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Randomized trials evaluating programmed cell death protein 1 (PD-1) inhibitors in metastatic melanoma either permitted treatment for 2 years (pembrolizumab) or more (nivolumab).,The optimal duration of therapy is currently unknown due to limited data, and shorter therapies may be effective.,Data of patients with metastatic cutaneous melanoma treated with single-agent PD-1 inhibitors at Huntsman Cancer Institute from January 1, 2015, to December 31, 2018, was reviewed to identify a continuous series of patients who made the joint decision with their provider to electively discontinue therapy at 1 year (>6 months and <18 months) in the setting of ongoing treatment response or disease stability.,Patients were excluded if they received PD-1 inhibitors with other systemic therapy, had prior exposure to PD-1 therapy, or discontinued treatment due to disease progression or immune-related adverse event.,Best objective response (BOR) per RECIST V.1.1 at treatment discontinuation, progression-free survival (PFS), and retreatment characteristics was analyzed.,Of 480 patients who received PD-1 inhibitors, 52 met the inclusion criteria.,The median treatment duration from first to the last dose was 11.1 months (95% CI 10.5 to 11.4).,BOR was complete response in 13 (25%), partial response in 28 (53.8%), and stable disease in 11 (21.2%) patients.,After a median follow-up of 20.5 months (range 3-49.2) from treatment discontinuation, 39 (75%) patients remained without disease progression, while 13 (25%) had progression (median PFS 3.9 months; range 0.7-30.9).,On multivariable analysis, younger age, history of brain metastasis, and higher lactate dehydrogenase at the time of anti-PD-1 discontinuation were associated with recurrence.,Patients with recurrent melanoma were managed with localized treatment, anti-PD-1 therapies, and BRAF-MEK inhibitors.,All patients except one were alive at data cutoff.,In this large real-world, observational cohort study, the majority of patients with metastatic melanoma after 1 year of anti-PD-1 therapy remained without progression on long-term follow-up.,The risk of disease progression even in patients with residual disease on imaging was low.,After prospective validation, elective PD-1 discontinuation at 1 year may reduce financial and immunotherapy-related toxicity without sacrificing outcomes.

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

Cancer cell phenotype largely depends on oxygen availability.,The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue.,Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2).,A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines.,Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner.,Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells.,Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner.,In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

Outcomes of melanoma patient treatment remain unsatisfactory despite accessibility of oncoprotein-targeting drugs and immunotherapy.,Here, we reported that 17-aminogeldanamycin more potently activated caspase-3/7 in BRAFV600E melanoma cells than geldanamycin, another inhibitor of heat shock protein 90 (HSP90). 17-aminogeldanamycin alleviated self-triggered compensatory increase in HSP70 mRNA level and induced endoplasmic reticulum (ER) stress, which was followed by selective diminution of cytoprotective IRE1α-XBP1s pathway activity of unfolded protein response (UPR), inhibition of ERK1/2 activity and induction of apoptosis.,Concomitantly, ATF6/p50 level and expression of PERK-dependent genes, CHOP and BIM, remained unaltered.,This might result from an inframe deletion in EIF2AK3 leading to a PERKL21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRASQ61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1P187S).,In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAFV600E) in induction of apoptosis in melanoma cell lines as evidenced by in-cell caspase-3/7 activation and PARP cleavage that occurred earlier compared with either drug used alone.,As trametinib and vemurafenib did not significantly affect HSP70 and GRP78 transcript levels, cooperation of MEK/BRAFV600E inhibitors and 17-aminogeldanamycin might result from a concurrent inhibition of the RAS/RAF/MEK/ERK cascade and IRE1α-dependent signaling, and cell-intrinsic ER homeostasis can determine the extent of the drug cooperation.,Our study indicates that 17-aminogeldanamycin takes several advantages compared with other HSP90-targeting compounds, and can complement activity of BRAF/MEK inhibitors in melanoma cells of different genetic subtypes.,The online version of this article (10.1007/s10495-019-01542-y) contains supplementary material, which is available to authorized users.

Immunotherapy can be used for cutaneous, mucosal, uveal and conjunctival melanoma.,Nevertheless, we cannot expect the same benefit from checkpoint inhibitors for all the types of melanoma.,The different biological features can explain the variable efficacy.,The main results obtained with immune checkpoint inhibitors in the various types of melanoma were reviewed.

Ipilimumab, a fully human monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4, has demonstrated an improvement in overall survival in two phase III trials of patients with advanced melanoma.,The primary objective of the current trial was to prospectively explore candidate biomarkers from the tumor microenvironment for associations with clinical response to ipilimumab.,In this randomized, double-blind, phase II biomarker study (ClinicalTrials.gov NCT00261365), 82 pretreated or treatment-naïve patients with unresectable stage III/IV melanoma were induced with 3 or 10 mg/kg ipilimumab every 3 weeks for 4 doses; at Week 24, patients could receive maintenance doses every 12 weeks.,Efficacy was evaluated per modified World Health Organization response criteria and safety was assessed continuously.,Candidate biomarkers were evaluated in tumor biopsies collected pretreatment and 24 to 72 hours after the second ipilimumab dose.,Polymorphisms in immune-related genes were also evaluated.,Objective response rate, response patterns, and safety were consistent with previous trials of ipilimumab in melanoma.,No associations between genetic polymorphisms and clinical activity were observed.,Immunohistochemistry and histology on tumor biopsies revealed significant associations between clinical activity and high baseline expression of FoxP3 (p = 0.014) and indoleamine 2,3-dioxygenase (p = 0.012), and between clinical activity and increase in tumor-infiltrating lymphocytes (TILs) between baseline and 3 weeks after start of treatment (p = 0.005).,Microarray analysis of mRNA from tumor samples taken pretreatment and post-treatment demonstrated significant increases in expression of several immune-related genes, and decreases in expression of genes implicated in cancer and melanoma.,Baseline expression of immune-related tumor biomarkers and a post-treatment increase in TILs may be positively associated with ipilimumab clinical activity.,The observed pharmacodynamic changes in gene expression warrant further analysis to determine whether treatment-emergent changes in gene expression may be associated with clinical efficacy.,Further studies are required to determine the predictive value of these and other potential biomarkers associated with clinical response to ipilimumab.

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.

Immune checkpoint blockade has shown significant promise as an anti-cancer treatment, yet the determinants of response are not completely understood.,Here, we show that somatic mutations in SERPINB3 and SERPINB4 are associated with survival following anti-CTLA4 immunotherapy in two independent cohorts of melanoma patients (n=174).,Interestingly, serpins are homologues of the well-known ovalbumin antigen and are associated with autoimmunity.,Our findings have implications for the personalization of immunotherapy.

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months.,Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue.,Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment.,Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells.,We also observed these findings in clinical melanoma specimens.,Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling.,Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence.,Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation.,Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib.,We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape.,Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.

Hyaluronan is an extracellular matrix glycosaminoglycan involved in invasion, proliferation and metastasis of various types of carcinomas.,In many cancers, aberrant hyaluronan expression implicates disease progression and metastatic potential.,Melanoma is an aggressive skin cancer.,The role of hyaluronan in melanoma progression including benign nevi and lymph node metastases has not been investigated earlier, nor the details of its synthesis and degradation.,The melanocytic and dysplastic nevi, in situ melanomas, superficially and deeply invasive melanomas and their lymph node metastases were analysed immunohistochemically for the amount of hyaluronan, its cell surface receptor CD44, hyaluronan synthases 1-3 and hyaluronidases 1-2.,Hyaluronan content of tumoral cells in deeply invasive melanomas and metastatic lesions was clearly reduced compared to superficial melanomas or benign lesions.,Furthermore, hyaluronan content in the stromal cells of benign nevi was higher than in the premalignant or malignant tumors.,The immunopositivity of hyaluronidase 2 was significantly increased in the premalignant and malignant lesions indicating its specific role in the degradation of hyaluronan during tumor progression.,Similarly, the expression of hyaluronan synthases 1-2 and CD44 receptor was decreased in the metastases compared to the primary melanomas.,These findings suggest that the reciprocal relationship between the degrading and synthesizing enzymes account for the alterations in hyaluronan content during the growth of melanoma.,These results provide new information about hyaluronan metabolism in benign, premalignant and malignant melanocytic tumors of the skin.

Optimal treatment of brain metastases is often hindered by limitations in diagnostic capabilities.,To meet this challenge, here we profile DNA methylomes of the three most frequent types of brain metastases: melanoma, breast, and lung cancers (n = 96).,Using supervised machine learning and integration of DNA methylomes from normal, primary, and metastatic tumor specimens (n = 1860), we unravel epigenetic signatures specific to each type of metastatic brain tumor and constructed a three-step DNA methylation-based classifier (BrainMETH) that categorizes brain metastases according to the tissue of origin and therapeutically relevant subtypes.,BrainMETH predictions are supported by routine histopathologic evaluation.,We further characterize and validate the most predictive genomic regions in a large cohort of brain tumors (n = 165) using quantitative-methylation-specific PCR.,Our study highlights the importance of brain tumor-defining epigenetic alterations, which can be utilized to further develop DNA methylation profiling as a critical tool in the histomolecular stratification of patients with brain metastases.,The treatment of brain metastases is often limited by the ability to diagnose their origins.,Here the authors generate DNA methylomes from the three most frequent types of brain metastases, identify epigenetic signatures specific to each type of metastasis and construct a DNA methylation-based classifier (BrainMETH) to advance brain metastasis diagnosis.

