed by CAST-PCR.,The median survival times were estimated using the Kaplan-Meier curves and were stratified according to the clinicopathological characteristics of patients and lesions.,The overall median survival time was 17 months.,The 1-year, 3-year, and 5-year survival rates were 66.7, 18.5, and 7.4%, respectively.,Disease stage, burden, and percentage of MGMT methylation significantly affected survival.,We estimated an MGMT promoter methylation cut-off of at least 14%, which was significantly associated with a longer survival after melphalan regional chemotherapy.,Our data suggest that MGMT promoter methylation could be an important factor in determining which melanoma patients should receive melphalan regional chemotherapy, but its prognostic significance in the routine clinical setting needs to be clarified in a larger study.

Sorafenib, a multikinase inhibitor of cell proliferation and angiogenesis, inhibits the mitogen-activated protein kinase pathway that is activated in most uveal melanoma tumors.,This phase II study was conducted by the SWOG cooperative group to evaluate the efficacy of sorafenib in combination with carboplatin and paclitaxel (CP) in metastatic uveal melanoma.,Twenty-five patients with stage IV uveal melanoma who had received 0-1 prior systemic therapy were enrolled.,Treatment included up to 6 cycles of carboplatin (AUC = 6) and paclitaxel (225 mg/m2) administered IV on day 1 plus sorafenib (400 mg PO twice daily), followed by sorafenib monotherapy until disease progression.,The primary endpoint was objective response rate (ORR); a two-stage design was used with the study to be terminated if no confirmed responses were observed in the first 20 evaluable patients.,Secondary efficacy endpoints included progression-free survival (PFS) and overall survival (OS).,No confirmed objective responses occurred among the 24 evaluable patients (ORR = 0% [95% CI: 0-14%]) and the study was terminated at the first stage.,Minor responses (tumor regression less than 30%) were seen in eleven of 24 (45%) patients.,The median PFS was 4 months [95% CI: 1-6 months] and the 6-month PFS was 29% [95% CI: 13%-48%].,The median OS was 11 months [95% CI: 7-14 months].,In this study, the overall efficacy of CP plus sorafenib in metastatic uveal melanoma did not warrant further clinical testing when assessed by ORR, although minor tumor responses and stable disease were observed in some patients.,ClinicalTrials.govNCT00329641

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types.,Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas.,PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation.,In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells.,PEDF was regulated at the protein level.,Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia.,Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells.,Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells.,Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells.,Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.

Obesity is a risk factor for malignancy; however, its prognostic role in patients with metastatic melanoma is controversial.,We aim to investigate the prognostic role of body mass index (BMI) in patients with metastatic melanoma receiving mitogen-activated pathway kinase inhibitors (MAPKi), immune checkpoint inhibitors (ICIs) alone or their sequence.,Data on patients with metastatic melanoma receiving ≥1 line of systemic treatment were retrieved from prospectively collected databases.,Progression-free survival (PFS) and overall survival (OS) were analyzed by means of multivariable stratified Cox regression models; disease control rate (DCR) was analyzed by multivariable stratified logistic regression models.,Subgroup analyzes according to the type of treatments received, and in BRAF-mutated patients were pre-planned.,All multivariable models included BMI, age, gender, American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment sequencing strategy as covariates.,Between November 2010 and November 2018, 688 patients from three Italian and two Polish centers were enrolled. 379 (57%) patients had M1c/d disease, 273 (41%) were female and the mean BMI was 27.1 (SD=4.9).,Considering first-line treatment, 446 patients (66.8%) received ICIs and 222 MAPKi.,No impact of BMI on OS was detected either considering the first line of ICIs, or ICIs sequencing (HR=1.02, 95% CI: 0.99 to 1.05, p=0.202, and HR=1.02, 95% CI: 0.99 to 1.04, p=0.237, respectively).,A late effect of BMI on OS was found in patients treated with MAPKi: for five units increment, a 51% of risk reduction at 18 months and a 76% of risk reduction at 30 months were observed.,No significant effect of BMI on PFS and DCR was found in any of the subgroup analyzes.,In patients with metastatic melanoma receiving ICIs, there is no impact of BMI on DCR, PFS and OS.,The late prognostic effect of BMI in patients treated with MAPKi should be considered hypothesis generating and needs to be further investigated.

Coffee contains biologically-active substances that suppress carcinogenesis in vivo, and coffee consumption has been associated with a lower risk of malignant melanoma.,We studied the impact of total coffee consumption and of different brewing methods on the incidence of malignant melanoma in a prospective cohort of Norwegian women.,We had baseline information on total coffee consumption and consumption of filtered, instant, and boiled coffee from self-administered questionnaires for 104,080 women in the Norwegian Women and Cancer (NOWAC) Study.,We also had follow-up information collected 6-8 years after baseline.,Multiple imputation was used to deal with missing data, and multivariable Cox regression models were used to calculate hazard ratios (HR) for malignant melanoma by consumption category of total, filtered, instant, and boiled coffee.,During 1.7 million person-years of follow-up, 762 cases of malignant melanoma were diagnosed.,Compared to light consumers of filtered coffee (≤1 cup/day), we found a statistically significant inverse association with low-moderate consumption (>1-3 cups/day, HR = 0.80; 95 % confidence interval [CI] 0.66-0.98) and high-moderate consumption of filtered coffee (>3-5 cups/day, HR = 0.77; 95 % CI 0.61-0.97) and melanoma risk (ptrend = 0.02).,We did not find a statistically significant association between total, instant, or boiled coffee consumption and the risk of malignant melanoma in any of the consumption categories.,The data from the NOWAC Study indicate that a moderate intake of filtered coffee could reduce the risk of malignant melanoma.,The online version of this article (doi:10.1186/s12885-016-2586-5) contains supplementary material, which is available to authorized users.

Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour.,How PTEN loss mediates these effects is unknown.,Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of β-catenin independent of the PI3K-AKT-GSK3β axis.,The absence of PTEN leads to caveolin-1 (CAV1)-dependent β-catenin transcriptional modulation in vitro, cooperates with NRASQ61K to initiate melanomagenesis in vivo and induces efficient metastasis formation associated with E-cadherin internalization.,The CAV1-β-catenin axis is mediated by a feedback loop in which β-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells.,These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of β-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.,The mechanism by which PTEN mutation is melanomagenic is complicated by different PTEN functions in different cellular locations.,Here, the authors identify an alternative to membrane PI3K-AKT signalling, a caveolin-1-dependent PTEN pathway that induces nuclear localization and activation of β-catenin.

MAPK and AKT pathways are frequently co-activated in melanoma through overexpression of receptor tyrosine kinases, mutations in their signaling surrogates, such as RAS and BRAF, or loss of negative regulators such as PTEN.,Since RAS can be a positive upstream regulator of PI3-K, it has been proposed that the loss of PTEN and the activation of RAS are redundant events in melanoma pathogenesis (Tsao et al., 2000).,Here, in genetically engineered mouse models of cutaneous melanomas, we sought to better understand the genetic interactions between HRAS activation and PTEN inactivation in melanoma genesis and progression in vivo.,We showed that HRAS activation cooperates with Pten+/- and Ink4a/Arf-/- to increase melanoma penetrance and promote metastasis.,Correspondingly, gain- and loss-of-function studies established that Pten loss increases invasion and migration of melanoma cells and non-transformed melanocytes, and that such biological activity correlates with a shift to phosphorylation of AKT2 isoform and E-cadherin down-regulation.,Thus, Pten inactivation can drive the genesis and promote the metastatic progression of RAS activated Ink4a/Arf deficient melanomas.

The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic.,The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance.,Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer.,This specific inflammatory setting is evident in the affected tissue.,However, certain mediators can leak into the systemic circulation and affect the whole organism.,The present study analysed the complex inflammatory response in the sera of patients with MM of various stages.,Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed.,These targets were observed in immunohistochemical profiles of primary tumours from the same patients.,Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer-associated fibroblasts.,Growth factors such as hepatocyte growth factor, granulocyte-colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)-8, and cytokines IL-6, interferon-α and IL-1 receptor antagonist significantly differed in these patients compared with the healthy controls.,Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases.,Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood‐brain barrier.,By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps.,We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia‐like membrane protrusions towards the tumour cells.,Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route.,On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells.,When co‐culturing melanoma cells with cerebral endothelial cells, we observed N‐cadherin enrichment at melanoma‐melanoma and melanoma‐endothelial cell borders.,However, for breast cancer cells N‐cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo.,Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.

Melanin possess radioprotective and scavenging properties, and its presence can affect the behavior of melanoma cells, its surrounding environment and susceptibility to the therapy, as showed in vitro experiments.,To determine whether melanin presence in melanoma affects the efficiency of radiotherapy (RTH) we evaluated the survival time after RTH treatment in metastatic melanoma patients (n = 57).,In another cohort of melanoma patients (n = 84), the relationship between melanin level and pT and pN status was determined.,A significantly longer survival time was found in patients with amelanotic metastatic melanomas in comparison to the melanotic ones, who were treated with either RTH or chemotherapy (CHTH) and RTH.,These differences were more significant in a group of melanoma patients treated only with RTH.,A detailed analysis of primary melanomas revealed that melanin levels were significantly higher in melanoma cells invading reticular dermis than the papillary dermis.,A significant reduction of melanin pigmentation in pT3 and pT4 melanomas in comparison to pT1 and T2 tumors was observed.,However, melanin levels measured in pT3-pT4 melanomas developing metastases (pN1-3, pM1) were higher than in pN0 and pM0 cases.,The presence of melanin in metastatic melanoma cells decreases the outcome of radiotherapy, and melanin synthesis is related to higher disease advancement.,Based on our previous cell-based and clinical research and present research we also suggest that inhibition of melanogenesis can improve radiotherapy modalities.,The mechanism of relationship between melanogenesis and efficacy of RTH requires additional studies, including larger melanoma patients population and orthotopic, imageable mouse models of metastatic melanoma.

Thirteen common susceptibility loci have been reproducibly associated with cutaneous malignant melanoma (CMM).,We report the results of an international two-stage meta-analysis of 11 genome-wide association studies (GWAS, five unpublished) of CMM and Stage two datasets, totaling 15,990 cases and 26,409 controls.,Five loci not previously associated with CMM risk reached genome-wide significance (P < 5×10−8) as did two previously-reported but un-replicated loci and all thirteen established loci.,Novel SNPs fall within putative melanocyte regulatory elements, and bioinformatic and eQTL data highlight candidate genes including one involved in telomere biology.

Metastatic melanoma is characterized by a poor response to chemotherapy.,Furthermore, there is a lack of established predictive and prognostic markers.,In this single institution study, we correlated mutation status and expression levels of BRAF and NRAS to dacarbazine (DTIC) treatment response as well as progression-free and overall survival in a cohort of 85 patients diagnosed with advanced melanoma.,Neither BRAF nor NRAS mutation status correlated to treatment response.,However, patients with tumors harboring NRAS mutations had a shorter overall survival (p < 0.001) compared to patients with tumors wild-type for NRAS.,Patients having a clinical benefit (objective response or stable disease at 3 months) on DTIC therapy had lower BRAF and NRAS expression levels compared to patients progressing on therapy (p = 0.037 and 0.003, respectively).,For BRAF expression, this association was stronger among patients with tumors wild-type for BRAF (p = 0.005).,Further, low BRAF as well as NRAS expression levels were associated with a longer progression-free survival in the total population (p = 0.004 and <0.001, respectively).,Contrasting low NRAS expression levels, which were associated with improved overall survival in the total population (p = 0.01), low BRAF levels were associated with improved overall survival only among patients with tumors wild-type for BRAF (p = 0.013).,These findings indicate that BRAF and NRAS expression levels may influence responses to DTIC as well as prognosis in patients with advanced melanoma.,The online version of this article (doi:10.1007/s10585-013-9587-4) contains supplementary material, which is available to authorized users.

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas.,Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas.,Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS.,Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas.,This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain.,Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration.,These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

Epidemiological studies propose that obesity increases the risk of several cancers, including melanoma.,Obesity increases the expression of leptin, a multifunctional peptide produced predominantly by adipocytes which may promote tumor growth.,Several recently experiments have suggested that the tumors growth is in need of endothelial progenitor cell (EPC) dependent generation of new blood vessels.,Our objectives in the present study were to examine the effects of leptin on melanoma growth, circulating EPCs number and plasma levels of nitric oxide metabolites (NOx).,2 × 106 B16F10 melanoma cells were injected to thirty two C57BL6 mice subcutaneously.,The mice were randomly divided into 4 groups (n = 8) in 8th day.,Two groups were received twice daily intraperitoneal(i.p) injections of either PBS or recombinant murine leptin (1 μg/g initial body weight).,Two groups were received i.p. injections of either 9F8 an anti leptin receptor antibody or the control mouse IgG at 50 μg/mouse every 3 consecutive days.,By the end of the second week the animals were euthanized and blood samples and tumors were analyzed.,The tumor weight, EPC numbers and NOx level in leptin, PBS, 9F8, and IgG group were (3.2 ± 0.6, 1.7 ± 0.3, 1.61 ± 0.2,1.7 ± 0.3 g), (222.66 ± 36.5, 133.33 ± 171, 23.33 ± 18, 132.66 ± 27.26/ml of blood), and (22.47 ± 5.5, 12.30 ± 1.5, 6.26 ± 0.84, 15.75 ± 6.3 μmol/L) respectively.,Tumors weight and size, circulating EPC numbers and plasma levels of NOx were significantly more in the leptin than 9f8 and both control groups (p < 0.05).,The plasma concentration of NOx significantly decreased in 9f8 treated mice compare to control group (p < 0.05).,In conclusion, our observations indicate that leptin causes melanoma growth likely through increased NO production and circulating EPC numbers and consequently vasculogenesis.

A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells.,Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability.,In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported.,The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.,We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal.,Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated.,Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells.,In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells.,CXCR6+ cells produced more aggressive tumors.,CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.,The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology.,Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development.,Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type.,To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors.,Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2.,Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8.,SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas.,TERT aberrations and ATRX mutations are associated with alterations in telomere length.,Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.,Mucosal melanomas are challenging to treat partly because so little is known about the genetic drivers underpinning them.,Here, the authors perform a genomic landscape analysis of samples collected from three continents, revealing a potential role for CDK4/6 or MEK inhibition in the treatment of the disease.

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2.,BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors.,Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5.,For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events.,Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma.,SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish.,Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1.,Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Uveal melanoma (UM) is a severe human malignancy with a high mortality rate that demands continued research into new and alternative forms of prevention and treatment.,The emerging field of epigenetics is beginning to unfold an era of contemporary approaches to reducing the risk and improving the clinical treatment of UM.,Epigenetic changes have a high prevalence rate in cancer, are reversible in nature, and can lead to cancer characteristics even in mutation-free cells.,The information contained in this review highlights and expands on the main mechanisms of epigenetic dysregulation in UM tumorigenesis, progression and metastasis, including microRNA expression, hypermethylation of genes and histone modification.,Epigenetic drugs have been shown to enhance tumor suppressor gene expression and drug sensitivity in many other cancer cell lines and animal models.,An increased understanding of epigenetic mechanisms in UM will be invaluable in the design of more potent epigenetic drugs, which when used in combination with traditional therapies, may permit improved therapeutic outcomes.

We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas.,In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter.,Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+.,Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001).,PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively).,In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004).,PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter.,Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status.,Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA.,PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes.,Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Skin cancer is the most commonly diagnosed cancer type worldwide, and 80 % of skin cancers are basal cell carcinoma (BCC).,The main risk factor for developing BCC is exposure to ultraviolet radiation (UVR), particularly high-dose exposure at a young age.,Outdoor workers, particularly farmers, are at high risk of developing BCC.,However, studies of BCC in this population are scant.,To comprehensively evaluate all cases of BCC of the head and neck region treated during the years 2007-2013 at our hospital in Poland, and to compare the tumour characteristics in farmers to non-farmers.,Retrospective analysis of 312 patients treated for head and neck BCC during the study period (2007-2013).,Most patients (198 cases; 63 %) were males, with 114 females (37 %).,Median age was 73 years (range 32-96 years).,The most common tumour location was the nose and cheek (114 pts; 37 %) followed by the auricle (82 pts; 26 %), lips (54 pts; 18 %), scalp (26 pts; 8 %), and eye (36 pts; 12 %).,The most common disease stage on presentation was stage T2 (104 pts, 33 %), followed by stage T1 (79 pts; 25 %), stage T3 (89 pts; 28 %), and stage T4 (40 pts; 14 %).,By occupation, farmers accounted for 33 % of all patients (102 of 312 pts).,The most common tumour localisations in the farmer subgroup were the nose and cheek (50 pts; 49 %; p < 0.001; odds ratio [OR] 2.19; 95 % confidence interval [CI] 1.35-3.57), followed by the auricle (32 pts; 31 %), scalp (16 pts; 16 %), ocular region (3 pts; 3 %), and lips (1 pt; 1 %).,Patients in the farmer group were significantly younger than non-farmers (62 vs. 73 years; p < 0.001; OR 0.90, 95 % CI 0.88-0.93).,Farmers were significantly more likely to present disease recurrence (27 vs. 12 % of cases; p < 0.001; OR 5.94; 95 % CI 2.86-12.33).,The results highlight the increased incidence and risk of recurrence of BCC in farmers.,It is therefore necessary to consider enhancing educational programmes and other preventative measures in this occupational group and to evaluate the effectiveness of such programmes.

To estimate the proportion and numbers of cancers occurring in Australia attributable to solar ultraviolet radiation (UVR) and the proportion and numbers prevented by regular sun protection factor (SPF) 15+ sunscreen use.,We estimated the population attributable fraction (PAF) and numbers of melanomas and keratinocyte cancers (i.e. basal cell carcinomas and squamous cell carcinomas) due to exposure to ambient UVR resulting from residing in Australia versus residing in the UK (for melanoma) or Scandinavia (for keratinocyte cancers).,We also estimated the prevented fraction (PF): the proportion of cancers that would have occurred but were likely prevented by regular sunscreen use.,An estimated 7,220 melanomas (PAF 63%) and essentially all keratinocyte cancers occurring in Australia were attributable to high ambient UVR levels in Australia.,We estimated that regular sunscreen use prevented around 14,190 (PF 9.3%) and 1,730 (PF 14%) people from developing SCC and melanoma, respectively.,Although our approach was conservative, a high proportion of skin cancers in Australia are attributable to high ambient levels of UVR.,Prevailing levels of sunscreen use probably reduced skin cancer incidence by 10-15%.,Most skin cancers are preventable.,Sunscreen should be a component of a comprehensive sun protection strategy.

