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Melanin, the pigment produced by specialized cells, melanocytes, is responsible for skin and hair color.,Skin pigmentation is an important protective mechanism against the DNA damaging and mutagenic effects of solar ultraviolet radiation (UV).,It is acknowledged that exposure to UV is the main etiological environmental factor for all forms of skin cancer, including melanoma.,DNA repair capacity is another major factor that determines the risk for skin cancer.,Human melanocytes synthesize eumelanin, the dark brown form of melanin, as well as pheomelanin, which is reddish-yellow in color.,The relative rates of eumelanin and pheomelanin synthesis by melanocytes determine skin color and the sensitivity of skin to the drastic effects of solar UV.,Understanding the complex regulation of melanocyte function and how it responds to solar UV has a huge impact on developing novel photoprotective strategies to prevent skin cancer, particularly melanoma, the most fatal form, which originates from melanocytes.,This review provides an overview of the known differences in the photoprotective effects of eumelanin versus pheomelanin, how these two forms of melanin are regulated genetically and biochemically, and their impact on the DNA damaging effects of UV exposure.,Additionally, this review briefly discusses the role of paracrine factors, focusing on α-melanocortin (α-melanocyte stimulating hormone; α-MSH), in regulating melanogenesis and the response of melanocytes to UV, and describes a chemoprevention strategy based on targeting the melanocortin 1 receptor (MC1R) by analogs of its physiological agonist α-MSH.

To elucidate the complex molecular mechanisms underlying the adverse effects UV radiation (UVR) on skin homeostasis, we performed multi-omics studies to characterize UV-induced genetic and epigenetic changes.,Human keratinocytes from a single donor treated with or without UVR were analyzed by RNA-seq, exome-seq, and H3K27ac ChIP-seq at 4 h and 72 h following UVR.,Compared to the relatively moderate mutagenic effects of UVR, acute UV exposure induced substantial epigenomic and transcriptomic alterations, illuminating a previously underappreciated role of epigenomic and transcriptomic instability in skin pathogenesis.,Integration of the multi-omics data revealed that UVR-induced transcriptional dysregulation of a subset of genes was attributable to either genetic mutations or global redistribution of H3K27ac.,H3K27ac redistribution further led to the formation of distinctive super enhancers in UV-irradiated cells.,Our analysis also identified several new UV target genes, including CYP24A1, GJA5, SLAMF7 and ETV1, which were frequently dysregulated in human squamous cell carcinomas, highlighting their potential as new molecular targets for prevention or treatment of UVR-induced skin cancers.,Taken together, our concurrent multi-omics analyses provide new mechanistic insights into the complex molecular networks underlying UV photobiological effects, which have important implications in understanding its impact on skin homeostasis and pathogenesis.

Hypoxia, a condition of oxygen deprivation, is considered a hallmark of tumor microenvironment regulating several pathways and promoting cancer progression and resistance to therapy.,Semaphorins, a family of about 20 secreted, transmembrane and GPI-linked glycoproteins, and their cognate receptors (plexins and neuropilins) play a pivotal role in the crosstalk between cancer and stromal cells present in the tumor microenvironment.,Many studies reported that some semaphorins are involved in the development of a permissive tumor niche, guiding cell-cell communication and, consequently, the development and progression, as well as the response to therapy, of different cancer histotypes, including melanoma.,In this review we will summarize the state of art of semaphorins regulation by hypoxic condition in cancer with different origin.,We will also describe evidence about the ability of semaphorins to affect the expression and activity of transcription factors activated by hypoxia, such as hypoxia-inducible factor-1.,Finally, we will focus our attention on findings reporting the role of semaphorins in melanocytes transformation, melanoma progression and response to therapy.,Further studies are necessary to understand the mechanisms through which semaphorins induce their effect and to shed light on the possibility to use semaphorins or their cognate receptors as prognostic markers and/or therapeutic targets in melanoma or other malignancies.

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.

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.

Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes.,Melanoma provides a recent example of this paradigm.,We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.,The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5-10% mutant allele frequency) and minimal amounts of DNA (10-20 nanograms).,The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue.,Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.,Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively.,Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.,We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma.,Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.

Staphylococcus aureus,and its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL).,CD8+ T cells play a crucial role in anti-cancer responses and high CD8+ T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL.,Here, we show that CD8+ T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not.,Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8+ T cells, leading to their escape from induced cell death and continued proliferation.,These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivo by inhibiting CD8+ T cell cytotoxicity.,Thus, we propose a novel mechanism by which colonization with Staphylococcus aureus may contribute to cancer immune evasion and disease progression in CTCL.

In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency.,Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus.,Bacterial toxins such as staphylococcal enterotoxins (SE) have long been suspected to be involved in the pathogenesis in CTCL.,Here, we review links between bacterial infections and CTCL with focus on earlier studies addressing a direct role of SE on malignant T cells and recent data indicating novel indirect mechanisms involving SE- and cytokine-driven cross-talk between malignant- and non-malignant T cells.

The mechanisms by which some melanoma cells adapt to BRAF inhibitor therapy are incompletely understood.,In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF-mutant and PTEN-null.,Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN-dependent.,These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs.,Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment.,Analysis of a melanoma TMA showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis.,Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin.,This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1.,The protection conveyed by the induction of fibronectin expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAFV600E melanoma.,Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors.,We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC50) and maximal effect (i.e., Emax ≪ 1).,Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing.,This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug Emax.,Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.

This study was to investigate the synergistic effect of NB/Cur on growth and apoptosis in A375 human melanoma cell line by MTT assay, flow cytometry and Western blotting.,Our results demonstrated that NB effectively synergized with Cur to enhance its antiproliferative activity on A375 human melanoma cells by induction of apoptosis, as evidenced by an increase in sub-G1 cell population, DNA fragmentation, PARP cleavage and caspase activation.,Further mechanistic studies by Western blotting showed that after treatment of the cells with NB/Cur, up-regulation of the expression level of phosphorylated JNK and down-regulation of the expression level of phosphorylated ERK and Akt contributed to A375 cells apoptosis.,Moreover, NB also potentiated Cur to trigger intracellular ROS overproduction and the DNA damage with up-regulation of the expression level of phosphorylated ATM, phosphorylated Brca1 and phosphorylated p53.,The results indicate the combinational application potential of NB and Cur in treatments of cancers.

Atmospheric gas plasmas (AGPs) up-regulate intracellular ROS levels and induce apoptosis in melanoma cells.,Evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.,Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity.,However, the mechanism of AGP-induced cancer cell death is unknown.,In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells.,By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells.,TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis.,Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis.,Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis.,The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Melanoma is the deadliest form of skin cancer and one of few cancers with a growing incidence.,A thorough understanding of its pathogenesis is fundamental to developing new strategies to combat mortality and morbidity.,Zebrafish-due in large part to their tractable genetics, conserved pathways, and optical properties-have emerged as an excellent system to model melanoma.,Zebrafish have been used to study melanoma from a single tumor initiating cell, through metastasis, remission, and finally into relapse.,In this review, we examine seminal zebrafish studies that have advanced our understanding of melanoma.

Fascin is a F-actin bundling protein and its overexpression is correlated with poor prognosis and increases metastatic potential in a number of cancers.,But underlying function and mechanism of fascin on tumorigenesis in melanoma remain elusive.,The melanoma cell lines WM793 and WM39 were employed for the soft agar and sphere formation assay.,Quantitative RT-PCR and Western blot were performed for identifying the gene expression at mRNA and protein levels, respectively.,Co-IP and in vitro GST pulldown experiments were used to test the interaction between fascin and MST2.,Fascin regulates tumorigenesis and cancer cell stemness in melanoma through inhibition of the Hippo pathway kinase MST2 and the activation of transcription factor TAZ.,Our data showed that fascin interacts with the kinase domain of MST2 to inhibit its homodimer formation and kinase activity.,Depletion of fascin led to increase of p-LATS level and decrease of TAZ, but not YAP.,We also demonstrated that fascin regulates melanoma tumorigenesis independent of its actin-bundling activity.,Fascin is a new regulator of the MST2-LATS-TAZ pathway and plays a critical role in melanoma tumorigenesis.,Inhibition of fascin reduces melanoma tumorigenesis and stemness, and thus fascin could be a potential therapeutic target for this malignancy.,The online version of this article (10.1186/s12964-018-0250-1) contains supplementary material, which is available to authorized users.

Anoikis resistance is one of the abilities acquired along tumor progression.,This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process.,In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition.,After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively.,These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells.,After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained.,Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype.,Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice.,However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.,The β1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies.,To analyze the interaction among Timp1, CD63 and β1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002.,Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.,Differential association among Timp1, CD63 and β1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells.,Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation.,In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.,Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and β1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation.,In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.

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.

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.

The molecular mechanisms mediating CYLD tumor suppressor function appear to be manifold.,Here, we demonstrated that, in contrast to the increased levels of pJNK, CYLD was decreased in a majority of melanoma cell lines and tissues examined.,Exogenous expression of CYLD but not its catalytically deficient mutant markedly inhibited melanoma cell proliferation and migration in vitro and subcutaneous tumor growth in vivo.,In addition, the melanoma cells expressing exogenous CYLD were unable to form pulmonary tumor nodules following tail-vein injection.,At the molecular level, CYLD decreased β1-integrin and inhibited pJNK induction by TNFα or cell-attachment to collagen IV.,Moreover, CYLD induced an array of other molecular changes associated with modulation of the ‘malignant’ phenotype, including a decreased expression of cyclin D1, N-cadherin and nuclear Bcl3, and an increased expression of p53 and E-cadherin.,Most interestingly, co-expression of the constitutively active MKK7 or c-Jun mutants with CYLD prevented the above molecular changes, and fully restored melanoma growth and metastatic potential in vivo.,Our findings demonstrate that JNK/AP-1 signaling pathway underlies the melanoma growth and metastasis that is associated with CYLD loss-of-function.,Thus, restoration of CYLD and inhibition of JNK and β1-integrin function represent potential therapeutic strategies for treatment of malignant melanoma.

Human malignant melanoma shows a high rate of mortality after metastasization, and its incidence is continuously rising worldwide.,Several studies have suggested that MCAM/MUC18/CD146 plays an important role in the progression of this malignant disease.,MCAM/MUC18/CD146 is a typical single-spanning transmembrane glycoprotein, existing as two membrane isoforms, long and short, and an additional soluble form, sCD146.,We previously documented that molecular MCAM/MUC18/CD146 expression is strongly associated with disease progression.,Recently, we showed that MCAM/MUC18/CD146 and ABCB5 can serve as melanoma-specific-targets in the selection of highly primitive circulating melanoma cells, and constitute putative proteins associated with disease spreading progression.,Here, we analyzed CD146 molecular expression at onset or at disease recurrence in an enlarged melanoma case series.,For some patients, we also performed the time courses of molecular monitoring.,Moreover, we explored the role of soluble CD146 in different cohorts of melanoma patients at onset or disease progression, rather than in clinical remission, undergoing immune therapy or free from any clinical treatment.,We showed that MCAM/MUC18/CD146 can be considered as: (1) a membrane antigen suitable for identification and enrichment in melanoma liquid biopsy; (2) a highly effective molecular “warning” marker for minimal residual disease monitoring; and (3) a soluble protein index of inflammation and putative response to therapeutic treatments.

Background.,Dendritic cells could be involved in immune surveillance of highly immunogenic tumors such as melanoma.,Their role in the progression melanocytic nevi to melanoma is however a matter of controversy.,Methods.,The number of dendritic cells within epidermis, in peritumoral zone, and within the lesion was counted on slides immunohistochemically stained for CD1a, CD1c, DC-LAMP, and DC-SIGN in 21 of dysplastic nevi, 27 in situ melanomas, and 21 invasive melanomas.,Results.,We found a significant difference in the density of intraepidermal CD1c+ cells between the examined lesions; the mean CD1c cell count was 7.00/mm2 for invasive melanomas, 2.94 for in situ melanomas, and 13.35 for dysplastic nevi.,The differences between dysplastic nevi and melanoma in situ as well as between dysplastic nevi and invasive melanoma were significant.,There was no correlation in number of positively stained cells between epidermis and dermis.,We did not observe any intraepidermal DC-LAMP+ cells neither in melanoma in situ nor in invasive melanoma as well as any intraepidermal DC-SIGN+ cells in dysplastic nevi.,Conclusion.,It was shown that the number of dendritic cells differs between dysplastic nevi, in situ melanomas, and invasive melanomas.,This could eventually suggest their participation in the development of melanoma.

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.

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.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting, and association with ultraviolet (UV) exposure early in life1.,To obtain a comprehensive genomic view of melanoma, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA.,A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-UV exposed hairless skin of the extremities (3 and 14 per Mb genome), intermediate in those originating from hair-bearing skin of the trunk (range = 5 to 55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb).,Analysis of whole-genome sequence data identified PREX2 - a PTEN-interacting protein and negative regulator of PTEN in breast cancer2 - as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas.,PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumor formation of immortalized human melanocytes in vivo.,Thus, whole-genome sequencing of human melanoma tumors revealed genomic evidence of UV pathogenesis and discovered a new recurrently mutated gene in melanoma.

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.

Melanoma is an aggressive malignancy with a poor prognosis.,Current studies show that imatinib treatment is a promising approach in treating advanced melanoma patients harboring c-Kit mutations or amplifications.,We retrospectively analyzed the clinical medical records of 78 patients with metastatic melanoma harboring c-Kit mutations or amplifications.,These patients were treated with imatinib at a dose of 400 mg/day continuously unless intolerable toxicities or disease progression occurred.,Endpoints for exploration included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease of control rate (DCR).,The median OS and PFS of all patients were 13.1 and 4.2 months, respectively.,ORR and DCR were 21.8% and 60.3%, respectively.,The survival time of patients who achieved partial response or stable disease was significantly superior to those with disease progression.,Cox regression analysis showed that patients with M1c stage, subtype of cutaneous melanoma, or elevated LDH level (>upper limit of normal) had higher hazard ratios for overall survival.,Our study, combined with those studies targeting patients with a c-Kit alteration, validates the role of imatinib as an important and promising therapeutic agent in the treatment of patients with advanced melanoma.

Immune checkpoint inhibitors have improved the prognosis of advanced melanoma.,Although anti-programmed death ligand‐1 (PD‐L1) is a well‐studied biomarker for response to anti-programmed death‐1 PD‐1 therapy in melanoma, its clinical relevance remains unclear.,It has been established that the high expression of indoleamine 2,3‐dioxygenase (IDO) is correlated to a response to anti-CTLA‐4 treatment in melanoma.,However, it is still unknown whether the IDO expression is associated with response to anti-PD‐1 therapy in advanced melanoma.,In addition, acral and mucosal melanomas, which comprise a great proportion of all melanomas in Asians, are genetically different subtypes from cutaneous melanomas; however, they have not been independently analyzed due to their low frequency in Western countries.,To evaluate the association of IDO and PD‐L1 expression with response to anti-PD‐1 antibody in acral and mucosal melanoma patients, we analyzed 32 Japanese patients with acral and mucosal melanomas treated with anti-PD‐1 antibody from the perspective of IDO and PD‐L1 expression levels by immunohistochemistry (IHC).,Multivariate Cox regression models showed that the low expression of IDO in tumors was associated with poor progression‐free survival (HR = 0.33, 95% CI = 0.13‐0.81, P = 0.016), whereas PD‐L1 expression on tumors was not associated with progression‐free survival.,Significantly lower expression of IDO in tumors was found in non-responders compared to responders.,Assessment of the IDO expression could be useful for the identification of suitable candidates for anti-PD‐1 therapy among acral and mucosal melanomas patients.,Further validation study is needed to estimate the clinical utility of our findings.

Human telomerase reverse transcriptase (hTERT) plays a crucial role in cancer development.,We previously identified paired-like homeodomain1 (PITX1) as an hTERT suppressor gene.,However, the underlying mechanisms that are involved in the regulation of PITX1 remain unknown.,Here, we report that the microRNA-19b (miR-19b) regulates hTERT expression and cell proliferation through inhibition of PITX1.,Compared with normal melanocyte cells, miR-19b expression was higher in most melanoma cells and was accompanied by downregulation of PITX1.,Moreover, overexpression of miR-19b inhibited PITX1 mRNA translation through a miR-19b binding site within the 3′UTR of the PITX1 mRNA.,Our combined findings indicate the participation of miR-19b as a novel upstream effector of hTERT transcription via direct targeting of PITX1.

The Microphthalmia associated transcription factor (Mitf) is an important regulator in melanocyte development and has been shown to be involved in melanoma progression.,The current model for the role of Mitf in melanoma assumes that the total activity of the protein is tightly regulated in order to secure cell proliferation.,Previous research has shown that regulation of Mitf is complex and involves regulation of expression, splicing, protein stability and post-translational modifications.,Here we show that microRNAs (miRNAs) are also involved in regulating Mitf in melanoma cells.,Sequence analysis revealed conserved binding sites for several miRNAs in the Mitf 3′UTR sequence.,Furthermore, miR-148 was shown to affect Mitf mRNA expression in melanoma cells through a conserved binding site in the 3′UTR sequence of mouse and human Mitf.,In addition we confirm the previously reported effects of miR-137 on Mitf.,Other miRNAs, miR-27a, miR-32 and miR-124 which all have conserved binding sites in the Mitf 3′UTR sequence did not have effects on Mitf.,Our data show that miR-148 and miR-137 present an additional level of regulating Mitf expression in melanocytes and melanoma cells.,Loss of this regulation, either by mutations or by shortening of the 3′UTR sequence, is therefore a likely factor in melanoma formation and/or progression.

Spatial profiling of cutaneous melanoma at the single-cell level provided molecular features of progression and immunosuppression, which include paracrine cytokine signaling and direct immune cell-cell contact.,Cutaneous melanoma is a highly immunogenic malignancy that is surgically curable at early stages but life-threatening when metastatic.,Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially resolved microregion transcriptomics to study immune evasion and immunoediting in primary melanoma.,We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis involving precursor states, melanoma in situ, and invasive tumor.,Hallmarks of immunosuppression are already detectable in precursor regions.,When tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary.,This environment is established by cytokine gradients that promote expression of MHC-II and IDO1, and by PD1-PDL1-mediated cell contacts involving macrophages, dendritic cells, and T cells.,A few millimeters away, cytotoxic T cells synapse with melanoma cells in fields of tumor regression.,Thus, invasion and immunoediting can coexist within a few millimeters of each other in a single specimen.,The reorganization of the tumor ecosystem in primary melanoma is an excellent setting in which to study immunoediting and immune evasion.,Guided by classic histopathology, spatial profiling of proteins and mRNA reveals recurrent morphologic and molecular features of tumor evolution that involve localized paracrine cytokine signaling and direct cell-cell contact.,This article is highlighted in the In This Issue feature, p.,1397

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment.,We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort.,We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival.,This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin-mediated failure to recruit CD141+ DCs.,A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles.,The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation.,The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants.,In summary, we report evidence for a β-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome.,We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.

Fourier transform infrared (FTIR) microspectroscopy was used to evaluate the growth of human melanoma cells (SK-MEL-2) in two-dimensional (2D) versus three-dimensional (3D) spheroid culture systems.,FTIR microspectroscopy, coupled with multivariate analysis, could be used to monitor the variability of spheroid morphologies prepared from different cell densities.,The characteristic shift in absorbance bands of the 2D cells were different from the spectra of cells from 3D spheroids.,FTIR microspectroscopy can also be used to monitor cell death similar to fluorescence cell staining in 3D spheroids.,A change in the secondary structure of protein was observed in cells from the 3D spheroid versus the 2D culture system.,FTIR microspectroscopy can detect specific alterations in the biological components inside the spheroid, which cannot be detected using fluorescence cell death staining.,In the cells from 3D spheroids, the respective lipid, DNA, and RNA region content represent specific markers directly proportional to the spheroid size and central area of necrotic cell death, which can be confirmed using unsupervised PCA and hierarchical cluster analysis.,FTIR microspectroscopy could be used as an alternative tool for spheroid cell culture discrimination, and validation of the usual biochemical technique.