Until 50% of patients with renal cancer or melanoma, develop brain metastases during the course of their disease.,Stereotactic radiotherapy has become a standard of care for patients with a limited number of brain metastases.,Given the radioresistant nature of melanoma and renal cancer, optimization of the fractionation of stereotactic radiotherapy is needed.,The purpose of this retrospective study was to elucidate if hypofractionated stereotactic radiotherapy (HFSRT) impacts local control of brain metastases from radioresistant tumors such as melanoma and renal cancer, in comparison with radiosurgery (SRS).,Between 2012 and 2016, 193 metastases, smaller than 3 cm, from patients suffering from radioresistant primaries (melanoma and renal cancer) were treated with HFSRT or SRS.,The primary outcome was local progression free survival (LPFS) at 6, 12 and 18 months.,Overall survival (OS) and cerebral progression free survival (CPFS) were secondary outcomes, and were evaluated per patient.,Objective response rate and radionecrosis incidence were also reported.,The statistical analysis included a supplementary propensity score analysis to deal with bias induced by non-randomized data.,After a median follow-up of 7.4 months, LPFS rates at 6, 12 and 18 months for the whole population were 83, 74 and 70%, respectively.,With respect to fractionation, LPFS rates at 6, 12 and 18 months were 89, 79 and 73% for the SRS group and 80, 72 and 68% for the HFSRT group.,The fractionation schedule was not statistically associated with LPFS (HR = 1.39, CI95% [0.65-2.96], p = 0.38).,Time from planning MRI to first irradiation session longer than 14 days was associated with a poorer local control rate.,Over this time, LPFS at 12 months was reduced from 86 to 70% (p = 0.009).,Radionecrosis occurred in 7.1% for HFSRT treated metastases to 9.6% to SRS treated metastases, without any difference according to fractionation (p = 0.55).,The median OS was 9.6 months.,Six, 12 and 18 months CPFS rates were 54, 24 and 17%, respectively.,Fractionation does not decrease LPFS.,Even for small radioresistant brain metastases (< 3 cm), HFSRT, with 3 or 6 fractions, leads to an excellent local control rate of 72% at 1 year with a rate of 7.1% of radionecrosis.,HFSRT is a safe and efficient alternative treatment to SRS.,The online version of this article (10.1186/s13014-018-1083-1) contains supplementary material, which is available to authorized users.

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

Melanoma is the most aggressive and lethal form of skin cancer.,Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.,Using the combinatorial Gal4 -UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter.,Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth.,By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish.,The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line.,Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors.,In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.,This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors.,Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

Cutaneous SCC (cSCC) is the most frequent skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors.,Here we use massively parallel exome and targeted level sequencing 132 sporadic cSCC, 39 squamoproliferative lesions and cSCC arising in patients receiving the BRAF inhibitor vemurafenib, as well as 10 normal skin samples to identify significant NOTCH1 mutation as an early event in squamous cell carcinogenesis.,Bisected vemurafenib induced lesions revealed surprising heterogeneity with different activating HRAS and NOTCH1 mutations identified in two halves of the same cSCC suggesting polyclonal origin.,Immunohistochemical analysis using an antibody specific to nuclear NOTCH1 correlates with mutation status in sporadic cSCC and regions of NOTCH1 loss or down-regulation are frequently observed in normal looking skin.,Our data indicate that NOTCH1 acts as a gatekeeper in human cSCC.

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells.,The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair.,However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known.,We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin.,The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6).,Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF.,KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen.,Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1).,KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3).,Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2.,Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression.,These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes.

Melanoma is an extremely aggressive tumor and is considered to be an extremely immunogenic tumor because compared to other cancers it usually presents a well-expressed lymphoid infiltration.,The aim of this paper is to perform a multidisciplinary comprehensive review of the evidence available about the combination of radiotherapy and immunotherapy for melanoma.,Radiation, in fact, can increase tumor antigens visibility and promote priming of T cells but can also exert immunosuppressive action on tumor microenvironment.,Combining radiotherapy with immunotherapy provides an opportunity to increase immunostimulatory potential of radiation.,We therefore provide the latest clinical evidence about radiobiological rationale, radiotherapy techniques, timing, and role both in advanced and systemic disease (with a special focus on ocular melanoma and brain, liver, and bone metastases) with a particular attention also in geriatric patients.,The combination of immunotherapy and radiotherapy seems to be a safe therapeutic option, supported by a clear biological rationale, even though the available data confirm that radiotherapy is employed more for metastatic than for non-metastatic disease.,Such a combination shows promising results in terms of survival outcomes; however, further studies, hopefully prospective, are needed to confirm such evidence.

Growing evidence has revealed that abnormal alternative splicing (AS) events are closely related to carcinogenic processes.,However, the comprehensive study on the prognostic value of splicing events involved in uveal melanoma (UM) is still lacking.,Therefore, splicing data of 80 UM patients were obtained from the Cancer Genome Atlas (TCGA) SpliceSeq and RNA sequence data of UM and patient clinical features were downloaded from the Cancer Genome Atlas (TCGA) database to identify survival related splicing events in UM.,As a result, a total of 37996 AS events of 17911 genes in UM were detected, among which 5299 AS events of 3529 genes were significantly associated with UM patients’ survival.,Functional enrichment analysis revealed that this survival related splicing genes are corelated with mRNA catabolic process and ribosome pathway.,Based on survival related splicing events, seven types of prognostic markers and the final overall prognostic signature could independently predict the overall survival of UM patients.,Finally, an 11 spliced gene was identified in the final signature.,On the basis of these 11 genes, we constructed a Support Vector Machine (SVM) classifier and evaluated it with leave-one-out cross-validation.,The results showed that the 11 genes could determine short- and long-term survival with a predicted accuracy of 97.5%.,Besides, the splicing factors and alternative splicing events correlation network was constructed to serve as therapeutic targets for UM treatment.,Thus, our study depicts a comprehensive landscape of alternative splicing events in the prognosis of UM.,The correlation network and associated pathways would provide additional potential targets for therapy and prognosis.

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 remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities.,Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses.,Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas.,Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology.,Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype.,Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed.,Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas.,Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

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.

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.

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.

A cardinal feature of malignant melanoma is its metastatic propensity.,An incomplete view of the genetic events driving metastatic progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients.,In this study, we conducted global genomic characterization of primary and metastatic melanomas to examine the genomic landscape associated with metastatic progression.,In addition to uncovering three genomic subclasses of metastastic melanomas, we delineated 39 focal and recurrent regions of amplification and deletions, many of which encompassed resident genes that have not been implicated in cancer or metastasis.,To identify progression-associated metastasis gene candidates, we applied a statistical approach, Integrative Genome Comparison (IGC), to define 32 genomic regions of interest that were significantly altered in metastatic relative to primary melanomas, encompassing 30 resident genes with statistically significant expression deregulation.,Functional assays on a subset of these candidates, including MET, ASPM, AKAP9, IMP3, PRKCA, RPA3, and SCAP2, validated their pro-invasion activities in human melanoma cells.,Validity of the IGC approach was further reinforced by tissue microarray analysis of Survivin showing significant increased protein expression in thick versus thin primary cutaneous melanomas, and a progression correlation with lymph node metastases.,Together, these functional validation results and correlative analysis of human tissues support the thesis that integrated genomic and pathological analyses of staged melanomas provide a productive entry point for discovery of melanoma metastases genes.

We appraise the role of screening for distress as part of health psychology assessment of patients newly diagnosed with cancer.,We reviewed records of consecutive patients who accepted a health psychologist’s assessment over 4 years, examining convergence and divergence of the result of screening (whether patients reached threshold as ‘cases’) with the psychologist’s clinical judgment of need for intervention.,Of 261 patients, 88 (33.7%) were ‘cases’.,Of these, need for psychological intervention was identified in 70 (79.5%).,Of the 173 (66.3%) ‘non-cases’, need was identified in 59 (34.1%).,Examination of cases where the psychologist’s judgment diverged from screening showed that ‘caseness’ can arise from distress that patients can manage themselves and, conversely, that psychological needs arise in the absence of overt distress.,Formal screening may not identify need for psychological intervention.,The psychologist’s role is to make expert judgments of patients’ current and future needs.,Dialogue with patients should be the vehicle for assessment.

Metastatic uveal melanoma is a deadly disease with no proven standard of care.,Here we present a metastatic uveal melanoma patient with an exceptional high sensitivity to a PD-1 inhibitor associated with outlier CpG>TpG mutation burden, MBD4 germline deleterious mutation, and somatic MBD4 inactivation in the tumor.,We identify additional tumors in The Cancer Genome Atlas (TCGA) cohorts with similar hypermutator profiles in patients carrying germline deleterious MBD4 mutations and somatic loss of heterozygosity.,This MBD4-related hypermutator phenotype may explain unexpected responses to immune checkpoint inhibitors.,Hypermutated tumors respond more favorably to checkpoint inhibitor-based immune therapy.,Here, the authors describe a new hypermutated phenotype due to germline mutations and subsequent somatic loss of heterozygosity of MBD4, and a dramatic response to the PD-1 inhibitor pembrolizumab in a patient with a MBD4-inactivated hypermutated uveal melanoma.