The long noncoding RNA, LINC00518, is highly expressed in various types of cancers and is involved in cancer progression.,Although LINC00518 promotes the metastasis of cutaneous malignant melanoma (CMM), the mechanism underlaying its effects on CMM radiosensitivity remains unclear.,In this study, LINC00518 expression was significantly upregulated in CMM samples, and LINC00518 levels were associated with poor prognosis of patients with CMM.,Knockdown of LINC00518 in CMM cells significantly inhibited cell invasion, migration, proliferation, and clonogenicity.,LINC00518-mediated invasion, migration, proliferation, and clonogenicity were negatively regulated by the microRNA, miR-33a-3p, in vitro, which increased sensitivity to radiotherapy via inhibition of the hypoxia-inducible factor 1α (HIF-1α)/lactate dehydrogenase A glycolysis axis.,Additionally, HIF-1α recognized the miR-33a-3p promoter region and recruited histone deacetylase 2, which decreased the expression of miR-33a-3p and formed an LINC00518/miR-33a-3p/HIF-1α negative feedback loop.,Furthermore, signaling with initially activated glycolysis and radioresistance in CMM cells was impaired by Santacruzamate A, a histone deacetylase inhibitor, and 2-deoxy-D-glucose, a glycolytic inhibitor.,Lastly, knockdown of LINC00518 expression sensitized CMM cancer cells to radiotherapy in an in vivo subcutaneously implanted tumor model.,In conclusion, LINC00518 was confirmed to be an oncogene in CMM, which induces radioresistance by regulating glycolysis through an miR-33a-3p/HIF-1α negative feedback loop.,Our study, may provide a potential strategy to improve the treatment outcome of radiotherapy in CMM.

In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma.,However, despite their growing implication in cancer, little is known about the role of long non-coding RNAs in melanoma progression.,We hypothesized that copy number alterations of intergenic non-protein coding domains could help identify long intergenic non-coding RNAs (lincRNAs) associated with metastatic cutaneous melanoma.,Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines.,The locus was actively transcribed in metastatic melanoma cells, and up-regulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model.,In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC stage III lymph node metastasis.,Moreover, siRNA knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states.,Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes.,These findings support a key role for CASC15 in metastatic melanoma.

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.

Activated RAS promotes dimerization of members of the RAF kinase family1-3.,ATP-competitive RAF inhibitors activate ERK signaling4-7 by transactivating RAF dimers4.,In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity.,This tumor-specific inhibition of ERK signaling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E)8.,However, resistance invariably develops.,Here, we identify a novel resistance mechanism.,We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E).,In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor.,Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib.,Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumors of six of 19 patients with acquired resistance to vemurafenib.,These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood.,Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment.,We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun.,MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression.,This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction.,Consistently, inflammatory MITFlow/c-Junhigh syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts.,Our study suggests myeloid cell-directed therapies may be useful for MITFlow/c-Junhigh melanomas to counteract their growth-promoting and immunosuppressive functions.,The c-Jun transcription factor can mediate a cell's response to TNFa.,Here, Riesenberg et al. show in melanoma cells that c-Jun has an inverse relationship with the key melanocyte transcription factor MITF and that high c-Jun levels contribute to melanoma heterogeneity and an inflammatory microenvironment.

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading.,Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event.,This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively.,Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors.,Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma.,Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.,The key regulators that allow transition from proliferative to invasive phenotype in melanoma cells have not been identified yet.,The authors perform chromatin and transcriptome profiling followed by comprehensive bioinformatics analysis identifying new candidate regulators for two distinct cell states of melanoma.

The heterogeneous behavior of patients with melanoma makes prognostication challenging.,To address this, a gene expression profile (GEP) test to predict metastatic risk was previously developed.,This study evaluates the GEP’s prognostic accuracy in an independent cohort of cutaneous melanoma patients.,This multi-center study analyzed primary melanoma tumors from 523 patients, using the GEP to classify patients as Class 1 (low risk) and Class 2 (high risk).,Molecular classification was correlated to clinical outcome and assessed along with AJCC v7 staging criteria.,Primary endpoints were recurrence-free (RFS) and distant metastasis-free (DMFS) survival.,The 5-year RFS rates for Class 1 and Class 2 were 88% and 52%, respectively, and DMFS rates were 93% versus 60%, respectively (P < 0.001).,The GEP was a significant predictor of RFS and DMFS in univariate analysis (hazard ratio [HR] = 5.4 and 6.6, respectively, P < 0.001 for each), along with Breslow thickness, ulceration, mitotic rate, and sentinel lymph node (SLN) status (P < 0.001 for each).,GEP, tumor thickness and SLN status were significant predictors of RFS and DMFS in a multivariate model that also included ulceration and mitotic rate (RFS HR = 2.1, 1.2, and 2.5, respectively, P < 0.001 for each; and DMFS HR = 2.7, 1.3 and 3.0, respectively, P < 0.01 for each).,The GEP test is an objective predictor of metastatic risk and provides additional independent prognostic information to traditional staging to help estimate an individual’s risk for recurrence.,The assay identified 70% of stage I and II patients who ultimately developed distant metastasis.,Its role in consideration of patients for adjuvant therapy should be examined prospectively.,The online version of this article (10.1186/s12885-018-4016-3) contains supplementary material, which is available to authorized users.

The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis.,This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM.,Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients.,Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles.,Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm.,The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses.,Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients.,Patients with a “favorable” methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis.,Median OS of stage IIIC patients with a “favorable” vs. “unfavorable” methylation profile were 31.5 and 10.4 months, respectively.,The 5 year OS for stage IIIC patients with a “favorable” methylation profile was 41.2% as compared to 0% for patients with an “unfavorable” methylation profile.,Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for “unfavorable” methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045).,A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.,A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients.,Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma.,In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy.,Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins.,Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma.,We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status.,Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry.,Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line.,Immunoblot was applied to analyze the IFN-ɣ signaling pathway.,For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed.,Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53.,Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53L22Q,W23S, a transcriptionally-impaired variant, in TP53-wt cells.,Accordingly, expression of p53L22Q,W23S in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression.,The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression.,While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression.,Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.

DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma.,We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi.,Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray.,Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥0.2.,Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of >0.95.,Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons.,This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.

Drug resistance remains a vexing problem in the treatment of cancer patients.,While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy.,Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anti-cancer drugs.,We found that stroma-mediated resistance is surprisingly common - particularly to targeted agents.,We further characterized the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most of these patients exhibit some degree of innate resistance1-4.,Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition.,Immunohistochemistry confirmed stromal HGF expression in patients with BRAF-mutant melanoma and a statistically significant correlation between stromal HGF expression and innate resistance to treatment.,Dual inhibition of RAF and MET resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma.,A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines.,More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown.,Using whole exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy.,Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance.,Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of CDKN2A mutations in this population.,These variants were not found in public databases or in 2,038 genotyped Italian controls.,We also identified two rare recurrent POT1 variants in American and French familial melanoma cases.,Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Previous studies have reported an association between sun exposure and improved cutaneous melanoma (CM) survival.,We analysed the association of UV exposure with prognostic factors and outcome in a large melanoma cohort.,A questionnaire was given to 289 (42%) CM patients at diagnosis (Group 1) and to 402 CM patients (58%) during follow-up (Group 2).,Analyses were carried out to investigate the associations between sun exposure and melanoma prognostic factors and survival.,Holidays in the sun two years before CM diagnosis were significantly associated with lower Breslow thickness (p=0.003), after multiple adjustment.,Number of weeks of sunny holidays was also significantly and inversely associated with thickness in a dose-dependent manner (p=0.007).,However when stratifying by gender this association was found only among women (p=0.0004) the risk of CM recurrence in both sexes was significantly lower in patients (n=271) who had holidays in the sun after diagnosis, after multiple adjustment including education: HR=0.30 (95%CI:0.10-0.87; p=0.03) conclusions: Holidays in the sun were associated with thinner melanomas in women and reduced rates of relapse in both sexes.,However, these results do not prove a direct causal effect of sun exposure on survival since other confounding factors, such as vitamin D serum levels and socio-economic status, may play a role.,Other factors in sun seeking individuals may also possibly affect these results.

Melanoma incidence rates have continued to increase in the United States, and risk behaviors remain high.,Melanoma is responsible for the most skin cancer deaths, with about 9,000 persons dying from it each year.,CDC analyzed current (2011) melanoma incidence and mortality data, and projected melanoma incidence, mortality, and the cost of treating newly diagnosed melanomas through 2030.,Finally, CDC estimated the potential melanoma cases and costs averted through 2030 if a comprehensive skin cancer prevention program was implemented in the United States.,In 2011, the melanoma incidence rate was 19.7 per 100,000, and the death rate was 2.7 per 100,000.,Incidence rates are projected to increase for white males and females through 2019.,Death rates are projected to remain stable.,The annual cost of treating newly diagnosed melanomas was estimated to increase from $457 million in 2011 to $1.6 billion in 2030.,Implementation of a comprehensive skin cancer prevention program was estimated to avert 230,000 melanoma cases and $2.7 billion in initial year treatment costs from 2020 through 2030.,If additional prevention efforts are not undertaken, the number of melanoma cases is projected to increase over the next 15 years, with accompanying increases in health care costs.,Much of this morbidity, mortality, and health care cost can be prevented.,Substantial reductions in melanoma incidence, mortality, and cost can be achieved if evidence-based comprehensive interventions that reduce ultraviolet (UV) radiation exposure and increase sun protection are fully implemented and sustained.

In a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours.,In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells.,To shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition.,A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound.,The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms.,In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways.,Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis.,Our findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.

Accumulating evidences indicated that plasmacytoma variant translocation 1 (PVT1) plays vital roles in several cancers.,However, the expression, functions, and clinical values of PVT1 in melanoma are still unknown.,In this study we measured the expression of PVT1 in clinical tissues and serum samples and explored the diagnostic value of PVT1 for melanoma and the effects of PVT1 on melanoma cell proliferation, cell cycle, and migration.,Our results, combined with publicly available PVT1 expression data, revealed that PVT1 is upregulated in melanoma tissues compared with nonneoplastic nevi tissues.,Serum PVT1 level is significantly increased in melanoma patients compared with age and gender-matched nonmelanoma controls with melanocytic nevus.,Receiver operating characteristic curve analyses revealed that serum PVT1 level could sensitively discriminate melanoma patients from controls.,Furthermore, serum PVT1 level indicted melanoma dynamics.,Functional experiments showed that overexpression of PVT1 promotes melanoma cells proliferation, cell cycle progression, and migration, while depletion of PVT1 significantly inhibits melanoma cells proliferation, cell cycle progression, and migration.,Collectively, our results indicate that PVT1 functions as an oncogene in melanoma and could be a potential diagnostic biomarker and therapeutic target for melanoma.

Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ∼50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized.,Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAFV600E protein.,To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAFV600E expression.,To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAFV600E-mutant human melanomas.,BRAFV600E regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts.,These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription.,Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs.,BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration.,BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11.,Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.

Malignant melanoma is a life-threatening form of skin cancer with a low response rate to single-agent chemotherapy.,Although combined therapies of metformin (MET) and doxorubicin (DOX) are effective in treating a variety of cancers, including breast cancer, their different physicochemical properties and administration routines reduce the effective co-accumulation of both drugs in tumors.,Nanoparticles (NPs) have been demonstrated to potentially improve drug delivery efficiency in cancer therapy of, for example, liver and lung cancers.,Hence, in this study, we prepared pH-sensitive, biocompatible, tumor-targeting NPs based on the conjugation of biomaterials, including sodium alginate, cholesterol, and folic acid (FCA).,As expected, since cholesterol and folic acid are two essentials, but insufficient, substrates for melanoma growth, we observed that the FCA NPs specifically and highly effectively accumulated in xenograft melanoma tumors.,Taking advantage of the FCA NP system, we successfully co-delivered a combination of MET and DOX into melanoma tumors to trigger pyroptosis, apoptosis, and necroptosis (PANoptosis) of the melanoma cells, thus blocking melanoma progression.,Combined, the establishment of such an FCA NP system provides a promising vector for effective drug delivery into melanoma and increases the possibility and efficiency of drug combinations for cancer treatment.

People with pale skin, red hair, freckles, and an inability to tan-the “redhair/fairskin” phenotype- are at highest risk of developing melanoma, compared to all other pigmentation types1.,Genetically, this phenotype is frequently the product of inactivating polymorphisms in the Melanocortin 1 receptor (MC1R) gene.,MC1R encodes a cAMP stimulating G-protein coupled receptor that controls pigment production.,Minimal receptor activity, as in redhair/fairskin polymorphisms, produces red/yellow pheomelanin pigment, while increasing MC1R activity stimulates production of black/brown eumelanin2.,Pheomelanin has weak UV shielding capacity relative to eumelanin and has been shown to amplify UVA-induced reactive oxygen species (ROS) 3-5.,Several observations, however, complicate the assumption that melanoma risk is completely UV dependent.,For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and UV signature mutations are infrequently oncogenic drivers6.,While linkage of melanoma risk to UV exposure is beyond doubt, UV-independent events are also likely to play a significant role1,7.,Here, we introduced into mice carrying an inactivating mutation in the Mc1r gene (who exhibit a phenotype analogous to redhair/fairskin humans), a conditional, melanocyte-targeted allele of the most commonly mutated melanoma oncogene, BRafV600E.,We observed a high incidence of invasive melanomas without providing additional gene aberrations or UV exposure.,To investigate the mechanism of UV-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r e/e background.,Selective absence of pheomelanin synthesis was protective against melanoma development.,In addition, normal Mc1re/e mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1re/e mouse skin.,These data suggest that the pheomelanin pigment pathway produces UV-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage.,While UV protection remains important, additional strategies may be required for optimal melanoma prevention.

Long noncoding RNAs (lncRNAs) are recognized as a new area for cancer therapy.,B-cell lymphoma-2 (Bcl-2)-mediated suppression of apoptosis is an important molecular hallmark of cancer.,However, the influence of lncRNA on the regulation of oncogenic Bcl-2 in cancer stem cells has not been explored.,In this study, our findings revealed that the lncRNA LHFPL3-AS1-long, generated from the polypyrimidine tract binding protein 1 (PTBP1)-mediated splicing of the LHFPL3-AS1 precursor, upregulated BCL2 protein to contribute to tumorigenesis of melanoma stem cells.,The in vitro and in vivo results showed that LHFPL3-AS1-long directly interacted with miR-181a-5p to inhibit the mRNA degradation of Bcl-2 (the target of miR-181), thus suppressing apoptosis of melanoma stem cells.,The splicing factor PTBP1 regulated the alternative splicing of LHFPL3-AS1 transcript by preferentially binding to the motifs located in exon3 of LHFPL3-AS1 precursor, leading to the biogenesis of LHFPL3-AS1-long in melanoma stem cells.,In patients with melanoma, the expressions of PTBP1 and LHFPL3-AS1 were significantly upregulated compared with the healthy donors.,Therefore, our study revealed a mechanistic crosstalk among an onco-splicing factor, lncRNA and tumorigenesis of melanoma stem cells, enabling PTBP1 and LHFPL3-AS1 to serve as the attractive therapeutic targets for melanoma.

The aim of this study was to identify long non-coding RNAs (lncRNAs) which may prove useful for risk-classifying patients with melanoma.,For this purpose, based on a dataset from The Cancer Genome Atlas (TCGA), we selected and analyzed samples from melanoma stages I, II, III and IV, from which differentially expressed lncRNAs were identified.,The lncRNAs were classified using two-way hierarchical clustering analysis and analysis of support vector machine (SVM), followed by Kaplan-Meier survival analysis.,The prognostic capacity of the signature was verified on an independent dataset. lncRNA-mRNA networks were built using signature lncRNAs and corresponding target genes.,The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was conducted on the target genes.,A total of 48 differentially expressed lncRNAs were identified, from which 6 signature lncRNAs (AL050303 and LINC00707, LINC01324, RP11-85G21, RP4-794I6.4 and RP5-855F16) were identified.,Two-way hierarchical clustering analysis revealed that the accuracy of the six-lncRNA signature in risk-stratifying samples was 84.84%, and the accuracy of the SVM classifier was 85.9%.,This predictive signature performed well on the validation dataset [accuracy, 86.76; area under the ROC curve (AUROC), 0.816].,A total of 720 target genes of the 6 lncRNAs were selected for the lncRNA-mRNA networks.,These genes were significantly related to mitogen-activated protein kinase (MAPK), the neurotrophin signaling pathway, focal adhesion pathways, and several immune and inflammation-related pathways.,On the whole, we identified a six-lncRNA prognostic signature for risk-stratifying patients with melanoma.,These lncRNAs may affect prognosis by regulating the MAPK pathway, immune and inflammation-related pathways, the neurotrophin signaling pathway and focal adhesion pathways.

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients.,Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi.,In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively.,In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi.,These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib.,In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF.,Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.

Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50-70% of malignant melanomas1.,Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1-5-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6-8.,However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9-11.,Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12.,Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor.,We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines.,COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling.,Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition.,We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting.,Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50-70% of malignant melanomas1.,Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1-5-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6-8.,However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9-11.,Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12.,Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor.,We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines.,COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling.,Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition.,We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting.,Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

B-RAF is the most frequently mutated protein kinase in human cancers.1 The finding that oncogenic mutations in BRAF are common in melanoma2 followed by the demonstration that these tumors are dependent on the RAF/MEK/ERK pathway3 offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients.,Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity.,Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts.4 Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032.5 In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment.,This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumor regressions.,At higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of ERK phosphorylation in the tumors of patients correlated with clinical response.,Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

We conducted the phase III double-blind European Organisation for Research and Treatment of Cancer (EORTC) 1325/KEYNOTE-054 trial to evaluate pembrolizumab versus placebo in patients with resected high-risk stage III melanoma.,On the basis of 351 recurrence-free survival (RFS) events at a 1.25-year median follow-up, pembrolizumab prolonged RFS (hazard ratio [HR], 0.57; P < .0001) compared with placebo.,This led to the approval of pembrolizumab adjuvant treatment by the European Medicines Agency and US Food and Drug Administration.,Here, we report an updated RFS analysis at the 3.05-year median follow-up.,A total of 1,019 patients with complete lymph node dissection of American Joint Committee on Cancer Staging Manual (seventh edition; AJCC-7), stage IIIA (at least one lymph node metastasis > 1 mm), IIIB, or IIIC (without in-transit metastasis) cutaneous melanoma were randomly assigned to receive pembrolizumab at a flat dose of 200 mg (n = 514) or placebo (n = 505) every 3 weeks for 1 year or until disease recurrence or unacceptable toxicity.,The two coprimary end points were RFS in the overall population and in those with programmed death-ligand 1 (PD-L1)-positive tumors.,Pembrolizumab (190 RFS events) compared with placebo (283 RFS events) resulted in prolonged RFS in the overall population (3-year RFS rate, 63.7% v 44.1% for pembrolizumab v placebo, respectively; HR, 0.56; 95% CI, 0.47 to 0.68) and in the PD-L1-positive tumor subgroup (HR, 0.57; 99% CI, 0.43 to 0.74).,The impact of pembrolizumab on RFS was similar in subgroups, in particular according to AJCC-7 and AJCC-8 staging, and BRAF mutation status (HR, 0.51 [99% CI, 0.36 to 0.73] v 0.66 [99% CI, 0.46 to 0.95] for V600E/K v wild type).,In resected high-risk stage III melanoma, pembrolizumab adjuvant therapy provided a sustained and clinically meaningful improvement in RFS at 3-year median follow-up.,This improvement was consistent across subgroups.

Talimogene laherparepvec is an oncolytic immunotherapy approved in the US, Europe, Australia and Switzerland.,We report the final planned analysis of OPTiM, a randomized open-label phase III trial in patients with unresectable stage IIIB-IVM1c melanoma.,Patients were randomized 2:1 to receive intratumoral talimogene laherparepvec or subcutaneous recombinant GM-CSF.,In addition to overall survival (OS), durable response rate (DRR), objective response rate (ORR), complete responses (CR), and safety are also reported.,All final analyses are considered to be descriptive and treatment responses were assessed by the investigators.,Of 436 patients in the intent-to-treat population, 295 were allocated to talimogene laherparepvec and 141 to GM-CSF.,Median follow-up in the final OS analysis was 49 months.,Median OS was 23.3 months (95% confidence interval [CI], 19.5-29.6) and 18.9 months (95% CI, 16.0-23.7) in the talimogene laherparepvec and GM-CSF arms, respectively (unstratified hazard ratio, 0.79; 95% CI, 0.62-1.00; p = 0.0494 [descriptive]).,DRR was 19.0 and 1.4% (unadjusted odds ratio, 16.6; 95% CI, 4.0-69.2; p < 0.0001); ORR was 31.5 and 6.4%.,Fifty (16.9%) and 1 (0.7%) patient in the talimogene laherparepvec and GM-CSF arms, respectively, achieved CR.,In talimogene laherparepvec-treated patients, median time to CR was 8.6 months; median CR duration was not reached.,Among patients with a CR, 88.5% were estimated to survive at a 5-year landmark analysis.,Talimogene laherparepvec efficacy was more pronounced in stage IIIB-IVM1a melanoma as already described in the primary analysis.,The safety reporting was consistent with the primary OPTiM analysis.,In this final planned OPTiM analysis, talimogene laherparepvec continued to result in improved longer-term efficacy versus GM-CSF and remained well tolerated.,The final analysis also confirms that talimogene laherparepvec was associated with durable CRs that were associated with prolonged survival.,ClinicalTrials.gov identifier: NCT00769704.,The online version of this article (10.1186/s40425-019-0623-z) contains supplementary material, which is available to authorized users.

In recent years, a significant number of studies have investigated the preventive role of vitamin D in a number of different neoplasms.,In this study, we analyze various components of the vitamin D signaling pathways in the human uveal tract and uveal melanoma, including analysis of the expression of vitamin D receptors (VDR), the activating and inactivating hydroxylases, respectively, CYP27B1 and CYP24A1, and the retinoic acid-related orphan receptors (ROR) α (RORα) and γ (RORγ) in these tissues.,We further analyzed the expression of VDR, CYP27B1, CYP24A1, and ROR in relation to melanin levels, clinical stage and prognosis.,Our study indicated that the uveal melanoma melanin level inversely correlated with VDR expression.,We further showed that vitamin D is metabolized in uveal melanoma.,This is significant because until now there has been no paper published, that would describe presence of VDR, hydroxylases CYP27B1 and CYP24A1, and RORα and RORγ in the human uveal tract and uveal melanomas.,The outcomes of our research can contribute to the development of new diagnostic and therapeutic methods in uveal tract disorders, especially in uveal melanoma.,The presented associations between vitamin D signaling elements and uveal melanoma in comparison to uveal tract encourage future clinical research with larger patients’ population.

Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance.,Nuclear factor-κB (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action.,Cultured melanoma cells were used for mechanistic studies on NF-κB activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays.,Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-κB activity in situ.,Novel 20-hydroxyvitamin (20(OH)D3) and classical 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) secosteroids inhibited melanoma cell proliferation.,Active forms of vitamin D were found to inhibit NF-κB activity in nonpigmented cells, while having no effect on pigmented cells.,Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-κB DNA binding and NF-κB-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-κB subunit and its accumulation in the cytoplasm.,Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-κB p65 expression than highly pigmented melanomas.,Classical 1,25(OH)2D3 and novel 20(OH)D3 hydroxyderivatives of vitamin D3 can target NF-κB and regulate melanoma progression in nonpigmented melanoma cells.,Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D3 and 1,25(OH)2D3 treatment.

Nonmelanoma skin cancers (NMSCs), are among the most common human malignancies.,Current methods for their prevention include avoidance of natural and artificial sources of UV radiation, photoprotective clothing and sunscreens.,However, these methods have proven to be inadequate in stemming the rise in skin cancer incidence over the past several years.,There is accumulating evidence that cyclooxygenase-2 (COX-2), an enzyme involved in prostaglandin synthesis, may be involved in the pathogenesis of NMSC.,In preclinical studies, animals genetically deficient in the COX-2 enzyme or that have been treated with pharmacological inhibitors of COX-2 develop significantly fewer tumors when subjected to a UV-induced skin carcinogenesis protocol than control mice.,Several epidemiological studies in humans support the concept that this enzyme is intimately involved in UV-induced skin cancer development, and UV radiation is known to augment COX-2 expression in human skin.,Recent studies suggest that drugs that block COX-2 expression may prevent the development of NMSCs.,Thus, pharmacologic agents that inhibit the enzyme cyclooxygenase-2 may be effective chemopreventive agents for NMSCs.

Merkel cell carcinoma is a very rare and aggressive neoplasm.,Due to its rarity, therapeutic guidelines are not well established, especially for regionally advanced disease.,Articles in English, French, Italian, Portuguese, and Spanish from the last 20 years were identified in MEDLINE and reviewed.,The key word “Merkel” was used for the search, relevant articles were selected, and their references were examined.,The most important articles related to epidemiology, genesis and treatment were reviewed.,The incidence of Merkel cell carcinoma is increasing due to the advancing age of the population, higher rates of sun exposure and an increasing number of immunocompromised individuals.,With regard to etiology, the recently described Merkel Cell polyomavirus is thought to play a role.,Either local or regional surgical intervention remains the standard of care, but adjuvant radiotherapy or radiotherapy as a primary treatment have been discussed as reasonable therapeutic options.,An update on this rare neoplasia is essential because of its increasing incidence and changing treatment options.

Uveal melanoma (UM), the most common ocular malignancy, is characterized by GNAQ/11 mutations.,Hippo/YAP and Ras/mitogen-activated protein kinase (MAPK) emerge as two important signaling pathways downstream of G protein alpha subunits of the Q class (GαQ/11)-mediated transformation, although whether and how they contribute to UM genesis in vivo remain unclear.,Here, we adapt an adeno-associated virus (AAV)-based ocular injection method to directly deliver Cre recombinase into the mouse uveal tract and demonstrate that Lats1/2 kinases suppress UM formation specifically in uveal melanocytes.,We find that genetic activation of YAP, but not Kras, is sufficient to initiate UM.,We show that YAP/TAZ activation induced by Lats1/2 deletion cooperates with Kras to promote UM progression via downstream transcriptional reinforcement.,Furthermore, dual inhibition of YAP/TAZ and Ras/MAPK synergizes to suppress oncogenic growth of human UM cells.,Our data highlight the functional significance of Lats-YAP/TAZ in UM initiation and progression in vivo and suggest combination inhibition of YAP/TAZ and Ras/MAPK as a new therapeutic strategy for UM.

Metastatic uveal melanoma (UM) has a very poor prognosis and no effective therapy.,Despite remarkable advances in treatment of cutaneous melanoma, UM remains recalcitrant to chemotherapy, small-molecule kinase inhibitors, and immune-based therapy.,We assessed two sets of oxidative phosphorylation (OxPhos) genes within 9858 tumors across 31 cancer types.,An OxPhos inhibitor was used to characterize differential metabolic programming of highly metastatic monosomy 3 (M3) UM.,Seahorse analysis and global metabolomics profiling were done to identify metabolic vulnerabilities.,Analyses of UM TCGA data set were performed to determine expressions of key OxPhos effectors in M3 and non-M3 UM.,We used targeted knockdown of succinate dehydrogenase A (SDHA) to determine the role of SDHA in M3 UM in conferring resistance to OxPhos inhibition.,We identified UM to have among the highest median OxPhos levels and showed that M3 UM exhibits a distinct metabolic profile.,M3 UM shows markedly low succinate levels and has highly increased levels of SDHA, the enzyme that couples the tricarboxylic acid cycle with OxPhos by oxidizing (lowering) succinate.,We showed that SDHA-high M3 UM have elevated expression of key OxPhos molecules, exhibit abundant mitochondrial reserve respiratory capacity, and are resistant to OxPhos antagonism, which can be reversed by SDHA knockdown.,Our study has identified a critical metabolic program within poor prognostic M3 UM.,In addition to the heightened mitochondrial functional capacity due to elevated SDHA, M3 UM SDHA-high mediate resistance to therapy that is reversible with targeted treatment.

Uveal melanoma represents ∼85% of all ocular melanomas and up to 50% of patients develop metastatic disease.,Metastases are most frequently localised to the liver and, as few patients are candidates for potentially curative surgery, this is associated with a poor prognosis.,There is currently little published evidence for the optimal management and treatment of metastatic uveal melanoma and the lack of effective therapies in this setting has led to the widespread use of systemic treatments for patients with cutaneous melanoma.,Uveal and cutaneous melanomas are intrinsically different diseases and so dedicated management strategies and therapies for uveal melanoma are much needed.,This review explores the biology of uveal melanoma and how this relates to ongoing trials of targeted therapies in the metastatic disease setting.,In addition, we consider the options to optimise patient management and care.

Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM).,While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated.,Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM.,UM cells were examined for expression of HIF-1α, VEGF, and ANGPTL4.,Their contribution to the angiogenic potential of UM cells was assessed using the endothelial cell tubule formation and directed in vivo angiogenesis assays.,These results were corroborated in tissue from UM animal models and in tissue from patients with UM.,Inhibition of VEGF partially reduced tubule formation promoted by conditioned medium from UM cells.,Inhibition of ANGPTL4, which was highly expressed in hypoxic UM cells, a UM orthotopic transplant model, a UM tumor array, and vitreous samples from UM patients, inhibited the angiogenic potential of UM cells in vitro and in vivo; this effect was additive to VEGF inhibition.,Targeting both ANGPTL4 and VEGF may be required for the effective inhibition of angiogenesis in UM.

Primary cutaneous lymphoepithelioma-like carcinoma is a rare disease with low metastatic potential.,Its morphologic and pathological features are similar to those of nasopharyngeal lymphoepithelial carcinoma.,We report the case of a 60-year-old man with an infrapalpebral pearly papule, measuring 0.6 cm in diameter.,The lesion was excised with a clinical hypothesis of basal cell carcinoma or squamous cell carcinoma.,Histopathological analysis revealed a malignant neoplasm with syncytial arrangement of cells with vesicular nuclei, associated with dense lymphocytic infiltrate.,Immunohistochemistry revealed cytokeratin-positive cells (AE1/AE3) and p63 protein, indicating epithelial histogenesis and squamous differentiation.,A negative Epstein-Barr virus test result was achieved by immunohistochemistry.,Primary lymphoepithelioma-like carcinoma of the skin is a differential diagnosis of lesions with prominent inflammatory infiltrates.

Aquaporin 3 (AQP3) and phospholipase D2 (PLD2) are abnormally expressed and/or localized in squamous cell carcinoma (SCC).,AQP3 transports glycerol to PLD2 for the synthesis of lipid second messenger, which can mediate the effect of the AQP3/PLD2 signaling module in the regulation of keratinocyte proliferation and differentiation.,However, the role of the AQP3/PLD2 signaling module in the pathogenesis of SCC remains to be fully elucidated.,In the present study, the expression levels of AQP3 and PLD2 in tissue samples were examined using immunohistochemistry, it was found that the expression levels of AQP3 and PLD2 in tissue samples of actinic keratosis (AK), Bowen's disease (BD) and SCC were significantly increased.,AQP3 small interfering RNA (siRNA) and PLD2 siRNA were constructed and used for transfection into the human A431 SCC cell line, and their anticancer effect on SCC was examined.,The mRNA expression and protein expression levels of AQP3 and PLD2 were significantly downregulated following siRNA transfection.,AQP3 siRNA and PLD2 siRNA inhibited the proliferation and promoted the apoptosis of A431 cells.,Taken together, the findings of the present study suggested that increased levels of AQP3 and PLD2 were correlated with tumor progression and development in SCC.,AQP3 siRNA and PLD2 siRNA significantly downregulated the mRNA and protein levels of AQP3 and PLD2 in the A431 cells; inhibiting proliferation and promoting apoptosis in vitro.,The concomitant effects of AQP3/PLD2 signaling by inhibiting the expression of siRNA may be important for the treatment of SCC in the future.

The development of brain metastases in patients with advanced stage melanoma is common, but the molecular mechanisms responsible for their development are poorly understood.,Melanoma brain metastases cause significant morbidity and mortality and confer a poor prognosis; traditional therapies including whole brain radiation, stereotactic radiotherapy, or chemotherapy yield only modest increases in overall survival (OS) for these patients.,While recently approved therapies have significantly improved OS in melanoma patients, only a small number of studies have investigated their efficacy in patients with brain metastases.,Preliminary data suggest that some responses have been observed in intracranial lesions, which has sparked new clinical trials designed to evaluate the efficacy in melanoma patients with brain metastases.,Simultaneously, recent advances in our understanding of the mechanisms of melanoma cell dissemination to the brain have revealed novel and potentially therapeutic targets.,In this review, we provide an overview of newly discovered mechanisms of melanoma spread to the brain, discuss preclinical models that are being used to further our understanding of this deadly disease and provide an update of the current clinical trials for melanoma patients with brain metastases.

Here, we retrospectively review imaging of 68 consecutive unselected patients with BRAF V600‐mutant metastatic melanoma for organ‐specific response and progression on vemurafenib.,Complete or partial responses were less often seen in the central nervous system (CNS) (36%) and bone (16%) compared to lung (89%), subcutaneous (83%), spleen (71%), liver (85%) and lymph nodes/soft tissue (83%), P < 0.001.,CNS was also the most common site of progression.,Based on this, we tested in vitro the efficacy of the BRAF inhibitors PLX4720 and dabrafenib in the presence of cerebrospinal fluid (CSF).,Exogenous CSF dramatically reduced cell death in response to both BRAF inhibitors.,Effective cell killing was restored by co‐administration of a PI‐3 kinase inhibitor.,We conclude that the efficacy of vemurafenib is variable in different organs with CNS being particularly prone to resistance.,Extrinsic factors, such as ERK‐ and PI3K‐activating factors in CSF, may mediate BRAF inhibitor resistance in the CNS.

Metastatic melanoma is the most aggressive and difficult to treat type of skin cancer, with a survival rate of less than 10%.,Metastatic melanoma has conventionally been considered very difficult to treat; however, recent progress in understanding the cellular and molecular mechanisms involved in the tumorigenesis, metastasis and immune escape have led to the introduction of new therapies.,These include targeted molecular therapy and novel immune-based approaches such as immune checkpoint blockade (ICB), tumor-infiltrating lymphocytes (TILs), and genetically engineered T-lymphocytes such as chimeric antigen receptor (CAR) T cells.,Among these, CAR T cell therapy has recently made promising strides towards the treatment of advanced hematological and solid cancers.,Although CAR T cell therapy might offer new hope for melanoma patients, it is not without its shortcomings, which include off-target toxicity, and the emergence of resistance to therapy (e.g., due to antigen loss), leading to eventual relapse.,The present review will not only describe the basic steps of melanoma metastasis, but also discuss how CAR T cells could treat metastatic melanoma.,We will outline specific strategies including combination approaches that could be used to overcome some limitations of CAR T cell therapy for metastatic melanoma.

The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts.,Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively.,The strong antimetastatic activity of MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10 melanoma lung metastases in drug-treated mice.,Our data also show that MC3181 affects the lactate production in the high glycolytic WM266.4 cell line.,To unveil the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the degree of activation of different MAPK pathways, as well as the expression/activity levels of several proteins involved in angiogenesis, invasion, and survival (i.e.,AP2, MCAM/MUC18, N-cadherin, VEGF and MMP-2).,Our data disclosed both a decrease of the phospho-active form of JNK and an increased expression of the transcription factor AP2, events that occur in the very early phase of drug treatment and may be responsible of the antimetastatic effects of MC3181.

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes.,EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer.,The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized.,We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology.,In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration.,At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility.,Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition.,Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies.,This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2.,BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors.,Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5.,For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events.,Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma.,SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish.,Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1.,Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma.,Five-year outcomes in all patients and treatment-naive patients are reported herein.,Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed.,Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw.,Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated.,Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017).,KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months.,Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively.,Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively.,Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months.,Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab.,One patient each achieved CR and partial response (after data cut-off).,Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE.,This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma.,ClinicalTrials.gov, NCT01295827.

Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge.,We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF.,A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression.,Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors.,Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors.,We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.,•MITF is a driver of a reversible non-mutational drug-tolerance phase in melanoma•Drug repositioning identifies nelfinavir mesylate as a suppressor of MITF expression•Nelfinavir sensitizes BRAF and NRAS mutant melanoma to MAPK inhibitor treatment•A nelfinavir combination therapy overcomes NRAS-driven acquired resistance,MITF is a driver of a reversible non-mutational drug-tolerance phase in melanoma,Drug repositioning identifies nelfinavir mesylate as a suppressor of MITF expression,Nelfinavir sensitizes BRAF and NRAS mutant melanoma to MAPK inhibitor treatment,A nelfinavir combination therapy overcomes NRAS-driven acquired resistance,Smith et al. discover PAX3-mediated overexpression of MITF as a reversible resistance mechanism to MAPK-pathway inhibition in BRAF mutant melanomas and identify nelfinavir, which inhibits this mechanism and sensitizes not only BRAF mutant but also BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors.

Metastatic melanoma is the most aggressive skin cancer.,Recently, phenotypically distinct subpopulations of tumor cells were identified.,Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties.,In addition, ABCB5+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5.,Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified.,Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells.,Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression.,These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine.,In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs.,Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5.,This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.

The question whether tumorigenic cancer stem cells exist in human melanomas has arisen recently1.,Here we show that in melanomas, tumor stem cells (MTSC) can be isolated prospectively as a highly enriched CD271+ MTSC population using a process that maximizes viable cell transplantation1,6.,In this study the tumors sampled were taken from a broad spectrum of sites and stages.,High viability FACS isolated cells resuspended in a matrigel vehicle were implanted into T, B, and NK deficient Rag2−/− γc−/− mice (RG) mice.,The CD271+ subset of cells was the tumor initiating population in 9/10 melanomas tested.,Transplantation of isolated melanoma cells into engrafted human skin or bone in RG mice resulted in melanoma from CD271+ but not CD271− cells.,We also showed that tumors transplanted by CD271+ patient cells were capable of metastasis in-vivo.,Importantly, CD271+ melanoma cells lacked expression of TYR, MART and MAGE in 86%, 69% and 68% of melanoma patients respectively suggesting why T cell therapies directed at these antigens usually result in only temporary tumor shrinkage.

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors.,In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model.,We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4′,6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways).,Further, the anticancer effect in vivo was confirmed through a xenograft model.,Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner.,In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis.,Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins.,Additionally, shikonin was overexpressed in MAPK pathways.,Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.,Furthermore, pathologic changes were not observed in the liver and kidney of mice.,Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.

Genetically engineered T cells expressing a T-cell receptor (TCR) are powerful tools for cancer treatment and have shown significant clinical effects in sarcoma patients.,However, mismatch of the introduced TCR α/β chains with endogenous TCR may impair the expression of transduced TCR, resulting in an insufficient antitumor capacity of modified T cells.,Here, we report the development of immunotherapy using human lymphocytes transduced with a codon-optimized melanoma-associated antigen (MAGE)-A4 and HLA-A*2402-restricted TCR, which specifically downregulate endogenous TCR by small interfering RNA (si-TCR).,We evaluated the efficacy of this immunotherapy in both NOD-SCID mice and uterine leiomyosarcoma patients.,Our results revealed that transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR, enhanced cytotoxic activity against antigen-expressing tumor cells, and increased interferon-γ production by specific MAGE-A4 peptide stimulation.,Retarded tumor growth was also observed in NOD-SCID mice inoculated with human tumor cell lines expressing both MAGE-A4 and HLA-A*2402.,Furthermore, we report the successful management of a case of uterine leiomyosarcoma treated with MAGE-A4 si-TCR/HLA-A*2402 gene-modified T cells.,Our results indicate that the TCR-modified T cell therapy is a promising novel strategy for cancer treatment.

Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases.,Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment.,The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients.,A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein.,Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank.,The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences.,In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues.,Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.

The identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity.,This has been well documented in colon cancer.,However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes of cancer, suggesting that galectin-7 may have a dual role in cancer progression.,In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung.,In the present work, we investigated the expression and function of galectin-7 in melanoma.,An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma.,This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis.,Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis.,Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression.,Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells.,It also failed to modulate the dissemination of B16F1 cells to the lung.

The presence of T cells in tumors predicts overall survival for cancer patients.,However, why most tumors are poorly infiltrated by T cells is barely understood.,T-cell recruitment towards the tumor requires a chemokine gradient of the critical IFNγ-induced chemokines CXCL9/10/11.,Here, we describe how tumors can abolish IFNγ-induced chemokines, thereby reducing T-cell attraction.,This mechanism requires extracellular galectin-3, a lectin secreted by tumors.,Galectins bind the glycans of glycoproteins and form lattices by oligomerization.,We demonstrate that galectin-3 binds the glycans of the extracellular matrix and those decorating IFNγ.,In mice bearing human tumors, galectin-3 reduces IFNγ diffusion through the tumor matrix.,Galectin antagonists increase intratumoral IFNγ diffusion, CXCL9 gradient and tumor recruitment of adoptively transferred human CD8+ T cells specific for a tumor antigen.,Transfer of T cells reduces tumor growth only if galectin antagonists are injected.,Considering that most human cytokines are glycosylated, galectin secretion could be a general strategy for tumor immune evasion.,Most tumours are poorly infiltrated by T cells.,Here the authors show that galectin-3 secreted by tumours binds both glycosylated IFNγ and glycoproteins of the tumour extracellular matrix, thus avoiding IFNγ diffusion and the formation of an IFNγ-induced chemokine gradient required for T cell infiltration.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, known to play important roles in melanoma formation and progression.,Recent advances in genome-wide methylation methods have provided the means to identify differentially methylated genes, methylation signatures, and potential biomarkers.,However, despite considerable effort and advances in cataloging methylation changes in melanoma, many questions remain unanswered.,The aim of this review is to summarize recent developments, emerging trends, and important unresolved questions in the field of aberrant DNA methylation in melanoma.,In addition to reviewing recent developments, we carefully synthesize the findings in an effort to provide a framework for understanding the current state and direction of the field.,To facilitate clarity, we divided the review into DNA methylation changes in melanoma, biomarker opportunities, and therapeutic developments.,We hope this review contributes to accelerating the utilization of the diagnostic, prognostic, and therapeutic potential of DNA methylation for the benefit of melanoma patients.

While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined.,Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion.,In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively.,Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear.,Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells.,Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2.,A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype.,Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.,•NFIB mediates a slow cycling, highly invasive/migratory melanoma cell phenotype downstream of BRN2.,•NFIB increases EZH2 expression downstream of BRN2, which further decreases MITF levels.,•NFIB expression is defined by an invasive gene signature and colocalises with BRN2 in primary and metastatic human melanoma tumours.,NFIB mediates a slow cycling, highly invasive/migratory melanoma cell phenotype downstream of BRN2.,NFIB increases EZH2 expression downstream of BRN2, which further decreases MITF levels.,NFIB expression is defined by an invasive gene signature and colocalises with BRN2 in primary and metastatic human melanoma tumours.,Melanoma is a heterogeneous cancer, made up of many cellular populations that differ in their ability to induce tumour growth or invasion throughout the body (metastasis).,These populations have been found to switch back and forth to drive invasion and progression.,This process appears to be controlled by an inverse axis between two genes, MITF and BRN2.,BRN2 drives metastatic spread, but the process by which it acts is not well characterized and cannot be targeted clinically.,This study has uncovered a role for the gene NFIB in driving invasion downstream of BRN2.,Importantly, it appears to drive this process through EZH2, which can be targeted therapeutically to reduce metastasis.

Oncogene-driven metabolic rewiring is an adaptation to low nutrient and oxygen conditions in the tumor microenvironment that enables cancer cells of diverse origin to hyperproliferate.,Aerobic glycolysis and enhanced reliance on glutamine utilization are prime examples of such rewiring.,However, tissue of origin as well as specific genetic and epigenetic changes determines gene expression profiles underlying these metabolic alterations in specific cancers.,In melanoma, activation of the MAPK pathway driven by mutant BRAF or NRAS is a primary cause of malignant transformation.,Activity of the MAPK pathway, as well as other factors, such as HIF1α, Myc and MITF, are among those that control the balance between non-oxidative and oxidative branches of central carbon metabolism.,Here, we discuss the nature of metabolic alterations that underlie melanoma development and affect its response to therapy.

N6-methylation of adenosine (m6A) RNA modification plays important roles in development and tumorigenesis.,The functions and mechanisms of m6A demethylases during cancer immunotherapy is still unclear.,Here we employed melanoma and colon syngeneic mouse models to study the roles of m6A demethylases ALKBH5 and FTO during anti-PD-1 antibody and GVAX vaccination therapy.,We found that ALKBH5 knockout in tumor cells enhances efficacy of immunotherapy and prolonged mouse survival.,ALKBH5 modulates target gene expression and gene splicing, leading to changes of metabolite contents, such as lactate in tumor microenvironment, which regulates suppressive lymphocytes Treg and myeloid-derived suppressor cell accumulations.,Importantly, by using ALKBH5-specific inhibitor, we observed the similar phenotype, indicating future translational application of our findings.,Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB.,N6-methylation of adenosine (m6A) in RNA regulates many pathophysiological processes.,Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy.,Alkbh5 has effects on m6A density and splicing events in tumors during ICB.,Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells.,Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy.,Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy.,Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.

Melanoma is one of the most deadly and therapy-resistant cancers.,Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy.,FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice.,FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway.,Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2.,Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity.,Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.,FTO is an m6A demethylase.,Here, the authors show that FTO promotes melanoma tumorigenicity and contributes to resistance to anti-PD1 blockade, while FTO inhibition sensitizes melanoma to anti-PD1 blockade.

Little is known about whether UVB can directly influence epigenetic regulatory pathways to induce cutaneous squamous cell carcinoma (CSCC).,This study aimed to identify epigenetic-regulated signalling pathways through global methylation and gene expression profiling and to elucidate their function in CSCC development.,Global DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) and genome-wide gene expression analysis by RNA sequencing (RNA-seq) in eight pairs of matched CSCC and adjacent normal skin tissues were used to investigate the potential candidate gene(s).,Clinical samples, animal models, cell lines, and UVB irradiation were applied to validate the mechanism and function of the genes of interest.,We identified the downregulation of the TGF-β/BMP-SMAD-ID4 signalling pathway in CSCC and increased methylation of inhibitor of DNA binding/differentiation 4 (ID4).,In normal human and mouse skin tissues and cutaneous cell lines, UVB exposure induced ID4 DNA methylation, upregulated DNMT1 and downregulated ten-eleven translocation (TETs).,Similarly, we detected the upregulation of DNMT1 and downregulation of TETs accompanying ID4 DNA methylation in CSCC tissues.,Silencing of DNMT1 and overexpression of TET1 and TET2 in A431 and Colo16 cells led to increased ID4 expression.,Finally, we showed that overexpression of ID4 reduced cell proliferation, migration, and invasion, and increased apoptosis in CSCC cell lines and reduced tumourigenesis in mouse models.,The results indicate that ID4 is downregulated by UVB irradiation via DNA methylation.,ID4 acts as a tumour suppressor gene in CSCC development.,CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-3-021, 2017-I2M-1-017), the Natural Science Foundation of Jiangsu Province (BK20191136), and the Fundamental Research Funds for the Central Universities (3332019104).

Cutaneous squamous cell carcinoma (CSCC) is a common type of skin malignancy.,MicroRNA-221 (miRNA-221) is a critical non-coding RNA in tumor initiation and progression.,However, the molecular mechanisms of miRNA-221 in the development of CSCC remain unknown.,This study investigated the expression of miRNA-221 in CSCC and its potential tumor biological functions.,MTT assay, colony assay, PCR, and Western blot were adopted.,In this study, miRNA-221 expression was significantly higher in CSCC tissues and cell lines than in normal tissues and cells (P < 0.05).,Further functional experiments indicated that miRNA-221 knockdown inhibited the proliferation and cell cycle, while upregulation of miRNA-221 presented the opposite role.,The dual reporter gene assays indicated that PTEN is a direct target gene of miRNA-221.,PTEN protein or mRNA levels were decreased after the cells were transfected with miR-221 mimics.,Taken together, the obtained results indicated that miR-221 plays an oncogenic function in CSCC by targeting PTEN and further suggest that miR-221 may be a potential target for CSCC diagnosis and treatment.

Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized.,In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors.,We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation.,Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB.,Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data.,Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response.,Analysis of fully clinically annotated and sequenced melanoma tumor samples collected before anti-PD1 treatment suggests that determinants of response differ on the basis of previous anti-CTLA4 therapy, and that tumor mutational burden may not be a strong predictor of response across melanoma subtypes.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology.,We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses.,We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases.,No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes.,MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive.,Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations.,Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition.,This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes.,Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments.,Here we genomically profile metastatic tumors and infiltrating lymphocytes.,BAP1 alterations are overrepresented and found in 29/32 of cases.,Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease.,One outlier tumor has a high mutational burden associated with UV-damage.,CDKN2A deletions also occur, which are rarely present in primaries.,A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains.,Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant.,This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease.,The genetics of uveal melanoma has mainly been studied in primary tumours.,In this study, the authors perform whole genome sequencing as well as immune cell profiling of biopsy samples obtained from metastatic uveal melanoma patients, providing an updated genomic landscape of these advanced lesions.

Metastatic uveal melanoma (UM) carries a poor prognosis; liver is the most frequent and often solitary site of recurrence.,Available systemic treatments have not improved outcomes.,Melphalan percutaneous hepatic perfusion (M‐PHP) allows selective intrahepatic delivery of high dose cytotoxic chemotherapy.,Retrospective analysis of outcomes data of UM patients receiving M‐PHP at two institutions was performed.,Tumor response and toxicity were evaluated using RECIST 1.1 and Common Terminology Criteria for Adverse Events (CTCAE) v4.03, respectively.,A total of 51 patients received 134 M‐PHP procedures (median of 2 M‐PHPs). 25 (49%) achieved a partial (N = 22, 43.1%) or complete hepatic response (N = 3, 5.9%).,In 17 (33.3%) additional patients, the disease stabilized for at least 3 months, for a hepatic disease control rate of 82.4%.,After median follow‐up of 367 days, median overall progression free (PFS) and hepatic progression free survival (hPFS) was 8.1 and 9.1 months, respectively and median overall survival was 15.3 months.,There were no treatment related fatalities.,Non‐hematologic grade 3‐4 events were seen in 19 (37.5%) patients and were mainly coagulopathic (N = 8) and cardiovascular (N = 9).,M‐PHP results in durable intrahepatic disease control and can form the basis for an integrated multimodality treatment approach in appropriately selected UM patients.

Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance.,The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors.,We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification).,These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, in vitro sphere formation, and in vivo xenograft tumor formation by small amount of tumor cells.,The selected populations would be melanoma stem-like cells with high expression of stem cell markers and altered kinase activation.,Microarray and bioinformatics analysis highlighted the high expression of angiopoietin-like 4 protein in drug-selected melanoma stem-like cells.,Further validation by specific shRNA demonstrated the role of angiopoietin-like 4 protein in drug-selected subpopulation associated with enhanced drug-resistance, sphere formation, reduced kinase activation, in vitro tube-forming ability correlated with heparan-sulfate proteoglycans.,Our finding would be applicable to explore the mechanism of melanoma stemness and use angiopoietin-like 4 as potential biomarkers to identify melanoma stem-like cells.

Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response.,Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks.,Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane.,This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells.,These features resemble the initial stages of invasive melanoma.,Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts.,This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment.,Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade.,Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163+PD-L1+ M2-like phenotype upon IL-10 blockade.,Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.,The online version of this article (10.1007/s00262-020-02626-4) contains supplementary material, which is available to authorized users.

Metastatic melanoma is an aggressive skin cancer and is one of the global malignancies with high mortality and morbidity.,It is essential to identify and verify diagnostic biomarkers of early metastatic melanoma.,Previous studies have systematically assessed protein biomarkers and mRNA-based expression characteristics.,However, molecular markers for the early diagnosis of metastatic melanoma have not been identified.,To explore potential regulatory targets, we have analyzed the gene microarray expression profiles of malignant melanoma samples by co-expression analysis based on the network approach.,The differentially expressed genes (DEGs) were screened by the EdgeR package of R software.,A weighted gene co-expression network analysis (WGCNA) was used for the identification of DEGs in the special gene modules and hub genes.,Subsequently, a protein-protein interaction network was constructed to extract hub genes associated with gene modules.,Finally, twenty-four important hub genes (RASGRP2, IKZF1, CXCR5, LTB, BLK, LINGO3, CCR6, P2RY10, RHOH, JUP, KRT14, PLA2G3, SPRR1A, KRT78, SFN, CLDN4, IL1RN, PKP3, CBLC, KRT16, TMEM79, KLK8, LYPD3 and LYPD5) were treated as valuable factors involved in the immune response and tumor cell development in tumorigenesis.,In addition, a transcriptional regulatory network was constructed for these specific modules or hub genes, and a few core transcriptional regulators were found to be mostly associated with our hub genes, including GATA1, STAT1, SP1, and PSG1.,In summary, our findings enhance our understanding of the biological process of malignant melanoma metastasis, enabling us to identify specific genes to use for diagnostic and prognostic markers and possibly for targeted therapy.

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis.,Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development.,Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma.,Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation.,Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR.,Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER).,This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.,The interaction of DOT1L with MLL oncogenic fusion proteins has been implicated in leukemogenesis.,Here, the authors show a contrasting role for DOT1L in protecting UVR-induced melanomagenesis by facilitating DNA repair through interaction with XPC.