Melanoma is the most aggressive form of skin cancer.,Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy.,To increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1).,With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro.,In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft.,We demonstrated two to five times higher binding and internalization of these immunoliposomes by M1+/A1+ melanoma cells in vitro in comparison with nontargeted liposomes.,Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes.,In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively.,Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1+/A1+ expressing tumors in mice compared with the treatment of M1−/A1+ tumors.,Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo.,Liposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma.,We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response.,Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy.,Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

Cutaneous melanoma is a complex disorder characterized by an elevated degree of heterogeneity, features that place it among the most aggressive types of cancer.,Although significant progress was recorded in both the understanding of melanoma biology and genetics, and in therapeutic approaches, this malignancy still represents a major problem worldwide due to its high incidence and the lack of a curative treatment for advanced stages.,This review offers a survey of the most recent information available regarding the melanoma epidemiology, etiology, and genetic profile.,Also discussed was the topic of cutaneous melanoma murine models outlining the role of these models in understanding the molecular pathways involved in melanoma initiation, progression, and metastasis.

The major genetic determinants of cutaneous melanoma risk in the general population are disruptive variants (R alleles) in the melanocortin 1 receptor (MC1R) gene.,These alleles are also linked to red hair, freckling, and sun sensitivity, all of which are known melanoma phenotypic risk factors.,Here we report that in melanomas and for somatic C>T mutations, a signature linked to sun exposure, the expected single-nucleotide variant count associated with the presence of an R allele is estimated to be 42% (95% CI, 15-76%) higher than that among persons without an R allele.,This figure is comparable to the expected mutational burden associated with an additional 21 years of age.,We also find significant and similar enrichment of non-C>T mutation classes supporting a role for additional mutagenic processes in melanoma development in individuals carrying R alleles.,Deleterious germline variants in the MC1R gene are associated with red hair and freckles, and with an increased risk of developing melanoma.,Here, the authors investigate melanoma samples from patients with and without these variants and find that their presence is predictive of a higher overall mutation prevalence.

Tenascin-C (TNC) is highly expressed in melanoma; however, little is known about its functions.,Recent studies indicate that TNC plays a role within the stem cell niche.,We hypothesized that TNC creates a specific environment for melanoma cells to exhibit a stem cell-like phenotype, driving tumor growth and evading conventional therapies.,TNC expression was strongly up-regulated in melanoma cells grown as 3D spheres (enriched for stem-like cells) when compared to adherent cells.,Down-modulation of TNC by shRNA-lentiviruses significantly decreased the growth of melanoma spheres.,The incidence of pulmonary metastases after intravenous injection of TNC knockdown cells was significantly lower in NOD/SCID IL2Rγnull mice compared to control cells.,Melanoma spheres contain and increased number of side population (SP) cells, which exhibited stem cell characteristics and the potential for drug resistance due to their high efflux capacity.,Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely due to the down-regulation of multiple ABC transporters, including ABCB5.,These data suggest that TNC plays a critical role in melanoma progression by mediating protective signals in the therapy-resistant population of melanoma.

Malignant melanoma, generally described as incurable, is notoriously refractory to chemotherapy.,The mechanisms contributing to this have not yet been defined and the contributions of drug efflux pumps, implicated in chemo-resistance of many other cancer types, have not been extensively investigated in melanoma.,In this study, expression of multi-drug resistant (MDR1/P-gp and MRP-1) proteins was examined, by immunohistochemistry, in archival specimens from 134 melanoma patients.,This included 92 primary tumours and 42 metastases.,On assessing all specimens, MRP-1 and MDR1/P-gp expression was found to be common, with the majority (81%) of melanomas expressing at least one of these efflux pumps.,Although there is significant association between expression of these pumps (P=0.007), MRP-1 was found to be the predominant (67% of cases) form detected. χ2 analysis showed significant associations between expression of MRP-1 and/or MDR1/P-gp and the aggressive nature of this disease specifically increased Breslow's depth, Clark's level and spread to lymph nodes.,This association with aggressiveness and spread is further supported by the observation that a significantly higher percentage of metastases, than primary tumours, express MRP-1 (91% vs 57% P<0.0001) and MDR1/P-gp (74% vs 50% P=0.010).,The predominant expression of these pumps and, in particular, MRP-1 suggests that they may be important contributors to the inherent aggressive and resistant nature of malignant melanoma.

Patients with late stage resected cutaneous melanoma have poor overall survival (OS) and experience irreversible adverse events from systemic therapy.,There is a clinical need to identify biomarkers to predict outcome.,Performing germline/tumour whole-exome sequencing of 44 stage III/IV melanoma patients we identified pathogenic germline mutations in CDKN2A, CDK4, ATM, POLH, MRE11A, RECQL4 and XPC, affecting 7/44 patients.,These mutations were associated with poor OS (p = 0.0082).,We confirmed our findings in The Cancer Genome Atlas (TCGA) human skin cutaneous melanoma cohort where we identified pathogenic variants in 40/455 patients (p = 0.0203).,Combining these cohorts (n = 499) further strengthened these findings showing germline carriers had worse OS (p = 0.0009).,Additionally, we determined whether tumour mutation burden (TMB) or BRAF status were prognostic markers of survival.,Low TMB rate (< 20 Mut/Mb; p = 0.0034) and BRAF p.V600 mutation (p = 0.0355) were associated with worse progression-free survival.,Combining these biomarkers indicated that V600 mutant patients had significantly lower TMB (p = 0.0155).,This was confirmed in the TCGA (n = 443, p = 0.0007).,Integrative analysis showed germline mutation status conferred the highest risk (HR 5.2, 95% CI 1.72-15.7).,Stage IV (HR 2.5, 0.74-8.6) and low TMB (HR 2.3, 0.57-9.4) were similar, whereas BRAF V600 status was the weakest prognostic biomarker (HR 1.5, 95% CI 0.44-5.2).

The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas.,MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.,We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth.,Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression.,Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression.,RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish.,All statistical tests were two-sided.,Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition.,Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF.,High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity.,Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis.,Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells.,Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).,Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

Conjunctival melanoma (CJM) is a rare but potentially lethal and highly-recurrent cancer of the eye.,Similar to cutaneous melanoma (CM), it originates from melanocytes.,Unlike CM, however, CJM is relatively poorly characterized from a genomic point of view.,To fill this knowledge gap and gain insight into the genomic nature of CJM, we performed whole-exome (WES) or whole-genome sequencing (WGS) of tumor-normal tissue pairs in 14 affected individuals, as well as RNA sequencing in a subset of 11 tumor tissues.,Our results show that, similarly to CM, CJM is also characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions.,This, as well as the presence of a UV light-induced mutational signature, are clear signs of the role of sunlight in CJM tumorigenesis.,In addition, the genomic classification of CM proposed by TCGA seems to be well-applicable to CJM, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, NF1, RAS, and triple wild-type.,In line with these results, transcriptomic analyses revealed similarities with CM as well, namely the presence of a transcriptomic subtype enriched for immune genes and a subtype enriched for genes associated with keratins and epithelial functions.,Finally, in seven tumors we detected somatic mutations in ACSS3, a possible new candidate oncogene.,Transfected conjunctival melanoma cells overexpressing mutant ACSS3 showed higher proliferative activity, supporting the direct involvement of this gene in the tumorigenesis of CJM.,Altogether, our results provide the first unbiased and complete genomic and transcriptomic classification of CJM.

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.

Although p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene.,Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway.,Expression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes.,Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b.,The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group).,Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing.,Expression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment.,MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation.,The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression.,Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition.,Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors.

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.

Cutaneous melanoma is a malignant neoplasm with a constantly increasing incidence, the prognosis of which is largely dependent on early diagnosis.,The appropriateness of requests for ultrasound (US) tests during melanoma follow-up of patients referred to our institute was evaluated.,The requests for US tests of all patients referred to our institute over a four-month period were assessed.,In order to correctly evaluate the appropriateness of requests, patients were split into two groups on the basis of melanoma thickness: > 1 mm (Group A) and < 1 mm (Group B).,546 patients were enrolled in our study out of a total of 1240 US tests performed.,Out of 290 Group A patients, 104 patients (35%) did not meet the established congruity criteria.,Group B was composed of 256 individuals, 92 patients (35.9%) of which were found to have at least one inappropriate request.,In our study, more than 30% of the requests for US tests were found to be inappropriate, to the detriment of those with a real need for diagnostic testing.,This lengthens waiting lists and it may also increase public healthcare costs.,Therefore, it is mandatory to adopt new, widely accepted and easily applicable guidelines.

Kaposi Sarcoma (KS) is a malignancy of endothelial skin cells with multifocal localization on the skin, lymph nodes and visceral organs.,Although all clinical variants are associated with HHV-8 infection, specific differences in the clinical onset and in the natural history of AIDS-KS and Classic-KS have been described.,The present randomised prospective-observational study aimed to investigate whether the ultrasound pattern and color Doppler flow imaging of vascularisation of skin lesions of patients with Classic KS (CKS) or AIDS-KS could provide useful information to the evaluation of clinical activity of the disease.,Cutaneous lesions of 24 patients with histologically confirmed KS were investigated using very high frequency ultrasound probes; 16 patients had CKS and 8 had AIDS-KS.,HHV-8 infection was confirmed in all patients by investigating the specific humoral response to viral antigens.,Immunological and virological parameters were also assessed to monitor HIV or HHV-8 viral infection.,For each patient, a target skin lesion was selected on the basis of size (diameter from 0.4 to 2 cm).,Each lesion was analyzed in terms of size, depth and color Doppler pattern.,The B-mode ultrasound patterns of skin lesions did not differ when comparing CKS patients to AIDS-KS patients, whereas the color Doppler signal, which is associated with vascular activity, was detected in the KS lesions of 6/8 AIDS-KS patients (75.0%) and in 2/16 CKS (16,7%); the latter two patients showed a clinically progressive and extensive disease stage (IV B).,Our preliminary results suggest that small cutaneous KS lesions - in both CKS and AIDS-KS patients- display similar B-mode ultrasound patterns ( hypoechoic, well defined, superficial lesions).,However, the color Doppler signal, which is associated with endothelial activity and angiogenesis, which play a substantial role in KS progression, could constitute a useful tool for evaluating disease activity.

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.

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway.,About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E).,BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors.,The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3).,The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression.,Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment.,Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation.,In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations.,Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD).,Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy.,For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months.,A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab.,In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2.,Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations.,Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

The resistance of melanoma to current treatment modalities represents a major obstacle for durable therapeutic response, and thus, the elucidation of mechanisms of resistance is urgently needed.,The crucial functions of Activating Transcription Factor-2 (ATF2) in the development and therapeutic resistance of melanoma have been previously reported, although the precise underlying mechanisms remain unclear.,Here, we report a protein kinase C epsilon (PKCε)- and Activating Transcription Factor-2 (ATF2)-mediated mechanism that facilitates resistance by transcriptionally repressing the expression of IFNβ1 and downstream type-I IFN signaling, which is otherwise induced upon exposure to chemotherapy.,Treatment of early stage melanomas expressing low levels of PKCε with chemotherapies relieves its transcriptional repression of IFNB1, resulting in impaired S-phase progression, a senescence-like phenotype, and increased cell death.,This response is lost in late stage metastatic melanomas expressing high levels of PKCε.,Notably, nuclear ATF2 and low expression of IFNβ1 in melanoma tumor samples correlates with poor patient responsiveness to biochemotherapy or neoadjuvant IFN-α2a.,Conversely, cytosolic ATF2 and induction of IFNβ1 coincides with therapeutic responsiveness.,Collectively, we identify an IFNβ1-dependent, cell autonomous mechanism that contributes to the therapeutic resistance of melanoma via the PKCε-ATF2 regulatory axis.

Deltex-3-like (DTX3L), an E3 ligase, is a member of the Deltex (DTX) family and is also called B-lymphoma and BAL-associated protein (BBAP).,Previously, we established RFP/RET-transgenic mice, in which systemic hyperpigmented skin, benign melanocytic tumor(s) and melanoma(s) develop stepwise.,Here we showed that levels of Dtx3l/DTX3L in spontaneous melanoma in RFP/RET-transgenic mice and human melanoma cell lines were significantly higher than those in benign melanocytic cells and primarily cultured normal human epithelial melanocytes, respectively.,Immunohistochemical analysis of human tissues showed that more than 80% of the melanomas highly expressed DTX3L.,Activity of FAK/PI3K/AKT signaling, but not that of MEK/ERK signaling, was decreased in Dtx3l/DTX3L-depleted murine and human melanoma cells.,In summary, we demonstrated not only increased DTX3L level in melanoma cells but also DTX3L-mediated regulation of invasion and metastasis in melanoma through FAK/PI3K/AKT but not MEK/ERK signaling.,Our analysis in human BRAFV600E inhibitor-resistant melanoma cells showed about 80% decreased invasion in the DTX3L-depleted cells compared to that in the DTX3L-intact cells.,Thus, DTX3L is clinically a potential therapeutic target as well as a potential biomarker for melanoma.

BAP1 regulates developmental switch in lineages commonly affected by BAP1-mutant cancers.,The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome.,It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression.,Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development.,Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma.,The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4.,Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion.,These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

Chemokine receptors have been documented to exert critical functions in melanoma progression.,However, current drugs targeting these receptors have limited efficacy in clinical applications, suggesting the urgency to further explore the roles of chemokine receptors in melanoma.,Here we found that C-X-C chemokine receptor 7 (CXCR7) was the most highly expressed chemokine receptor in murine melanoma cell lines.,In addition, the expression level of CXCR7 was positively correlated with melanoma progression in the clinical samples.,High CXCR7 expression was associated with shorter overall survival in melanoma patients.,Increased expression of CXCR7 augmented melanoma proliferation in vitro and tumor growth in vivo, whereas knockout of CXCR7 exhibited significant inhibitory effects.,Moreover, our data elucidated that CXCR7 activated Src kinase phosphorylation in a β-arrestin2-dependent manner.,The administration of the Src kinase inhibitor PP1 or siRNA specific for β-arrestin2 abolished CXCR7-promoted cell proliferation.,Importantly, CXCR7 also regulated melanoma angiogenesis and the secretion of vascular endothelial growth factor (VEGF).,Subsequent investigations revealed a novel event that the activation of the CXCR7-Src axis stimulated the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) to accelerate the translation of hypoxia-inducible factor 1α (HIF-1α), which enhanced the secretion of VEGF from melanoma cells.,Collectively, our results illuminate the crucial roles of CXCR7 in melanoma tumorigenesis, and indicate the potential of targeting CXCR7 as new therapeutic strategies for melanoma treatment.

Despite recent advances in therapy, liver metastasis from melanoma is still associated with poor prognosis.,Although targeting the mTOR signaling pathway exerts potent anti-tumor activity, little is known about specific mTORC2 inhibition regarding liver metastasis.,Using the novel mTORC2 specific inhibitor JR-AB2-011, we show significantly reduced migration and invasion capacity by impaired activation of MMP2 in melanoma cells.,In addition, blockade of mTORC2 induces cell death by non-apoptotic pathways and reduces tumor cell proliferation rate dose-dependently.,Furthermore, a significant reduction of liver metastasis was detected in a syngeneic murine metastasis model upon therapy with JR-AB2-011 as determined by in vivo imaging and necropsy.,Hence, our study for the first time highlights the impact of the pharmacological blockade of mTORC2 as a potent novel anti-cancer approach for liver metastasis from melanoma.

FKBP51 immunophilin is abundantly expressed by immune cells.,Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s.,Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response.,An expansion of FKBP51s+PD-L1+ monocytes was measured in a group of non-responding patients to anti-CTLA-4.,The aim of this work was to confirm the predictive value of response of FKBP51s+Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s.,Co-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s.,PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s+Treg and FKBP51s+PD-L1+ monocytes in 22 advanced melanoma patients treated with anti-PD1.,Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages’ behaviour.,FKBP51s+Tregs count was increased in responders and had a prognostic value.,Non-responders showed an early increase in FKBP51s+ PD-L1+ monocytes during anti-PD1 treatment.,Manipulation of FKBP51s modulated the macrophage-phenotype, with forced protein expression promoting aspects associated with tolerance.,FKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy.,Manipulation of FKBP51s may overcome resistance.

Our understanding of how checkpoint inhibitors (CPI) affect T cell evolution is incomplete, limiting our ability to achieve full clinical benefit from these drugs.,Here we analyzed peripheral T cell populations after one cycle of CPI and identified a dynamic awakening of the immune system revealed by T cell evolution in response to treatment.,We sequenced T cell receptors (TCR) in plasma cell-free DNA (cfDNA) and peripheral blood mononuclear cells (PBMC) and performed phenotypic analysis of peripheral T cell subsets from metastatic melanoma patients treated with CPI.,We found that early peripheral T cell turnover and TCR repertoire dynamics identified which patients would respond to treatment.,Additionally, the expansion of a subset of immune-effector peripheral T cells we call TIE cells correlated with response.,These events are prognostic and occur within 3 weeks of starting immunotherapy, raising the potential for monitoring patients responses using minimally invasive liquid biopsies.”

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.

Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner.,Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E.,To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy.,This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation.,Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide.,When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation.,Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

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 MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis.,Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated.,Here we identify 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions.,Our work suggests that one of these, Disrupted In Renal Carcinoma 3 (DIRC3), may be a clinically important MITF-SOX10 regulated tumour suppressor.,DIRC3 depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by low DIRC3 expression have decreased survival.,DIRC3 is a nuclear lncRNA that activates expression of its neighbouring IGFBP5 tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within the DIRC3 locus.,In turn, DIRC3 dependent regulation of IGFBP5 impacts the expression of genes involved in cancer associated processes and is needed for DIRC3 control of anchorage-independent growth.,Our work indicates that lncRNA components of MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets that can act not only as downstream mediators of MITF-SOX10 function but as feedback regulators of MITF-SOX10 activity.

Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF‐mutant melanoma.,In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching.,The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas.,Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAFV 600‐mutated cell lines and tumors.,ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment.,ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITF low/p75high stem‐like and tumorigenic phenotype.,ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells.,Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi.

Despite recent advances in therapy, liver metastasis from melanoma is still associated with poor prognosis.,Although targeting the mTOR signaling pathway exerts potent anti-tumor activity, little is known about specific mTORC2 inhibition regarding liver metastasis.,Using the novel mTORC2 specific inhibitor JR-AB2-011, we show significantly reduced migration and invasion capacity by impaired activation of MMP2 in melanoma cells.,In addition, blockade of mTORC2 induces cell death by non-apoptotic pathways and reduces tumor cell proliferation rate dose-dependently.,Furthermore, a significant reduction of liver metastasis was detected in a syngeneic murine metastasis model upon therapy with JR-AB2-011 as determined by in vivo imaging and necropsy.,Hence, our study for the first time highlights the impact of the pharmacological blockade of mTORC2 as a potent novel anti-cancer approach for liver metastasis from melanoma.

Since metastatic melanoma is deadly, early diagnosis thereof is crucial for managing the disease.,We recently developed α-melanocyte-stimulating hormone (αMSH) derivatives, [68Ga]Ga-CCZ01048 and [18F]CCZ01064, that target the melanocortin 1 receptor (MC1R) for mouse melanoma imaging.,In this study, we aim to evaluate [18F]CCZ01064 as well as a novel dual-ammoniomethyl-trifluoroborate (AmBF3) derivative, [18F]CCZ01096, for targeting human melanoma xenograft using μPET imaging.,The peptides were synthesized on solid phase using Fmoc chemistry.,Radiolabeling was achieved in a one-step 18F-19F isotope-exchange reaction. μPET imaging and biodistribution studies were performed in NSG mice bearing SK-MEL-1 melanoma xenografts.,The MC1R density on the SK-MEL-1 cell line was determined to be 972 ± 154 receptors/cell (n = 4) via saturation assays.,Using [18F]CCZ01064, moderate tumor uptake (3.05 ± 0.47%ID/g) and image contrast were observed at 2 h post-injection.,Molar activity was determined to play a key role.,CCZ01096 with two AmBF3 motifs showed comparable sub-nanomolar binding affinity to MC1R and much higher molar activity.,This resulted in improved tumor uptake (6.46 ± 1.42%ID/g) and image contrast (tumor-to-blood and tumor-to-muscle ratios were 30.6 ± 5.7 and 85.7 ± 11.3, respectively) at 2 h post-injection.,[18F]CCZ01096 represents a promising αMSH-based μPET imaging agent for human melanoma and warrants further investigation for potential clinical translation.