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.

Recent studies have shown that immunotherapies and molecular targeted therapies are effective for advanced melanoma.,Non-antigen-specific immunotherapies such as immunocheckpoint blockades have been shown to be effective in the treatment of advanced melanoma.,However, the response rates remain low.,To improve their efficacy, they should be combined with antigen-specific immunotherapy.,Elevated expression of the transcription factor, Forkhead box M1 (FOXM1), has been reported in various human cancers, and it has been shown to have potential as a target for immunotherapy.,The purpose of this study was to investigate the FOXM1 expression in human melanoma samples and cell lines, to evaluate the relationship between the FOXM1 expression and the clinical features of melanoma patients and to investigate the association between the FOXM1 and MAPK and PI3K/AKT pathways in melanoma cell lines.,We conducted the quantitative reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines, and investigated melanoma and nevus tissue samples by qRT-PCR and immunohistochemistry.,We performed MEK siRNA and PI3K/AKT inhibitor studies and FOXM1 siRNA studies in melanoma cell lines.,We found that FOXM1 was expressed in all of the melanoma cell lines, and was expressed in 49% of primary melanomas, 67% of metastatic melanomas and 10% of nevi by performing immunohistochemical staining.,Metastatic melanoma samples exhibited significantly higher mRNA levels of FOXM1 (p = 0.004).,Primary melanomas thicker than 2 mm were also more likely to express FOXM1.,Patients whose primary melanoma expressed FOXM1 had a significantly poorer overall survival compared to patients without FOXM1 expression (p = 0.024).,Downregulation of FOXM1 by siRNA significantly inhibited the proliferation of melanoma cells, and blockade of the MAPK and PI3K/AKT pathways decreased the FOXM1 expression in melanoma cell lines.,In conclusion, FOXM1 is considered to be a new therapeutic target for melanoma.

The incidence of cutaneous melanoma (CM) has increased in the past few decades.,The biology of melanoma is characterized by a complex interaction between genetic, environmental and phenotypic factors.,A greater understanding of the molecular mechanisms that promote melanoma cell growth and dissemination is crucial to improve diagnosis, prognostication, and treatment of CM.,Both small and long non‐coding RNAs (lncRNAs) have been identified to play a role in melanoma biology; microRNA and lncRNA expression is altered in transformed melanocytes and this in turn has functional effects on cell proliferation, apoptosis, invasion, metastasis, and immune response.,Moreover, specific dysregulated ncRNAs were shown to have a diagnostic or prognostic role in melanoma and to drive the establishment of drug resistance.,Here, we review the current literature on small and lncRNAs with a role in melanoma, with the aim of putting into some order this complex jigsaw puzzle.

Response to targeted therapies varies significantly despite shared oncogenic mutations.,Nowhere is this more apparent than in BRAF(V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace.,Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs) though the underlying mechanisms have been largely uncharacterized.,Here, we found that EGFR induced vemurafenib resistance is ligand dependent.,We then employed whole-genome expression analysis and discovererd that vemurafenib resistance correlated with the loss of MITF, along with its melanocyte lineage program, and with the activation of EGFR signaling.,An inverse relationship between MITF, vemurafenib resistance and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients’ tumor specimens.,Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype.,In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors.,These findings indicate that an “autocrine drug resistance loop” is suppressed by melanocyte lineage signal(s), such as MITF.,This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition.

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) homeostasis.,Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities.,Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas.,Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases.,In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis.,The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas.,With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases.,Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity.,Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes.,Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival.,In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.

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.

Ipilimumab (Ipi) improves the survival of advanced melanoma patients with an incremental long-term benefit in 10-15 % of patients.,A tumor signature that correlates with this survival benefit could help optimizing individualized treatment strategies.,Freshly frozen melanoma metastases were collected from patients treated with either Ipi alone (n: 7) or Ipi combined with a dendritic cell vaccine (TriMixDC-MEL) (n: 11).,Samples were profiled by immunohistochemistry (IHC), whole transcriptome (RNA-seq) and methyl-DNA sequencing (MBD-seq).,Patients were divided in two groups according to clinical evolution: durable benefit (DB; 5 patients) and no clinical benefit (NB; 13 patients). 20 metastases were profiled by IHC and 12 were profiled by RNA- and MBD-seq. 325 genes were identified as differentially expressed between DB and NB.,Many of these genes reflected a humoral and cellular immune response.,MBD-seq revealed differences between DB and NB patients in the methylation of genes linked to nervous system development and neuron differentiation.,DB tumors were more infiltrated by CD8+ and PD-L1+ cells than NB tumors.,B cells (CD20+) and macrophages (CD163+) co-localized with T cells.,Focal loss of HLA class I and TAP-1 expression was observed in several NB samples.,Combined analyses of melanoma metastases with IHC, gene expression and methylation profiling can potentially identify durable responders to Ipi-based immunotherapy.,The online version of this article (doi:10.1186/s12967-016-0990-x) contains supplementary material, which is available to authorized users.

Approximately 75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known.,We established a melanoma cell line from a tumor with none of the common driver mutations.,This cell line demonstrated a signaling profile similar to BRAF-mutants, but lacked sensitivity to the BRAF inhibitor vemurafenib.,RNA-seq mutation data implicated CRAF R391W as the alternative driver mutation of this melanoma.,CRAF R391W was homozygous and over expressed.,These melanoma cells were highly sensitive to CRAF, but not BRAF knockdown.,In reconstitution experiments, CRAF R391W, but not CRAF WT, transformed NIH3T3 cells in soft-agar colony formation assays, increased kinase activity in vitro, induced MAP kinase signaling and conferred vemurafenib resistance.,MAP kinase inducing activity was dependent on CRAF dimerization.,Thus, CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild type melanomas.

The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation.,It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states.,Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions.,Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model.,The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas.,Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas.,Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

In this study, Falletta et al. show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,Their results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.,The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance.,Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown.,In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance.,However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood.,Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors.,However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion.,Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance.,Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated.,We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures.,We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression.,Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally.,These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures.,We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma.,Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.,A significant proportion of patients develop innate or acquired resistance to immune checkpoint inhibitors.,Here, the authors show that resistance to anti-PD-1 blockade is associated with TGF-beta driven major histocompatibility complex I (MHCI) down-regulation and a de-differentiated phenotype in melanoma patients.

PD-1 blocking agents, such as nivolumab, have demonstrated clear anti-tumor effects and clinical benefits in a subset of patients with advanced malignancies.,Nonetheless, more efforts are needed to identify reliable biomarkers for outcome, to correctly select patients who will benefit from anti-PD-1 treatment.,The aim of this study was to investigate the role of peripheral CD8+T cells expressing CD73, involved in the generation of the immune suppressive molecule adenosine, in predicting outcome after nivolumab treatment in advanced melanoma patients.,PBMCs from 100 melanoma patients treated with nivolumab were collected at National Cancer Institute “G.,Pascale” of Naples.,Frequencies of CD8+ lymphocytes phenotypes were assessed by flow cytometry at baseline before nivolumab treatment, along with clinical characteristics and blood count parameters.,Healthy controls (n = 20) were also analysed.,Percentages of baseline T cells expressing PD-1 and CD73 were correlated with outcome after nivolumab treatment.,Melanoma patients presented a lower frequency of total circulating CD8+ lymphocytes than control subjects (p = 0.008).,Patients with low baseline percentage of circulating CD8+PD-1+CD73+ lymphocytes (< 2.3%) had better survival (22.4 months vs 6.9 months, p = 0.001).,Patients (39%) with clinical benefit from nivolumab therapy presented a significantly lower frequency of circulating CD8+PD-1+CD73+ lymphocytes than patients who progressed to nivolumab treatment (p = 0.02).,Our observations suggest that baseline CD73 expression on circulating CD8+PD-1+ lymphocytes appear a promising biomarker of response to anti-PD-1 treatment in melanoma patients.,Further investigations are needed for validation and for clarifying its role as prognostic or predictive marker.

Several melanoma-specific dermoscopic features have been described, some of which have been reported as indicative of in situ or invasive melanomas.,To assess the usefulness of these features to differentiate between these 2 categories, a retrospective, single-centre investigation was conducted.,Dermoscopic images of melanomas were reviewed by 7 independent dermatologists.,Fleiss’ kappa (κ) was used to analyse interobserver agreement of predefined features.,Logistic regression and odds ratios were used to assess whether specific features correlated with melanoma in situ or invasive melanoma.,Overall, 182 melanomas (101 melanoma in situ and 81 invasive melanomas) were included.,The interobserver agreement for melanoma-specific features ranged from slight to substantial.,Atypical blue-white structures (κ=0.62, 95% confidence interval 0.59-0.65) and shiny white lines (κ=0.61, 95% confidence interval 0.58-0.64) had a substantial interobserver agreement.,These 2 features were also indicative of invasive melanomas >1.0 mm in Breslow thickness.,Furthermore, regression/peppering correlated with thin invasive melanomas.,The overall agreement for classification of the lesions as invasive or melanoma in situ was moderate (κ=0.52, 95% confidence interval 0.49-0.56).