Adoptive cell therapy may be based on isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL), in vitro activation and expansion and the reinfusion of these cells into patients upon chemotherapy induced lymphodepletion.,Together with high-dose interleukin (IL)-2 this treatment has been given to patients with advanced malignant melanoma and impressive response rates but also significant IL-2 associated toxicity have been observed.,Here we present data from a feasibility study at a Danish Translational Research Center using TIL adoptive transfer in combination with low-dose subcutaneous IL-2 injections.,This is a pilot trial (ClinicalTrials.gov identifier: NCT00937625) including patients with metastatic melanoma, PS ≤1, age <70, measurable and progressive disease and no involvement of the central nervous system.,Six patients were treated with lymphodepleting chemotherapy, TIL infusion, and 14 days of subcutaneous low-dose IL-2 injections, 2 MIU/day.,Low-dose IL-2 considerably decreased the treatment related toxicity with no grade 3-4 IL-2 related adverse events.,Objective clinical responses were seen in 2 of 6 treated patients with ongoing complete responses (30+ and 10+ months), 2 patients had stable disease (4 and 5 months) and 2 patients progressed shortly after treatment.,Tumor-reactivity of the infused cells and peripheral lymphocytes before and after therapy were analyzed.,Absolute number of tumor specific T cells in the infusion product tended to correlate with clinical response and also, an induction of peripheral tumor reactive T cells was observed for 1 patient in complete remission.,Complete and durable responses were induced after treatment with adoptive cell therapy in combination with low-dose IL-2 which significantly decreased toxicity of this therapy.

BAP1 has been shown to be a target of both somatic alteration in high-risk ocular melanomas (OM) and germline inactivation in a few individuals from cancer-prone families.,These findings suggest that constitutional BAP1 changes may predispose individuals to metastatic OM and that familial permeation of deleterious alleles could delineate a new cancer syndrome.,To characterize BAP1's contribution to melanoma risk, we sequenced BAP1 in a set of 100 patients with OM, including 50 metastatic OM cases and 50 matched non-metastatic OM controls, and 200 individuals with cutaneous melanoma (CM) including 7 CM patients from CM-OM families and 193 CM patients from CM-non-OM kindreds.,Germline BAP1 mutations were detected in 4/50 patients with metastatic OM and 0/50 cases of non-metastatic OM (8% vs. 0%, p = 0.059).,Since 2/4 of the BAP1 carriers reported a family history of CM, we analyzed 200 additional hereditary CM patients and found mutations in 2/7 CM probands from CM-OM families and 1/193 probands from CM-non-OM kindreds (29% vs.,0.52%, p = .003).,Germline mutations co-segregated with both CM and OM phenotypes and were associated with the presence of unique nevoid melanomas and highly atypical nevoid melanoma-like melanocytic proliferations (NEMMPs).,Interestingly, 7/14 germline variants identified to date reside in C-terminus suggesting that the BRCA1 binding domain is important in cancer predisposition.,Germline BAP1 mutations are associated with a more aggressive OM phenotype and a recurrent phenotypic complex of cutaneous/ocular melanoma, atypical melanocytic proliferations and other internal neoplasms (ie.,COMMON syndrome), which could be a useful clinical marker for constitutive BAP1 inactivation.

Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments.,Here we genomically profile metastatic tumors and infiltrating lymphocytes.,BAP1 alterations are overrepresented and found in 29/32 of cases.,Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease.,One outlier tumor has a high mutational burden associated with UV-damage.,CDKN2A deletions also occur, which are rarely present in primaries.,A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains.,Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant.,This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease.,The genetics of uveal melanoma has mainly been studied in primary tumours.,In this study, the authors perform whole genome sequencing as well as immune cell profiling of biopsy samples obtained from metastatic uveal melanoma patients, providing an updated genomic landscape of these advanced lesions.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy.,Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples.,Tumor cells reveal novel subclonal genomic complexity and transcriptional states.,Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4.,V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response.,An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity.,This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.,Uveal melanoma is highly metastatic and unresponsive to checkpoint immunotherapy.,Here, the authors present single-cell transcriptomics of 59,915 cells in 8 primary and 3 metastatic samples, highlighting the diversity of the tumour microenvironment.

In patients with advanced melanoma the detection of BRAF mutations is considered mandatory before the initiation of an expensive treatment with BRAF/MEK inhibitors.,Sometimes it is difficult to perform such an analysis if archival tumor tissue is not available and fresh tissue has to be collected.,514 of 1170 patients (44%) carried a BRAF mutation.,All models revealed age and histological subtype of melanoma as the two major predictive variables.,Accuracy ranged from 0.65-0.71, being best in the random forest model.,Sensitivity ranged 0.76-0.84, again best in the random forest model.,Specificity was low in all models ranging 0.51-0.55.,We collected the clinical data and mutational status of 1170 patients with advanced melanoma and established three different predictive models (binary logistic regression, classification and regression trees, and random forest) to forecast the BRAF status.,Up to date statistical models are not able to predict BRAF mutations in an acceptable accuracy.,The analysis of the mutational status by sequencing or immunohistochemistry must still be considered as standard of care.

Almost 50% of metastatic melanoma patients harbor a BRAFV600 mutation andthe introduction of BRAF inhibitors has improved their treatment options.,BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma.,However, most patients develop mechanisms of acquired resistance and about 15% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy.,Moreover, early adaptive responses limit the initial efficacy of BRAF inhibition, leading mostly to incomplete responses that may favor the selection of a sub-population of resistant clones and the acquisition of alterations that cause tumor regrowth and progressive disease.,The purpose of this paper is to review the mechanisms of resistance to therapy with BRAF inhibitors and to discuss the strategies to overcome them based on pre-clinical and clinical evidences.

Talimogene laherparepvec is an oncolytic immunotherapy approved in the US, Europe, Australia and Switzerland.,We report the final planned analysis of OPTiM, a randomized open-label phase III trial in patients with unresectable stage IIIB-IVM1c melanoma.,Patients were randomized 2:1 to receive intratumoral talimogene laherparepvec or subcutaneous recombinant GM-CSF.,In addition to overall survival (OS), durable response rate (DRR), objective response rate (ORR), complete responses (CR), and safety are also reported.,All final analyses are considered to be descriptive and treatment responses were assessed by the investigators.,Of 436 patients in the intent-to-treat population, 295 were allocated to talimogene laherparepvec and 141 to GM-CSF.,Median follow-up in the final OS analysis was 49 months.,Median OS was 23.3 months (95% confidence interval [CI], 19.5-29.6) and 18.9 months (95% CI, 16.0-23.7) in the talimogene laherparepvec and GM-CSF arms, respectively (unstratified hazard ratio, 0.79; 95% CI, 0.62-1.00; p = 0.0494 [descriptive]).,DRR was 19.0 and 1.4% (unadjusted odds ratio, 16.6; 95% CI, 4.0-69.2; p < 0.0001); ORR was 31.5 and 6.4%.,Fifty (16.9%) and 1 (0.7%) patient in the talimogene laherparepvec and GM-CSF arms, respectively, achieved CR.,In talimogene laherparepvec-treated patients, median time to CR was 8.6 months; median CR duration was not reached.,Among patients with a CR, 88.5% were estimated to survive at a 5-year landmark analysis.,Talimogene laherparepvec efficacy was more pronounced in stage IIIB-IVM1a melanoma as already described in the primary analysis.,The safety reporting was consistent with the primary OPTiM analysis.,In this final planned OPTiM analysis, talimogene laherparepvec continued to result in improved longer-term efficacy versus GM-CSF and remained well tolerated.,The final analysis also confirms that talimogene laherparepvec was associated with durable CRs that were associated with prolonged survival.,ClinicalTrials.gov identifier: NCT00769704.,The online version of this article (10.1186/s40425-019-0623-z) contains supplementary material, which is available to authorized users.

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2.,BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors.,Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5.,For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events.,Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma.,SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish.,Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1.,Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Uveal melanoma (UM) represents the most common intraocular malignancy in adults and accounts for about 5% of all melanomas.,Primary disease can be effectively controlled by several local therapy options, but UM has a high potential for metastatic spread, especially to the liver.,Despite its clinical and genetic heterogeneity, therapy of metastatic UM has largely been adopted from cutaneous melanoma (CM) with discouraging results until now.,The introduction of antibodies targeting CTLA-4 and PD-1 for immune checkpoint blockade (ICB) has revolutionized the field of cancer therapy and has achieved pioneering results in metastatic CM.,Thus, expectations were high that patients with metastatic UM would also benefit from these new therapy options.,This review provides a comprehensive and up-to-date overview on the role of ICB in UM.,We give a summary of UM biology, its clinical features, and how it differs from CM.,The results of several studies that have been investigating ICB in metastatic UM are presented.,We discuss possible reasons for the lack of efficacy of ICB in UM compared to CM, highlight the pitfalls of ICB in this cancer entity, and explain why other immune-modulating therapies could still be an option for future UM therapies.

Locally advanced cutaneous melanoma has marked quality-of-life implications; however, the patient experience of symptom management and subsequent impact on quality of life has not been well described.,This study aims to address the impact on patients of advanced cutaneous melanoma through qualitative interviews.,Adults with stage IIIB, IIIC, or IV (M1a) cutaneous melanoma were recruited from two cancer centers in the USA and one in Australia.,Telephone interviews were conducted to assess how locoregionally advanced cutaneous melanoma impacted everyday life.,Interviews were recorded, transcribed, and coded for qualitative analysis.,Twenty-two melanoma patients were interviewed, mean age 69.7 years (range: 52-83), 64% male.,The study included stage IIIB (36%), stage IIIC (59%), and stage IV M1a (5%) patients.,Emotional health/self-perception issues were the most commonly identified (41% of patient impact expressions), including worry, concern, embarrassment, self-consciousness, fear, and thoughts of death.,Limitations of lifestyle and activities were also identified (28% of expressions) including leisure and social activities, physical functioning, general functioning, and personal care.,Coping strategies such as modified clothing choices, increased use of pain and/or anti-inflammatory medications, and avoidance/protection from the sun represented 20% of all impact expressions.,Ratings of the degree of difficulty patients experienced (using an 11-point numerical rating scale) ranged from 0.0 to 10.0 (mean 5.7, SD 2.9).,Condition-related and treatment-related factors were well characterized in patients with locally advanced cutaneous melanoma.,This provides a strong foundation for assessment of how cutaneous melanoma impacts quality of life.

Cutaneous malignant melanoma is among the deadliest human cancers, broadly resistant to most clinical therapies.,A majority of patients with BRAFV600E melanomas respond well to inhibitors such as vemurafenib, but all ultimately relapse.,Moreover, there are no viable treatment options available for other non-BRAF melanoma subtypes in the clinic.,A key to improving treatment options lies in a better understanding of mechanisms underlying melanoma progression, which are complex and heterogeneous.,In this study we integrated gene and microRNA (miRNA) expression data from genetically engineered mouse models of highly and poorly malignant melanocytic tumors, as well as available human melanoma databases, and discovered an important role for a pathway centered on a tumor suppressor miRNA, miR-32.,Malignant tumors frequently exhibited poor expression of miR-32, whose targets include NRAS, PI3K and notably, MCL-1.,Accordingly, MCL-1 was often highly expressed in melanomas, and when knocked down diminished oncogenic potential.,Forced MCL-1 overexpression transformed immortalized primary mouse melanocytes, but only when also expressing activating mutations in BRAF, CRAF or PI3K.,Importantly, both miR-32 replacement therapy and the MCL-1-specific antagonist sabutoclax demonstrated single-agent efficacy, and acted synergistically in combination with vemurafenib in preclinical melanoma models.,We here identify miR-32/MCL-1 pathway members as key early genetic events driving melanoma progression, and suggest that their inhibition may be an effective anti-melanoma strategy irrespective of NRAS, BRAF, and PTEN status.

MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs).,To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs.,Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines.,We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients.,Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p).,Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment.,Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment.,Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity.,Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1.,Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer.,Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration.,Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies.,Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.,There are many patients who do not respond to immune checkpoint inhibitor (ICI) immunotherapy.,Here, the authors show a significant negative correlation between sphingosine kinase-1 (SK1) expression and survival for ICI-treated melanoma patients, and further show that targeting SK1 improves response to ICI in mouse cancer models.

The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events.,There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations.,However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs.,The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes.,Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system.,Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogenesis of melanoma.

Identifying soluble factors that influence epidermal integrity is critical for the development of preventative and therapeutic strategies for disorders such as ichthyosis, psoriasis, dermatitis and epidermal cancers.,The transcription factor Grainyhead-like 3 (GRHL3) is essential for maintaining barrier integrity and preventing development of cutaneous squamous cell carcinoma (SCC); however, how loss of this factor, which in the skin is expressed exclusively within suprabasal epidermal layers triggers proliferation of basal keratinocytes, had thus far remained elusive.,Our present study identifies thymus and activation-regulated chemokine (TARC) as a novel soluble chemokine mediator of keratinocyte proliferation following loss of GRHL3.,Knockdown of GRHL3 in human keratinocytes showed that of 42 cytokines examined, TARC was the only significantly upregulated chemokine.,Mouse skin lacking Grhl3 presented an inflammatory response with hallmarks of TARC activation, including heightened induction of blood clotting, increased infiltration of mast cells and pro-inflammatory T cells, increased expression of the pro-proliferative/pro-inflammatory markers CD3 and pSTAT3, and significantly elevated basal keratinocyte proliferation.,Treatment of skin cultures lacking Grhl3 with the broad spectrum anti-inflammatory 5-aminosalicylic acid (5ASA) partially restored epidermal differentiation, indicating that abnormal keratinocyte proliferation/differentiation balance is a key driver of barrier dysfunction following loss of Grhl3, and providing a promising therapeutic avenue in the treatment of GRHL3-mediated epidermal disorders.

Zhang et al. show that miR-125b is abundantly expressed, particularly at early stages of malignant progression to squamous cell carcinoma.,When elevated in normal murine epidermis, miR-125b promotes tumor initiation and contributes to malignant progression. mir-125b directly represses stress-responsive MAPK genes and indirectly prolongs activated EGFR signaling by repressing Vps4B, encoding a protein implicated in negatively regulating the endosomal sorting complexes that are necessary for the recycling of active EGFR.,Previously, we identified miR-125b as a key regulator of the undifferentiated state of hair follicle stem cells.,Here, we show that in both mice and humans, miR-125b is abundantly expressed, particularly at early stages of malignant progression to squamous cell carcinoma (SCC), the second most prevalent cancer worldwide.,Moreover, when elevated in normal murine epidermis, miR-125b promotes tumor initiation and contributes to malignant progression.,We further show that miR-125b can confer “oncomiR addiction” in early stage malignant progenitors by delaying their differentiation and favoring an SCC cancer stem cell (CSC)-like transcriptional program.,To understand how, we systematically identified and validated miR125b targets that are specifically associated with tumors that are dependent on miR-125b.,Through molecular and genetic analysis of these targets, we uncovered new insights underlying miR-125b’s oncogenic function.,Specifically, we show that, on the one hand, mir-125b directly represses stress-responsive MAP kinase genes and associated signaling.,On the other hand, it indirectly prolongs activated (phosphorylated) EGFR signaling by repressing Vps4b (vacuolar protein-sorting 4 homolog B), encoding a protein implicated in negatively regulating the endosomal sorting complexes that are necessary for the recycling of active EGFR.,Together, these findings illuminate miR-125b as an important microRNA regulator that is shared between normal skin progenitors and their early malignant counterparts.

Melanoma accounts for only a small portion of skin cancer but it is associated with high mortality.,Melanoma serum biomarkers that may aid early diagnosis or guide therapy are needed clinically.,However, studies of serum biomarkers have often been hampered by the serum interference that causes false readouts in immunological tests.,Here we show that, after using a special buffer to eliminate the serum interference, IL-8 and cathepsin B levels were significantly elevated in melanoma patients (p < 0.05).,More importantly, the combination of IL-8 and cathepsin B were also studied as a prognosis marker for melanoma mortality.,Our study provides a novel approach to examine serum biomarkers.

Reflectance-mode confocal microscopy (RCM) is a new in vivo skin imaging technique.,We present our one-year experience in RCM examinations in skin tumors and the retrospective analysis of patients enrolled in the Dermatological Department of ‘N.,Paulescu’ Institute using the Fotofinder Dermoscope IIŴ for the dermatoscopy analysis and VivaScope 1500Ŵ for in vivo RCM.,We established the rank of RCM in the complex algorithm of skin cancer diagnose, showing that the presented experience can open new possibilities to implement this automated image analyzing system in the routine practice.,Our analyzed cases clearly showed that confocal microscopy, therefore, optical biopsy, could guide the clinician towards an accurate diagnosis before surgical removal.,Moreover, we emphasized that the development of this technique increases the potential of future teledermatologic applications.

The discovery of BRAF mutations in the majority of patients with metastatic melanoma combined with the identification of highly selective BRAF inhibitors have revolutionized the treatment of patients with metastatic melanoma.,The first highly specific BRAF inhibitor, vemurafenib, began clinical testing in 2008 and moved towards a rapid approval in 2011.,Vemurafenib induced responses in ~50% of patients with metastatic BRAF-mutant melanoma and demonstrated improved overall survival in a randomized Phase III trial.,Furthermore, vemurafenib is well-tolerated with a low toxicity profile and rapid onset of action.,Finally, vemurafenib is active even in patients with widely metastatic disease.,Despite the success of vemurafenib in treating patients with BRAF-mutant metastatic melanoma, most, if not all, patients ultimately develop resistance resulting in disease progression at a median time of ~6 months.,Multiple mechanisms of resistance have been described and rationale strategies are underway to combat resistance.,This review highlights the development, clinical utility, resistance mechanisms, and future use of vemurafenib both in melanoma and other malignancies.,We consulted PubMed, Scopus, MEDLINE, ASCO annual symposium abstracts, and http://clinicaltrials.gov/ for the purpose of this review.

Uveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential.,We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1.,The aim of this study was to investigate the role of BAP1 in uveal melanoma progression.,Uveal melanoma cells were studied following RNAi-mediated depletion of BAP1 using proliferation, BrdU incorporation, flow cytometry, migration, invasion, differentiation and clonogenic assays, as well as in vivo tumorigenicity experiments in NOD-SCID-Gamma mice.,Depletion of BAP1 in uveal melanoma cells resulted in a loss of differentiation and gain of stem-like properties, including expression of stem cell markers, increased capacity for self-replication, and enhanced ability to grow in stem cell conditions.,BAP1 depletion did not result in increased proliferation, migration, invasion or tumorigenicity.,BAP1 appears to function in the uveal melanocyte lineage primarily as a regulator of differentiation, with cells deficient for BAP1 exhibiting stem-like qualities.,It will be important to elucidate how this effect of BAP1 loss promotes metastasis and how to reverse this effect therapeutically.