Cutaneous malignant melanoma is the most fatal skin cancer and although improved comprehension of its pathogenic pathways allowed to realize some effective molecular targeted therapies, novel targets and drugs are still needed.,Aiming to add genetic information potentially useful for novel targets discovery, we performed an extensive genomic characterization by whole-exome sequencing and SNP array profiling of six cutaneous melanoma cell lines derived from metastatic patients.,We obtained a total of 3,325 novel coding single nucleotide variants, including 2,172 non-synonymous variants.,We catalogued the coding mutations according to Sanger COSMIC database and to a manually curated list including genes involved in melanoma pathways identified by mining recent literature.,Besides confirming the presence of known melanoma driver mutations (BRAFV600E, NRASQ61R), we identified novel mutated genes involved in signalling pathways crucial for melanoma pathogenesis and already addressed by current targeted therapies (such as MAPK and glutamate pathways).,We also identified mutations in four genes (MUC19, PAICS, RBMXL1, KIF23) never reported in melanoma, which might deserve further investigations.,All data are available to the entire research community in our Melanoma Exome Database (at https://155.253.6.64/MExDB/).,In summary, these cell lines are valuable biological tools to improve the genetic comprehension of this complex cancer disease and to study functional relevance of individual mutational events, and these findings could provide insights potentially useful for identification of novel therapeutic targets for cutaneous malignant melanoma.

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.

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance.,Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms.,Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance.,Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines.,There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described.,This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

Vasculogenic mimicry (VM) a microvascular system consisting of non-endothelial cells that is newly formed by aggressive tumors, has been proposed as an important therapeutic target in malignant melanoma (MM).,We performed a systematic literature review to evaluate the diagnostic and prognostic accuracy of VM status for overall survival of MM patients.,The quality of the included studies was evaluated using the QUADAS-2 tool.,Diagnostic capacity of VM variables, including sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and the area under summary receiver operating characteristic (SROC), were pooled using Meta-DiSc software.,A retrospective observational study was conducted based on twelve clinical studies including 978 clinically confirmed melanoma patients with proportion (P).,VM+ melanoma cells were associated with poor prognosis in 38% of MM group (P = 0.35, 95% confidence intervals (CI): 0.27-0.42, p < 0.001).,The pooled sensitivity and specificity were 0.82 (95% CI: 0.79-0.84) and 0.69 (95% CI: 0.66-0.71), respectively.,Furthermore, the pooled PLR, NLR, and DOR were 2.56 (95% CI: 1.94-3.93), 0.17 (95% CI: 0.07-0.42), and 17.75 (95% CI: 5.30-59.44), respectively.,Furthermore, the AUC of SROC was 0.63, indicating high reliability of VM status as a biomarker.,Importantly, subgroup results suggested that VM+ status is a significantly accurate prognostic biomarker when diagnosed by the CD31−/PAS+ staining methods in Asian MM samples (p < 0.001).,Our findings support the potential of VM status of tumors as a promising prognostic biomarker and emphasize an effective adjuvant therapeutic strategy in the prognosis of Asian MM patients.

Approximately 85% of melanoma patients who undergo a sentinel lymph node biopsy (SLNB) are node‐negative.,Melanoma incidence is highest in patients ≥65 years, but their SLNB positivity rate is lower than in younger patients.,CP‐GEP, a model combining clinicopathologic and gene expression variables, identifies primary cutaneous melanoma (CM) patients who may safely forgo SLNB due to their low risk for nodal metastasis.,Here, we validate CP‐GEP in a U.S. melanoma patient cohort.,A cohort of 208 adult patients with primary CM from the Mayo Clinic and West Virginia University was used.,Patients were stratified according to their risk for nodal metastasis: CP‐GEP High Risk and CP‐GEP Low Risk.,The main performance measures were SLNB reduction rate (RR) and negative predictive value (NPV).,SLNB positivity rate for the entire cohort was 21%.,Most patients had a T1b (34%) or T2a (31%) melanoma.,In the T1‐T2 group (153 patients), CP‐GEP achieved an SLNB RR of 41.8% (95% CI: 33.9‐50.1) at an NPV of 93.8% (95% CI: 84.8‐98.3).,Subgroup analysis showed similar performance in T1‐T2 patients ≥65 years of age (51 patients; SLNB positivity rate, 9.8%): SLNB RR of 43.1% (95% CI: 29.3‐57.8) at an NPV of 95.5% (95% CI: 77.2‐99.9).,We confirmed the potential of CP‐GEP to reduce negative SLNB in all relevant age groups.,Our findings are especially relevant to patients ≥65 years, where surgery is often elective.,CP‐GEP may guide SLNB decision‐making in clinical practice.

The Clinicopathological and Gene Expression Profile (CP‐GEP) model was developed to accurately identify patients with T1-T3 primary cutaneous melanoma at low risk for nodal metastasis.,To validate the CP‐GEP model in an independent Dutch cohort of patients with melanoma.,Patients (aged ≥ 18 years) with primary cutaneous melanoma who underwent sentinel lymph node biopsy (SLNB) between 2007 and 2017 at the Erasmus Medical Centre Cancer Institute were eligible.,The CP‐GEP model combines clinicopathological features (age and Breslow thickness) with the expression of eight target genes involved in melanoma metastasis (ITGB3, PLAT, SERPINE2, GDF15, TGFBR1, LOXL4, CXCL8 and MLANA).,Using the pathology result of SLNB as the gold standard, performance measures of the CP‐GEP model were calculated, resulting in CP‐GEP high risk or low risk for nodal metastasis.,In total, 210 patients were included in the study.,Most patients presented with T2 (n = 94, 45%) or T3 (n = 70, 33%) melanoma.,Of all patients, 27% (n = 56) had a positive SLNB, with nodal metastasis in 0%, 30%, 54% and 16% of patients with T1, T2, T3 and T4 melanoma, respectively.,Overall, the CP‐GEP model had a negative predictive value (NPV) of 90·5% [95% confidence interval (CI) 77·9-96.2], with an NPV of 100% (95% CI 72·2-100) in T1, 89·3% (95% CI 72·8-96·3) in T2 and 75·0% (95% CI 30·1-95·4) in T3 melanomas.,The CP‐GEP indicated high risk in all T4 melanomas.,The CP‐GEP model is a noninvasive and validated tool that accurately identified patients with primary cutaneous melanoma at low risk for nodal metastasis.,In this validation cohort, the CP‐GEP model has shown the potential to reduce SLNB procedures in patients with melanoma.,What is already known about this topic?,The majority (70-85%) of patients with cutaneous melanoma who undergo a sentinel lymph node biopsy (SLNB) procedure have no metastasis in the SLN.To identify patients at low risk for nodal metastasis, the Clinicopathological and Gene Expression Profile (CP‐GEP) model was developed (n = 754 patients, US cohort).,The CP‐GEP model combines age, Breslow thickness and the expression of eight target genes involved in melanoma metastasis, and has the potential to reduce SLNB procedures in patients with T1-T3 cutaneous melanoma.,The majority (70-85%) of patients with cutaneous melanoma who undergo a sentinel lymph node biopsy (SLNB) procedure have no metastasis in the SLN.,To identify patients at low risk for nodal metastasis, the Clinicopathological and Gene Expression Profile (CP‐GEP) model was developed (n = 754 patients, US cohort).,The CP‐GEP model combines age, Breslow thickness and the expression of eight target genes involved in melanoma metastasis, and has the potential to reduce SLNB procedures in patients with T1-T3 cutaneous melanoma.,What does this study add?,This is the first independent validation of the CP‐GEP model in European patients with primary cutaneous melanoma.The CP‐GEP model can be applied to full‐excision tumour tissue and does not require macrodissection of tumour tissue.The CP‐GEP model has a negative predictive value of 90·5% (95% confidence interval 77·9-96·2) in T1-T3 melanomas.The CP‐GEP model is a promising tool in patient care.,In this validation cohort, the CP‐GEP model has shown the potential to reduce SLNB procedures in patients with primary cutaneous melanoma.,This is the first independent validation of the CP‐GEP model in European patients with primary cutaneous melanoma.,The CP‐GEP model can be applied to full‐excision tumour tissue and does not require macrodissection of tumour tissue.,The CP‐GEP model has a negative predictive value of 90·5% (95% confidence interval 77·9-96·2) in T1-T3 melanomas.,The CP‐GEP model is a promising tool in patient care.,In this validation cohort, the CP‐GEP model has shown the potential to reduce SLNB procedures in patients with primary cutaneous melanoma.

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate.,Therefore, the discovery of novel drug leads is an important goal.,In a previous study, we have isolated several shikonin derivatives from the roots of Onosma paniculata Bureau & Franchet (Boraginaceae) which evolved as promising anticancer candidates. β,β-Dimethylacrylshikonin (1) was the most cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards melanoma cells.,In this study, we synthesized eighteen novel shikonin derivatives in order to obtain compounds which exhibit a higher cytotoxicity than 1.,We investigated their cytotoxic potential against various melanoma cell lines and juvenile skin fibroblasts.,The most active compound was (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (cyclopropylacetylshikonin) (6).,It revealed significant stronger tumor growth inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and MUG-Mel2).,Further investigations have shown that 6 induced apoptosis caspase-dependently, increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by phosphorylation of H2AX.,Cell membrane damage and cell cycle arrest were not observed.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis.,Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma.,While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics.,In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2.,We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p.,Q61R mutated melanoma.,Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present.,These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets.,The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

Skin melanoma remains a highly prevalent and yet deadly form of cancer, with the exact degree of melanoma-associated mortality being strongly dependent upon the local tumor microenvironment.,The exact composition of stromal and immune cells within this microenvironmental region has the potential to profoundly impact melanoma progression and prognosis.,As such, the present study was designed with the goal of clarifying the predictive relevance of stromal and immune cell-related genes in melanoma patients through comprehensive bioinformatics analyses.,We therefore analyzed melanoma sample gene expression within The Cancer Genome Atlas database and employed the ESTIMATE algorithm as a means of calculating both stromal and immune scores that were in turn used for identifying differentially expressed genes (DEGs).,Subsequently, univariate analyses were used to detect DEGs associated with melanoma patient survival, and through additional functional enrichment analyses, we determined that these survival-related DEGs are largely related to inflammatory and immune responses.,A prognostic signature comprised of 10 genes (IL15, CCL8, CLIC2, SAMD9L, TLR2, HLA.DQB1, IGHV1-18, RARRES3, GBP4, APOBEC3G) was generated.,This 10-gene signature effectively separated melanoma patients into low- and high-risk groups based upon their survival.,These low- and high-risk groups also exhibited distinct immune statuses and differing degrees of immune cell infiltration.,In conclusion, our results offer novel insights into a number of microenvironment-associated genes that impact survival outcomes in melanoma patients, potentially highlighting these genes as viable therapeutic targets.

The metastatic Skin Cutaneous Melanoma (SKCM) has been associated with diminished survival rates and high mortality rates worldwide.,Thus, segregating metastatic melanoma from the primary tumors is crucial to employ an optimal therapeutic strategy for the prolonged survival of patients.,The SKCM mRNA, miRNA and methylation data of TCGA is comprehensively analysed to recognize key genomic features that can segregate metastatic and primary tumors.,Further, machine learning models have been developed using selected features to distinguish the same.,The Support Vector Classification with Weight (SVC-W) model developed using the expression of 17 mRNAs achieved Area under the Receiver Operating Characteristic (AUROC) curve of 0.95 and an accuracy of 89.47% on an independent validation dataset.,This study reveals the genes C7, MMP3, KRT14, LOC642587, CASP7, S100A7 and miRNAs hsa-mir-205 and hsa-mir-203b as the key genomic features that may substantially contribute to the oncogenesis of melanoma.,Our study also proposes genes ESM1, NFATC3, C7orf4, CDK14, ZNF827, and ZSWIM7 as novel putative markers for cutaneous melanoma metastasis.,The major prediction models and analysis modules to predict metastatic and primary tumor samples of SKCM are available from a webserver, CancerSPP (http://webs.iiitd.edu.in/raghava/cancerspp/).

Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-MET) signaling is involved in complex cellular programs that are important for embryonic development and tissue regeneration, but its activity is also utilized by cancer cells during tumor progression.,HGF and c-MET usually mediate heterotypic cell-cell interactions, such as epithelial-mesenchymal, including tumor-stroma interactions.,In the skin, dermal fibroblasts are the main source of HGF.,The presence of c-MET on keratinocytes is crucial for wound healing in the skin.,HGF is not released by normal melanocytes, but as melanocytes express c-MET, they are receptive to HGF, which protects them from apoptosis and stimulates their proliferation and motility.,Dissimilar to melanocytes, melanoma cells not only express c-MET, but also release HGF, thus activating c-MET in an autocrine manner.,Stimulation of the HGF/c-MET pathways contributes to several processes that are crucial for melanoma development, such as proliferation, survival, motility, and invasiveness, including distant metastatic niche formation.,HGF might be a factor in the innate and acquired resistance of melanoma to oncoprotein-targeted drugs.,It is not entirely clear whether elevated serum HGF level is associated with low progression-free survival and overall survival after treatment with targeted therapies.,This review focuses on the role of HGF/c-MET signaling in melanoma with some introductory information on its function in skin and melanocytes.

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 mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas.,MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.,We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth.,Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression.,Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression.,RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish.,All statistical tests were two-sided.,Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition.,Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF.,High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity.,Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis.,Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells.,Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).,Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway.,About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E).,BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors.,The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3).,The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression.,Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment.,Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation.,In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations.,Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD).,Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy.,For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months.,A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab.,In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2.,Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations.,Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

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.

The CheckMate 066 trial investigated nivolumab monotherapy as first-line treatment for patients with previously untreated BRAF wild-type advanced melanoma.,Five-year results are presented herein.,In this multicenter, double-blind, phase III study, 418 patients with previously untreated, unresectable, stage III/IV, wild-type BRAF melanoma were randomly assigned 1:1 to receive nivolumab 3 mg/kg every 2 weeks or dacarbazine 1,000 mg/m2 every 3 weeks.,The primary end point was overall survival (OS), and secondary end points included progression-free survival (PFS), objective response rate (ORR), and safety.,Patients were followed for a minimum of 60 months from the last patient randomly assigned (median follow-up, 32.0 months for nivolumab and 10.9 months for dacarbazine).,Five-year OS rates were 39% with nivolumab and 17% with dacarbazine; PFS rates were 28% and 3%, respectively.,Five-year OS was 38% in patients randomly assigned to dacarbazine who had subsequent therapy, including nivolumab (n = 37).,ORR was 42% with nivolumab and 14% with dacarbazine; among patients alive at 5 years, ORR was 81% and 39%, respectively.,Of 42 patients treated with nivolumab who had a complete response (20%), 88% (37 of 42) were alive as of the 5-year analysis.,Among 75 nivolumab-treated patients alive and evaluable at the 5-year analysis, 83% had not received subsequent therapy; 23% were still on study treatment, and 60% were treatment free.,Safety analyses were similar to the 3-year report.,Results from this 5-year analysis confirm the significant benefit of nivolumab over dacarbazine for all end points and add to the growing body of evidence supporting long-term survival with nivolumab mono-therapy.,Survival is strongly associated with achieving a durable response, which can be maintained after treatment discontinuation, even without subsequent systemic therapies.

Therapies targeting signaling molecules mutated in cancers can often have striking short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures1,2.,Resistance can result from a secondary mutations3,4, but other times there is no clear genetic cause, raising the possibility of non-genetic rare cell variability5-11.,Here, we show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug treatment.,This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells.,The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state.,This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD.,Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells.,We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general rare-cell expression program.

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.

Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level.,Here we show that melanoma cells exhibit an oxidative stress phenotype compared with normal melanocytes, as evidenced by increased total cellular ROS, KEAP1/NRF2 pathway activity, protein damage, and elevated oxidized glutathione.,Our overall objective was to test whether augmenting this high oxidative stress level in melanoma cells would inhibit their dependence on oncogenic PI3K/AKT/mTOR-mediated survival.,We report that NexrutineR augmented the constitutively elevated oxidative stress markers in melanoma cells, which was abrogated by N-acetyl cysteine (NAC) pre-treatment.,NexrutineR disrupted growth homeostasis by inhibiting proliferation, survival, and colony formation in melanoma cells without affecting melanocyte cell viability.,Increased oxidative stress in melanoma cells inhibited PI3K/AKT/mTOR pathway through disruption of mTORC1 formation and phosphorylation of downstream targets p70S6K, 4EBP1 and rpS6.,NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism.,Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR.

Actinic keratoses (AKs) are lesions of epidermal keratinocyte dysplasia and are precursors for invasive cutaneous squamous cell carcinoma (cSCC).,Identifying the specific genomic alterations driving the progression from normal skin to skin with AK to skin with invasive cSCC is challenging because of the massive UVR-induced mutational burden characteristic at all stages of this progression.,In this study, we report the largest AK whole-exome sequencing study to date and perform a mutational signature and candidate driver gene analysis on these lesions.,We demonstrate in 37 AKs from both immunosuppressed and immunocompetent patients that there are significant similarities between AKs and cSCC in terms of mutational burden, copy number alterations, mutational signatures, and patterns of driver gene mutations.,We identify 44 significantly mutated AK driver genes and confirm that these genes are similarly altered in cSCC.,We identify azathioprine mutational signature in all AKs from patients exposed to the drug, providing further evidence for its role in keratinocyte carcinogenesis. cSCCs differ from AKs in having higher levels of intrasample heterogeneity.,Alterations in signaling pathways also differ, with immune-related signaling and TGFβ signaling significantly more mutated in cSCC.,Integrating our findings with independent gene expression datasets confirms that dysregulated TGFβ signaling may represent an important event in AK-cSCC progression.

Cutaneous squamous cell carcinoma (cSCC) has a high tumour mutational burden (50 mutations per megabase DNA pair).,Here, we combine whole-exome analyses from 40 primary cSCC tumours, comprising 20 well-differentiated and 20 moderately/poorly differentiated tumours, with accompanying clinical data from a longitudinal study of immunosuppressed and immunocompetent patients and integrate this analysis with independent gene expression studies.,We identify commonly mutated genes, copy number changes and altered pathways and processes.,Comparisons with tumour differentiation status suggest events which may drive disease progression.,Mutational signature analysis reveals the presence of a novel signature (signature 32), whose incidence correlates with chronic exposure to the immunosuppressive drug azathioprine.,Characterisation of a panel of 15 cSCC tumour-derived cell lines reveals that they accurately reflect the mutational signatures and genomic alterations of primary tumours and provide a valuable resource for the validation of tumour drivers and therapeutic targets.,It is known cutaneous squamous cell carcinoma (cSCC) involves a high tumour mutation burden.,Here the authors identify common cSCC mutated genes, copy number changes, altered pathways and report the presence of a novel mutation signature associated with chronic exposure to the immunosuppressive drug azathioprine.

Previous studies have shown that histone deacetylase 6 (HDAC6) plays critical roles in many cellular processes related to cancer.,However, its biological roles in the development of melanoma remain unexplored.,Our aim was to investigate whether HDAC6 has a biological role in human melanoma development and to understand its underlying mechanism.,In the present study, HDAC6 expression was up-regulated in melanoma tissues and cell lines.,Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of A375.,S2 cells, promoted cell arrest at G0/G1 phase and apoptosis.,Additionally, western blotting assay showed that HDAC6 silencing suppressed Bcl-2 level and enhanced Bax level, then activated caspase-9 and caspase-3, and further activated the release of cytochrome c from mitochondria to cytoplasm, finally induced cell apoptosis involving the mitochondrial pathway.,Knockdown of HDAC6 triggered a significant generation of ROS and disruption of mitochondrial membrane potential (MMP).,Furthermore, ROS inhibitor, NAC reduced HDAC6 siRNA-induced ROS production, and blocked HDAC6 siRNA-induced loss of MMP and apoptosis.,NAC also significantly blocked HDAC6 siRNA-induced mtDNA copy number decrease and mitochondrial biogenesis and degradation imbalance.,In conclusion, the results showed that knockdown of HDAC6 induced apoptosis in human melanoma A375.,S2 cells through a ROS-dependent mitochondrial pathway.

This study was to investigate the synergistic effect of NB/Cur on growth and apoptosis in A375 human melanoma cell line by MTT assay, flow cytometry and Western blotting.,Our results demonstrated that NB effectively synergized with Cur to enhance its antiproliferative activity on A375 human melanoma cells by induction of apoptosis, as evidenced by an increase in sub-G1 cell population, DNA fragmentation, PARP cleavage and caspase activation.,Further mechanistic studies by Western blotting showed that after treatment of the cells with NB/Cur, up-regulation of the expression level of phosphorylated JNK and down-regulation of the expression level of phosphorylated ERK and Akt contributed to A375 cells apoptosis.,Moreover, NB also potentiated Cur to trigger intracellular ROS overproduction and the DNA damage with up-regulation of the expression level of phosphorylated ATM, phosphorylated Brca1 and phosphorylated p53.,The results indicate the combinational application potential of NB and Cur in treatments of cancers.