Objective To quantify the accuracy and reproducibility of pathologists’ diagnoses of melanocytic skin lesions.,Design Observer accuracy and reproducibility study.,Setting 10 US states.,Participants Skin biopsy cases (n=240), grouped into sets of 36 or 48.,Pathologists from 10 US states were randomized to independently interpret the same set on two occasions (phases 1 and 2), at least eight months apart.,Main outcome measures Pathologists’ interpretations were condensed into five classes: I (eg, nevus or mild atypia); II (eg, moderate atypia); III (eg, severe atypia or melanoma in situ); IV (eg, pathologic stage T1a (pT1a) early invasive melanoma); and V (eg, ≥pT1b invasive melanoma).,Reproducibility was assessed by intraobserver and interobserver concordance rates, and accuracy by concordance with three reference diagnoses.,Results In phase 1, 187 pathologists completed 8976 independent case interpretations resulting in an average of 10 (SD 4) different diagnostic terms applied to each case.,Among pathologists interpreting the same cases in both phases, when pathologists diagnosed a case as class I or class V during phase 1, they gave the same diagnosis in phase 2 for the majority of cases (class I 76.7%; class V 82.6%).,However, the intraobserver reproducibility was lower for cases interpreted as class II (35.2%), class III (59.5%), and class IV (63.2%).,Average interobserver concordance rates were lower, but with similar trends.,Accuracy using a consensus diagnosis of experienced pathologists as reference varied by class: I, 92% (95% confidence interval 90% to 94%); II, 25% (22% to 28%); III, 40% (37% to 44%); IV, 43% (39% to 46%); and V, 72% (69% to 75%).,It is estimated that at a population level, 82.8% (81.0% to 84.5%) of melanocytic skin biopsy diagnoses would have their diagnosis verified if reviewed by a consensus reference panel of experienced pathologists, with 8.0% (6.2% to 9.9%) of cases overinterpreted by the initial pathologist and 9.2% (8.8% to 9.6%) underinterpreted.,Conclusion Diagnoses spanning moderately dysplastic nevi to early stage invasive melanoma were neither reproducible nor accurate in this large study of pathologists in the USA.,Efforts to improve clinical practice should include using a standardized classification system, acknowledging uncertainty in pathology reports, and developing tools such as molecular markers to support pathologists’ visual assessments.

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies.,Mechanisms underlying tumor cell plasticity remain poorly understood.,SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas.,Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors.,We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells.,Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties.,Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo.,These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance.,Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells.,Mechanisms underlying tumor cell plasticity remain poorly understood.,Here, the authors show the presence of a dormant-invasive SOX10- subpopulation in cutaneous melanoma that can be targeted by cIAP1/2 inhibitors.

DNA methylation at CpG dinucleotides is modified in tumorigenesis with potential impact on transcriptional activity.,We used the Illumina 450 K platform to evaluate DNA methylation patterns of 50 metastatic melanoma tumors, with matched gene expression data.,We identified three different methylation groups and validated the groups in independent data from The Cancer Genome Atlas.,One group displayed hypermethylation of a developmental promoter set, genome-wide demethylation, increased proliferation and activity of the SWI/SNF complex.,A second group had a methylation pattern resembling stromal and leukocyte cells, over-expressed an immune signature and had improved survival rates in metastatic tumors (p < 0.05).,A third group had intermediate methylation levels and expressed both proliferative and immune signatures.,The methylation groups corresponded to some degree with previously identified gene expression phenotypes.,Melanoma consists of divergent methylation groups that are distinguished by promoter methylation, proliferation and content of immunological cells.,The online version of this article (doi:10.1186/s12920-015-0147-4) contains supplementary material, which is available to authorized users.

Melanoma is known as the most aggressive and lethal type of cutaneous cancer due to its rapid development of drug resistance to chemotherapy drugs.,In our study, we conducted a variety of studies, including quantitative PCR, Western blot, and autophagy and apoptosis assays to investigate the involvement of miR-26a and HMGB1 in modulation of dabrafenib sensitivity in human melanoma cell lines.,Our studies revealed that the expressions of miR-26a and HMGB1 were altered in two melanoma cell lines after dabrafenib treatment.,Additionally, dabrafenib caused autophagy in melanoma and this autophagic process was regulated by miR-26a via modifying HMGB1 expression.,Furthermore, silencing HMGB1-inhibited autophagy induced by dabrafenib in melanoma cells.,Last, we verified that treatment with a miR-26a mimic and HMGB1 shRNA could increase the efficacy of dabrafenib in melanoma cells.,Taken together, we showed that miR-26a is involved in the regulation of dabrafenib efficacy via a HMGB1-dependent autophagy pathway in melanoma cells.,These results shed light on a novel treatment for conventional dabrafenib-based chemotherapy for melanoma.

Autophagy has been linked with melanoma risk and survival, but no polymorphisms in autophagy‐related (ATG) genes have been investigated in relation to melanoma progression.,We examined five single‐nucleotide polymorphisms (SNPs) in three ATG genes (ATG5;ATG10; and ATG16L) with known or suspected impact on autophagic flux in an international population‐based case-control study of melanoma.,DNA from 911 melanoma patients was genotyped.,An association was identified between (GG) (rs2241880) and earlier stage at diagnosis (OR 0.47; 95% Confidence Intervals (CI) = 0.27-0.81, P = 0.02) and a decrease in Breslow thickness (P = 0.03).,The ATG16L heterozygous genotype (AG) (rs2241880) was associated with younger age at diagnosis (P = 0.02).,Two SNPs in ATG5 were found to be associated with increased stage (rs2245214 CG, OR 1.47; 95% CI = 1.11-1.94, P = 0.03; rs510432 CC, OR 1.84; 95% CI = 1.12-3.02, P = 0.05).,Finally, we identified inverse associations between ATG5 (GG rs2245214) and melanomas on the scalp or neck (OR 0.20, 95% CI = 0.05-0.86, P = 0.03); ATG10 (CC) (rs1864182) and brisk tumor infiltrating lymphocytes (TILs) (OR 0.42; 95% CI = 0.21-0.88, P = 0.02), and ATG5 (CC) (rs510432) with nonbrisk TILs (OR 0.55; 95% CI = 0.34-0.87, P = 0.01).,Our data suggest that ATG SNPs might be differentially associated with specific host and tumor characteristics including age at diagnosis, TILs, and stage.,These associations may be critical to understanding the role of autophagy in cancer, and further investigation will help characterize the contribution of these variants to melanoma progression.

Malignant melanoma is a highly metastatic type of cancer, which arises frequently from transformed pigment cells and melanocytes as a result of long-term UV radiation exposure.,In recent years, the incidence of newly diagnosed melanoma patients reached 5% of all cancer cases.,Despite the development of novel targeted therapies directed against melanoma-specific markers, patients’ response to treatment is often weak or short-term due to a rapid acquisition of drug resistance.,Among the factors affecting therapy effectiveness, elements of the tumor microenvironment play a major role.,Melanoma niche encompasses adjacent cells, such as keratinocytes, cancer-associated fibroblasts (CAFs), adipocytes, and immune cells, as well as components of the extracellular matrix and tumor-specific physicochemical properties.,In this review, we summarize the current knowledge concerning the influence of cancer-associated cells (keratinocytes, CAFs, adipocytes) on the process of melanomagenesis, tumor progression, invasiveness, and the emergence of drug resistance in melanoma.,We also address how melanoma can alter the differentiation and activation status of cells present in the tumor microenvironment.,Understanding these complex interactions between malignant and cancer-associated cells could improve the development of effective antitumor therapeutic strategies.

Apoptosis-associated speck-like protein containing a CARD (ASC) was originally named because it triggered apoptosis in certain tumors.,More recently, however, ASC was found to be a central adaptor protein of inflammasome which mediates the secretion of pro-tumorigenic inflammation.,Here we examined the role of ASC in tumorigenesis of human melanoma.,Compared with primary melanoma, ASC protein expression was generally downregulated in metastatic melanoma.,While overexpressing ASC in metastatic melanoma showed no effects on cell viability, silencing ASC with short hairpin RNA induced G1 cell cycle arrest, reduced cell viability and suppressed tumorigenesis in metastatic melanoma.,On the other hand, silencing ASC in primary melanoma reduced cell death, increased cell viability and enhanced tumorigenesis.,In primary and metastatic melanoma cells, ASC knockdown inhibited inflammasome-mediated caspase-1 activity and IL-1β secretion.,However, phosphorylated IKKα/β expression and NF-κB activity were suppressed in metastatic melanoma and enhanced in primary melanoma after ASC knockdown.,These findings suggest stage-dependent dual roles of ASC in tumorigenesis.,ASC expression in primary melanoma inhibits tumorigenesis, by reducing IKKα/β phosphorylation and inhibiting NF-κB activity.,In metastatic melanoma, on the other hand, this inhibitory effect is diminished, and ASC induces tumorigenic pathways through enhanced NF-κB activity and inflammasome-mediated IL-1β secretion.