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.

In the current issue, two articles highlight the impact of MITF on melanoma development.,In the first, Lister et al. (2013) reveal in vivo proof of MITF directly regulating tumor development in BRAFV600E melanomas.,In the second, Sturm et al. (2013) present a clinical trial that emphasizes the importance of the recently discovered E318K MITF germline mutation in patients with multiple primary melanomas.

Uveal melanoma (UM), a rare cancer of the eye, is distinct from cutaneous melanoma by its etiology, the mutation frequency and profile, and its clinical behavior including resistance to targeted therapy and immune checkpoint blockers.,Primary disease is efficiently controlled by surgery or radiation therapy, but about half of UMs develop distant metastasis mostly to the liver.,Survival of patients with metastasis is below 1 year and has not improved in decades.,Recent years have brought a deep understanding of UM biology characterized by initiating mutations in the G proteins GNAQ and GNA11.,Cytogenetic alterations, in particular monosomy of chromosome 3 and amplification of the long arm of chromosome 8, and mutation of the BRCA1-associated protein 1, BAP1, a tumor suppressor gene, or the splicing factor SF3B1 determine UM metastasis.,Cytogenetic and molecular profiling allow for a very precise prognostication that is still not matched by efficacious adjuvant therapies.,G protein signaling has been shown to activate the YAP/TAZ pathway independent of HIPPO, and conventional signaling via the mitogen-activated kinase pathway probably also contributes to UM development and progression.,Several lines of evidence indicate that inflammation and macrophages play a pro-tumor role in UM and in its hepatic metastases.,UM cells benefit from the immune privilege in the eye and may adopt several mechanisms involved in this privilege for tumor escape that act even after leaving the niche.,Here, we review the current knowledge of the biology of UM and discuss recent approaches to UM treatment.

Variation in the melanocortin-1receptor (MC1R) gene is associated with pigmentary phenotypes and risk of malignant melanoma.,Few studies have reported on MC1R variation with respect to tumor characteristics, especially clinically important prognostic features.,We examined associations between MC1R variants and histopathological melanoma characteristics.,Study participants were enrolled from nine geographic regions in Australia, Canada, Italy and the United States and were genotyped for MC1R variants classified as high-risk [R] (D84E, R142H, R151C, R160W, and D294H, all nonsense and insertion/deletion) or low-risk [r] (all other nonsynonymous) variants.,Tissue was available for 2,160 white participants of the Genes, Environment and Melanoma (GEM) Study with a first incident primary melanoma diagnosis, and underwent centralized pathologic review.,No statistically significant associations were observed between MC1R variants and AJCC established prognostic tumor characteristics: Breslow thickness, presence of mitoses or presence of ulceration.,However, MC1R was significantly associated with anatomic site of melanoma (p = 0.002) and a positive association was observed between carriage of more than one [R] variant and melanomas arising on the arms (OR = 2.39; 95% CI: 1.40, 4.09).,We also observed statistically significant differences between sun-sensitive and sun-resistant individuals with respect to associations between MC1R genotype and AJCC prognostic tumor characteristics.,Our results suggest inherited variation in MC1R may play an influential role in anatomic site presentation of melanomas and may differ with respect to skin pigmentation phenotype.

To describe the pharmacological properties, preclinical and clinical data of the novel V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-inhibitor encorafenib (LGX818) and to compare these with established BRAF-inhibitors in the treatment of locally advanced or metastatic melanoma.,Encorafenib has shown improved efficacy in the treatment of metastatic melanoma in comparison with vemurafenib.,Combination with the MEK inhibitor (MEKi) binimetinib allows for higher dose intensities of encorafenib further improving response rates (RRs).,Combination therapy with BRAF and MEKi has evolved as a standard of care in the treatment of locally advanced or metastatic BRAFV600-mutated melanoma.,Despite compelling initial RRs, development of treatment resistance eventually leads to tumor progression in the majority of BRAF/MEK-inhibitor treated patients.,Moreover, treatment-related adverse events are frequent, resulting in a substantial proportion of dose modifications and/or treatment discontinuations.,The second-generation BRAF inhibitor encorafenib has been developed aiming at improved efficacy and tolerability through modifications in pharmacological properties.,Clinical phase 3 data show improved progression-free survival both for encorafenib monotherapy and combination therapy with binimetinib compared with vemurafenib.,Overall survival data and regulatory approval of this novel substance are eagerly awaited.

B-RAF is the most frequently mutated protein kinase in human cancers.1 The finding that oncogenic mutations in BRAF are common in melanoma2 followed by the demonstration that these tumors are dependent on the RAF/MEK/ERK pathway3 offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients.,Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity.,Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts.4 Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032.5 In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment.,This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumor regressions.,At higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of ERK phosphorylation in the tumors of patients correlated with clinical response.,Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.

Despite recent genetic advances and numerous ongoing therapeutic trials, malignant melanoma remains fatal, and prognostic factors as well as more efficient treatments are needed.,The development of such research strongly depends on the availability of appropriate models recapitulating all the features of human melanoma.,The concept of comparative oncology, with the use of spontaneous canine models has recently acquired a unique value as a translational model.,Canine malignant melanomas are naturally occurring cancers presenting striking homologies with human melanomas.,As for many other cancers, dogs present surprising breed predispositions and higher frequency of certain subtypes per breed.,Oral melanomas, which are much more frequent and highly severe in dogs and cutaneous melanomas with severe digital forms or uveal subtypes are subtypes presenting relevant homologies with their human counterparts, thus constituting close models for these human melanoma subtypes.,This review addresses how canine and human melanoma subtypes compare based on their epidemiological, clinical, histological, and genetic characteristics, and how comparative oncology approaches can provide insights into rare and poorly characterized melanoma subtypes in humans that are frequent and breed-specific in dogs.,We propose canine malignant melanomas as models for rare non-UV-induced human melanomas, especially mucosal melanomas.,Naturally affected dogs offer the opportunity to decipher the genetics at both germline and somatic levels and to explore therapeutic options, with the dog entering preclinical trials as human patients, benefiting both dogs and humans.

The dominant phenotype of greying with age in horses, caused by a 4.6-kb duplication in intron 6 of STX17, is associated with a high incidence of melanoma and vitiligo-like skin depigmentation.,However, the progressive greying and the incidence of melanoma, vitiligo-like depigmentation, and amount of speckling in these horses do not follow a simple inheritance pattern.,To understand their inheritance, we analysed the melanoma grade, grey level, vitiligo grade, and speckling grade of 1,119 Grey horses (7,146 measurements) measured in six countries over a 9-year period.,We estimated narrow sense heritability (h2), and we decomposed this parameter into polygenic heritability (h2 POLY), heritability due to the Grey (STX17) mutation (h2 STX17), and heritability due to agouti (ASIP) locus (h2 ASIP).,A high heritability was found for greying (h2 = 0.79), vitiligo (h2 = 0.63), and speckling (h2 = 0.66), while a moderate heritability was estimated for melanoma (h2 = 0.37).,The additive component of ASIP was significantly different from zero only for melanoma (h2 ASIP = 0.02).,STX17 controlled large proportions of phenotypic variance (h2 STX17 = 0.18-0.55) and overall heritability (h2 STX17/h2 = 0.28-0.83) for all traits.,Genetic correlations among traits were estimated as moderate to high, primarily due to the effects of the STX17 locus.,Nevertheless, the correlation between progressive greying and vitiligo-like depigmentation remained large even after taking into account the effects of STX17.,We presented a model where four traits with complex inheritance patterns are strongly influenced by a single mutation.,This is in line with evidence of recent studies in domestic animals indicating that some complex traits are, in addition to the large number of genes with small additive effects, influenced by genes of moderate-to-large effect.,Furthermore, we demonstrated that the STX17 mutation explains to a large extent the moderate to high genetic correlations among traits, providing an example of strong pleiotropic effects caused by a single gene.

The presence of immune cells in the tumor microenvironment has been associated with response to immunotherapies across several cancer types, including melanoma.,Despite its therapeutic relevance, characterization of the melanoma immune microenvironments remains insufficiently explored.,To distinguish the immune microenvironment in a cohort of 180 metastatic melanoma clinical specimens, we developed a method using promoter CpG methylation of immune cell type‐specific genes extracted from genome‐wide methylation arrays.,Unsupervised clustering identified three immune methylation clusters with varying levels of immune CpG methylation that are related to patient survival.,Matching protein and gene expression data further corroborated the identified epigenetic characterization.,Exploration of the possible immune exclusion mechanisms at play revealed likely dependency on MITF protein level and PTEN loss‐of‐function events for melanomas unresponsive to immunotherapies (immune‐low).,To understand whether melanoma tumors resemble other solid tumors in terms of immune methylation characteristics, we explored 15 different solid tumor cohorts from TCGA.,Low‐dimensional projection based on immune cell type‐specific methylation revealed grouping of the solid tumors in line with melanoma immune methylation clusters rather than tumor types.,Association of survival outcome with immune cell type‐specific methylation differed across tumor and cell types.,However, in melanomas immune cell type‐specific methylation was associated with inferior patient survival.,Exploration of the immune methylation patterns in a pan‐cancer context suggested that specific immune microenvironments might occur across the cancer spectrum.,Together, our findings underscore the existence of diverse immune microenvironments, which may be informative for future immunotherapeutic applications.,The importance of the tumor immune microenvironment has been highlighted in multiple cancers, but information regarding the contribution of individual immune cell types is lacking for melanoma.,In this study, we have sought to fill the gap by identifying distinct DNA methylation patterns for specifc immune cells.,These were corroborated by other molecular correlates and found to be shared across other types of solid tomors.

Clonal selection and transcriptional reprogramming (e.g., epithelial-mesenchymal transition or phenotype switching) are the predominant theories thought to underlie tumor progression.,However, a “division of labor” leading to cooperation among tumor-cell subpopulations could be an additional catalyst of progression.,Using a zebrafish-melanoma xenograft model, we found that in a heterogeneous setting, inherently invasive cells, which possess protease activity and deposit extracellular matrix (ECM), co-invade with subpopulations of poorly invasive cells, a phenomenon we term “cooperative invasion”.,Whereas the poorly invasive cells benefit from heterogeneity, the invasive cells switch from protease-independent to an MT1-MMP-dependent mode of invasion.,We did not observe changes in expression of the melanoma phenotype determinant MITF during cooperative invasion, thus ruling out the necessity for phenotype switching for invasion.,Altogether, our data suggest that cooperation can drive melanoma progression without the need for clonal selection or phenotype switching and can account for the preservation of heterogeneity seen throughout tumor progression.,•Inherently invasive and poorly invasive melanoma subpopulations co-invade•Leader cells are inherently invasive and provide MT1-MMP and deposit ECM•Follower cells affect the mode of invasion and thereby elicit protease dependency•Cooperative invasion bypasses phenotype switching and maintains tumor heterogeneity,Inherently invasive and poorly invasive melanoma subpopulations co-invade,Leader cells are inherently invasive and provide MT1-MMP and deposit ECM,Follower cells affect the mode of invasion and thereby elicit protease dependency,Cooperative invasion bypasses phenotype switching and maintains tumor heterogeneity,Tumor heterogeneity has been viewed as the outcome of adaptive competition or differential transcriptional reprogramming (phenotype switching).,Alternatively, heterogeneity may reflect a division of labor between tumor cell subpopulations with complementary acquired characteristics.,Chapman et al. now show reciprocal interactions between heterogeneous melanoma cell subpopulations that lead to cooperative invasion without competition and without phenotype switching.,Thus, cooperative behavior emerges as a property of heterogeneity relevant for tumor biology and worth targeting therapeutically.

In a relatively short period of time, treatment strategies for metastatic melanoma have radically changed leading to an unprecedented improvement in patient survival.,In this period, immunotherapy options have evolved from cytokine-based approaches to antibody-mediated inhibition of immune checkpoints, cancer vaccines and pharmacological modulation of the melanoma microenvironment.,Combination of immunotherapy strategies and the association of immune checkpoint inhibitors (ICIs) with BRAF V600 targeted therapy show encouraging results.,The future of drug development in this field is promising.,The comprehension of primary and acquired resistance mechanisms to ICIs and the dissection of melanoma immunobiology will be instrumental for the development of new treatment strategies and to improve clinical trial design.,Moreover, biomarker discovery will help patient stratification and management during immunotherapy treatment.,In this review, we summarize landmark clinical trials of immune checkpoint inhibitors in advanced melanoma and discuss the rational for immunotherapy combinations.,Immunotherapy approaches at early stage of clinical development and recent advances in melanoma immunotherapy biomarker development are also discussed.

Previous work identified RMEL3 as a lncRNA with enriched expression in melanoma.,Analysis of The Cancer Genome Atlas (TCGA) data confirmed RMEL3 enriched expression in melanoma and demonstrated its association with the presence of BRAFV600E.,RMEL3 siRNA-mediated silencing markedly reduced (95%) colony formation in different BRAFV600E melanoma cell lines.,Multiple genes of the MAPK and PI3K pathways found to be correlated with RMEL3 in TCGA samples were experimentally confirmed.,RMEL3 knockdown led to downregulation of activators or effectors of these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2.,RMEL3 knockdown induces gain of protein levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1.,Consistently, knockdown resulted in an accumulation of cells in G1 phase and subG0/G1 in an asynchronously growing population.,Thus, TCGA data and functional experiments demonstrate that RMEL3 is required for MAPK and PI3K signaling, and its knockdown decrease BRAFV600E melanoma cell survival and proliferation.

During embryonic development Wnt family members and bone morphogenetic proteins (BMPs) cooperatively induce epithelial-mesenchymal transition (EMT) in the neural crest.,Wnt and BMPs are reactivated during malignant transformation in melanoma.,We previously demonstrated that the BMP-antagonist noggin blocked the EMT phenotype of melanoma cells in the neural crest and malignant invasion of melanoma cells in the chick embryo; vice-versa, malignant invasion was induced in human melanocytes in vivo by pre-treatment with BMP-2.,Although there are conflicting results in the literature about the role of β-catenin for invasion of melanoma cells, we found Wnt/β-catenin signaling to be analogously important for the EMT-like phenotype of human metastatic melanoma cells in the neural crest and during invasion: β-catenin was frequently expressed at the invasive front of human primary melanomas and Wnt3a expression was inversely correlated with survival of melanoma patients.,Accordingly, cytoplasmic β-catenin levels were increased during invasion of melanoma cells in the rhombencephalon of the chick embryo.,Fibroblast derived Wnt3a reduced melanoma cell adhesion and enhanced migration, while the β-catenin inhibitor PKF115-584 increased adhesion and reduced migration in vitro and in the chick embryonic neural crest environment in vivo.,Similarly, knockdown of β-catenin impaired intradermal melanoma cell invasion and PKF115-584 efficiently reduced liver metastasis in a chick chorioallantoic membrane model.,Our observations were accompanied by specific alterations in gene expression which are linked to overall survival of melanoma patients.,We present a novel role for Wnt-signaling in neural crest like melanoma cell invasion and metastasis, stressing the crucial role of embryonic EMT-inducing neural crest signaling for the spreading of malignant melanoma.,The online version of this article (10.1186/s12943-018-0773-5) contains supplementary material, which is available to authorized users.

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts.,By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease.,In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival.,Loss of both copies of B2M is found only in non-responders.,B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.,Resistance to immune-checkpoint blockade often occurs in treated patients.,Here, the authors demonstrate that B2M loss is a mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1 in melanoma patients.

Knowledge of the genotype of melanoma is important to guide patient management.,Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.,While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time.,We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest.,We have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status.,There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR.,However, these are often rare and of unknown significance for treatment.,PCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup.,The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.,The online version of this article (doi:10.1186/s12885-017-3149-0) contains supplementary material, which is available to authorized users.

Patients with BRAF mutation-positive advanced melanoma respond well to matched therapy with BRAF or MEK inhibitors, but often quickly develop resistance.,Tumor tissue from ten patients with advanced BRAF mutation-positive melanoma who achieved partial response (PR) or complete response (CR) on BRAF and/or MEK inhibitors was analyzed using next generation sequencing (NGS) assay.,Genomic libraries were captured for 3230 exons in 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to average median depth of 734X with 99% of bases covered >100X.,Three of the ten patients (median number of prior therapies = 2) attained prolonged CR (duration = 23.6+ to 28.7+ months); seven patients achieved either a PR or a short-lived CR.,One patient who achieved CR ongoing at 28.7+ months and had tissue available close to the time of initiating BRAF inhibitor therapy had only a BRAF mutation.,Abnormalities in addition to BRAF mutation found in other patients included: mutations in NRAS, APC and NF1; amplifications in BRAF, aurora kinase A, MYC, MITF and MET; deletions in CDKN2A/B and PAX5; and, alterations in RB1 and ATM.,Heterogeneity between patients and molecular evolution within patients was noted.,NGS identified potentially actionable DNA alterations that could account for resistance in patients with BRAF mutation-positive advanced melanoma who achieved a PR or CR but whose tumors later progressed.,A subset of patients with advanced melanoma may harbor only a BRAF mutation and achieve a durable CR on BRAF pathway inhibitors.,The online version of this article (doi:10.1186/s12885-015-1029-z) contains supplementary material, which is available to authorized users.

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements.,However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs.,Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements.,Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not.,The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis.,Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.,•Reducing transcription factor DNA-binding affinity increases activity in vivo•Acetylation is triggered by MAPK signaling•Acetylation leads to genome-wide transcription factor redistribution•Acetylation of MITF drives tumorigenesis and melanocyte development,Reducing transcription factor DNA-binding affinity increases activity in vivo,Acetylation is triggered by MAPK signaling,Acetylation leads to genome-wide transcription factor redistribution,Acetylation of MITF drives tumorigenesis and melanocyte development,Decreasing the DNA-binding affinity of a transcription factor via MAPK-stimulated p300/CBP-mediated acetylation of a lysine contacting the DNA-phosphate backbone paradoxically increases its functional output by promoting release from a genome-wide reservoir of low-affinity binding sites.