In melanoma, the presence of promoter related hypermethylation has previously been reported, however, no methylation-based distinction has been drawn among the diverse melanoma subtypes.,Here, we investigated DNA methylation changes associated with melanoma progression and links between methylation patterns and other types of somatic alterations, including the most frequent mutations and DNA copy number changes.,Our results revealed that the methylome, presenting in early stage samples and associated with the BRAFV600E mutation, gradually decreased in the medium and late stages of the disease.,An inverse relationship among the other predefined groups and promoter methylation was also revealed except for histologic subtype, whereas the more aggressive, nodular subtype melanomas exhibited hypermethylation as well.,The Breslow thickness, which is a continuous variable, allowed for the most precise insight into how promoter methylation decreases from stage to stage.,Integrating our methylation results with a high-throughput copy number alteration dataset, local correlations were detected in the MYB and EYA4 genes.,With regard to the effects of DNA hypermethylation on melanoma patients' survival, correcting for clinical cofounders, only the KIT gene was associated with a lower overall survival rate.,In this study, we demonstrate the strong influence of promoter localized DNA methylation changes on melanoma initiation and show how hypermethylation decreases in melanomas associated with less favourable clinical outcomes.,Furthermore, we establish the methylation pattern as part of an integrated apparatus of somatic DNA alterations.

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide.,Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition.,In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions.,In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients.,On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

Vitamin D deficiency (≤20 ng/mL) is associated with an increased incidence and worse prognosis of various types of cancer including melanoma.,A retrospective, single-center study of individuals diagnosed with melanoma from January 2007 through June 2013 who had a vitamin D (25(OH)D3) level measured within one year of diagnosis was performed to determine whether vitamin D deficiency and repletion are associated with melanoma outcome.,A total of 409 individuals diagnosed with histopathology-confirmed melanoma who had an ever measured serum 25(OH)D3 level were identified. 252 individuals with a 25(OH)D3 level recorded within one year after diagnosis were included in the study and the individual and melanoma characteristics such as age, sex, Breslow thickness, ulceration, stage, mitotic rate, and LDH were obtained from the medical record.,A worse melanoma prognosis was associated with vitamin D deficiency (P=0.012), higher stage (P<0.001), ulceration (P=0.001), and higher mitotic rate (P=0.001) (HR 1.93, 95% CI 1.15-3.22).,In patients with stage IV metastatic melanoma, vitamin D deficiency was associated with significantly worse melanoma-specific mortality (adjusted HR 2.06, 95% CI 1.10-3.87).,Patients with metastatic melanoma who were initially vitamin D deficient and subsequently had a decrease or ≤20 ng/mL increase in their 25(OH)D3 concentration had significantly worse outcomes (HR 4.68, 95% CI 1.05-20.88) compared to non-deficient patients who had a >20 ng/mL increase.,Our results suggest that initial vitamin D deficiency and insufficient repletion is associated with a worse prognosis in patients with metastatic melanoma.

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.

Historically, melanoma with brain metastases has a poor prognosis.,In this retrospective medical record review, we report the outcome of patients with stage IV melanoma with brain metastases treated with ipilimumab and brain stereotactic radiosurgery (SRS).,All patients with metastatic melanoma treated with ipilimumab from June 2010 to September 2012 were identified and stratified by presence (A) or absence (B) of brain metastases at the time of ipilimumab administration.,All patients with brain metastases received SRS.,Overall survival (OS) was defined as time from the date of stage IV diagnosis and the time of ipilimumab administration to death or last follow-up.,Survival curves were estimated using the Kaplan-Meier method, and Cox proportional hazards model was employed to compute the hazard ratios (HR).,Results: Five out of 10 patients in Cohort A and 10 out of 21 patients in Cohort B died as of last follow-up.,In Cohort A, median number of lesions treated with SRS was 3.,Median survivals from date of stage IV for Cohorts A and B were 29.3 and 33.1 months, respectively (HR = 0.93, P = 0.896).,Median survival from cycle 1 ipilimumab was 16.5 and 24.5 months for Cohort A and B, respectively (HR = 1.05, P = 0.931).,The 3-year survival rates from the date of cycle one of ipilimumab administration for Cohort A and B were 50% (95% CI: 27-93%) and 39% (95% CI: 19-81%), respectively.,Eight of 10 patients in Cohort A maintained a good PS.,Survival of patients with melanoma brain metastases treated with ipilimumab combined with SRS may be comparable to patients without brain metastases.

Ipilimumab, an antibody that enhances T-cell activation, may augment immunogenicity of tumor cells that are injured by radiation therapy.,We hypothesized that patients with melanoma brain metastasis treated with both ipilimumab and radiotherapy would have improved overall survival, and that the sequence of treatments may affect disease control in the brain.,We analyzed the clinical and radiographic records of melanoma patients with brain metastases who were treated with whole brain radiation therapy or stereotactic radiosurgery between 2005 and 2012.,The hazard ratios for survival were estimated to assess outcomes as a function of ipilimumab use and radiation type.,Seventy patients were identified, 33 of whom received ipilimumab and 37 who did not.,The patients who received ipilimumab had a censored median survival of 18.3 months (95% confidence interval 8.1-25.5), compared with 5.3 months (95% confidence interval 4.0-7.6) for patients who did not receive ipilimumab.,Ipilimumab and stereotactic radiosurgery were each significant predictors of improved overall survival (hazard ratio = 0.43 and 0.45, with P = 0.005 and 0.008, respectively).,Four of 10 evaluable patients (40.0%) who received ipilimumab prior to radiotherapy demonstrated a partial response to radiotherapy, compared with two of 22 evaluable patients (9.1%) who did not receive ipilimumab.,Ipilimumab is associated with a significantly reduced risk of death in patients with melanoma brain metastases who underwent radiotherapy, and this finding supports the need for multimodality therapy to optimize patient outcomes.,Prospective studies are needed and are underway.

Some studies have reported a possible association between exposure to tumour necrosis factor (TNF) inhibitors and an increased risk of melanoma.,The aim of this study was to investigate the incidence of invasive cutaneous melanomas in patients with rheumatoid arthritis (RA) treated with TNF inhibitors (TNFi), other biologic disease modifying drugs and non-biologic therapy.,Eleven biologic registers from nine European countries participated in this collaborative project.,According to predefined exposure definitions, cohorts of patients with RA were selected.,Using the country-specific general population of each register as reference, age, sex and calendar year standardised incidence ratios (SIRs) of invasive histology-confirmed cutaneous melanoma were calculated within each register.,Pooled SIR and incidence rate ratios (IRRs) comparing biologic cohorts to biologic-naïve were calculated across countries by taking the size of the register into account.,Overall 130 315 RA patients with a mean age of 58 years contributing 579 983 person-years were available for the analysis and 287 developed a first melanoma.,Pooled SIRs for biologic-naïve, TNFi and rituximab-exposed patients were 1.1 (95% CI 0.9 to 1.4), 1.2 (0.99 to 1.6) and 1.3 (0.6 to 2.6), respectively.,Incidence rates in tocilizumab and abatacept-exposed patients were also not significantly increased.,IRR versus biologic-naïve patients were: TNFi 1.1 (95% CI 0.8 to 1.6); rituximab 1.2 (0.5 to 2.9).,This large European collaborative project did not confirm an overall increased risk of melanoma following exposure to TNFi.

Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%.,The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAFV600E.,However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments.,It is therefore critical to elucidate alternative mechanisms regulating melanoma progression.,Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition.,We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAFWT) and WM35 (BRAFV600E), with in vitro environmental stress and nutrient deprivation.,Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells.,Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo.,Our results demonstrate that, in BRAFWT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAFWT melanomas.,Implications: Expression of CXCR3 on BRAFWT melanoma cells may be a mediator of melanoma progression.

CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma.,Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype.,All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL).,Phenotypic data related to primary melanoma and pigmentation characteristics were collected.,The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced.,Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation.,The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years.,Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM).,A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects.,CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p<0.001).,MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010).,Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM.,In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene located on chromosome 3p21.,Germline BAP1 mutations have been recently associated with an increased risk of malignant mesothelioma, atypical melanocytic tumors and other neoplasms.,To answer the question if different germline BAP1 mutations may predispose to a single syndrome with a wide phenotypic range or to distinct syndromes, we investigated the presence of melanocytic tumors in two unrelated families (L and W) with germline BAP1 mutations and increased risk of malignant mesothelioma.,Suspicious cutaneous lesions were clinically and pathologically characterized and compared to those present in other families carrying BAP1 mutations.,We then conducted a meta-analysis of all the studies reporting BAP1-mutated families to survey cancer risk related to the germline BAP1 mutation (means were compared using t-test and proportions were compared with Pearson χ2 test or two-tailed Fisher’s exact test).,Melanocytic tumors: of the five members of the L family studied, four (80%) carried a germline BAP1 mutation (p.Gln684*) and also presented one or more atypical melanocytic tumors; of the seven members of W family studied, all carried a germline BAP1 mutation (p.Pro147fs*48) and four of them (57%) presented one or more atypical melanocytic tumors, that we propose to call “melanocytic BAP1-mutated atypical intradermal tumors” (MBAITs).,Meta-analysis: 118 individuals from seven unrelated families were selected and divided into a BAP1-mutated cohort and a BAP1-non-mutated cohort.,Malignant mesothelioma, uveal melanoma, cutaneous melanoma, and MBAITs prevalence was significantly higher in the BAP1-mutated cohort (p ≤ 0.001).,Germline BAP1 mutations are associated with a novel cancer syndrome characterized by malignant mesothelioma, uveal melanoma, cutaneous melanoma and MBAITs, and possibly by other cancers.,MBAITs provide physicians with a marker to identify individuals who may carry germline BAP1 mutations and thus are at high risk of developing associated cancers.

Women diagnosed with cutaneous melanoma have a survival advantage compared to men, which has been hypothesized to be due to difference in behavior and/or biology (sex hormones).,It remains controversial whether this advantage is dependent on age or stage of disease.,We sought to compare melanoma‐specific survival between females in pre, peri, and postmenopausal age groups to males in the same age group, adjusting for stage of disease.,This is a retrospective population‐based cohort study using the Surveillance, Epidemiology, and End Results (SEER) database.,Patients diagnosed from 1 January 1992 through 31 January 2011 with primary invasive cutaneous melanoma were included in our cohort.,Melanoma‐specific survival was the main outcome studied.,Of the 106,511 subjects that were included, 45% were female.,Females in all age groups (18-45, 46-54, and ≥55) with localized and regional disease, were less likely to die from melanoma compared to males in the same age group.,Among patients with localized and regional disease, the relative risk of death due to melanoma increased with advancing age at diagnosis; this increase was more pronounced among females than males.,In contrast, we observed no female survival advantage among patients with distant disease and no effect of age on relative risk of death from melanoma.,Females with localized and regional melanoma have a decreased risk of death compared to males within all age groups.,Our data show no differences in survival between men and women with metastatic melanoma, indicating that the influence of sex on survival is limited to early stage disease but not confined to pre or perimenopausal age groups.

Acral melanoma (AM), as a peculiar subgroup of melanoma, is rare in Caucasians but has higher incidence in Asians.,Large series of study on AM with clinicopathological features and prognostic factors is still limited, especially in Asian population.,We retrospectively collected clinical, pathological and follow-up data of 142 AM cases.,All patients were Chinese, with the age ranging from 24 to 87 years (mean 62.0; median 62.0).,The Breslow thickness of primary lesions ranged from 0.6 to 16.3 mm (mean 4.9; median 3.7).,85.9% of the patients had acral lentiginous histologic subtype.,Plantar was the most frequently involved site, followed by heels.,Statistically, duration of the lesion before diagnosis (≤2.5 years), Breslow thickness >4.0 mm (T4), high mitotic index (>15 mm−2), presence of vascular invasion, regional lymph node metastasis at diagnosis and pathologic stage (II/III/IV) were found to be independent prognostic factors in both univariate and multivariate analyses.,The prognosis of AM in Chinese is extremely poor.,Our 5- and 10-year disease-specific survival (DSS) rates were 53.3% and 27.4%, respectively.,Therefore, AM in Asians represents a more biologically aggressive melanoma subtype and is thought to carry a worse prognosis when compared with other races or cutaneous melanomas in other anatomic sites.

Inflammatory, angiogenic, and immune processes have been associated with uveal melanoma (UM).,The aim of the present study was to evaluate the presence of some specific aqueous humor (AH) soluble biomarkers in eyes affected by UM.,Thirty-five eyes affected by primary UM and 35 control eyes, scheduled for cataract surgery, underwent full ophthalmic examination and AH sampling at time of surgery (brachytherapy or cataract surgery, respectively).,AH samples were analyzed by means of ELISA, to detect the concentration of selected cytokines, chemokines, and growth factors.,Compared with the control group, higher levels of IL-6 (P = .049), IL-8 (P = .006), RANTES (P = .008), EGF (P = .032), bFGF (P = .016), MIF (P = .007), and MCP (P = .020) were detected in eyes with UM.,VEGF concentration between the two groups was statistically borderline (P = .058).,Comparison between clinical characteristics and cytokine concentrations showed a positive correlation between tumor thickness and IL-8 (P = .032), and degree of serous retinal detachment and IL-6 (P = .021).,UM is characterized by the presence of retinal neuroinflammatory, angiogenic, and immune biomarkers in AH.,The proteomic analysis of AH could characterize UM microenvironment, allowing to better understand its pathophysiology.

Imatinib mesylate (IM) is a compound that inhibits both BCR-ABL tyrosine kinase and c-kit receptors.,Tyrosine kinases are important in cellular signaling and mediate major cellular processes such as proliferation, differentiation, apoptosis, attachment, and migration.,Twenty-six albino rabbits were injected with 1 × 106 human uveal melanoma (UM) cells (92.1) into the suprachoroidal space.,Animals were immunosuppressed (cyclosporin A) over the course of the 12-week experiment and divided into two groups (n = 13).,The experimental group received IM once daily by gavage while the control group received a placebo.,One animal per group was sacrificed every week after the 2nd week.,Upon necropsy, organs were harvested for histopathological examination.,Cells from the primary tumors were recultured and tested in proliferation and invasion assays.,A PCR array was used to investigate the differences in expression of 84 genes related to tumor metastasis.,In the treated group, 4 rabbits developed intraocular tumors, with an average largest tumor dimension (LTD) of 2.5 mm and 5 animals reported metastatic disease.,Whereas 6 rabbits in the control group developed intraocular tumors, with an average LTD of 5.8 mm and 6 animals reported metastatic disease.,The recultured cells from the treated group demonstrated lower proliferation rates and were less invasive (p < 0.001 The PCR array showed differences in expression of genes related to metastasis.,Notably, there was 290-fold increase in SERPINB5, a tumor suppressor gene, and a 10-fold higher expression of KISS1, a metastasis suppressor gene, in the treated group.,Proangiogenic genes such as VEGFA, PDGFA and PDGFB were downregulated in the treated group.,To the best of our knowledge, this is the first report detailing the altered expression of specific genes in UM cells after treatment with IM.

Immunotherapy is among the most rapidly evolving treatment strategies in oncology.,The therapeutic potential of immune-checkpoint inhibitors is exemplified by the recent hail of Food and Drug Administration (FDA) approvals for their use in various malignancies.,Continued efforts to enhance outcomes with immunotherapy agents have led to the formulation of advanced treatment strategies.,Recent evidence from pre-clinical studies evaluating immune-checkpoint inhibitors in various cancer cell-lines has suggested that combinatorial approaches may have superior survival outcomes compared to single-agent immunotherapy regimens.,Preliminary trials assessing combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 immune-checkpoint inhibitors have documented considerable advantages in survival indices over single-agent immunotherapy.,The therapeutic potential of combinatorial approaches is highlighted by the recent FDA approval of nivolumab plus ipilimumab for patients with advanced melanoma.,Presently, dual-immune checkpoint inhibition with anti-programmed death receptor-1/programmed cell death receptor- ligand-1 (anti-PD-1/PD-L1) plus anti-cytotoxic T lymphocyte associated antigen-4 (anti-CTLA-4) monoclonal antibodies (MoAbs) is being evaluated for a wide range of tumor histologies.,Furthermore, several ongoing clinical trials are investigating combination checkpoint inhibition in association with traditional treatment modalities such as chemotherapy, surgery, and radiation.,In this review, we summarize the current landscape of combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 MoAbs for patients with melanoma and non-small cell lung cancer (NSCLC).,We present a synopsis of the prospects for expanding the indications of dual immune-checkpoint inhibition therapy to a more diverse set of tumor histologies.

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.

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment.,We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort.,We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival.,This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin-mediated failure to recruit CD141+ DCs.,A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles.,The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation.,The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants.,In summary, we report evidence for a β-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome.,We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.

We have investigated the role of vascular-endothelial (VE)-cadherin in melanoma and breast cancer metastasis.,We found that VE-cadherin is expressed in highly aggressive melanoma and breast cancer cell lines.,Remarkably, inactivation of VE-cadherin triggered a significant loss of malignant traits (proliferation, adhesion, invasion and transendothelial migration) in melanoma and breast cancer cells.,These effects, except transendothelial migration, were induced by the VE-cadherin RGD motifs.,Co-immunoprecipitation experiments demonstrated an interaction between VE-cadherin and α2β1 integrin, with the RGD motifs found to directly affect β1 integrin activation.,VE-cadherin-mediated integrin signaling occurred through specific activation of SRC, ERK and JNK, including AKT in melanoma.,Knocking down VE-cadherin suppressed lung colonization capacity of melanoma or breast cancer cells inoculated in mice, while pre-incubation with VE-cadherin RGD peptides promoted lung metastasis for both cancer types.,Finally, an in silico study revealed the association of high VE-cadherin expression with poor survival in a subset of melanoma patients and breast cancer patients showing low CD34 expression.,These findings support a general role for VE-cadherin and other RGD cadherins as critical regulators of lung and liver metastasis in multiple solid tumours.,These results pave the way for cadherin-specific RGD targeted therapies to control disseminated metastasis in multiple cancers.

Adjuvant systemic therapies are now routinely used following resection of stage III melanoma, however accurate prognostic information is needed to better stratify patients.,We use differential expression analyses of primary tumours from 204 RNA-sequenced melanomas within a large adjuvant trial, identifying a 121 metastasis-associated gene signature.,This signature strongly associated with progression-free (HR = 1.63, p = 5.24 × 10−5) and overall survival (HR = 1.61, p = 1.67 × 10−4), was validated in 175 regional lymph nodes metastasis as well as two externally ascertained datasets.,The machine learning classification models trained using the signature genes performed significantly better in predicting metastases than models trained with clinical covariates (pAUROC = 7.03 × 10−4), or published prognostic signatures (pAUROC < 0.05).,The signature score negatively correlated with measures of immune cell infiltration (ρ = −0.75, p < 2.2 × 10−16), with a higher score representing reduced lymphocyte infiltration and a higher 5-year risk of death in stage II melanoma.,Our expression signature identifies melanoma patients at higher risk of metastases and warrants further evaluation in adjuvant clinical trials.,The identification of prognostic biomarkers can help stratify cancer patients.,Here, the authors apply deep RNA sequencing from primary melanomas coupled with long-term clinical outcome data from a prospective multicentre phase III trial, to develop and validate a 121 metastasis-associated gene signature identifying early-stage melanoma patients at higher risk of metastasis and worse survival.

Gene expression profiles of cutaneous melanoma were analyzed to identify critical genes associated with metastasis.,Two gene expression datasets were downloaded from Gene Expression Omnibus (GEO) and another dataset was obtained from The Cancer Genome Atlas (TCGA).,Differentially expression genes (DEGs) between metastatic and non-metastatic melanoma were identified by meta-analysis.,A protein-protein interaction (PPI) network was constructed for the DEGs using information from BioGRID, HPRD and DIP.,Betweenness centrality (BC) was calculated for each node in the network and the top feature genes ranked by BC were selected to construct the support vector machine (SVM) classifier using the training set.,The SVM classifier was then validated in another independent dataset.,Pathway enrichment analysis was performed for the feature genes using Fisher's exact test.,A total of 798 DEGs were identified and a PPI network including 337 nodes and 466 edges was then constructed.,Top 110 feature genes ranked by BC were included in the SVM classifier.,The prediction accuracies for the three datasets were 96.8, 100 and 94.4%, respectively.,A total of 11 KEGG pathways and 13 GO biological pathways were significantly over-represented in the 110 feature genes, including endometrial cancer, regulation of actin cytoskeleton, focal adhesion, ubiquitin mediated proteolysis, regulation of apoptosis and regulation of cell proliferation.,A SVM classifier of high prediction accuracy was acquired.,Several critical genes implicated in melanoms metastasis were also revealed.,These results may advance understanding of the molecular mechanisms underlying metastasis, and also provide potential therapeutic targets.