Cerium (Ce) oxide nanoparticles (CNP; nanoceria) are reported to have cytotoxic effects on certain cancerous cell lines, while at the same concentration they show no cytotoxicity on normal (healthy) cells.,Redox-active CNP exhibit both selective prooxidative as well as antioxidative properties.,The former is proposed to be responsible for impairment of tumor growth and invasion and the latter for rescuing normal cells from reactive oxygen species (ROS)-induced damage.,Here we address possible underlying mechanisms of prooxidative effects of CNP in a metastatic human melanoma cell line.,Malignant melanoma is the most aggressive form of skin cancer, and once it becomes metastatic the prognosis is very poor.,We have shown earlier that CNP selectively kill A375 melanoma cells by increasing intracellular ROS levels, whose basic amount is significantly higher than in the normal (healthy) counterpart, the melanocytes.,Here we show that CNP initiate a mitochondrial increase of ROS levels accompanied by an increase in mitochondrial thiol oxidation.,Furthermore, we observed CNP-induced changes in mitochondrial bioenergetics, dynamics, and cristae morphology demonstrating mitochondrial dysfunction which finally led to tumor cell death.,CNP-induced cell death is abolished by administration of PEG-conjugated catalase.,Overall, we propose that cerium oxide nanoparticles mediate cell death via hydrogen peroxide production linked to mitochondrial dysfunction.

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.

Data on the KIT mutation rate in melanoma in the central European region is missing.,Accordingly, in a cohort of 79 BRAF/NRAS double wild type cutaneous melanoma and 17 mucosal melanoma KIT mutation was assessed by Sanger sequencing of exons 9,11,13,17 and 18.,In this cutaneous melanoma cohort KIT mutation frequency was found to be 34/79 (43.04%) with a significantly higher rate in acrolentiginous melanoma (ALM) as compared to UV-induced common variants (20/34, 58.8% versus 14/45, 31.1%, p = 0.014).,In the double wild type mucosal melanoma cohort the KIT mutation frequency was found to be comparable (41.2%).,The actual frequency of KIT mutation in the original 227 patient cutaneous melanoma cohort was 34/227, 14.9%.,Exon 11 was the most frequent mutation site (44.7%) followed by exon 9 (21.1%) equally characterizing UV-induced common histotypes and ALM tumors.,In mucosal melanoma exon 9 was the most frequently involved exon followed by exon 13 and 17.,KIT mutation hotspots were identified in exon 9 (c482/491/492), in exon 11 (c559,c572, c570), in exon 13 (c642), in exon 17 (c822) and in exon 18 (c853).,The relatively high KIT mutation rate in cutaneous melanoma in this central-European cohort justifies regular testing of this molecular target in this entity, not only in mucosal variants.

Melanoma is the deadliest form of skin cancer.,In the early stages, melanoma can be treated successfully with surgery alone and survival rates are high, but after metastasis survival rates drop significantly.,Therefore, early and correct diagnosis is key for ensuring patients have the best possible prognosis.,Melanoma misdiagnosis accounts for more pathology and dermatology malpractice claims than any cancer other than breast cancer, as an early misdiagnosis can significantly reduce a patient’s chances of survival.,As far as treatment for metastatic melanoma goes, there have been several new drugs developed over the last 10 years that have greatly improved the prognosis of patients with metastatic melanoma, however, a majority of patients do not show a lasting response to these treatments.,Thus, new biomarkers and drug targets are needed to improve the accuracy of melanoma diagnosis and treatment.,This article will discuss the major advancements of melanoma diagnosis and treatment from antiquity to the present day.

Combination therapy with BRAF and MEK inhibitors significantly improves survival in BRAF mutated melanoma patients but is unable to prevent disease recurrence due to the emergence of drug resistance.,Cancer stem cells (CSCs) have been involved in these long-term treatment failures.,We previously reported in lung cancer that CSCs maintenance is due to altered lipid metabolism and dependent upon Stearoyl-CoA-desaturase (SCD1)-mediated upregulation of YAP and TAZ.,On this ground, we investigated the role of SCD1 in melanoma CSCs.,SCD1 gene expression data of melanoma patients were downloaded from TCGA and correlated with disease progression by bioinformatics analysis and confirmed on patient’s tissues by qRT-PCR and IHC analyses.,The effects of combination of BRAF/MEKi and the SCD1 inhibitor MF-438 were monitored by spheroid-forming and proliferation assays on a panel of BRAF-mutated melanoma cell lines grown in 3D and 2D conditions, respectively.,SCD1, YAP/TAZ and stemness markers were evaluated in melanoma cells and tissues by qRT-PCR, WB and Immunofluorescence.,We first observed that SCD1 expression increases during melanoma progression.,BRAF-mutated melanoma 3D cultures enriched for CSCs overexpressed SCD1 and were more resistant than 2D differentiated cultures to BRAF and MEK inhibitors.,We next showed that exposure of BRAF-mutated melanoma cells to MAPK pathway inhibitors enhanced stemness features by upregulating the expression of YAP/TAZ and downstream genes but surprisingly not SCD1.,However, SCD1 pharmacological inhibition was able to downregulate YAP/TAZ and to revert at the same time CSC enrichment and resistance to MAPK inhibitors.,Our data underscore the role of SCD1 as prognostic marker in melanoma and promote the use of SCD1 inhibitors in combination with MAPK inhibitors for the control of drug resistance.,The online version of this article (10.1186/s13046-018-0989-7) contains supplementary material, which is available to authorized users.

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments.,The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells.,The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy.,LAN has shown to counteract drug tumor resistance.,We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects.,We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models.,We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments.,The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem.,LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.,This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging.,Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood.,The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers.,The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis.,The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches.,We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy.,We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

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.

Effective management of melanoma depends heavily on early diagnosis.,When detected in early non-metastatic stages, melanoma is almost 100% curable by surgical resection, however when detected in late metastatic stages III and IV, 5-year survival rates drop to ~50% and 10-25%, respectively, due to limited efficacy of current treatment options.,This presents a pressing need to identify biomarkers that can detect patients at high risk of recurrence and progression to metastatic disease, which will allow for early intervention and survival benefit.,Accumulating evidence over the past few decades has highlighted the potential use of circulating molecular biomarkers for melanoma diagnosis and prognosis, including lactate dehydrogenase (LDH), S100 calcium-binding protein B (S100B) and circulating tumor DNA (ctDNA) fragments.,Since 2010, circulating microRNAs (miRNAs) have been increasingly recognised as more robust non-invasive biomarkers for melanoma due to their structural stability under the harsh conditions of the blood and different conditions of sample processing and isolation.,Several pre-analytical and analytical variables challenge the accurate quantification of relative miRNA levels between serum samples or plasma samples, leading to conflicting findings between studies on circulating miRNA biomarkers for melanoma.,In this review, we provide a critical summary of the circulating miRNA biomarkers for melanoma published to date.

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

The low efficiency of currently-used anti-cancer therapies poses a serious challenge, especially in the case of malignant melanoma, a cancer characterized by elevated invasiveness and relatively high mortality rate.,The role of the tumor microenvironment in the progression of melanoma and its acquisition of resistance to treatment seems to be the main focus of recent studies.,One of the factors that, in normal conditions, aids the organism in its fight against the cancer and, following the malignant transformation, adapts to facilitate the development of the tumor is the immune system.,A variety of cell types, i.e., T and B lymphocytes, macrophages, and dendritic and natural killer cells, as well as neutrophils, support the growth and invasiveness of melanoma cells, utilizing a plethora of mechanisms, including secretion of pro-inflammatory molecules, induction of inhibitory receptors expression, or depletion of essential nutrients.,This review provides a comprehensive summary of the processes regulated by tumor-associated cells that promote the immune escape of melanoma cells.,The described mechanisms offer potential new targets for anti-cancer treatment and should be further studied to improve currently-employed therapies.

MCL-1 (BCL-2 family anti-apoptotic protein) is responsible for melanoma's resistance to therapy.,Cancer initiating cells also contribute to resistance and relapse from treatments.,Here we examined the effects of the MCL-1 inhibitor SC-2001 in killing non melanoma-initiating-cells (bulk of melanoma), and melanoma-initiating-cells (MICs).,By itself, SC-2001 significantly kills melanoma cells under monolayer conditions in vitro and in a conventional mouse xenograft model.,However, even at high doses (10μM), SC-2001 does not effectively eliminate MICs.,In contrast, the combination of SC-2001 with ABT-737 (a BCL-2/BCL-XL/BCL-W inhibitor) significantly decreases ALDH+ cells, disrupts primary spheres, and inhibits the self-renewability of MICs.,These results were observed in multiple melanomas, including short term cultures of relapsed tumors from current treatments, independent of the mutation status of BRAF or NRAS.,Using a low-cell-number mouse xenograft model, we examined the effects of these treatments on the tumor initiating ability of MIC-enriched cultures.,The combination therapy reduces tumor formation significantly compared to either drug alone.,Mechanistic studies using shRNA and the CRISPR-Cas9 technology demonstrated that the upregulation of pro-apoptotic proteins NOXA and BIM contribute to the combination-induced cell death.,These results indicate that the MCL-1 inhibitor SC-2001 combined with ABT-737 is a promising treatment strategy for targeting melanoma.