Molecular signatures specific to particular tumor types are required to design treatments for resistant tumors.,However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures.,We developed similarity core analysis (SCA), a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines.,We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases.,The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion.,About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7) and miRNAs (211-5p, 221-3p, and 10a-5p).,The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.,Cancer cell lines are at the forefront of drug discovery but are often limited in representing the tumor of origin due to the artificial culture conditions.,Rambow et al. develop a computational approach for identifying tumor cell lineage expression cores.,These core genes reveal relevant molecular dependencies linking aggressiveness, patient survival, and drug sensitivity.

Basal cell carcinomas are the most frequent skin cancers in the fair-skinned adult population over 50 years of age.,Their incidence is increasing throughout the world.,Ultraviolet (UV) exposure is the major carcinogenic factor.,Some genodermatosis can predispose to formation of basal cell carcinomas at an earlier age.,Basal cell carcinomas are heterogeneous, from superficial or nodular lesions of good prognosis to very extensive difficult-to-treat lesions that must be discussed in multidisciplinary committees.,Recent guidelines have updated the management of basal cell carcinoma.,The prognosis is linked to the risk of recurrence of basal cell carcinoma or its local destructive capacity.,Characteristic molecular events in these tumours are: (i) activation of the hedgehog pathway, which has allowed the development of hedgehog inhibitors for difficult-to-treat lesions that are not accessible to surgery or radiotherapy; (ii) high mutational burden, which suggests that hedgehog inhibitor refractory tumours could be offered immunotherapy; some trials are ongoing.,The standard treatment for most basal cell carcinomas is surgery, as it allows excision margin control and shows a low risk of recurrence.,Superficial lesions can be treated by non-surgical methods with significant efficacy.

The incidence of keratinocyte carcinomas is increasing worldwide and currently there is no standardised strategy for the follow-up of patients with multiple tumours.,The objective of this study was to assess the prevalence of premalignant lesions, i.e., actinic keratosis and Bowen’s disease, as well as basal cell carcinoma (BCC) and cutaneous melanoma (CM) among patients with cutaneous squamous cell carcinoma (cSCC).,Pathology database search was performed to identify all cSCC patients diagnosed in the Pirkanmaa region of Finland in 2006-2015.,Details of the patients and tumours were obtained through medical record review.,The cohort consisted of 774 patients with 1131 cSCC tumours.,Overall 559 patients (72%) had premalignant lesions.,A total of 316 patients (41%) had BCC and 52% of these (n = 164) had more than one BCC tumour. 50 patients (6%) had CM.,Overall 180 cSCC patients (23%) had no premalignant changes, BCC or CM.,The median age of these patients was 6 years less than that of the patients with premalignant lesions (p < 0.001) or BCC (p < 0.001).,The invasion depth of the tumours was deeper in the patients with only cSCC (median 3 mm, interquartile range 2-6) than in those with premalignant lesions or BCC (median 2 mm, interquartile range 1-3), p < 0.001.,CSCC patients have a high risk of developing multiple skin cancers and need long-term follow-up.

The management of melanoma has evolved due to improved understanding of its molecular drivers.,To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed.,Mutations were identified in 43 of the 46 genes on the panel.,The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%).,Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype.,Concurrent TP53 mutations were the most frequent event in tumors with BRAFV600 and NRAS mutations.,Melanomas with BRAFNon-V600 mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%).,The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location.,Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600 and NRAS mutations.,These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma.

Alterations in key-regulator genes of disease pathogenesis (BRAF, cKIT, CyclinD1) have been evaluated in patients with multiple primary melanoma (MPM).,One hundred twelve MPM patients (96 cases with two primary melanomas, 15 with three, and 1 with four) were included into the study.,Paired synchronous/asynchronous MPM tissues (N = 229) were analyzed for BRAF mutations and cKIT/CyclynD1 gene amplifications.,BRAF mutations were identified in 109/229 (48%) primary melanomas, whereas cKIT and CyclinD1 amplifications were observed in 10/216 (5%) and 29/214 (14%) tumor tissues, respectively.,While frequency rates of BRAF mutations were quite identical across the different MPM lesions, a significant increase of cKIT (p < 0.001) and CyclinD1 (p = 0.002) amplification rates was observed between first and subsequent primary melanomas.,Among the 107 patients with paired melanoma samples, 53 (49.5%) presented consistent alteration patterns between first and subsequent primary tumors.,About one third (40/122; 32.8%) of subsequent melanomas presented a discrepant pattern of BRAF mutations as compared to incident primary tumors.,The low consistency in somatic mutation patterns among MPM lesions from same patients provides further evidence that melanomagenesis is heterogeneous and different cell types may be involved.,This may have implications in clinical practice due to the difficulties in molecularly classifying patients with discrepant primary melanomas.

This diagnostic study evaluates whether fellowship training and board certification of physicians in dermatopathology affect the diagnostic reliability of second-opinion strategies for difficult-to-diagnose cutaneous melanocytic lesions.,For cutaneous melanocytic lesions that are difficult to diagnose, do second opinions rendered by pathologists who have board certification and/or fellowship training in dermatopathology improve overall reliability of diagnosis?,In this diagnostic study of 240 melanocytic lesions from the Melanoma Pathology Study data set, misclassification of melanocytic lesions was lowest when first, second, and third consulting reviewers were subspecialty-trained dermatopathologists and when all lesions were subject to second opinions; misclassification was highest when reviewers were all general pathologists lacking subspecialty training.,Variability of in situ and thin invasive melanoma was relatively intractable to all examined strategies.,The findings suggest that second opinions rendered by dermatopathologists improve overall reliability of diagnosis of melanocytic lesions but do not eliminate or substantially reduce misclassification.,Histopathologic criteria have limited diagnostic reliability for a range of cutaneous melanocytic lesions.,To evaluate the association of second-opinion strategies by general pathologists and dermatopathologists with the overall reliability of diagnosis of difficult melanocytic lesions.,This diagnostic study used samples from the Melanoma Pathology Study, which comprises 240 melanocytic lesion samples selected from a dermatopathology laboratory in Bellevue, Washington, and represents the full spectrum of lesions from common nevi to invasive melanoma.,Five sets of 48 samples were evaluated independently by 187 US pathologists from July 15, 2013, through May 23, 2016.,Data analysis was performed from April 2016 through November 2017.,Accuracy of diagnosis, defined as concordance with an expert consensus diagnosis of 3 experienced pathologists, was assessed after applying 10 different second-opinion strategies.,Among the 187 US pathologists examining the 24 lesion samples, 113 were general pathologists (65 men [57.5%]; mean age at survey, 53.7 years [range, 33.0-79.0 years]) and 74 were dermatopathologists (49 men [66.2%]; mean age at survey, 46.4 years [range, 33.0-77.0 years]).,Among the 8976 initial case interpretations, physicians desired second opinions for 3899 (43.4%), most often for interpretation of severely dysplastic nevi.,The overall misclassification rate was highest when interpretations did not include second opinions and initial reviewers were all general pathologists lacking subspecialty training (52.8%; 95% CI, 51.3%-54.3%).,When considering different second opinion strategies, the misclassification of melanocytic lesions was lowest when the first, second, and third consulting reviewers were subspecialty-trained dermatopathologists and when all lesions were subject to second opinions (36.7%; 95% CI, 33.1%-40.7%).,When the second opinion strategies were compared with single interpretations without second opinions, the reductions in misclassification rates for some of the strategies were statistically significant, but none of the strategies eliminated diagnostic misclassification.,Melanocytic lesions in the middle of the diagnostic spectrum had the highest misclassification rates (eg, moderately or severely dysplastic nevus, Spitz nevus, melanoma in situ, and pathologic stage [p]T1a invasive melanoma).,Variability of in situ and thin invasive melanoma was relatively intractable to all examined strategies.,The results of this study suggest that second opinions rendered by dermatopathologists improve reliability of melanocytic lesion diagnosis.,However, discordance among pathologists remained high.

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression.,Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors.,However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.,The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically.,There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS.,In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.,Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation.,Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs.,This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor.,These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties.,Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors.,Notably, several viral oncoproteins are capable of binding and inhibiting PPs.,Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells.,Therefore, we analyzed the interaction of MCPyV-LT with the PPs.,Co-IP experiments indicate that MCPyV-LT binds potently only to RB1.,Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown.,Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression.,Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent.,Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.

Merkel cell carcinoma (MCC) is a virally associated cancer characterized by its aggressive behavior and strong immunogenicity.,Both viral infection and malignant transformation induce expression of MHC class I chain-related protein (MIC) A and B, which signal stress to cells of the immune system via Natural Killer group 2D (NKG2D) resulting in elimination of target cells.,However, despite transformation and the continued presence of virally-encoded proteins, MICs are only expressed in a minority of MCC tumors in situ and are completely absent on MCC cell lines in vitro.,This lack of MIC expression was due to epigenetic silencing via MIC promoter hypo-acetylation; indeed, MIC expression was re-induced by pharmacological inhibition of histone deacetylases (HDACs) both in vitro and in vivo.,This re-induction of MICs rendered MCC cells more sensitive to immune-mediated lysis.,Thus, epigenetic silencing of MICs is an important immune escape mechanism of MCCs.

Although patients with unresectable or metastatic melanoma can experience long-term survival with BRAF- and MEK-targeted agents or immune checkpoint inhibitors over 5 years, resistance develops in most patients.,There is a distinct lack of pretherapeutic biomarkers to identify which patients are likely to benefit from each therapy type.,Most research has focused on the predictive role of T cells in antitumor responses as opposed to B cells.,We conducted prespecified exploratory biomarker analysis using gene expression profiling and digital pathology in 146 patients with previously untreated BRAF V600-mutant metastatic melanoma from the randomized, phase III COMBI-v trial and treated with dabrafenib plus trametinib who had available tumor specimens from screening.,Baseline cell-cycle gene expression signature was associated with progression-free survival (P = 0.007).,Patients with high T-cell/low B-cell gene signatures had improved median overall survival (not reached [95% confidence interval (CI), 33.8 months-not reached]) compared with patients with high T-cell/high B-cell signatures (19.1 months; 95% CI, 13.4-38.6 months).,Patients with high B-cell signatures had high B-cell infiltration into the tumor compartment, corresponding with decreased MAPK activity and increased expression of immunosuppressive markers.,B cells may serve as a potential biomarker to predict clinical outcome in patients with advanced melanoma treated with dabrafenib plus trametinib.,As separate studies have shown an opposite effect for B-cell levels and response to immunotherapy, B cells may serve as a potential biomarker to facilitate treatment selection.,Further validation in a larger patient cohort is needed.

There is far less information available about the tumor infiltrating B (TIL-B) cells, than about the tumor infiltrating T cells.,We focused on discovering the features and potential role of B lymphocytes in solid tumors.,Our project aimed to develop innovative strategies to define cancer membrane structures.,We chose two solid tumor types, with variable to considerable B cell infiltration.,The strategy we set up with invasive breast carcinoma, showing medullary features, has been introduced and standardized in metastatic melanoma.,After detecting B lymphocytes by immunohistochemistry, VH-JH, Vκ-Jκ immunoglobulin rearranged V region genes were amplified by RT-PCR, from TIL-B cDNA.,Immunoglobulin variable-region genes of interest were cloned, sequenced, and subjected to a comparative DNA analysis.,Single-chain variable (scFv) antibody construction was performed in selected cases to generate a scFv library and to test tumor binding capacity.,DNA sequence analysis revealed an overrepresented VH3-1 cluster, represented both in the breast cancer and the melanoma TIL-B immunoglobulin repertoire.,We observed that our previously defined anti GD3 ganglioside-binder antibody-variable region genes were present in melanoma as well.,Our antibody fragments showed binding potential to disialylated glycosphingolipids (GD3 ganglioside) and their O acetylated forms on melanoma cancer cells.,We conclude that our results have a considerable tumor immunological impact, as they reveal the power of TIL-B cells to recognize strong tumor-associated glycosphingolipid structures on melanomas and other solid tumors.,As tumor-derived gangliosides affect immune cell functions and reduce the B lymphocytes' antibody production, we suspect an important B lymphocyte and cancer cell crosstalk mechanism.,We not only described the isolation and specificity testing of the tumor infiltrating B cells, but also showed the TIL-B cells' highly tumor-associated GD3 ganglioside-revealing potential in melanomas.,The present data help to identify new cancer-associated biomarkers that may serve for novel cancer diagnostics.,The two-direction regulation mechanism between immune B cells and the tumor could eventually be developed into an innovative cancer treatment strategy.

DNA methylation is the most well studied epigenetic modification in cancer biology. 5-hydroxymethylcytosine is an epigenetic mark that can be converted from 5-methylcytosine by the ten-eleven translocation gene family.,We recently reported the loss of 5-hydroxymethylcytosine in melanoma compared to benign nevi and suggested that loss of this epigenetic marker is correlated with tumor virulence based on its association with a worse prognosis.,In this study we further characterize the immunoreactivity patterns of 5-hydroxymethylcytosine in the full spectrum of melanocytic lesions to further validate the potential practical application of this epigenetic marker. 175 cases were evaluated: 18 benign nevi, 20 dysplastic nevi (10 low-grade and 10 high-grade lesions), 10 atypical Spitz nevi, 20 borderline tumors, 5 melanomas arising within nevi, and 102 primary melanomas.,Progressive loss of 5-hydroxymethylcytosine from benign dermal nevi to high-grade dysplastic nevi to borderline melanocytic neoplasms to melanoma was observed.,In addition, an analysis of the relationship of nuclear diameter to 5-hydroxymethylcytosine staining intensity within lesional cells revealed a significant correlation between larger nuclear diameter and decreased levels of 5-hydroxymethylcytosine.,Furthermore, borderline lesions uniquely exhibited a diverse spectrum of staining of each individual case.,This study further substantiates the association of 5-hydroxymethylcytosine loss with dysplastic cytomorphologic features and tumor progression and supports the classification of borderline lesions as a biologically distinct category of melanocytic lesions.

In this study, Klein and colleagues investigated the impact of minimal cancer sentinel lymph node spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.,The authors found that cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death and the best predictor of outcome was a model based on combined quantitative effects of DCCD, tumor thickness, and ulceration.,Please see later in the article for the Editors' Summary,Sentinel lymph node spread is a crucial factor in melanoma outcome.,We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.,We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology.,For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100.,None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells.,We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations.,At a median follow-up of 49 mo (range 3-123 mo), 138 patients (13.4%) had died from melanoma.,Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61-2.01, for a 10-fold increase in DCCD + 1).,Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02-2.58).,Upon multivariable testing DCCD was a stronger predictor of death than histopathology.,The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.,Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death.,A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up.,If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.,Please see later in the article for the Editors' Summary,Because the skin contains many different cell types, there are many types of skin cancer.,The most dangerous type-melanoma-develops when mutations occur in melanocytes, the cells that produce the pigment melanin.,Less than 5% of skin cancers are melanomas, but melanoma causes most skin cancer deaths.,Early signs of melanoma are a change in the appearance of a mole (a pigmented skin blemish) or the development of a new and unusual pigmented lesion.,If these signs are noticed and the melanoma is diagnosed before it has spread from the skin into nearby lymph nodes and other tissues, surgery often provides a cure.,For advanced melanomas, the outlook is generally poor, although novel therapies may prolong a patient's life.,When a person is diagnosed with melanoma, it is important to “stage” the tumor.,Knowing the extent and severity of the melanoma helps oncologists plan treatments and estimate their patients' likely outcomes.,The detection of isolated melanoma cells in sentinel lymph nodes (the nodes to which cancer cells are most likely to spread from a primary tumor) is included in melanoma staging recommendations.,However, finding rare tumor cells in sentinel lymph node biopsies by examining the tissue requires the analysis of many slides from each node removed from the patient and is extremely time-consuming.,In this study, the researchers investigate the predictive value of a quantitative immunocytological assay that involves disaggregation of the sentinel node and detection of disseminated cancer cells (DCCs) by immunostaining for gp100 (a marker for melanoma cells).,They also use this new assay to examine the effect of increasing numbers of DCCs on melanoma-specific survival.,The researchers used routine histopathology and immunocytology to analyze 1,834 sentinel lymph nodes from 1,027 patients with melanoma who underwent sentinel lymph node biopsy at one German hospital.,For immunocytology, the researchers recorded the number of gp100-positive cells per million lymph node cells (the DCC density).,During follow-up, 138 patients (13.4%) died from melanoma.,The results indicated that increased DCC density was associated with an increased risk of death due to melanoma.,Specifically, every 10-fold increase in DCC density + 1 was associated with a near doubling of the risk of death from melanoma (a hazard ratio of 1.81).,Even patients with three or fewer gp100-positive cells per million lymph node cells had an increased risk of dying from melanoma compared to patients with no gp100-positive cells (hazard ratio 1.63).,When other predictors of outcome such as age and primary tumor location were taken into account, DCC density was a stronger predictor of death than histopathology.,Finally, a survival model that included tumor thickness, tumor ulceration, and DCC density provided survival prediction superior to that of a model based on the current standard staging recommendations.,These findings show that quantification of cancer cell dissemination from melanomas to sentinel lymph nodes is feasible and can be combined with other characteristics of the primary tumor to provide an accurate prediction of outcomes for individual patients with melanoma.,Notably, the new prediction model identifies a group of patients at high risk of progression for whom the current clinical standard underestimates the risk of death.,These patients may benefit from adjuvant therapies, so the new analysis presented in this study may help to stratify patients for clinical trials.,Importantly, quantitative immunocytology and the new model, although internally validated in this study, need to be validated in an independent group of patients before they can be considered for routine clinical use.,If external validation is successful, quantitative immunocytology, which is much less labor-intensive than histopathology, has the potential to change the routine clinical care of patients with melanoma and probably with other solid tumors, conclude the researchers.,Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001604.,The US National Cancer Institute provides information for patients and professionals on melanoma, cancer staging, and sentinel lymph node biopsy (in English and Spanish),The nonprofit organization American Cancer Society provides information in several languages on cancer and how it develops and specific information on melanoma, including the AJCC system for staging and personal stories,The UK National Health Service Choices website includes an introduction to cancer and a page on melanoma that includes personal stories,The nonprofit organization Cancer Research UK provides basic information about cancer and detailed information on melanoma

Although next-generation sequencing has demonstrated great potential for novel gene discovery, confirming disease-causing genes after initial discovery remains challenging.,Here, we applied a network analysis approach to prioritize candidate genes identified from whole-exome sequencing analysis of 98 cutaneous melanoma patients from 27 families.,Using a network propagation method, we ranked candidate genes by their similarity to known disease genes in protein-protein interaction networks and identified gene clusters with functional connectivity.,Using this approach, we identified several new candidate susceptibility genes that warrant future investigations such as NGLY1, IL1RN, FABP2, PRKDC, and PROSER2.,The propagated network analysis also allowed us to link families that did not have common underlying genes but that carried variants in genes that interact on protein-protein interaction networks.,In conclusion, our study provided an analysis perspective for gene prioritization in the context of genetic heterogeneity across families and prioritized top potential candidate susceptibility genes in our dataset.