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.

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.

New therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors.,Drug screening, IC50 determinations as well as synergy assays were detected by the MTT assay.,Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry.,TUNEL staining was performed using immunocytochemistry.,Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry.,To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used.,Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors.,Evaluation of immune infiltrates was carried out by flow cytometry.,Using a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines.,Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS).,TM’s anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition.,Specifically, TM blunted S6 phosphorylation in both BRAFV600E and BRAF wildtype (WT) melanoma cell lines.,TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model.,In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation.,Furthermore TM decreased the infiltration of immune suppressive regulatory CD4+CD25+ T cells and FOXP3 and ROR-γt positive CD4+ T cells.,Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAFV600E mutation and could be additively or synergistically combined with Cobimetinib in both BRAFV600E and BRAF WT melanoma cell lines in inducing anti-cancer effects.,Taken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAFV600E and BRAF WT melanoma.

Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients.,Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma.,We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo.,We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM.,Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner.,Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid.,The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM.,Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed.,In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity.,Cell cycle analysis indicated moderate changes in apoptotic indices.,Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression.,Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies.

Melanoma is one of the most immunologic malignancies based on its higher prevalence in immune-compromised patients, the evidence of brisk lymphocytic infiltrates in both primary tumors and metastases, the documented recognition of melanoma antigens by tumor-infiltrating T lymphocytes and, most important, evidence that melanoma responds to immunotherapy.,The use of immunotherapy in the treatment of metastatic melanoma is a relatively late discovery for this malignancy.,Recent studies have shown a significantly higher success rate with combination of immunotherapy and chemotherapy, radiotherapy, or targeted molecular therapy.,Immunotherapy is associated to a panel of dysimmune toxicities called immune-related adverse events that can affect one or more organs and may limit its use.,Future directions in the treatment of metastatic melanoma include immunotherapy with anti-PD1 antibodies or targeted therapy with BRAF and MEK inhibitors.

UVB exposure leads to DNA damage, which when unrepaired induces C>T transitions.,These mutations are found throughout the melanoma genome, particularly in non-transcribed regions.,The global genome repair (GGR) branch of nucleotide excision repair (NER) is responsible for repairing UV-induced DNA damage across non-transcribed and silent regions of the genome.,This study aimed to examine the relationship between UVB and GGR in melanoma.,DNA repair capacity and relative expression of NER in melanocytes and melanoma cell lines before and after treatment with UVB was quantified.,Transcript expression from 196 melanomas was compared to clinical parameters including solar elastosis and whole transcriptome data collected.,Melanoma cell lines showed significantly reduced DNA repair when compared to melanocytes, most significantly in the S phase of the cell cycle.,Expression of GGR components XPC, DDB1 and DDB2 was significantly lower in melanoma after UVB.,In the melanoma tumours, XPC expression correlated with age of diagnosis and low XPC conferred significantly poorer survival.,The same trend was seen in the TCGA melanoma dataset.,Reduced GGR in melanoma may contribute to the UV mutation spectrum of the melanoma genome and adds further to the growing evidence of the link between UV, NER and melanoma.

Using data from the Nurses' Health Study, Jiali Han and colleagues examine the association between number of cutaneous nevi and the risk for breast cancer.,Please see later in the article for the Editors' Summary,Cutaneous nevi are suggested to be hormone-related.,We hypothesized that the number of cutaneous nevi might be a phenotypic marker of plasma hormone levels and predict subsequent breast cancer risk.,We followed 74,523 female nurses for 24 y (1986-2010) in the Nurses' Health Study and estimate the relative risk of breast cancer according to the number of cutaneous nevi.,We adjusted for the known breast cancer risk factors in the models.,During follow-up, a total of 5,483 invasive breast cancer cases were diagnosed.,Compared to women with no nevi, women with more cutaneous nevi had higher risks of breast cancer (multivariable-adjusted hazard ratio, 1.04, 95% confidence interval [CI], 0.98-1.10 for 1-5 nevi; 1.15, 95% CI, 1.00-1.31 for 6-14 nevi, and 1.35, 95% CI, 1.04-1.74 for 15 or more nevi; p for continuous trend = 0.003).,Over 24 y of follow-up, the absolute risk of developing breast cancer increased from 8.48% for women without cutaneous nevi to 8.82% (95% CI, 8.31%-9.33%) for women with 1-5 nevi, 9.75% (95% CI, 8.48%-11.11%) for women with 6-14 nevi, and 11.4% (95% CI, 8.82%-14.76%) for women with 15 or more nevi.,The number of cutaneous nevi was associated with increased risk of breast cancer only among estrogen receptor (ER)-positive tumors (multivariable-adjusted hazard ratio per five nevi, 1.09, 95% CI, 1.02-1.16 for ER+/progesterone receptor [PR]-positive tumors; 1.08, 95% CI, 0.94-1.24 for ER+/PR− tumors; and 0.99, 95% CI, 0.86-1.15 for ER−/PR− tumors).,Additionally, we tested plasma hormone levels according to the number of cutaneous nevi among a subgroup of postmenopausal women without postmenopausal hormone use (n = 611).,Postmenopausal women with six or more nevi had a 45.5% higher level of free estradiol and a 47.4% higher level of free testosterone compared to those with no nevi (p for trend = 0.001 for both).,Among a subgroup of 362 breast cancer cases and 611 matched controls with plasma hormone measurements, the multivariable-adjusted odds ratio for every five nevi attenuated from 1.25 (95% CI, 0.89-1.74) to 1.16 (95% CI, 0.83-1.64) after adjusting for plasma hormone levels.,Key limitations in this study are that cutaneous nevi were self-counted in our cohort and that the study was conducted in white individuals, and thus the findings do not necessarily apply to other populations.,Our results suggest that the number of cutaneous nevi may reflect plasma hormone levels and predict breast cancer risk independently of previously known factors.,Please see later in the article for the Editors' Summary,One woman in eight will develop breast cancer during her lifetime.,Breast cancer begins when cells in the breast acquire genetic changes that allow them to divide uncontrollably (which leads to the formation of a lump in the breast) and to move around the body (metastasize).,The treatment of breast cancer, which is diagnosed using mammography (a breast X-ray) or manual breast examination and biopsy, usually involves surgery to remove the lump, or the whole breast (mastectomy) if the cancer has started to metastasize.,After surgery, women often receive chemotherapy or radiotherapy to kill any remaining cancer cells and may also be given drugs that block the action of estrogen and progesterone, female sex hormones that stimulate the growth of some breast cancer cells.,Globally, half a million women die from breast cancer each year.,However, in developed countries, nearly 90% of women affected by breast cancer are still alive five years after diagnosis.,Several sex hormone-related factors affect breast cancer risk, including at what age a woman has her first child (pregnancy alters sex hormone levels) and her age at menopause, when estrogen levels normally drop.,Moreover, postmenopausal women with high circulating levels of estrogen and testosterone (a male sex hormone) have an increased breast cancer risk.,Interestingly, moles (nevi)-dark skin blemishes that are a risk factor for the development of melanoma, a type of skin cancer-often darken or enlarge during pregnancy.,Might the number of nevi be a marker of hormone levels, and could nevi counts therefore be used to predict an individual's risk of breast cancer?,In this prospective cohort study, the researchers look for an association between number of nevi and breast cancer risk among participants in the US Nurses' Health Study (NHS).,A prospective cohort study enrolls a group of people, determines their baseline characteristics, and follows them over time to see which characteristics are associated with the development of certain diseases.,The NHS, which enrolled 121,700 female nurses aged 30-55 years in 1976, is studying risk factors for cancer and other chronic diseases in women.,In 1986, nearly 75,000 NHS participants (all of whom were white) reported how many nevi they had on their left arm.,Over the next 24 years, 5,483 invasive breast cancers were diagnosed in these women.,Compared to women with no nevi, women with increasing numbers of nevi had a higher risk of breast cancer after adjustment for known breast cancer risk factors.,Specifically, among women with 1-5 nevi, the hazard ratio (HR) for breast cancer was 1.04, whereas among women with 15 or more nevi the HR was 1.35.,An HR compares how often a particular event occurs in two groups with different characteristics; an HR greater than one indicates that a specific characteristic is associated with an increased risk of the event.,Over 24 years of follow-up, the absolute risk of developing breast cancer was 8.48% in women with no nevi but 11.4% for women with 15 or more nevi.,Notably, postmenopausal women with six or more nevi had higher blood levels of estrogen and testosterone than women with no nevi.,Finally, in a subgroup analysis, the association between number of nevi and breast cancer risk disappeared after adjustment for hormone levels.,These findings support the hypothesis that the number of nevi reflects sex hormone levels in women and may predict breast cancer risk.,Notably, they show that the association between breast cancer risk and nevus number was independent of known risk factors for breast cancer, and that the risk of breast cancer increased with the number of nevi in a dose-dependent manner.,These findings also suggest that a hormonal mechanism underlies the association between nevus number and breast cancer risk.,Because this study involved only white participants, these findings may not apply to non-white women.,Moreover, the use of self-reported data on nevus numbers may affect the accuracy of these findings.,Finally, because this study is observational, these findings are insufficient to support any changes in clinical recommendations for breast cancer screening or diagnosis.,Nevertheless, these data and those in an independent PLOS Medicine Research Article by Kvaskoff et al. support the need for further investigation of the association between nevi and breast cancer risk and of the mechanisms underlying this relationship.,Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001659.,An independent PLOS Medicine Research Article by Kvaskoff et al. also investigates the relationship between nevi and breast cancer risk,The US National Cancer Institute provides comprehensive information about cancer (in English and Spanish), including detailed information for patients and professionals about breast cancer; it also has a fact sheet on moles,Cancer Research UK, a not-for profit organization, provides information about cancer, including detailed information on breast cancer,The UK National Health Service Choices website has information and personal stories about breast cancer; the not-for profit organization Healthtalkonline also provides personal stories about dealing with breast cancer,More information about the Nurses' Health Study is available

The influence of variants at the 9p21 locus on melanoma risk has been reported through investigation of CDKN2A variants through candidate gene approach as well as by genome wide association studies (GWAS).,In the present study we genotyped, 25 SNPs that tag 273 variants on chromosome 9p21 in 837 melanoma cases and 1154 controls from Spain.,Ten SNPs were selected based on previous associations, reported in GWAS, with either melanocytic nevi or melanoma risk or both.,The other 15 SNPs were selected to fine map the CDKN2A gene region.,All the 10 variants selected from the GWAS showed statistically significant association with melanoma risk.,Statistically significant association with melanoma risk was also observed for the carriers of the variant T-allele of rs3088440 (540 C>T) at the 3’ UTR of CDKN2A gene with an OR 1.52 (95% CI 1.14-2.04).,Interaction analysis between risk associated polymorphisms and previously genotyped MC1R variants, in the present study, did not show any statistically significant association.,Statistical significant association was observed for the interaction between phototypes and the rs10811629 (located in intron 5 of MTAP).,The strongest association was observed between the homozygous carrier of the A-allele and phototype II with an OR of 15.93 (95% CI 5.34-47.54).,Our data confirmed the association of different variants at chromosome 9p21 with melanoma risk and we also found an association of a variant with skin phototypes.

Previously we have reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are impaired in their ability to perform A-to-I microRNA (miRNA) editing.,The effects of A-to-I miRNAs editing on melanoma growth and metastasis are yet to be determined.,Here we report that miR-378a-3p is undergoing A-to-I editing only in the non-metastatic but not in metastatic melanoma cells.,The function of the edited form is different from its wild-type counterpart.,The edited form of miR-378a-3p preferentially binds to the 3′-UTR of the PARVA oncogene and inhibits its expression, thus preventing the progression of melanoma towards the malignant phenotype.,Indeed, edited miR-378a-3p but not its WT form inhibits melanoma metastasis in vivo.,These results further emphasize the role of RNA editing in melanoma progression.,In melanoma, reduced ADAR1 impairs A-to-I microRNA editing.,Here, the authors show that miR-378a-3p undergoes this editing in non-metastatic cells and the edited form of miR-378a-3p binds to the PARVA oncogene, inhibiting its expression and preventing melanoma progression and metastasis.

Melanoma is an aggressive cancer that metastasizes rapidly, and is refractory to conventional chemotherapies.,Identifying miRNAs that are responsible for this pathogenesis is therefore a promising means of developing new therapies.,We identified miR-26a through microarray and qRT-PCR experiments as an miRNA that is strongly down-regulated in melanoma cell lines as compared to primary melanocytes.,Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared to a negative control in most melanoma cell lines tested.,In surveying targets of miR-26a, we found that protein levels of SMAD1 and BAG-4/SODD were strongly decreased in sensitive cells treated with miR-26a mimic compared to the control.,The luciferase reporter assays further demonstrated that miR-26a can repress gene expression through the binding site in the 3′UTR of SODD.,Knockdown of these proteins with siRNA showed that SODD plays an important role in protecting melanoma cells from apoptosis in most cell lines sensitive to miR-26a, while SMAD1 may play a minor role.,Furthermore, transfecting cells with a miR-26a inhibitor increased SODD expression.,Our findings indicate that miR-26a replacement is a potential therapeutic strategy for metastatic melanoma, and that SODD in particular is a potentially useful therapeutic target.

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.

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly.,We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC.,In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12).,LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells.,Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes.,These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.

Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance.,Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the meloe mRNA which is significantly expressed only in the melanocytic lineage.,In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of meloe in normal vs tumor cells.,This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells.,We first demonstrated that no splicing event or cryptic promoter could generate shorter meloe transcripts containing only one of the two ORFs.,Triggering meloe RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated.,Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length meloe cDNA in melanoma cells with eGFP constructs.,In conclusion, meloe is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity.

The melanoma antigens MELOE-1 and MELOE-2 are encoded by a messenger, called meloe, overexpressed in melanomas compared with other tumour cell types and healthy tissues.,They are both able to elicit melanoma-specific T cell responses in melanoma patients, and MELOE-1-specific CD8 T cells have been involved in melanoma immunosurveillance.,With the aim to develop immunotherapies targeting this antigen, we investigated the transcriptional mechanisms leading to the preferential expression of meloe messenger in the melanocytic lineage.,We defined the minimal promoter region of meloe gene and identified binding motifs for a set of transcription factors.,Using mutagenesis, co-transfection experiments and chromatin immunoprecipitation, we showed that transcription factors involved in meloe promoter activity in melanomas were the melanocytic specific SOX9 and SOX10 proteins together with the activated P-CREB protein.,Furthermore, we showed that meloe promoter was hypomethylated in melanomas and melanocytes, and hypermethylated in colon cancer cell lines and mesotheliomas, thus explaining the absence of P-CREB binding in these cell lines.,This was a second key to explain the overerexpression of meloe messenger in the melanocytic lineage.,To our knowledge, such a dual transcriptional control conferring tissue-specificity has never been described for the expression of tumour antigens.

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks.,It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment.,We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma.,We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and this response would correlate with disease-free survival.,We identified a rapid and potent anti-tumor response, with 8/27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease-free.,These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks with reinvigoration in the blood observed as early as 1 week.,Transcriptional analysis demonstrated a pre-treatment immune signature (Neoadjuvant Response Signature) that was associated with clinical benefit.,In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution.,Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma.,Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.

Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment.,Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells.,We sought to better understand the biology of IL32 in human melanoma.,We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset.,We evaluated global gene expression patterns associated with IL32 expression.,We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro.,In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5′ upstream region of the human IL32 gene into a luciferase reporter vector.,A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF).,Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32.,Cis-acting elements within this 5′ upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression.,In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature.,Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature.,Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5′ upstream region.,The online version of this article (10.1186/s12967-019-1862-y) contains supplementary material, which is available to authorized users.

Predicting metastasis in melanoma patients is important for disease management and could help to identify those who might benefit from adjuvant treatment.,The aim of this study was to investigate whether the tumor microenvironment-derived protein S100A8/A9 qualifies as prognostic marker for melanoma patients, also in the setting of immunotherapy.,S100A8/A9 gene and protein expression were analyzed on melanocytic nevi, primary melanomas and metastases using a cDNA library and three independent tissue-microarrays (TMA).,Serum levels of S100A8/A9 were measured using a specific ELISA in two independent cohorts of 354 stage III and stage IV melanoma patients as well as in two independent cohorts of patients treated with the PD-1 antibody pembrolizumab.,cDNA analysis revealed an upregulation of S100A8 and S100A9 gene expression in melanoma metastases compared to primary melanomas.,Significantly higher numbers of infiltrating S100A8/A9 positive cells were found in tissue samples of metastasizing primary melanomas compared to non-metastasizing melanomas (P < .0001) and in melanomas of short-term survivors compared to long-term survivors (P < .0001).,Serum S100A8/A9 levels > 5.5 mg/l were associated with impaired overall survival in two independent cohorts (both P < .0001).,Importantly, patients with serum elevated S100A8/A9 treated with pembrolizumab showed significantly impaired survival compared to patients with lower S100A8/A9 levels (cohort 1: P = .0051; cohort 2: P < .0001).,The tumor microenvironment-associated protein S100A8/A9 serves as a novel prognostic marker for metastasis and survival of metastatic melanoma patients and predicts response to immunotherapy with pembrolizumab.,These data underscore the significance of tumor microenvironment-derived factors as suitable biomarkers for melanoma.

The incidence of melanoma is increasing worldwide, and the prognosis for patients with high-risk or advanced metastatic melanoma remains poor despite advances in the field.,A systematic literature review of treatments for advanced, metastatic disease was conducted to present the success of current treatments and the promise of those still in clinical development that may yield incremental improvements in the treatment of advanced, metastatic melanoma.,Advances in the understanding of the mechanism of chemotherapy resistance offer the hope for improved results with chemotherapy, and the triumvirate of more effective chemotherapy, immunotherapy, and targeted therapy are likely to be combined with one another for significant advances in melanoma over the coming few years.,The incidence of melanoma is increasing worldwide, and the prognosis for patients with high-risk or advanced metastatic melanoma remains poor despite advances in the field.,Standard treatment for patients with thick (≥2.0 mm) primary melanoma with or without regional metastases to lymph nodes is surgery followed by adjuvant therapy or clinical trial enrollment.,Adjuvant therapy with interferon-α and cancer vaccines is discussed in detail.,Patients who progress to stage IV metastatic melanoma have a median survival of ≤1 year.,Standard treatment with chemotherapy yields low response rates, of which few are durable.,Cytokine therapy with IL-2 achieves durable benefits in a greater fraction, but it is accompanied by severe toxicities that require the patient to be hospitalized for support during treatment.,A systematic literature review of treatments for advanced, metastatic disease was conducted to present the success of current treatments and the promise of those still in clinical development that may yield incremental improvements in the treatment of advanced, metastatic melanoma.

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.

Clinical features of Asian melanoma patients are distinct from those of Western patients.,This study was designed to determine the molecular and clinical characteristics of Asian melanoma patients treated with anti-PD-1 antibody.,Patients with recurrent or metastatic melanoma who began anti-PD-1 antibody therapy between January 2015 and April 2018 were retrospectively reviewed.,Patients who underwent next-generation sequencing were also analyzed.,A total of 152 patients were included.,The median age was 61 years, and 53% of patients were female.,A total of 56 patients (37%) received immunotherapy as second-line or greater chemotherapy.,Primary sites were acral (38%), mucosal (31%), cutaneous (24%), uveal (2%), and unknown (5%).,The overall response rate was 17% (95% CI, 11-22%), and disease control rate was 60% (95% CI, 52-68%).,The median progression-free survival (PFS) was 4.2 months (95% CI, 1.8-6.6 months), and median overall survival (OS) was 32.9 months (95% CI, 20.0-45.7 months).,However, BRAFV600 and KIT mutational statuses were not associated with response or survival.,High neutrophil-lymphocyte ratio (NLR) was associated with poor PFS (median PFS 6.9 vs.,2.4 months, p = 0.015) and OS (median OS NR vs.,10.4 months, p < 0.001).,In multivariate analysis, high NLR independently predicted poor survival.,This study includes the largest set of integrated genomic data analyzing Asian patients with melanoma treated with immunotherapy.,BRAF V600 and KIT mutational statuses were not associated with response or survival, and high NLR was a strong predictor of poor response to and survival with anti-PD-1 therapy.