Despite recent improvements in prevention, diagnosis and treatment, vast differences in melanoma burden still exist between populations.,Comparative data can highlight these differences and lead to focused efforts to reduce the burden of melanoma.,To assess global, regional and national melanoma incidence, mortality and disability‐adjusted life year (DALY) estimates from the Global Burden of Disease Study 2015.,Vital registration system and cancer registry data were used for melanoma mortality modelling.,Incidence and prevalence were estimated using separately modelled mortality‐to‐incidence ratios.,Total prevalence was divided into four disease phases and multiplied by disability weights to generate years lived with disability (YLDs).,Deaths in each age group were multiplied by the reference life expectancy to generate years of life lost (YLLs).,YLDs and YLLs were added to estimate DALYs.,The five world regions with the greatest melanoma incidence, DALY and mortality rates were Australasia, North America, Eastern Europe, Western Europe and Central Europe.,With the exception of regions in sub‐Saharan Africa, DALY and mortality rates were greater in men than in women.,DALY rate by age was highest in those aged 75-79 years, 70-74 years and ≥ 80 years.,The greatest burden from melanoma falls on Australasian, North American, European, elderly and male populations, which is consistent with previous investigations.,These substantial disparities in melanoma burden worldwide highlight the need for aggressive prevention efforts.,The Global Burden of Disease Study results can help shape melanoma research and public policy.,What's already known about this topic?,Melanoma incidence and mortality has been assessed in the past for individual countries or world regions.,Melanoma incidence and mortality has been assessed in the past for individual countries or world regions.,What does this study add?,As part of the Global Burden of Disease Study, melanoma burden was estimated at the global, regional and country level for incidence, mortality, prevalence, years lived with disability, years of life lost and disability‐adjusted life years.These estimates can be used to guide prevention and treatment strategies, as well as resource allocation.,As part of the Global Burden of Disease Study, melanoma burden was estimated at the global, regional and country level for incidence, mortality, prevalence, years lived with disability, years of life lost and disability‐adjusted life years.,These estimates can be used to guide prevention and treatment strategies, as well as resource allocation.,Respond to this article,Plain language summary available online

In this paper, we combine kinetic modelling and patient gene expression data analysis to elucidate biological mechanisms by which melanoma becomes resistant to the immune system and to immunotherapy.,To this end, we systematically perturbed the parameters in a kinetic model and performed a mathematical analysis of their impact, thereby obtaining signatures associated with the emergence of phenotypes of melanoma immune sensitivity and resistance.,Our phenotypic signatures were compared with published clinical data on pretreatment tumor gene expression in patients subjected to immunotherapy against metastatic melanoma.,To this end, the differentially expressed genes were annotated with standard gene ontology terms and aggregated into metagenes.,Our method sheds light on putative mechanisms by which melanoma may develop immunoresistance.,Precisely, our results and the clinical data point to the existence of a signature of intermediate expression levels for genes related to antigen presentation that constitutes an intriguing resistance mechanism, whereby micrometastases are able to minimize the combined anti-tumor activity of complementary responses mediated by cytotoxic T cells and natural killer cells, respectively.,Finally, we computationally explored the efficacy of cytokines used as low-dose co-adjuvants for the therapeutic anticancer vaccine to overcome tumor immunoresistance.

Oncolytic immunotherapy is a research area of cancer immunotherapy investigating the use of modified viruses to target cancer cells.,A variety of different viral backbones (e.g., adenovirus, reovirus) with a diverse range of genetic modifications are currently being investigated for the treatment of a variety of cancers.,The oncolytic virus that has advanced the furthest in clinical development is talimogene laherparepvec, a recombinant HSV-1 virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF).,In a phase 3 study in patients with unresectable metastatic melanoma, intralesional talimogene laherparepvec treatment resulted in a higher durable response rate compared with subcutaneous GM-CSF treatment (16.3 versus 2.1%; P < 0.001).,Notably, responses were observed at uninjected lesions including visceral lesions, indicating a systemic antitumor response had occurred.,Studies evaluating combination treatments involving oncolytic viruses and immunologic agents are ongoing.,This review focuses on the mechanisms of action for oncolytic viruses and highlights select agents and combinations currently in development.

Talimogene laherparepvec (T-VEC) is an oncolytic immunotherapy designed to induce tumor regression of injected lesions through direct lytic effects, and of uninjected lesions through induction of systemic antitumor immunity.,In this study, we describe the patterns and time course of response to T-VEC from the phase III OPTiM trial of 436 patients with unresected stages IIIB-IV melanoma.,Lesion-level response analyses were performed based on the type of lesion (injected or uninjected cutaneous, subcutaneous, or nodal lesions; or visceral lesions [uninjected]), and the best percentage change from baseline of the sum of products of the longest diameters was calculated.,Patients randomized to T-VEC (n = 295) who experienced a durable response (continuous partial or complete response for ≥6 months) were evaluated for progression prior to response (PPR), defined as the appearance of a new lesion or >25 % increase in total baseline tumor area.,T-VEC resulted in a decrease in size by ≥50 % in 64 % of injected lesions (N = 2116), 34 % of uninjected non-visceral lesions (N = 981), and 15 % of visceral lesions (N = 177).,Complete resolution of lesions occurred in 47 % of injected lesions, 22 % of uninjected non-visceral lesions, and 9 % of visceral lesions.,Of 48 patients with durable responses, 23 (48 %) experienced PPR, including 14 who developed new lesions only.,No difference in overall survival was observed, and median duration of response was not reached in patients with PPR versus those without PPR.,Responses in uninjected lesions provide validation of T-VEC-induced systemic immunotherapeutic effects against melanoma.,PPR did not negatively impact the clinical effectiveness of T-VEC.,The online version of this article (doi:10.1245/s10434-016-5286-0) contains supplementary material, which is available to authorized users.

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.

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.

Blue rubber bleb nevus syndrome (BRBNS) is a rare vascular disorder consisting of multifocal venous malformations.,Delayed diagnosis or misdiagnosis frequently occurs in patients without typical cutaneous lesions or gastrointestinal bleeding symptoms.,This article reports a 10-year case of delayed diagnosis of BRBNS detected by capsule endoscopy.,A 15-year-old girl presented with refractory iron-deficiency anemia (IDA) for 10 years, without any hemorrhagic signs or noticeable cutaneous lesions, which led to her obvious physical growth retardation.,Capsule endoscopic examination revealed dozens of vascular blebs distributed from the jejunum to the ileum and a site of active bleeding.,Hence, she was diagnosed with BRBNS.,Laparotomy was performed with resection of the small bowel lesions, and iron supplementation was prescribed for 3 months.,Postoperatively, the patient had an uncomplicated course.,On follow-up after 3 years, IDA in this patient was cured and she did not require further blood transfusion and showed excellent vigor.,A high index of suspicion for BRBNS and adequate endoscopy examination will help to identify the origin of refractory IDA in older children, particularly in patients with vascular lesions of the skin.

Blue Rubber Bleb Nevus Syndrome (BRBNS) is an uncommon congenital disorder characterized by sporadic venous malformation which mainly occurs in skin and alimentary canal.,Here, we report a BRBNS patient with concomitant intestinal intussusception who diagnosed by intraoperative endoscopy and ultimately managed using surgical resection.,A 19-year-old boy was referred to urgent surgery for acute melena and stomachache.,He had used to be a long-term iron user for undiagnosed chronic anemia and papules.,Abdominal CT on admission demonstrated the presence of intestinal intussusception.,The following exploratory laparotomy and intraoperative endoscopy revealed multiple gastrointestinal hemangiomas.,The postoperative course was uneventful and pathological examination certified multiple cavernous hemangiomas in the resected gastrointestines.

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.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection.,Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival.,In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism.,Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting.,Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches.,•Anti-CTLA-4 of hIgG1 and hIgG2 isotypes promote depletion of intra-tumoral Treg cells•hIgG2 antibodies mediate in vivo depletion of intra-tumoral Treg cells via CD32a•Anti-CTLA-4 with enhanced Fc effector function improves therapeutic outcomes•The CD16-V158F SNP is associated with response to ipilimumab in inflamed tumors,Anti-CTLA-4 of hIgG1 and hIgG2 isotypes promote depletion of intra-tumoral Treg cells,hIgG2 antibodies mediate in vivo depletion of intra-tumoral Treg cells via CD32a,Anti-CTLA-4 with enhanced Fc effector function improves therapeutic outcomes,The CD16-V158F SNP is associated with response to ipilimumab in inflamed tumors,Arce Vargas et al. use a mouse model expressing human FcγRs to show that antibodies with isotypes equivalent to ipilimumab increase the CD8+ to Treg ratio by depleting intra-tumoral Tregs to promote tumor rejection.,In melanoma patients, response to ipilimumab is associated with a high affinity FcγR polymorphism.

A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1).,Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8+ and TH1 cells.,To address this therapeutic challenge, we invent bifunctional antibody-ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFβ receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFβ in the target cell microenvironment (a-CTLA4-TGFβRIIecd and a-PDL1-TGFβRIIecd). a-CTLA4-TGFβRIIecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab).,Likewise, a-PDL1-TGFβRIIecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab).,Our data demonstrate that Y-traps counteract TGFβ-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors.,Antitumor T cells can be inhibited by a TGFβ rich tumor microenvironment.,The authors develop bifunctional proteins comprising CTLA-4 or PD-L1 immune checkpoint-targeted antibodies fused to a “TGFβ trap” and show that they counteract tumor immune tolerance and enhance the efficacy of these antibodies.