CDKN2A is the main high-risk melanoma-susceptibility gene, but it has been poorly assessed in Latin America.,We sought to analyze CDKN2A and MC1R in patients from Latin America with familial and sporadic multiple primary melanoma (SMP) and compare the data with those for patients from Spain to establish bases for melanoma genetic counseling in Latin America.,Genet Med18 7, 727-736.,CDKN2A and MC1R were sequenced in 186 Latin American patients from Argentina, Brazil, Chile, Mexico, and Uruguay, and in 904 Spanish patients.,Clinical and phenotypic data were obtained.,Genet Med18 7, 727-736.,Overall, 24 and 14% of melanoma-prone families in Latin America and Spain, respectively, had mutations in CDKN2A.,Latin American families had CDKN2A mutations more frequently (P = 0.014) than Spanish ones.,Of patients with SMP, 10% of those from Latin America and 8.5% of those from Spain had mutations in CDKN2A (P = 0.623).,The most recurrent CDKN2A mutations were c.-34G>T and p.G101W.,Latin American patients had fairer hair (P = 0.016) and skin (P < 0.001) and a higher prevalence of MC1R variants (P = 0.003) compared with Spanish patients.,Genet Med18 7, 727-736.,The inclusion criteria for genetic counseling of melanoma in Latin America may be the same criteria used in Spain, as suggested in areas with low to medium incidence, SMP with at least two melanomas, or families with at least two cases among first- or second-degree relatives.,Genet Med18 7, 727-736.

Uveal melanoma (UM) is the most common intraocular tumor in adults.,Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking.,A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins.,Here, we have addressed the therapeutic potential of FR for UM.,We found that FR inhibited all oncogenic Gq/11 mutants reported in UM.,FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few.,Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors.,FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential.,FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models and reduced blood pressure transiently.,FR did not regress xenografted UM tumors or significantly affect heart rate, liver function, hematopoiesis, or behavior.,These results indicated the existence of a therapeutic window in which FR can be explored for treating UM and potentially other diseases caused by constitutively active Gq/11.

The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy.,Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas.,We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms.,Expression of all class-IA PI3K isoforms was readily detected in these cell lines.,A range of genetic changes in different components of the PI3K pathway was seen in different lines.,Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common.,Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common.,Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines.,Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes.,While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial.,This suggests that functional redundancy exists between PI3K isoforms.,Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition.,Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474.,Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.,The online version contains supplementary material available at 10.1186/s12885-021-07826-4.

In general, melanoma can be considered as a UV‐driven disease with an aggressive metastatic course and high mutational load, with only few tumors (acral, mucosal, and uveal melanomas) not induced by sunlight and possessing a lower mutational load.,The most commonly activated pathway in melanoma is the mitogen‐activated protein kinase (MAPK) pathway.,However, the prognostic significance of mutational stratification is unclear and needs further investigation.,Here, in silico we combined mutation data from 162 melanomas subjected to targeted deep sequencing with mutation data from three published studies.,Tumors from 870 patients were grouped according to BRAF,RAS,NF1 mutation or triple‐wild‐type status and correlated with tumor and patient characteristics.,We found that the NF1‐mutated subtype had a higher mutational burden and strongest UV mutation signature.,Searching for co‐occurring mutated genes revealed the RASopathy genes PTPN11 and RASA2, as well as another RAS domain‐containing gene RASSF2 enriched in the NF1 subtype after adjustment for mutational burden.,We found that a larger proportion of the NF1‐mutant tumors were from males and with older age at diagnosis.,Importantly, we found an increased risk of death from melanoma (disease‐specific survival, DSS; HR, 1.9; 95% CI, 1.21-3.10; P = 0.046) and poor overall survival (OS; HR, 2.0; 95% CI, 1.28-2.98; P = 0.01) in the NF1 subtype, which remained significant after adjustment for age, gender, and lesion type (DSS P = 0.03, OS P = 0.06, respectively).,Melanoma genomic subtypes display different biological and clinical characteristics.,The poor outcome observed in the NF1 subtype highlights the need for improved characterization of this group.

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear1,2.,The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event3.,To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a V600EBRAF mouse model.,In mice expressing V600EBRAF in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden.,We show that sunscreen (UVA superior: UVB SPF50) delayed the onset of UVR-driven melanoma, but only provided partial protection.,The UVR-exposed tumours presented increased numbers of single nucleotide variants (SNVs) and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in ~40% of cases.,TP53 is an accepted UVR target in non-melanoma skin cancer, but is not thought to play a major role in melanoma4.,However, we show that mutant Trp53 accelerated V600EBRAF-driven melanomagenesis and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma.,Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans5.,We identify TP53/Trp53 as a UVR-target gene that cooperates with V600EBRAF to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis.,Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

Metastatic melanoma (mM) and renal cell carcinoma (mRCC) are often treated with anti-PD-1 based therapy, however not all patients respond and further therapies are needed.,High dose interleukin-2 (HD IL-2) can lead to durable responses in a subset of mM and mRCC patients.,The efficacy and toxicity of HD IL-2 therapy following anti-PD-1 or anti-PD-L1 therapy have not yet been explored.,Reports on mM and mRCC patients who had received HD IL-2 after PD-1 or PD-L1 inhibition were queried from the PROCLAIMSM database.,Patient characteristics, toxicity and efficacy were analyzed.,A total of 57 patients (40 mM, 17 mRCC) were treated with high dose IL-2 after PD-1 or PD-L1 inhibition and had data recorded in the PROCLAIM database.,The best overall response rate to HD IL-2 was 22.5% for mM (4 complete response (CR), 5 partial responses (PRs)) and 24% for mRCC (2 CRs, 2 PRs).,The toxicity related to HD IL-2 observed in these patients was similar to that observed in patients treated with HD IL-2 without prior checkpoint blockade.,One patient who had received prior PD-L1 blockade developed drug induced pneumonitis with HD IL-2 requiring steroid therapy.,In this retrospective analysis, HD IL-2 therapy displayed durable antitumor activity in mM and mRCC patients who progressed following treatment with PD-1 and PD-L1 inhibition.,The toxicities were generally manageable and consistent with expectations from HD IL-2 but physicians should watch for immune related toxicities such as pneumonitis.,This analysis supports the development of randomized prospective trials to assess the proper sequencing and combination of immune checkpoint blockade and cytokine therapy.,The online version of this article (10.1186/s40425-019-0522-3) contains supplementary material, which is available to authorized users.

Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment.,Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples.,However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves.,With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information.,Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry.,Out of the ten patients, six had clinical follow-up data.,The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining.,Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease.,In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2.,The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content.,Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB.,A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival.,In this feasibility study, promising results show the feasibility of relating proteomics to histopathology and clinical outcome, and insight thus can be gained into the molecular processes driving the disease.,The combined analysis of histological features including the sample cellular composition with protein expression of each metastasis enabled the identification of novel, differentially expressed proteins.,Further studies are necessary to determine whether these putative biomarkers can be utilized in diagnostics and prognostic prediction of metastatic melanoma.

Epigenetic changes influence various physiological and pathological conditions in the human body.,Recent advances in epigenetic studies of the skin have led to an appreciation of the importance of epigenetic modifications in skin diseases.,Cutaneous sarcomas are intractable skin cancers, and there are no curative therapeutic options for the advanced forms of cutaneous sarcomas.,In this review, we discuss the detailed molecular effects of epigenetic modifications on skin sarcomas, such as dermatofibrosarcoma protuberans, angiosarcoma, Kaposi’s sarcoma, leiomyosarcoma, and liposarcoma.,We also discuss the application of epigenetic-targeted therapy for skin sarcomas.

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2.,BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors.,Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5.,For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events.,Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma.,SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish.,Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1.,Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Recently, it has been described that programmed cell death protein 1 (PD-1) overexpressing melanoma cells are highly aggressive.,However, until now it has not been defined which factors lead to the generation of PD-1 overexpressing subpopulations.,Here, we present that melanoma-derived exosomes, conveying oncogenic molecular reprogramming, induce the formation of a melanoma-like, PD-1 overexpressing cell population (mMSCPD-1+) from naïve mesenchymal stem cells (MSCs).,Exosomes and mMSCPD-1+ cells induce tumor progression and expression of oncogenic factors in vivo.,Finally, we revealed a characteristic, tumorigenic signaling network combining the upregulated molecules (e.g., PD-1, MET, RAF1, BCL2, MTOR) and their upstream exosomal regulating proteins and miRNAs.,Our study highlights the complexity of exosomal communication during tumor progression and contributes to the detailed understanding of metastatic processes.

BRAF inhibitors can extend progression‐free and overall survival in melanoma patients whose tumors harbor mutations in BRAF.,However, the majority of patients eventually develop resistance to these drugs.,Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival.,Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation.,We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first‐line setting.,We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.,Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.,We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.,Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.,Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.

Metastatic melanoma is challenging to manage.,Although targeted- and immune therapies have extended survival, most patients experience therapy resistance.,The adaptability of melanoma cells in nutrient- and therapeutically-challenged environments distinguishes melanoma as an ideal model for investigating therapy resistance.,In this review, we discuss the current available repertoire of melanoma models including two- and three-dimensional tissue cultures, organoids, genetically engineered mice and patient-derived xenograft.,In particular, we highlight how each system recapitulates different features of melanoma adaptability and can be used to better understand melanoma development, progression and therapy resistance.,Despite the new targeted and immunotherapies for metastatic melanoma, several patients show therapeutic plateau.,Here, the authors review the current pre-clinical models of cutaneous melanoma and discuss their strengths and limitations that may help with overcoming therapeutic plateau.

Malignant melanoma is one of the most metastatic cancer types, and despite recent success with novel treatment strategies, there is still a group of patients who do not respond to any therapies.,Earlier, the prenylation inhibitor hydrophilic bisphosphonate zoledronic acid (ZA) was found to inhibit melanoma growth in vitro, but only a weaker effect was observed in vivo due to its hydrophilic properties.,Recently, lipophilic bisphosphonates (such as BPH1222) were developed.,Accordingly, for the first time, we compared the effect of BPH1222 to ZA in eight melanoma lines using viability, cell-cycle, clonogenic and spheroid assays, videomicroscopy, immunoblot, and xenograft experiments.,Based on 2D and spheroid assays, the majority of cell lines were more sensitive to BPH.,The activation of Akt and S6 proteins, but not Erk, was inhibited by BPH.,Additionally, BPH had a stronger apoptotic effect than ZA, and the changes of Rheb showed a correlation with apoptosis.,In vitro, only M24met cells were more sensitive to ZA than to BPH; however, in vivo growth of M24met was inhibited more strongly by BPH.,Here, we present that lipophilic BPH is more effective on melanoma cells than ZA and identify the PI3K pathway, particularly Rheb as an important mediator of growth inhibition.

Hotspot mutations of the oncogenes BRAF and NRAS are the most common genetic alterations in cutaneous melanoma.,Specific inhibitors of BRAF and MEK have shown significant survival benefits in large phase III trials.,However, the prognostic significance of BRAF and NRAS mutations outside of clinical trials remains unclear.,The mutational status of BRAF (exon 15) and NRAS (exon 2 and 3) was determined in melanoma samples of 217 patients with pyrosequencing and Sanger sequencing.,The genotypes were correlated with clinical outcomes and pathologic features of the primary tumors.,Time to disease progression was calculated with the cumulative incidence function.,Survival analyses were performed with Kaplan-Meier estimates and Cox proportional hazards regression analysis.,Relative survival was calculated with the Ederer-II method.,Treatment with BRAF and MEK inhibitors and immune checkpoint blockade (ICB) was allowed.,Mutations in BRAF and NRAS were identified in 40.1 and 24.4% of cases, respectively.,Concurrent mutations in both genes were detected in further 2.3%.,The remaining 33.2% were wild type for the investigated exons (WT).,BRAF mutations were significantly associated with younger age at first diagnosis (p < 0.001) and truncal localization of the culprit primary (p = 0.002).,The nodular subtype was most common in the NRAS cohort.,In addition, NRAS-mutant melanoma patients showed a higher frequency of nodal relapse (p = 0.013) and development of metastatic disease (p = 0.021).,The time to loco-regional nodal relapse was shortest in NRAS-mutant melanoma (p = 0.002).,Presence of NRAS mutation was an independent risk factor for disease progression in multivariate analysis (HR 2.01; 95% CI 1.02 - 3.98).,BRAF-mutant melanoma patients showed a tendency for better overall and relative survival.,Genotype was not a consistent risk factor in multivariate analysis.,Instead, positive sentinel lymph node status (HR 2.65; 95% CI 1.15 - 6.10) and treatment with ICB in stage IV disease (HR 0.17; 95% CI 0.06-0.48) were significant multivariate risk factors.,NRAS-mutant tumors tended to behave more aggressively particularly in early stages of the disease in this high-risk melanoma population.,Treatment with immune checkpoint blockade improved survival in stage IV disease in a real-world setting.,The online version of this article (doi:10.1186/s12885-017-3529-5) contains supplementary material, which is available to authorized users.

The detection of V600E BRAF mutations has fundamental clinical consequences as the treatment option with BRAF inhibitors such as vemurafenib or dabrafenib yields response rates of ∼48%.,Heterogeneity with respect to BRAF mutation in different metastases has been described in single cases.,As this has important implications for the determination of BRAF status and treatment of patients, it is essential to acquire more data.,A total of 300 tumour samples from 187 melanoma patients were analysed for BRAF mutations by pyrosequencing.,Equivocal results were confirmed by capillary sequencing.,Clinical data with respect to melanoma type, tumour site and survival were summarised for 53 patients with multiple analysed tumour samples (2-13 per patient).,BRAF mutations were found in 84 patients (44.9%) and 144 tumour samples (48%) with BRAF mutations in 45.5% of primary tumours and 51.3% of metastases, respectively.,In 10 out of 53 patients (18.9%) where multiple samples were analysed results were discordant with respect to mutation findings with wild-type and mutated tumours in the same patient.,Mutations did not appear more frequently over the course of disease nor was its occurrence associated with a specific localisation of metastases.,As heterogeneity with respect to BRAF mutation status is detected in melanoma patients, subsequent testing of initially wild-type patients can yield different results and thus make BRAF inhibitor therapy accessible.,The role of heterogeneity in testing and for clinical response to therapy with a BRAF inhibitor needs to be further investigated.

Specific patterns of DNA mutations in normal skin show history of UV exposure and association with cancer risk.,In ultraviolet (UV) radiation-exposed skin, mutations fuel clonal cell growth.,The relationship between UV exposure and the accumulation of clonal mutations (CMs) and the correlation between CMs and skin cancer risk are largely unexplored.,We characterized 450 individual-matched sun-exposed (SE) and non-SE (NE) normal human skin samples.,The number and relative contribution of CMs were significantly different between SE and NE areas.,Furthermore, we identified hotspots in TP53, NOTCH1, and GRM3 where mutations were significantly associated with UV exposure.,In the normal skin from patients with cutaneous squamous cell carcinoma, we found that the cancer burden was associated with the UV-induced mutations, with the difference mostly conferred by the low-frequency CMs.,These findings provide previously unknown information on UV’s carcinogenic effect and pave the road for future development of quantitative assessment of subclinical UV damage and skin cancer risk.

Capsaicin is the main pungent in chili peppers, one of the most commonly used spices in the world; its analgesic and anti-inflammatory properties have been proven in various cultures for centuries.,It is a lipophilic substance belonging to the class of vanilloids and an agonist of the transient receptor potential vanilloid 1 receptor.,Taking into consideration the complex neuro-immune impact of capsaicin and the potential link between inflammation and carcinogenesis, the effect of capsaicin on muco-cutaneous cancer has aroused a growing interest.,The aim of this review is to look over the most recent data regarding the connection between capsaicin and muco-cutaneous cancers, with emphasis on melanoma and muco-cutaneous squamous cell carcinoma.

While multiple mechanisms of BRAFV600-mutant melanoma resistance to targeted MAPK signaling inhibitors (MAPKi) have been reported, the epigenetic regulation of this process remains undetermined.,Here, using a CRISPR-Cas9 screen targeting chromatin regulators, we discover that haploinsufficiency of the histone deacetylase SIRT6 allows melanoma cell persistence in the presence of MAPKi.,Haploinsufficiency, but not complete loss of SIRT6 promotes IGFBP2 expression via increased chromatin accessibility, H3K56 acetylation at the IGFBP2 locus, and consequent activation of the IGF-1 receptor (IGF-1R) and downstream AKT signaling.,Combining a clinically applicable IGF-1Ri with BRAFi overcomes resistance of SIRT6 haploinsufficient melanoma cells in vitro and in vivo.,Using matched melanoma samples derived from patients receiving dabrafenib + trametinib, we identify IGFBP2 as a potential biomarker for MAPKi resistance.,Our study has not only identified an epigenetic mechanism of drug resistance, but also provides insights into a combinatorial therapy that may overcome resistance to standard-of-care therapy for BRAFV600-mutant melanoma patients.,The epigenetic mechanisms of melanoma drug resistance are poorly understood.,Here, the authors develop a CRISPR-Cas9 screen targeting epigenetic regulators and discover that SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling.

Analysis of 501 melanoma exomes revealed RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas.,Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration.,RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival.,These findings reveal RASA2 inactivation as a melanoma driver and highlight the importance of Ras GAPs in cancer.