The common mutation BRAFV600 in primary melanomas activates the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway and the introduction of proto-oncogene B-Raf (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors (BRAFi and MEKi) was a breakthrough in the treatment of these cancers.,However, 15-20% of tumors harbor primary resistance to this therapy, and moreover, patients develop acquired resistance to treatment.,Understanding the molecular phenomena behind resistance to BRAFi/MEKis is indispensable in order to develop novel targeted therapies.,Most often, resistance develops due to either the reactivation of the MAPK/ERK pathway or the activation of alternative kinase signaling pathways including phosphatase and tensin homolog (PTEN), neurofibromin 1 (NF-1) or RAS signaling.,The hyperactivation of tyrosine kinase receptors, such as the receptor of the platelet-derived growth factor β (PDFRβ), insulin-like growth factor 1 receptor (IGF-1R) and the receptor for hepatocyte growth factor (HGF), lead to the induction of the AKT/3-phosphoinositol kinase (PI3K) pathway.,Another pathway resulting in BRAFi/MEKi resistance is the hyperactivation of epidermal growth factor receptor (EGFR) signaling or the deregulation of microphthalmia-associated transcription factor (MITF).

ILEI (FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell biological process that confers metastatic properties to a tumor cell.,Initially, we found that ILEI mRNA is highly expressed in melanoma metastases but not in primary tumors, suggesting that ILEI contributes to the malignant properties of melanoma.,While melanoma is not an epithelial cell-derived tumor and does not undergo a traditional EMT, melanoma undergoes a similar process known as phenotype switching in which high (micropthalmia-related transcription factor) MITF expressing (MITF-high) proliferative cells switch to a low expressing (MITF-low) invasive state.,We observed that MITF-high proliferative cells express low levels of ILEI (ILEI-low) and MITF-low invasive cells express high levels of ILEI (ILEI-high).,We found that inducing phenotype switching towards the MITF-low invasive state increases ILEI mRNA expression, whereas phenotype switching towards the MITF-high proliferative state decreases ILEI mRNA expression.,Next, we used in vitro assays to show that knockdown of ILEI attenuates invasive potential but not MITF expression or chemoresistance.,Finally, we used gene expression analysis to show that ILEI regulates several genes involved in the MITF-low invasive phenotype including JARID1B, HIF-2α, and BDNF.,Gene set enrichment analysis suggested that ILEI-regulated genes are enriched for JUN signaling, a known regulator of the MITF-low invasive phenotype.,In conclusion, we demonstrate that phenotype switching regulates ILEI expression, and that ILEI regulates partial phenotype switching in MITF-low melanoma cell lines.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood.,Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times.,Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases.,In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients.,These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.,The genetic changes that occur in late stage metastatic melanoma are not well delineated.,Here, the authors use rapid autopsy samples from metastatic melanoma patients and show that the late stage in the disease is characterised by whole genome doubling and aneuploidy.

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1.,To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines.,These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53).,As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated.,In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing.,The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM.,PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products.,Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life.,Treatment opportunities have been restricted until now and new therapy options are urgently required.,Our focus was to reveal the potential heterogeneity of melanoma brain metastasis.,We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers.,Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones.,Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model.,As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity.,Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

Approximately one-half of advanced (unresectable or metastatic) melanomas harbor a mutation in the BRAF gene, with V600E being the most common mutation.,Targeted therapy with BRAF and MEK inhibitors is associated with significant long-term treatment benefit in patients with BRAF V600-mutated melanoma.,Therefore, molecular testing for BRAF mutations is a priority in determining the course of therapy.,A literature search was performed using MEDLINE/PubMed and scientific congress databases using the terms ‘BRAF,’ ‘mutation,’ and ‘cancer/tumor.’,These results were filtered to include manuscripts that focused on diagnostic tests for determining BRAF mutation status.,Numerous BRAF testing methods were identified, including DNA-based companion diagnostic tests and DNA- and protein-based laboratory-developed tests.,Herein we review the characteristics of each method and highlight the strengths and weaknesses that should be considered before use and when interpreting results for each patient.,Molecular profiling has shown that mutation load increases with melanoma tumor progression and that unique patterns of genetic changes and evolutionary trajectories for different melanoma subtypes can occur.,Discordance in the BRAF mutational status between primary and metastatic lesions, as well as intratumoral heterogeneity, is known to occur.,Additionally, the development of acquired resistance to combination BRAF and MEK inhibitor therapy is still a formidable obstacle.,Therefore, tumor heterogeneity and the development of acquired resistance have important implications for molecular testing and ultimately the treatment of patients with advanced-stage melanoma.,Overall, this information may help community oncologists more accurately and effectively interpret results of diagnostic tests within the context of recent data characterizing melanoma tumor progression.

Growing evidence is showing that metastatic cell populations are able to transfer their characteristics to less malignant cells.,Exosomes (EXOs) are membrane vesicles of endocytic origin able to convey their cargo of mRNAs, microRNAs (miRs), proteins and lipids from donors to proximal as well as distant acceptor cells.,Our previous results indicated that miR-221&222 are key factors for melanoma development and dissemination.,The aim of this study was to verify whether the tumorigenic properties associated with miR-222 overexpression can be also propagated by miR-222-containing EXOs.,EXOs were isolated by UltraCentrifugation or Exoquick-TC® methods.,Preparations of melanoma-derived vesicles were characterized by using the Nanosight™ technology and the expression of exosome markers analyzed by western blot.,The expression levels of endogenous and exosomal miRNAs were examined by real time PCR.,Confocal microscopy was used to evaluate transfer and uptake of microvesicles from donor to recipient cells.,The functional significance of exosomal miR-222 was estimated by analyzing the vessel-like process formation, as well as cell cycle rates, invasive and chemotactic capabilities.,Besides microvesicle marker characterization, we evidenced that miR-222 exosomal expression mostly reflected its abundance in the cells of origin, correctly paralleled by repression of its target genes, such as p27Kip1, and induction of the PI3K/AKT pathway, thus confirming its functional implication in cancer.,The possible differential significance of PI3K/AKT blockade was assessed by using the BKM120 inhibitor in miR-222-transduced cell lines.,In addition, in vitro cultures showed that vesicles released by miR-222-overexpressing cells were able to transfer miR-222-dependent malignancy when taken-up by recipient primary melanomas.,Results were confirmed by antagomiR-221&222 treatments and by functional observations after internalization of EXOs devoid of these miRs.,All together these data, besides generally confirming the role of miR-222 in melanoma tumorigenesis, supported its responsibility in the exosome-associated melanoma properties, thus further indicating this miR as potential diagnostic and prognostic biomarker and its abrogation as a future therapeutic option.,The online version of this article (doi:10.1186/s12967-016-0811-2) contains supplementary material, which is available to authorized users.

BRAFV600 inhibitors have offered a new gateway for better treatment of metastatic melanoma.,However, the overall efficacy of BRAFV600 inhibitors has been lower than expected in clinical trials, and many patients have shown resistance to the drug’s effect.,We hypothesized that somatic mutations in the Phosphoinositide 3-Kinase (PI3K) pathway, which promotes proliferation and survival, may coincide with BRAFV600 mutations and contribute to chemotherapeutic resistance.,We performed a somatic mutation profiling study using the 454 FLX pyrosequencing platform in order to identify candidate cancer genes within the MAPK and PI3K pathways of melanoma patients.,Somatic mutations of theses candidate cancer genes were then confirmed using Sanger sequencing.,As expected, BRAFV600 mutations were seen in 51% of the melanomas, whereas NRAS mutations were seen in 19% of the melanomas.,However, PI3K pathway mutations, though more heterogeneous, were present in 41% of the melanoma, with PTEN being the highest mutated PI3K gene in melanomas (22%).,Interestingly, several novel PI3K pathway mutations were discovered in MTOR, IRS4, PIK3R1, PIK3R4, PIK3R5, and NFKB1.,PI3K pathway mutations co-occurred with BRAFV600 mutations in 17% of the tumors and co-occurred with 9% of NRAS mutant tumors, implying cooperativity between these pathways in terms of melanoma progression.,These novel PI3K pathway somatic mutations could provide alternative survival and proliferative pathways for metastatic melanoma cells.,They therefore may be potential chemotherapeutic targets for melanoma patients who exhibit resistance to BRAFV600 inhibitors.

Melanoma is the deadliest cutaneous neoplasm.,To prevent metastasis, early diagnosis and surgical treatment is vital.,Long non-coding RNAs (lncRNAs) may serve as biomarkers and therapeutic targets in tumors.,We investigated the molecular mechanisms of lncRNA KCNQ1OT1 in melanoma.,Real time PCR demonstrated that KCNQ1OT1 expression is up-regulated in melanoma tissues and cells.,KCNQ1OT1 promoted cell proliferation and metastasis in melanoma.,By directly bindin to miR-153, KCNQ1OT1 acted as a competing endogenous RNA (ceRNA) to de-repress MET expression.,Our results may provide the basis for a novel strategy for early detection and/or treatment of melanoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer.,Pathologic activation of PI3K/mTOR pathway and elevated expression of c-Myc are frequently detected in MCC.,Yet, there is no targeted therapy presently available for this lethal disease.,Recently, MLN0128, a second-generation dual TORC1/2 inhibitor is shown to have therapeutic efficacy in preclinical studies.,MLN0128 is currently in clinical trials as a potential therapy for advanced cancers.,Here we characterize the therapeutic efficacy of MLN0128 in the preclinical setting of MCC and delineate downstream targets of mTORC1/2 in MCC cellular systems.,MLN0128 significantly attenuates xenograft MCC tumor growth independent of Merkel cell polyomavirus.,Moreover, MLN0128 markedly diminishes MCC cell proliferation and induces apoptosis.,Further investigations indicate that senescence does not contribute to MLN0128-mediated repression of xenograft MCC tumor growth.,Finally, we also observe robust antitumor effects of MLN0128 when administered as a dual therapy with JQ1, a bromodomain protein BRD4 inhibitor.,These results suggest dual blockade of PI3K/mTOR pathway and c-Myc axis is effective in the control of MCC tumor growth.,Our results demonstrate that MLN0128 is potent as monotherapy or as a member of combination therapy with JQ1 for advanced MCC.

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.

Target-specific agents used in melanoma are not curative, and chemokines are being implicated in drug-resistance to target-specific agents.,Thus, the use of conventional agents in rationale combinations may result in optimization of therapy.,Because histone deacetylases participate in tumor development and progression, the combination of the pan-inhibitor SAHA and temozolomide might provide a therapeutic advantage.,Here, we show synergism between the two drugs in mutant BRAF cell lines, in association with decreased phosphorylation of cell survival proteins (e.g., C-Jun-N-terminal-kinase, JNK).,In the spontaneous ret transgenic mouse melanoma model, combination therapy produced a significant disease onset delay and down-regulation of Chemokine (C-C motif) ligand 2 (CCL2), JNK, and of Myeloid-derived suppressor cell recruitment.,Co-incubation with a CCL2-blocking-antibody enhanced in vitro cell sensitivity to temozolomide.,Conversely, recombinant CCL2 activated JNK in human tumor melanoma cells.,In keeping with these results, the combination of a JNK-inhibitor with temozolomide was synergistic.,By showing that down-regulation of CCL2-driven signals by SAHA and temozolomide via JNK contributes to reduce melanoma growth, we provide a rationale for the therapeutic advantage of the drug combination.,This combination strategy may be effective because of interference both with tumor cell and tumor microenvironment.

The current development of BRAF inhibitors has revolutionized the treatment of unresectable melanoma.,As the potential heterogeneity of BRAF mutations in melanoma has been reported, accurate detection of BRAF mutations are important.,However, the genetic heterogeneity of acral melanoma-a distinct type of melanoma with a unique genetic background-has not fully been investigated.,We conducted a retrospective review of our acral melanoma patients.,Of the 196 patients with acral melanoma, we retrieved 31 pairs of primary and matched metastatic melanomas.,We immunostained the 31 pairs with VE1, a BRAFV600E-mutation-specific monoclonal antibody.,Immunohistochemistry with VE1 showed a high degree of sensitivity and specificity for detecting BRAFV600E mutations compared with the real-time polymerase chain reaction method.,A total of nine primary (29.0%) and eight metastatic (25.8%) acral melanomas were positive for VE1.,In three patients (9.7%), we observed a discordance of VE1 staining between the primary and metastatic lesions.,Of note, VE1 immunohistochemical staining revealed a remarkable degree of intra-tumor genetic heterogeneity in acral melanoma.,Our study reveals that VE1 immunostaining is a useful ancillary method for detecting BRAFV600E mutations in acral melanoma and allows for a clear visualization of intra- and inter-tumor BRAF heterogeneity.

Molecular diagnostics are increasingly performed routinely in the diagnosis and management of patients with melanoma due to the development of novel therapies that target specific genetic mutations.,The development of next-generation sequencing (NGS) technologies has enabled to sequence multiple cancer-driving genes in a single assay, with improved sensitivity in mutation detection.,The main objective of this study was the design and implementation of a melanoma-specific sequencing panel, and the identification of the spectrum of somatic mutations in a series of primary melanoma samples.,A custom panel was designed to cover the coding regions of 35 melanoma-related genes.,Panel average coverage was 2,575.5 reads per amplicon, with 92,8% of targeted bases covered ≥500×.,Deep coverage enabled sensitive discovery of mutations in as low as 0.5% mutant allele frequency.,Eighty-five percent (85/100) of the melanomas had at least one somatic mutation.,The most prevalent mutated genes were BRAF (50%;50/199), NRAS (15%;15/100), PREX2 (14%;14/100), GRIN2A (13%;13/100), and ERBB4 (12%;12/100).,Turn-around-time and costs for NGS-based analysis was reduced in comparison to conventional molecular approaches.,The results of this study demonstrate the cost-effectiveness and feasibility of a custom-designed targeted NGS panel, and suggest the implementation of targeted NGS into daily routine practice.

Cutaneous basal cell carcinoma (cBCC) is the most common malignancy diagnosed in the human population. cBCC presents an increasing incidence which, in the near future, will be higher than all other cancers combined.,The majority of cBCC are located in the head and the neck.,A diversity of management modalities is currently available; nonetheless, surgical excision remains the main modality of treatment. cBCC rarely metastasises and presents a low mortality rate. cBCC morbidity is influenced by local invasion and destruction, especially in the face, where function and aesthetics are major issues.,Easy accessibility to the face and skin on the neck makes cBCC an important issue for otorhinolaryngology head and neck surgeons who must be aware and committed in its management, as the main modality of treatment continues to be surgical.,The aim of this review is to present a brief and practical overview of head and neck cBCC management for ear, nose and throat (ENT) surgeons, discussing key issues about its epidemiology, risk factors, diagnosis and pathogenesis.

Imiquimod is a synthetic imidazoquinolone amine, which has potent immune response modifier activity, when topically used.,This characteristic property of imiquimod has led to its use in a number of applications in dermatology, particularly in cutaneous malignancies, where it has been found to be effective and safe.,Currently, additional mechanisms for its activity in actinic keratosis, basal cell carcinoma, and invasive squamous cell carcinoma have been elucidated.,Its usage for cutaneous metastasis in breast cancer has been a further addition to its therapeutic armamentarium recently.

Drug tolerance brought about by reversible adaptive responses precedes the emergence of irreversible mutation-driven drug resistance and sustains tumor cells when at their most vulnerable.,Young et al. delineate a signaling relay incorporating IL-1 and CXCR2 ligands emanating from melanoma-associated macrophages and fibroblasts, respectively, that confer tolerance to MAPK inhibitors.,Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance.,Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance.,Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance.,In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands.,Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth.,In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors.,Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors.,We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment.

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.

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.

Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature.,While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells.,We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells.,We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001).,Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001).,Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells.,Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study.,This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity.,These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

Basal cell carcinoma (BCC) is the most common cancer worldwide with an annual incidence of 2.8 million cases in the United States alone.,Previous studies have demonstrated an association between 21 distinct genetic loci and BCC risk.,Here, we report the results of a two-stage genome-wide association study of BCC, totalling 17,187 cases and 287,054 controls.,We confirm 17 previously reported loci and identify 14 new susceptibility loci reaching genome-wide significance (P<5 × 10−8, logistic regression).,These newly associated SNPs lie within predicted keratinocyte regulatory elements and in expression quantitative trait loci; furthermore, we identify candidate genes and non-coding RNAs involved in telomere maintenance, immune regulation and tumour progression, providing deeper insight into the pathogenesis of BCC.,Basal cell carcinoma is a common skin lesion and the risk loci for this cancer are beginning to be understood.,In this study, the authors conduct a two-stage genome-wide association study and confirm known risk loci and identify an additional 14 loci.

Both environmental and host factors influence risk of cutaneous melanoma (CM), and worldwide, the incidence varies depending on constitutional determinants of skin type and pigmentation, latitude, and patterns of sun exposure.,We performed genetic analysis of CDKN2A, CDK4, BAP1, MC1R, and MITFp.E318K in Danish high-risk melanoma cases and found CDKN2A germline mutations in 11.3% of CM families with three or more affected individuals, including four previously undescribed mutations.,Rare mutations were also seen in CDK4 and BAP1, while MC1R variants were common, occurring at more than twice the frequency compared to Danish controls.,The MITF p.E318K variant similarly occurred at an approximately three-fold higher frequency in melanoma cases than controls.,To conclude, we propose that mutation screening of CDKN2A and CDK4 in Denmark should predominantly be performed in families with at least 3 cases of CM.,In addition, we recommend that testing of BAP1 should not be conducted routinely in CM families but should be reserved for families with CM and uveal melanoma, or mesothelioma.

Aiming to improve treatment options for BRAF wild-type melanoma, we previously conducted the DOC-MEK study of docetaxel with MEK inhibitor (MEKi) selumetinib or placebo, revealing trends to prolongation of progression-free survival (hazard ratio 0.75, P = 0.130), and improved response rates (32% vs 14%, P = 0.059) with docetaxel plus selumetinib.,NRAS status did not associate with outcome.,Here, the aim was to identify novel biomarkers of response to MEKi.,A MEK 6 gene signature was quantified using NanoString and correlated with clinical outcomes.,Two components of the gene signature were investigated by gene silencing in BRAF/NRAS wild-type melanoma cells.,In melanomas of patients on the selumetinib but not the placebo arm, two gene signature components, dual-specificity protein phosphatase 4 (DUSP4) and ETS translocation variant 4 (ETV4), were expressed more highly in responders than non-responders.,In vitro, ETV4 depletion inhibited cell survival but did not influence sensitivity to MEKi selumetinib or trametinib.,In contrast, DUSP4-depleted cells showed enhanced cell survival and increased resistance to both selumetinib and trametinib.,ETV4 and DUSP4 associated with clinical response to docetaxel plus selumetinib.,DUSP4 depletion induced MEKi resistance, suggesting that DUSP4 is not only a biomarker but also a mediator of MEKi sensitivity.,DOC-MEK (EudraCT no: 2009-018153-23).

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma.,They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit.,The master regulators driving the expression of resistance-genes remain poorly understood.,Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes.,Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse.,Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists.,We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation.,Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth.,We thus propose AhR-impairment as a strategy to overcome melanoma resistance.,Resistance to BRAF inhibitors limits their clinical benefit in melanoma patients.,Here, the authors show that the Aryl hydrocarbon Receptor (AhR) is a key mediator of resistant genes and use resveratrol, an AhR antagonist, to revert resistance in melanoma bearing mice.

Malignant melanoma is often used as a model tumor for the establishment of novel therapies.,It is known that two-dimensional (2D) culture methods are not sufficient to elucidate the various processes during cancer development and progression.,Therefore, it is of major interest to establish defined biofabricated three-dimensional (3D) models, which help to decipher complex cellular interactions.,To get an impression of their printability and subsequent behavior, we printed fluorescently labeled melanoma cell lines with Matrigel and two different types of commercially available bioinks, without or with modification (RGD (Arginine-Glycine-Aspartate)-sequence/laminin-mixture) for increased cell-matrix communication.,In general, we demonstrated the printability of melanoma cells in all tested biomaterials and survival of the printed cells throughout 14 days of cultivation.,Melanoma cell lines revealed specific differential behavior in the respective inks.,Whereas in Matrigel, the cells were able to spread, proliferate and form dense networks throughout the construct, the cells showed no proliferation at all in alginate-based bioink.,In gelatin methacrylate-based bioink, the cells proliferated in clusters.,Surprisingly, the modifications of the bioinks with RGD or the laminin blend did not affect the analyzed cellular behavior.,Our results underline the importance of precisely adapting extracellular matrices to individual requirements of specific 3D bioprinting applications.