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

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.

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

In this review, Goding and Arnheiter present the current understanding of MITF's role and regulation in development and disease and highlight key areas where our knowledge of MITF regulation and function is limited.,All transcription factors are equal, but some are more equal than others.,In the 25 yr since the gene encoding the microphthalmia-associated transcription factor (MITF) was first isolated, MITF has emerged as a key coordinator of many aspects of melanocyte and melanoma biology.,Like all transcription factors, MITF binds to specific DNA sequences and up-regulates or down-regulates its target genes.,What marks MITF as being remarkable among its peers is the sheer range of biological processes that it appears to coordinate.,These include cell survival, differentiation, proliferation, invasion, senescence, metabolism, and DNA damage repair.,In this article we present our current understanding of MITF's role and regulation in development and disease, as well as those of the MITF-related factors TFEB and TFE3, and highlight key areas where our knowledge of MITF regulation and function is limited.

Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis.,Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals.,Fully melanized melanosomes are delivered to keratinocytes of the skin and hair.,The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU).,Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation.,MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis.,Diseases involving alterations of EMU show various forms of pigmentation phenotypes.,This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade.

Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers.,Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks.,Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations.,We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds.,WP760 inhibited cell proliferation (IC50 = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM).,WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription.,Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway.,PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy.,Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines.,These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.,The online version of this article (doi:10.1007/s10637-017-0465-9) contains supplementary material, which is available to authorized users.

Despite recent advances in melanoma treatment, metastasis and resistance to therapy remain serious clinical challenges.,NME1 is a metastasis suppressor, a class of proteins which inhibits metastatic spread of cancer cells without impact on growth of the primary tumor.,We have identified a rare subpopulation of cells with markedly reduced expression of NME1 (NME1LOW) in human melanoma cell lines.,To enable isolation of viable NME1LOW cells for phenotypic analysis by fluorescence-activated cell sorting (FACS), a CRISPR-Cas9-mediated approach was used to attach an EGFP coding module to the C-terminus of the endogenous NME1 gene in melanoma cell lines.,NME1LOW cells displayed enhanced collective invasion in vitro when implanted as 3D aggregates in Matrigel.,NME1LOW cells were also highly metastatic to lung and liver when xenografted subcutaneously in immune-deficient NSG mice.,RNA-seq analysis revealed that NME1LOW cells express elevated levels of genes associated with tumor aggressiveness, as well as with morphogenesis of tissues of neural crest-like origin (melanocytes and neurons, bone and heart tissues; GO: 0009653).,The highly malignant NME1LOW variant of melanoma cells has potential to provide novel therapeutic targets and molecular markers for improved clinical management of patients with advanced melanoma.

The development of BRAF V600 and MEK inhibitors constitutes a breakthrough in the treatment of patients with BRAF-mutated metastatic melanoma.,However, although there is an increase in overall survival, these patients generally confront recurrence, and several resistance mechanisms have already been described.,In the present study we describe a different resistance mechanism.,After several weeks of long-term in vitro treatment of two different V600E BRAF-mutated melanoma cell lines with MARK inhibitors, PLX4032 and/or GDC-0973, the majority of the cells died whereas some remained viable and quiescent (SUR).,Markedly, discontinuing treatment of SUR cells with MAPK inhibitors allowed the population to regrow and these cells retained drug sensitivity equal to that of the parental cells.,SUR cells had increased expression levels of CD271 and ABCB5 and presented senescence-associated characteristics.,Notably, SUR cells were efficiently lysed by cytotoxic T lymphocytes recognizing MART-1 and gp100 melanoma differentiation antigens.,We propose quiescent plasticity as a mechanism of resistance to BRAF and MEK inhibitors while retaining sensitivity to immune effectors.

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system.,Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole‐exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel.,Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines.,Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer-testis antigens in the lines.,Together, these characteristics make the NZM panel a valuable resource for cell‐based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.

Whole-genome sequencing identifies a novel germline variant in the oncogene MITF, which is associated with the development of melanoma.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.,Two papers in this issue of Nature demonstrate that missense substitutions in the gene encoding for microphthalmia-associated transcription factor (MITF) are associated with susceptibility to melanoma and renal cell carcinoma.,Functional analysis shows that the variant has impaired sumoylation that leads to differential regulation of several MITF targets, and promotes tumour cell clonogenicity, migration and invasion.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.,So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families.,Mutations in CDKN2A account for approximately 40% of familial cases1, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds2.,Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma.,We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF).,Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant.,Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample.,Likewise, it was similarly associated in an independent case-control sample from the United Kingdom.,In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both.,The variant allele was also associated with increased naevus count and non-blue eye colour.,Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets.,These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.

Immunotherapy (IT) and radiotherapy (RT) have improved overall survival in patients with melanoma with brain metastasis (MBM).,We examined the real‐world survival impact of IT and RT combination and timing strategies.,From the facility‐based National Cancer Database (NCDB) data set, 3008 cases of MBM were identified between 2011 and 2015.,Six treatment cohorts were identified: stereotactic radiosurgery (SRS) + IT, SRS alone, whole brain radiotherapy (WBRT) + IT, WBRT alone, IT alone, and none.,Concurrent therapy was defined as IT given within 28 days before or after RT; nonconcurrent defined as IT administered within 28-90 days of RT.,The co‐primary outcomes were propensity score‐adjusted overall survival by treatment regimen and overall survival by RT and IT timing.,Median overall survival (mOS) was performed for each treatment category; SRS +IT 15.77 m; (95%CI 12.13-21.29), SRS alone (9.33 m; 95%CI: 8.0-11.3), IT alone (7.29 m; 95%CI: 5.35-12.91), WBRT +IT (4.89 m; 95%CI: 3.65-5.92), No RT or IT (3.29 m; 95%CI: 2.96-3.75), and WBRT alone (3.12 m; 95%CI 2.79-3.52).,By propensity score matching, mOS for SRS +IT (15.5 m; 95%CI: 11.5-20.2) was greater than SRS alone (10.1 m; 95%CI: 8.4-11.8) (p = 0.010), and median survival for WBRT +IT (4.6 m; 95%CI: 3.4-5.6) was greater than WBRT alone (2.9 m; 95%CI: 2.5-3.5) (p < 0.001).,In the SRS +IT group, 24‐month landmark survival was 47% (95%CI; 42-52) for concurrent versus 37% (95%CI; 30-44) for nonconcurrent (p = 0.40).,Those who received IT in addition to WBRT and SRS experienced longer survival compared to RT modalities alone, while those receiving concurrent SRS and IT trended toward improved survival versus nonconcurrent therapy.,In our study of the National Cancer Data Base (NCBD), addition of immunotherapy to radiation therapy improves survival in patients with melanoma with brain metastases when added to SBRT or WBRT, with the best survival observed for IT with SBRT.,Concurrrent versus Noncurrent immunotherapy combined with radiation had no significantly different survival.

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.

Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma.,However, many patients do not display therapeutic response or eventually relapse.,Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies.,Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown.,Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma.,Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers.,Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression.,TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens.,This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.,Most melanoma patients do not respond to anti-PD1 therapy.,Here, the authors show that TNFα blockade synergizes with anti-PD-1 by preventing anti-PD-1-induced CD8+ T cell death and TIM-3 expression on such cells.

Objective To estimate the burden of melanoma resulting from sunbed use in western Europe.,Design Systematic review and meta-analysis.,Data sources PubMed, ISI Web of Science (Science Citation Index Expanded), Embase, Pascal, Cochrane Library, LILACS, and MedCarib, along with published surveys reporting prevalence of sunbed use at national level in Europe.,Study selection Observational studies reporting a measure of risk for skin cancer (cutaneous melanoma, squamous cell carcinoma, basal cell carcinoma) associated with ever use of sunbeds.,Results Based on 27 studies ever use of sunbeds was associated with a summary relative risk of 1.20 (95% confidence interval 1.08 to 1.34).,Publication bias was not evident.,Restricting the analysis to cohorts and population based studies, the summary relative risk was 1.25 (1.09 to 1.43).,Calculations for dose-response showed a 1.8% (95% confidence interval 0% to 3.8%) increase in risk of melanoma for each additional session of sunbed use per year.,Based on 13 informative studies, first use of sunbeds before age 35 years was associated with a summary relative risk of 1.87 (1.41 to 2.48), with no indication of heterogeneity between studies.,By using prevalence data from surveys and data from GLOBOCAN 2008, in 2008 in the 15 original member countries of the European Community plus three countries that were members of the European Free Trade Association, an estimated 3438 cases of melanoma could be attributable to sunbed use, most (n=2341) occurring among women.,Conclusions Sunbed use is associated with a significant increase in risk of melanoma.,This risk increases with number of sunbed sessions and with initial usage at a young age (<35 years).,The cancerous damage associated with sunbed use is substantial and could be avoided by strict regulations.