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.

The number of treatment options for melanoma patients has grown in the past few years, leading to considerable improvements in both overall and progression-free survival.,Targeted therapies and immune checkpoint inhibitors have opened a new era in the management of melanoma patients.,Despite the clinical advances, further research efforts are needed to identify other “druggable” targets and new biomarkers to improve the stratification of melanoma patients who could really benefit from targeted and immunotherapies.,To this end, many studies have focused on the role of microRNAs (miRNAs) that are small non-coding RNAs (18-25 nucleotides in length), which post-transcriptionally regulate the expression of their targets.,In cancer, they can behave either as oncogenes or oncosuppressive genes and play a central role in many intracellular pathways involved in proliferation and invasion.,Given their modulating activity on the transcriptional landscape, their biological role is under investigation to study resistance mechanisms.,They are able to mediate the communication between tumor cells and their microenvironment and regulate tumor immunity through direct regulation of the genes involved in immune activation or suppression.,To date, a very promising miRNA-based strategy is to use them as prognosis and diagnosis biomarkers both as cell-free miRNAs and extracellular-vesicle miRNAs.,However, miRNAs have a complex role since they target different genes in different cellular conditions.,Thus, the ultimate aim of studies has been to recapitulate their role in melanoma in biological networks that account for miRNA/gene expression and mutational state.,In this review, we will provide an overview of current scientific knowledge regarding the oncogenic or oncosuppressive role of miRNAs in melanoma and their use as biomarkers, with respect to approved therapies for melanoma treatment.

Cutaneous melanoma (CM) is the most malignant tumor of skin cancers because of its rapid development and high mortality rate.,Long noncoding RNAs (lncRNAs), which play essential roles in the tumorigenesis and metastasis of CM and interplay with microRNAs (miRNAs) and mRNAs, are hopefully considered to be efficient biomarkers to detect deterioration during the progression of CM to improve the prognosis.,Bioinformatics analysis was fully applied to predict the vital lncRNAs and the associated miRNAs and mRNAs, which eventually constructed the competing endogenous RNA (ceRNA) network to explain the RNA expression patterns in the progression of CM.,Further statistical analysis emphasized the importance of these key genes, which were statistically significantly related to one or few clinical features from the ceRNA network.,The results showed the lncRNAs MGC12926 and LINC00937 were verified to be strongly connected with the prognosis of CM patients.

The MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis.,Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated.,Here we identify 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions.,Our work suggests that one of these, Disrupted In Renal Carcinoma 3 (DIRC3), may be a clinically important MITF-SOX10 regulated tumour suppressor.,DIRC3 depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by low DIRC3 expression have decreased survival.,DIRC3 is a nuclear lncRNA that activates expression of its neighbouring IGFBP5 tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within the DIRC3 locus.,In turn, DIRC3 dependent regulation of IGFBP5 impacts the expression of genes involved in cancer associated processes and is needed for DIRC3 control of anchorage-independent growth.,Our work indicates that lncRNA components of MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets that can act not only as downstream mediators of MITF-SOX10 function but as feedback regulators of MITF-SOX10 activity.

Melanoma is the deadliest form of skin cancer, and little is known about the impact of deregulated expression of long noncoding RNAs (lncRNAs) in the progression of this cancer.,In this study, we explored RNA-Seq data to search for lncRNAs associated with melanoma progression.,We found distinct lncRNA gene expression patterns across melanocytes, primary and metastatic melanoma cells.,Also, we observed upregulation of the lncRNA ZEB1-AS1 (ZEB1 antisense RNA 1) in melanoma cell lines.,Data analysis from The Cancer Genome Atlas (TCGA) confirmed higher ZEB1-AS1 expression in metastatic melanoma and its association with hotspot mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) gene and RAS family genes.,In addition, a positive correlation between ZEB1-AS1 and ZEB1 (zinc finger E-box binding homeobox 1) gene expression was verified in primary and metastatic melanomas.,Using gene expression signatures indicative of invasive or proliferative phenotypes, we found an association between ZEB1-AS1 upregulation and a transcriptional profile for invasiveness.,Enrichment analysis of correlated genes demonstrated cancer genes and pathways associated with ZEB1-AS1.,We suggest that the lncRNA ZEB1-AS1 could function by activating ZEB1 gene expression, thereby influencing invasiveness and phenotype switching in melanoma, an epithelial-to-mesenchymal transition (EMT)-like process, which the ZEB1 gene has an essential role.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF).,Accordingly, downregulation of MITF induces invasion in melanoma cells, however little is known about the underlying mechanisms.,Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C.,Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion.,We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells.,Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity, and lung colonization.,Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells.,Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

Despite the remarkable clinical response of melanoma harboring BRAF mutations to BRAF inhibitors (BRAFi), most tumors become resistant.,Here, we identified the downregulation of the ubiquitin ligase RNF125 in BRAFi-resistant melanomas and demonstrated its role in intrinsic and adaptive resistance to BRAFi in cultures as well as its association with resistance in tumor specimens.,Sox10/MITF expression correlated with and contributed to RNF125 transcription.,Reduced RNF125 was associated with elevated expression of receptor tyrosine kinases (RTKs), including EGFR.,Notably, RNF125 altered RTK expression through JAK1, which we identified as an RNF125 substrate.,RNF125 bound to and ubiquitinated JAK1, prompting its degradation and suppressing RTK expression.,Inhibition of JAK1 and EGFR signaling overcame BRAFi resistance in melanoma with reduced RNF125 expression, as shown in culture and in in vivo xenografts.,Our findings suggest that combination therapies targeting both JAK1 and EGFR could be effective against BRAFi-resistant tumors with de novo low RNF125 expression.

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma.,Current theories on regulation of inflammation center on anti-tumor T cell responses.,Here we show that tumor associated B cells are vital to melanoma associated inflammation.,Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro.,This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5.,Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers.,Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade.,Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.,The regulation of tumor inflammation is incompletely understood and the role of B cells is unclear.,Here, the authors show that a specific subtype of B cells is induced in melanoma and required to recruit T lymphocytes and elicit inflammation.

Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature.,While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells.,We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells.,We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001).,Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001).,Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells.,Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study.,This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity.,These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks.,It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment.,We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma.,We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and this response would correlate with disease-free survival.,We identified a rapid and potent anti-tumor response, with 8/27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease-free.,These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks with reinvigoration in the blood observed as early as 1 week.,Transcriptional analysis demonstrated a pre-treatment immune signature (Neoadjuvant Response Signature) that was associated with clinical benefit.,In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution.,Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma.,Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.

Checkpoint inhibitors are revolutionizing treatment options and expectations for patients with melanoma.,Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), was the first approved checkpoint inhibitor.,Emerging long-term data indicate that approximately 20% of ipilimumab-treated patients achieve long-term survival.,The first programmed death 1 (PD-1) inhibitor, pembrolizumab, was recently approved by the United States Food and Drug Administration for the treatment of melanoma; nivolumab was previously approved in Japan.,PD-1 inhibitors are also poised to become standard of care treatment for other cancers, including non-small cell lung cancer, renal cell carcinoma and Hodgkin's lymphoma.,Immunotherapy using checkpoint inhibition is a different treatment approach to chemotherapy and targeted agents: instead of directly acting on the tumor to induce tumor cell death, checkpoint inhibitors enhance or de novo stimulate antitumor immune responses to eliminate cancer cells.,Initial data suggest that objective anti-tumor response rates may be higher with anti-PD-1 agents compared with ipilimumab and the safety profile may be more tolerable.,This review explores the development and next steps for PD-1 pathway inhibitors, including discussion of their novel mechanism of action and clinical data to-date, with a focus on melanoma.

Preclinical data suggest the combination of an anti-programmed death receptor 1 antibody plus dabrafenib and trametinib to have superior antitumor activity compared with dabrafenib plus trametinib alone.,These observations are supported by translational evidence suggesting that immune checkpoint inhibitors plus targeted therapy may improve treatment outcomes in patients with BRAF V600-mutant metastatic melanoma.,COMBI-i is a phase III trial evaluating spartalizumab, an anti-programmed death receptor 1 antibody, in combination with dabrafenib and trametinib (sparta-DabTram), versus placebo plus dabrafenib and trametinib (placebo-DabTram) in patients with BRAF V600-mutant unresectable or metastatic melanoma.,Patients received spartalizumab 400 mg intravenously every 4 weeks plus dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily or placebo-DabTram.,Participants were age ≥ 18 years with unresectable or metastatic BRAF V600-mutant melanoma.,The primary end point was investigator-assessed progression-free survival.,Overall survival was a key secondary end point (ClinicalTrials.gov identifier: NCT02967692).,At data cutoff (July 1, 2020), the median progression-free survival was 16.2 months (95% CI, 12.7 to 23.9 months) in the sparta-DabTram arm versus 12.0 months (95% CI, 10.2 to 15.4 months) in the placebo-DabTram arm (hazard ratio, 0.82 [95% CI, 0.66 to 1.03]; P = .042 [one-sided; nonsignificant]).,The objective response rates were 69% (183 of 267 patients) versus 64% (170 of 265 patients), respectively.,Grade ≥ 3 treatment-related adverse events occurred in 55% (146 of 267) of patients in the sparta-DabTram arm and 33% (88 of 264) in the placebo-DabTram arm.,The study did not meet its primary end point; broad first-line use of sparta-DabTram is not supported by these results.,Further biomarker-driven investigation may identify patient subpopulations who could benefit from checkpoint inhibitor plus targeted therapy combinations.

Melanoma brain metastases (MBMs) are a challenging clinical problem with high morbidity and mortality.,Although first-line dabrafenib-trametinib and ipilimumab-nivolumab have similar intracranial response rates (50%-55%), central nervous system (CNS) resistance to BRAF-MEK inhibitors (BRAF-MEKi) usually occurs around 6 months, and durable responses are only seen with combination immunotherapy.,We sought to investigate the utility of ipilimumab-nivolumab after MBM progression on BRAF-MEKi and identify mechanisms of resistance.,Patients who received first-line ipilimumab-nivolumab for MBMs or second/third line ipilimumab-nivolumab for intracranial metastases with BRAFV600 mutations with prior progression on BRAF-MEKi and MRI brain staging from March 1, 2015 to June 30, 2018 were included.,Modified intracranial RECIST was used to assess response.,Formalin-fixed paraffin-embedded samples of BRAFV600 mutant MBMs that were naïve to systemic treatment (n=18) or excised after progression on BRAF-MEKi (n=14) underwent whole transcriptome sequencing.,Comparative analyses of MBMs naïve to systemic treatment versus BRAF-MEKi progression were performed.,Twenty-five and 30 patients who received first and second/third line ipilimumab-nivolumab, were included respectively.,Median sum of MBM diameters was 13 and 20.5 mm for the first and second/third line ipilimumab-nivolumab groups, respectively.,Intracranial response rate was 75.0% (12/16), and median progression-free survival (PFS) was 41.6 months for first-line ipilimumab-nivolumab.,Efficacy of second/third line ipilimumab-nivolumab after BRAF-MEKi progression was poor with an intracranial response rate of 4.8% (1/21) and median PFS of 1.3 months.,Given the poor activity of ipilimumab-nivolumab after BRAF-MEKi MBM progression, we performed whole transcriptome sequencing to identify mechanisms of drug resistance.,We identified a set of 178 differentially expressed genes (DEGs) between naïve and MBMs with progression on BRAF-MEKi treatment (p value <0.05, false discovery rate (FDR) <0.1).,No distinct pathways were identified from gene set enrichment analyses using Kyoto Encyclopedia of Genes and Genomes, Gene Ontogeny or Hallmark libraries; however, enrichment of DEG from the Innate Anti-PD1 Resistance Signature (IPRES) was identified (p value=0.007, FDR=0.03).,Second-line ipilimumab-nivolumab for MBMs after BRAF-MEKi progression has poor activity.,MBMs that are resistant to BRAF-MEKi that also conferred resistance to second-line ipilimumab-nivolumab showed enrichment of the IPRES gene signature.

Despite remarkable advances in the genomic characterization of adult melanoma, the molecular pathogenesis of pediatric melanoma remains largely unknown.,We analyzed 15 conventional melanomas (CMs), 3 melanomas arising in congenital nevi (CNMs), and 5 spitzoid melanomas (SMs), using various platforms, including whole genome or exome sequencing, the molecular inversion probe assay, and/or targeted sequencing.,CMs demonstrated a high burden of somatic single-nucleotide variations (SNVs), with each case containing a TERT promoter (TERT-p) mutation, 13/15 containing an activating BRAF V600 mutation, and >80% of the identified SNVs consistent with UV damage.,In contrast, the three CNMs contained an activating NRAS Q61 mutation and no TERT-p mutations.,SMs were characterized by chromosomal rearrangements resulting in activated kinase signaling in 40%, and an absence of TERT-p mutations, except for the one SM that succumbed to hematogenous metastasis.,We conclude that pediatric CM has a very similar UV-induced mutational spectrum to that found in the adult counterpart, emphasizing the need to promote sun protection practices in early life and to improve access to therapeutic agents being explored in adults in young patients.,In contrast, the pathogenesis of CNM appears to be distinct.,TERT-p mutations may identify the rare subset of spitzoid melanocytic lesions prone to disseminate.

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway.,About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E).,BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors.,The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3).,The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression.,Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment.,Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation.,In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations.,Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD).,Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy.,For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months.,A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab.,In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2.,Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations.,Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

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.

Melanoma is one of the most common skin malignancies.,Both microRNAs and long non-coding RNAs (lncRNAs) have critical roles in the progression of cancers, including melanoma.,However, the underlying molecular mechanism has not been fully characterized.,We demonstrated that miR-34a is negatively correlated with MALAT1 in melanoma cells and tumor specimens.,Interestingly, MALAT1, which contains functional sequence-specific miR-34a-binding sites, regulates miR-34a stability in melanoma cells and in vivo.,Importantly, MALAT1 was significantly enriched in the Ago2 complex, but not when the MALAT1-binding site of miR-34a was mutated.,Furthermore, MALAT1 could be shown to regulate c-Myc and Met expression by functioning as a miR-34a sponge.,Our results reveal an unexpected mode of action for MALAT1 as an important regulator of miR-34a.

microRNAs constitute a complex class of pleiotropic post-transcriptional regulators of gene expression involved in the control of several physiologic and pathologic processes.,Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5′ end of a 21-23 nt sequence with a partially complementary sequence located in the 3′ untranslated region of target mRNAs.,This leads to inhibition of mRNA translation and eventually to its degradation.,Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs.,In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells.,Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies.,In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs.,Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors.

Melanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward.,To improve diagnosis and prognosis, markers of malignancy are needed.,Receptor for activated C kinase 1 (RACK1) protein may be such a marker.,RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients.,Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses.,Using immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples.,Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes.,Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas.,Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features.,This study confirms that horses provide an interesting model for melanoma genesis studies.,It establishes MITF and PAX3 as markers of horse melanocytic cells.,RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.

Melanoma is a deadly disease affecting people worldwide.,Genetic studies have identified different melanoma subtypes characterized by specific recurrently mutated genes and led to the successful clinical introduction of targeted therapies.,Hotspot mutations in SF3B1 were recently reported in uveal melanoma.,Our aim was to see if these mutations also occur in cutaneous melanoma.,We analyzed a cohort of 85 cutaneous melanoma including 22 superficial spreading, 24 acral-lentiginous, 36 nodular, and 3 lentigo-maligna melanomas.,Exon 14 of SF3B1, containing the site of recurrent mutations described in uveal melanoma, was sequenced in all samples.,Additionally, NRAS exon 1 and 2 and BRAF exon 15 were sequenced in all, KIT exons 9, 11, 13, 17, and 18 in 30 samples.,High numbers of BRAF and NRAS mutations were identified with frequencies varying according to melanoma subtype.,None of the samples were found to harbor a SF3B1 mutation.,We conclude that recurrent mutations in codon 625 of SF3B1 as reported in uveal melanoma are not present in most types of cutaneous melanoma.,This highlights the genetic differences between cutaneous and uveal melanoma and the need for subtype specific therapeutic approaches.

Malignant melanoma is the most lethal form of skin cancer with a variable clinical course even in patients with thin melanomas and localized disease.,Despite increasing insights into melanoma biology, no prognostic biomarkers have yet been incorporated into clinical protocols.,Reduced expression of the RNA binding motif protein 3 (RBM3) has been shown to correlate with tumour progression and poor prognosis in melanoma and several other cancer forms.,In ovarian cancer, an inverse association was found between expression of RBM3 and the minichromosome maintenance 3 (MCM3) gene and protein.,In melanoma, gene expression analysis and immunohistochemical validation has uncovered MCM3 as a putative prognostic biomarker.,The aim of the present study was to examine the associations of MCM3 expression with clinical outcome and RBM3 expression in a prospective, population-based cohort of melanoma.,Immunohistochemical MCM3 expression was examined in 224 incident cases of primary melanoma from the Malmö Diet and Cancer Study, previously analysed for RBM3 expression.,Spearman´s Rho and Chi-Square tests were used to explore correlations between MCM3 expression, clinicopathological factors, and expression of RBM3 and Ki67.,Kaplan Meier analysis, the log rank test, and univariable and multivariable Cox proportional hazards modelling were used to assess the impact of MCM3 expression on disease-free survival (DFS) and melanoma-specific survival (MSS).,High MCM3 expression was significantly associated with unfavourable clinicopathological features and high Ki67 expression.,A significant inverse correlation was seen between expression of MCM3 and RBM3 (p = 0.025).,High MCM3 expression was associated with a reduced DFS (HR = 5.62) and MSS (HR = 6.03), and these associations remained significant in multivariable analysis, adjusted for all other factors (HR = 5.01 for DFS and HR = 4.96 for MSS).,RBM3 expression remained an independent prognostic factor for MSS but not DFS in the multivariable model.,These findings provide validation of the utility of MCM3 expression as an independent biomarker for prognostication of patients with primary melanoma.,Moreover, the inverse association and prognostic impact of MCM3 and RBM3 expression indicate a possible interaction of these proteins in melanoma progression, the functional basis for which merits further study.,The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1814908129755401

Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP,POT1), have also been implicated in melanomagenesis.,Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.,−57 T>G variant) has been reported in one family.,We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls.,Germline lymphocyte telomere length was estimated in carriers.,The c.,−57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls.,One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour.,The blood leukocyte telomere length of a carrier was similar to wild-type cases.,We provide evidence confirming that a rare promoter variant of TERT (c.,−57 T>G) is associated with high penetrance, early onset melanoma and potentially other cancers, and explains <1 % of UK melanoma multicase families.,The identification of POT1 and TERT germline mutations highlights the importance of telomere integrity in melanoma biology.,The online version of this article (doi:10.1007/s10689-015-9841-9) 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.

Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases.,Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies.,In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance.,Thus, combined treatment targeting both pathways is a promising strategy to overcome this.,Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines.,Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis.,Finally, an in vivo treatment experiment was carried out on NOD/SCID mice.,We show that combined therapy was more effective than monotherapy.,Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo.,This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.

NRAS and its effector BRAF are frequently mutated in melanoma.,Paradoxically, CRAF but not BRAF was shown to be critical for various RAS-driven cancers, raising the question of the role of RAF proteins in NRAS-induced melanoma.,Here, using conditional ablation of Raf genes in NRAS-induced mouse melanoma models, we investigate their contribution in tumour progression, from the onset of benign tumours to malignant tumour maintenance.,We show that BRAF expression is required for ERK activation and nevi development, demonstrating a critical role in the early stages of NRAS-driven melanoma.,After melanoma formation, single Braf or Craf ablation is not sufficient to block tumour growth, showing redundant functions for RAF kinases.,Finally, proliferation of resistant cells emerging in the absence of BRAF and CRAF remains dependent on ARAF-mediated ERK activation.,These results reveal specific and compensatory functions for BRAF and CRAF and highlight an addiction to RAF signalling in NRAS-driven melanoma.,The melanoma-driver mutations in NRAS and BRAF are mutually exclusive but the contribution of RAF signalling downstream of NRAS remains to be clarified.,Here, using mouse models, the authors show specific roles of each member of the RAF family at different stages of melanomagenesis.

Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide.,Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release.,We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway.,Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells.,Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET.,In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex.,Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI.,In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype.,Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.

Drug resistance remains an unsolved clinical issue in oncology.,Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients.,Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance.,The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays.,Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK.,Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis.,Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance.,To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells.,Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo.,These findings make ALK a promising clinical target in melanoma patients.,The online version of this article (10.1186/s12943-018-0886-x) contains supplementary material, which is available to authorized users.

Cancer-secreted exosomal miRNAs regulates the biological processes of many tumours.,The serum level of exosomal miR-106b-5p is significantly increased in melanoma patients.,However, the role and molecular mechanisms of exosomal miR-106b-5p in melanoma remains unclear.,Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-106b-5p and EphA4 in melanoma tissues.,Transmission electron microscopy (TEM) and western blotting were used to identify exosome.,QRT-qPCR and Cy3-labelled miR-106b-5p were used to demonstrated the transmission of melanoma cell-secreted exosomal miR-106b-5p.,Western blotting, Immunofluorescence, adhesion, transwell and scratch wound assay were used to explore the role of exosomal miR-106b-5p in melanocytes.,Luciferase reporter assays and RNA-Chromatin Immunoprecipitation (ChIP) assay were used to confirm whether erythropoietin-producing hepatocellular carcinoma receptor A4 (EphA4) was a direct target of miR-106b-5p.,We found that miR-106b-5p levels were increased in melanoma tissue, and high miR-106b-5p expression is an independent risk factor for the overall survival of patients with melanoma. miR-106b-5p is enriched in melanoma cell-secreted exosomes and transferred to melanocytes.,Exosomal miR-106b-5p promotes the epithelial-to-mesenchymal transition (EMT), migration, invasion and adhesion of melanocytes.,Exosomal miR-106b-5p exerted its role by targeting EphA4 to activate the ERK pathway.,We demonstrated that exosomal miR-106b-5p promoted melanoma metastasis in vivo through pulmonary metastasis assay.,Thus, melanoma cell-secreted exosomal miR-106b-5p may serve as a diagnostic indicator and potential therapeutic target in melanoma patients.,The online version contains supplementary material available at 10.1186/s13046-021-01906-w.

Stromal fibroblasts are an integral part of the tumor stroma and constantly interact with cancer cells to promote their initiation and progression.,However, the role and function of dermal fibroblasts during the early stage of melanoma development remain poorly understood.,We, therefore, designed a novel genetic approach to deactivate stromal fibroblasts at the onset of melanoma formation by targeted ablation of β‐catenin.,To our surprise, melanoma tumors formed from β‐catenin‐deficient group (B16F10 mixed with β‐catenin‐deficient fibroblasts) appeared earlier than tumors formed from control group (B16F10 mixed with normal dermal fibroblasts).,At the end point when tumors were collected, mutant tumors were bigger and heavier than control tumors.,Further analysis showed that there were fewer amounts of stromal fibroblasts and myofibroblasts inside mutant tumor stroma.,Melanoma tumors from control group showed reduced proliferation, down‐regulated expression of cyclin D1 and increased expression of cyclin‐dependent kinase inhibitor p16, suggesting dermal fibroblasts blocked the onset of melanoma tumor formation by inducing a cell cycle arrest in B16F10 melanoma cells.,Furthermore, we discovered that dermal fibroblasts prevented epithelial‐mesenchymal transition in melanoma cells.,Overall, our findings demonstrated that dermal fibroblasts crosstalk with melanoma cells to regulate in vivo tumor development via multiple mechanisms, and the outcomes of their reciprocal interactions depend on activation states of stromal fibroblasts and stages of melanoma development.

MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs.,Recent studies have demonstrated that miRNAs are implicated in the development of cancer.,However, the role of miR-144 in uveal melanoma metastasis remains largely unknown.,MiR-144 was downregulated in both uveal melanoma cells and tissues.,Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion.,After identification of two putative miR-144 binding sites within the 3' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites.,In addition, the expression of c-Met protein was inhibited by miR-144.,Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells.,In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.

Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis.,Here, we describe mutations occurring exclusively at arginine-625 in splicing factor 3B subunit 1 (SF3B1) in low-grade uveal melanomas with good prognosis.,Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutation, and these mutations denote a distinct molecular subset of uveal melanomas.

Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths.,Thus, searching for new melanoma treatment options is an important field of study.,The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation.,C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system.,The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells.,The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner.,This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug.,For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively.,Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation.,This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy.,The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.

Melanoma is the most aggressive form of skin cancer.,Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy.,To increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1).,With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro.,In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft.,We demonstrated two to five times higher binding and internalization of these immunoliposomes by M1+/A1+ melanoma cells in vitro in comparison with nontargeted liposomes.,Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes.,In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively.,Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1+/A1+ expressing tumors in mice compared with the treatment of M1−/A1+ tumors.,Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo.,Liposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma.,We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response.,Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy.,Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

Treatment options are limited for patients with recurrent and/or metastatic (R/M) cutaneous squamous cell carcinoma (cSCC); mortality rates exceed 70% in patients with distant metastases.,Here, we present the first interim analysis of the R/M cSCC cohort from the 2-cohort-locally advanced and R/M-phase II KEYNOTE-629 study.,Patients with R/M cSCC not amenable to surgery or radiation received pembrolizumab 200 mg every 3 weeks.,The primary end point was objective response rate per RECIST v1.1.,Secondary end points were duration of response, disease control rate, progression-free survival, overall survival, and safety.,At data cutoff (April 8, 2019), median follow-up of 105 enrolled patients in the R/M cohort was 11.4 months (range, 0.4 to 16.3 months).,Objective response rate was 34.3% (95% CI, 25.3% to 44.2%; 4 complete responses, 32 partial responses), and disease control rate was 52.4% (95% CI, 42.4% to 62.2%).,Median duration of response was not reached (range, 2.7 to 13.1+ months; ‘+’ refers to ongoing response at data cutoff).,Median progression-free survival was 6.9 months (95% CI, 3.1 months to 8.5 months).,Median overall survival was not reached (95% CI, 10.7 months to not reached).,Treatment-related adverse events occurred in 66.7% of patients (n = 70), the most common of which were pruritus (n = 15; 14.3%), asthenia (n = 14; 13.3%), and fatigue (n = 13; 12.4%).,Grade 3 to 5 treatment-related adverse events occurred in 5.7% (n = 6) of patients.,One patient died of treatment-related cranial nerve neuropathy.,Pembrolizumab demonstrated effective antitumor activity; clinically meaningful, durable responses; and acceptable safety in primarily elderly patients with R/M cSCC, supporting its use in clinical practice.,Pembrolizumab adverse events in this study were consistent with its established safety profile.

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.

Recent evidence suggests that immunotherapy efficacy in melanoma is modulated by gut microbiota.,Few studies have examined this phenomenon in humans, and none have incorporated metatranscriptomics, important for determining expression of metagenomic functions in the microbial community.,In melanoma patients undergoing immunotherapy, gut microbiome was characterized in pre-treatment stool using 16S rRNA gene and shotgun metagenome sequencing (n = 27).,Transcriptional expression of metagenomic pathways was confirmed with metatranscriptome sequencing in a subset of 17.,We examined associations of taxa and metagenomic pathways with progression-free survival (PFS) using 500 × 10-fold cross-validated elastic-net penalized Cox regression.,Higher microbial community richness was associated with longer PFS in 16S and shotgun data (p < 0.05).,Clustering based on overall microbiome composition divided patients into three groups with differing PFS; the low-risk group had 99% lower risk of progression than the high-risk group at any time during follow-up (p = 0.002).,Among the species selected in regression, abundance of Bacteroides ovatus, Bacteroides dorei, Bacteroides massiliensis, Ruminococcus gnavus, and Blautia producta were related to shorter PFS, and Faecalibacterium prausnitzii, Coprococcus eutactus, Prevotella stercorea, Streptococcus sanguinis, Streptococcus anginosus, and Lachnospiraceae bacterium 3 1 46FAA to longer PFS.,Metagenomic functions related to PFS that had correlated metatranscriptomic expression included risk-associated pathways of l-rhamnose degradation, guanosine nucleotide biosynthesis, and B vitamin biosynthesis.,This work adds to the growing evidence that gut microbiota are related to immunotherapy outcomes, and identifies, for the first time, transcriptionally expressed metagenomic pathways related to PFS.,Further research is warranted on microbial therapeutic targets to improve immunotherapy outcomes.

Despite major improvements in combatting metastatic melanoma since the advent of immunotherapy, the overall survival for patients with advanced disease remains low.,Recently, there is a growing number of reports supporting an “obesity paradox,” in which patients who are overweight or mildly obese may exhibit a survival benefit in patients who received immune checkpoint inhibitors.,We studied the relationship between body mass index and progression-free survival and overall survival in a cohort of 423 metastatic melanoma patients receiving immunotherapy, enrolled and prospectively followed up in the NYU Interdisciplinary Melanoma Cooperative Group database.,We analyzed this association stratified by first vs. second or greater-line of treatment and treatment type adjusting for age, gender, stage, lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, number of metastatic sites, and body mass index classification changes.,In our cohort, the patients who were overweight or obese did not have different progression-free survival than patients with normal body mass index.,Stratifying this cohort by first vs. non-first line immunotherapy revealed a moderate but insignificant association between being overweight or obese and better progression-free survival in patients who received first line.,Conversely, an association with worse progression-free survival was observed in patients who received non-first line immune checkpoint inhibitors.,Specifically, overweight and obese patients receiving combination immunotherapy had a statistically significant survival benefit, whereas patients receiving the other treatment types showed heterogeneous trends.,We caution the scientific community to consider several important points prior to drawing conclusions that could potentially influence patient care, including preclinical data associating obesity with aggressive tumor biology, the lack of congruence amongst several investigations, and the limited reproduced comprehensiveness of these studies.,The online version of this article (10.1186/s40425-019-0699-5) contains supplementary material, which is available to authorized users.

Despite several therapeutic advances, malignant melanoma still remains a fatal disease for which novel and long-term curative treatments are needed.,The successful development of innovative therapies strongly depends on the availability of appropriate pre-clinical models.,For this purpose, several mouse models holding the promise to provide insight into molecular biology and clinical behavior of melanoma have been generated.,The most relevant ones and their contribution for the advancement of therapeutic approaches for the treatment of human melanoma patients will be here summarized.,However, as models, mice do not recapitulate all the features of human melanoma, thus their strengths and weaknesses need to be carefully identified and considered for the translation of the results into the human clinics.,In this panorama, the concept of comparative oncology acquires a priceless value.,The revolutionary importance of spontaneous canine melanoma as a translational model for the pre-clinical investigation of melanoma progression and treatment will be here discussed, with a special consideration to the development of innovative immunotherapeutic approaches.

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.

Angiogenesis is important for the progression of cutaneous melanoma.,Here, we analyzed the prognostic impact of the angiogenic factor urokinase plasminogen activator resecptor (uPAR), vascular proliferation index (VPI) and tumor necrosis as a measure of hypoxia in a patient series of nodular melanomas (n = 255) and matched loco-regional metastases (n = 78).,Expression of uPAR was determined by immunohistochemistry and VPI was assessed by dual immunohistochemistry using Factor-VIII/Ki67 staining.,Necrosis was recorded based on HE-slides.,As novel findings, high uPAR expression and high VPI were associated with each other, and with increased tumor thickness, presence of tumor necrosis, tumor ulceration, increased mitotic count and reduced cancer specific survival in primary melanoma.,In matched cases, VPI was decreased in metastases, whereas the frequency of necrosis was increased.,Our findings demonstrate for the first time the impact on melanoma specific survival of uPAR expression and VPI in primary tumors, and of increased necrosis as an indicator of tumor hypoxia in loco-regional metastases.,These findings support the importance of tumor angiogenesis in melanoma aggressiveness, and suggest uPAR as an indicator of vascular proliferation and a potential biomarker in melanoma.

Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment.,In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases.,We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro.,B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection.,In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated.,Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages.,Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation.,Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures.,In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes.,In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation.,The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment.,We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort.,We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival.,This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin-mediated failure to recruit CD141+ DCs.,A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles.,The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation.,The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants.,In summary, we report evidence for a β-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome.,We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting, and association with ultraviolet (UV) exposure early in life1.,To obtain a comprehensive genomic view of melanoma, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA.,A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-UV exposed hairless skin of the extremities (3 and 14 per Mb genome), intermediate in those originating from hair-bearing skin of the trunk (range = 5 to 55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb).,Analysis of whole-genome sequence data identified PREX2 - a PTEN-interacting protein and negative regulator of PTEN in breast cancer2 - as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas.,PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumor formation of immortalized human melanocytes in vivo.,Thus, whole-genome sequencing of human melanoma tumors revealed genomic evidence of UV pathogenesis and discovered a new recurrently mutated gene in melanoma.

Cutaneous squamous cell carcinoma represents the second most common cutaneous malignancy, affecting 7-11% of Caucasians in the United States.,The genetic determinants of susceptibility to cutaneous squamous cell carcinoma remain largely unknown.,Here we report the results of a two-stage genome-wide association study of cutaneous squamous cell carcinoma, totalling 7,404 cases and 292,076 controls.,Eleven loci reached genome-wide significance (P<5 × 10−8) including seven previously confirmed pigmentation-related loci: MC1R, ASIP, TYR, SLC45A2, OCA2, IRF4 and BNC2.,We identify an additional four susceptibility loci: 11q23.3 CADM1, a metastasis suppressor gene involved in modifying tumour interaction with cell-mediated immunity; 2p22.3; 7p21.1 AHR, the dioxin receptor involved in anti-apoptotic pathways and melanoma progression; and 9q34.3 SEC16A, a putative oncogene with roles in secretion and cellular proliferation.,These susceptibility loci provide deeper insight into the pathogenesis of squamous cell carcinoma.,Cutaneous squamous cell carcinoma is the second most common type of skin cancer.,In this genome-wide association study, which includes over 7,000 cases, the authors identify 4 new susceptibility loci for this cancer and also provide independent replication of 9 previously reported susceptibility loci.

In an ongoing screen for DNA sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conduct a genome-wide association study (GWAS) of 24,988,228 SNPs and small indels detected through whole-genome sequencing of 2,636 Icelanders and imputed into 4,572 BCC patients and 266,358 controls.,Here we show the discovery of four new BCC susceptibility loci: 2p24 MYCN (rs57244888[C], OR=0.76, P=4.7 × 10−12), 2q33 CASP8-ALS2CR12 (rs13014235[C], OR=1.15, P=1.5 × 10−9), 8q21 ZFHX4 (rs28727938[G], OR=0.70, P=3.5 × 10−12) and 10p14 GATA3 (rs73635312[A], OR=0.74, P=2.4 × 10−16).,Fine mapping reveals that two variants correlated with rs73635312[A] occur in conserved binding sites for the GATA3 transcription factor.,In addition, expression microarrays and RNA-seq show that rs13014235[C] and a related SNP rs700635[C] are associated with expression of CASP8 splice variants in which sequences from intron 8 are retained.,Basal cell carcinoma is a common cancer among people of European ancestry, with associated high economic costs to monitor and treat.,Here Stacey et al. conduct a genome-wide association study on Icelandic and other European populations, identifying four novel loci associated with cancer susceptibility.

Approximately 20% of melanomas contain a mutation in NRAS.,However no direct inhibitor of NRAS is available.,One of the main signaling pathways downstream of NRAS is the MAPK pathway.,In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway.,Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated.,The majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination.,Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment.,Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1.,However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway.,Resistant cell lines also showed higher levels of p-GSK3β and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines.,The combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.,The online version of this article (doi:10.1186/s12943-015-0293-5) contains supplementary material, which is available to authorized users.

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.

Intrinsic and acquired resistance limit the therapeutic benefits of inhibitors of oncogenic BRAF in melanoma.,To identify microRNAs (miRNAs) associated with resistance to a BRAF inhibitor, we compared miRNA expression levels in three cell lines with different BRAF inhibitor sensitivity.,miRNA microarray analysis was conducted to compare miRNA expression levels.,Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to confirm the expression of differentially expressed miRNAs.,The cellular effects of miR-1246 were further examined by MTT assay, immunoblotting analysis, cell cycle analysis, flow cytometric assay of apoptosis, and autophagy assay.,The miRNA microarray analysis and qRT-PCR identified five miRNAs (miR-3617, miR-92a-1, miR-1246, miR-193b-3p, and miR-17-3p) with expression that was consistently altered in two BRAF inhibitor-resistant cell lines.,Among the five miRNAs, a miR-1246 mimic significantly reduced the antiproliferative effects of the BRAF inhibitor PLX4720 in BRAF inhibitor-resistant A375P (A375P/Mdr) cells, suggesting that miR-1246 upregulation confers acquired resistance to BRAF inhibition.,In particular, apoptosis was identified as a major type of cell death in miR-1246-transfected cells; however, necrosis predominated in mimic-control-transfected cells, indicating that the resistance to PLX4720 in miR-1246 mimic-transfected cells is predominantly due to a reduction in necrosis.,Furthermore, we found that miR-1246 promoted G2/M arrest through autophagy as a way to escape cell death by necrosis and apoptosis in response to PLX4720.,The promotion of BRAF inhibitor resistance by miR-1246 was associated with lowered levels of p-ERK.,These results suggest that miR-1246 may be a potential therapeutic target in melanoma with acquired resistance to BRAF inhibitors.

To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥2 fold, p < 0.05) miRNAs.,Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth.,We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers.,To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma.,NF1 was selected for functional validation because of its recent implication in acquired resistance to BRAFV600E-targeted therapy.,Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032.,These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

Despite the general focus on an invasive and de-differentiated phenotype as main driver of cancer metastasis, in melanoma patients many metastatic lesions display a high degree of pigmentation, indicative for a differentiated phenotype.,Indeed, studies in mice and fish show that melanoma cells switch to a differentiated phenotype at secondary sites, possibly because in melanoma differentiation is closely linked to proliferation through the lineage-specific transcriptional master regulator MITF.,Importantly, while a lot of effort has gone into identifying factors that induce the de-differentiated/invasive phenotype, it is not well understood how the switch to the differentiated/proliferative phenotype is controlled.,We identify collagen as a contributor to this switch.,We demonstrate that collagen stiffness induces melanoma differentiation through a YAP/PAX3/MITF axis and show that in melanoma patients increased collagen abundance correlates with nuclear YAP localization.,However, the interrogation of large patient datasets revealed that in the context of the tumour microenvironment, YAP function is more complex.,In the absence of fibroblasts, YAP/PAX3-mediated transcription prevails, but in the presence of fibroblasts tumour growth factor-β suppresses YAP/PAX3-mediated MITF expression and induces YAP/TEAD/SMAD-driven transcription and a de-differentiated phenotype.,Intriguingly, while high collagen expression is correlated with poorer patient survival, the worst prognosis is seen in patients with high collagen expression, who also express MITF target genes such as the differentiation markers TRPM1, TYR and TYRP1, as well as CDK4.,In summary, we reveal a distinct lineage-specific route of YAP signalling that contributes to the regulation of melanoma pigmentation and uncovers a set of potential biomarkers predictive for poor survival.

Cutaneous Malignant Melanoma is an aggressive form of skin cancer, arising in cutaneous melanocytes.,The transcription factor PAX3 regulates melanocyte specification from neural crest cells during development but expression in differentiated melanocytes is uncertain.,By contrast it is frequently found in melanomas and naevi and is a marker for melanoma staging and detection.,In this study we analysed the expression of PAX3 across the spectrum of melanocytic cells, from normal melanocytes to cells of benign and malignant lesions to better assess its function in these various tissues.,Pax3 and PAX3 (italicized) refer to the mouse and human gene, respectively; whereas Pax3 and PAX3 (non-italicized) refer to the corresponding mouse and human protein.,PAX3 expression was analysed by immunohistochemistry and qRT-PCR.,Immunofluorescence was used for co-expression with differentiation, migration and survival markers.,As expected PAX3 expression was observed in naevi and melanoma cells.,It was also found in melanocytes of normal skin where it co-expressed with melanocyte markers, MITF and MLANA.,Co-expression with its downstream target, antiapoptotic factor BCL2L1 confirms PAX3 as a cell survival regulator.,PAX3 was also co-expressed with melanoma cell migration marker MCAM in dermal naevi and melanoma cell nests, but this downstream target of PAX3 was not present in normal epidermal melanocytes, suggesting differential roles for PAX3 in normal epidermal melanocytes and melanoma cells.,Most interestingly, a proportion of PAX3-positive epidermal melanocytes in normal skin show HES1 and Ki67 co-expression, indicating their less differentiated proliferative phenotype.,Our results suggest that a previously identified role for PAX3, that of regulator of an undifferentiated plastic state, may operate in melanocytes of normal skin.,This role, possibly required for cellular response to environmental stimuli, may contribute to formation and development of melanocytic lesions in which PAX3 expression is prominent.