Malignant melanoma is a neoplasm of melanocytes, and the microphthalmia‐associated transcription factor (MITF) is essential for the existence of melanocytes.,MITF's relevance for this cell lineage is maintained in melanoma, where it is an important regulator of survival and balances melanoma cell proliferation with terminal differentiation (pigmentation).,The MITF gene is amplified in ~20% of melanomas and MITF mutation can predispose to melanoma development.,Furthermore, the regulation of MITF expression and function is strongly linked to the BRAF/MEK/ERK/MAP‐kinase (MAPK) pathway, which is deregulated in >90% of melanomas and central target of current therapies.,MITF expression in melanoma is heterogeneous, and recent findings highlight the relevance of this heterogeneity for the response of melanoma to MAPK pathway targeting drugs, as well as for MITF's role in melanoma progression.,This review aims to provide an updated overview on the regulation of MITF function and plasticity in melanoma with a focus on its link to MAPK signaling.

YAP and its paralog protein TAZ are downstream effectors of the Hippo pathway.,Both are amplified in many human cancers and promote cell proliferation and epithelial-mesenchymal transition.,Little is known about the status of the Hippo pathway in cutaneous melanoma.,We profiled Hippo pathway component expression in a panel of human melanoma cell lines and melanocytic lesions, and characterized the capacity of YAP and TAZ to control melanoma cell behavior.,YAP and TAZ immuno-staining in human samples revealed mixed cytoplasmic and nuclear staining for both proteins in benign nevi and superficial spreading melanoma.,TAZ was expressed at higher levels than YAP1/2 in all cell lines and in those with high invasive potential.,Stable YAP or TAZ knockdown dramatically reduced the expression of the classical Hippo target CCN2/connective-tissue growth factor (CTGF), as well as anchorage-independent growth, capacity to invade Matrigel, and ability form lung metastases in mice following tail-vein injection.,YAP knockdown also reduced invasion in a model of skin reconstruct.,Inversely, YAP overexpression increased melanoma cell invasiveness, associated with increased TEA domain-dependent transcription and CCN2/CTGF expression.,Together, these results demonstrate that both YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells and may represent worthy targets for therapeutic intervention.

In the KEYNOTE-022 study, pembrolizumab with dabrafenib and trametinib (triplet) improved progression-free survival (PFS) versus placebo with dabrafenib and trametinib (doublet) without reaching statistical significance.,Mature results on PFS, duration of response (DOR), and overall survival (OS) are reported.,The double-blind, phase 2 part of KEYNOTE-022 enrolled patients with previously untreated BRAF V600E/K-mutated advanced melanoma from 22 sites in seven countries.,Patients were randomly assigned 1:1 to intravenous pembrolizumab (200 mg every 3 weeks) or placebo plus dabrafenib (150 mg orally two times per day) and trametinib (2 mg orally one time a day).,Primary endpoint was PFS.,Secondary endpoints were objective response rate, DOR, and OS.,Efficacy was assessed in the intention-to-treat population, and safety was assessed in all patients who received at least one dose of study drug.,This analysis was not specified in the protocol.,Between November 30, 2015 and April 24, 2017, 120 patients were randomly assigned to triplet (n=60) or doublet (n=60) therapy.,With 36.6 months of follow-up, median PFS was 16.9 months (95% CI 11.3 to 27.9) with triplet and 10.7 months (95% CI 7.2 to 16.8) with doublet (HR 0.53; 95% CI 0.34 to 0.83).,With triplet and doublet, respectively, PFS at 24 months was 41.0% (95% CI 27.4% to 54.2%) and 16.3% (95% CI 8.1% to 27.1%); median DOR was 25.1 months (95% CI 14.1 to not reached) and 12.1 months (95% CI 6.0 to 15.7), respectively.,Median OS was not reached with triplet and was 26.3 months with doublet (HR 0.64; 95% CI 0.38 to 1.06).,With triplet and doublet, respectively, OS at 24 months was 63.0% (95% CI 49.4% to 73.9%) and 51.7% (95% CI 38.4% to 63.4%).,Grade 3-5 treatment-related adverse events (TRAEs) occurred in 35 patients (58%, including one death) receiving triplet and 15 patients (25%) receiving doublet.,In BRAF V600E/K-mutant advanced melanoma, pembrolizumab plus dabrafenib and trametinib substantially improved PFS, DOR, and OS with a higher incidence of TRAEs.,Interpretation of these results is limited by the post hoc nature of the analysis.

Targeted inhibitors elicit heterogeneous clinical responses in genetically stratified groups of patients.,While most studies focus on tumor intrinsic properties, factors in the tumor microenvironment were recently found to modulate the response to inhibitors.,Here, we show that in cutaneous BRAF V600E melanoma, the cytokine TNFα blocks RAF-inhibitor-induced apoptosis via activation of nuclear factor κB (NFκB).,Several NFκB-dependent factors are up-regulated following TNFα and RAF inhibitor treatment.,Of these factors, we show that death receptor inhibitor cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) is required for TNFα-induced protection against RAF inhibitor.,Overexpression of c-FLIP_S or c-FLIP_L isoform decreased RAF inhibitor-induced apoptosis in the absence of TNFα.,Importantly, targeting NFκB enhances response to RAF inhibitor in vitro and in vivo.,Together, our results show mechanistic evidence for cytokine-mediated resistance to RAF inhibitor and provide a preclinical rationale for the strategy of co-targeting the RAF-MEK-ERK1/2 pathway and the TNFα/NFκB axis to treat mutant BRAF melanomas.

The therapeutic landscape in metastatic melanoma has changed dramatically in the last decade, with the success of immune checkpoint inhibitors resulting in durable responses for a large number of patients.,For patients with BRAF mutations, combinations of BRAF and MEK inhibitors demonstrated response rates and benefit comparable to those from immune checkpoint inhibitors, providing the rationale for sequential treatment with targeted and immunotherapies and raising the question of optimal treatment sequencing.,Biomarkers for the selection of anti-PD-1 therapy in BRAF wild type (BRAF WT) and in BRAF mutated (BRAF MUT) patients help development of alternative treatments for patients unlikely to benefit, and might lead to better understanding of the interaction of checkpoint inhibition and targeted therapy.,In this paper we evaluate the performance of a previously developed serum proteomic test, BDX008, in metastatic melanoma patients treated with anti-PD-1 agents and investigate the role of BRAF mutation status.,BDX008, a pre-treatment proteomic test associated with acute phase reactants, wound healing and complement activation, stratifies patients into two groups, BDX008+ and BDX008-, with better and worse outcomes on immunotherapy.,Serum samples were available from 71 patients treated with anti-PD1 inhibitors; 25 patients had BRAF mutations, 39 were wild type.,Overall, BDX008+ patients had significantly better overall survival (OS) (HR = 0.50, P = 0.016) and a trend for better progression-free survival (PFS) (HR = 0.61, P = 0.060) than BDX008- patients.,BDX008 classification was statistically significant in the analyses adjusted for mutation status, LDH, and line of treatment (P = 0.009 for OS and 0.031 for PFS).,BRAF WT BDX008+ patients had markedly long median OS of 32.5 months and 53% landmark 2 years survival, with statistically significantly superior OS as compared to BDX008- patients (HR = 0.41, P = 0.032).,The difference between BDX008+ and BDX008- in PFS in BRAF WT patients and in OS and PFS in BRAF MUT patients did not reach statistical significance, though numerically was consistent with overall results.,The test demonstrated significant interaction with neutrophil-to-lymphocyte ratio (NLR) (PFS P = 0.041, OS P = 0.004).,BDX008 as a biomarker selecting for benefit from immune checkpoint blockade, especially in patients with wild type BRAF and in subgroups with low NLR, warrants further evaluation.,The online version of this article (10.1186/s40425-019-0569-1) contains supplementary material, which is available to authorized users.

An anti-programmed cell death protein 1 monoclonal antibody, nivolumab, is one of the most effective drugs for advanced melanoma.,Tumor cell-derived or immune cell-derived markers and clinical predictors such as serum lactate dehydrogenase (LDH) and cutaneous adverse events, have already been described as prognostic factors for advanced melanoma treated with nivolumab.,We sought to identify further clinical predictors that can be determined in routine clinical practice.,We retrospectively analyzed clinical findings of 98 consecutive patients with unresectable stage III or IV melanoma treated with nivolumab, at the National Cancer Center Hospital or at Keio University Hospital, in Tokyo, Japan, between July 2014 and July 2016.,These patients had been administered nivolumab at a dose of 2mg/kg every 3 weeks.,As for pretreatment prognostic factors, ECOG performance status (PS) ≥1, maximum tumor diameters of ≥30mm, elevated LDH and elevated C-reactive protein were significantly associated with poor overall survival (OS) (hazard ratio [HR] 0.29 [P<0.001], HR 0.40 [p=0.003], HR 0.29 [P<0.001], HR 0.42 [P=0.004], respectively) on univariate analysis.,Among these factors, PS and LDH were identified as independent variables by multivariate analysis.,As for early markers examined during therapy, patients with absolute lymphocyte count (ALC) ≥ 1000/μl (Week3: HR 0.40 [P=0.004], Week6: HR 0.33 [P=0.001]) and absolute neutrophil count (ANC) <4000/μl (Week3: HR 0.46 [P=0.014], Week6: HR 0.51 [P=0.046]) had significantly better OS.,ALC≥1000/μl and ANC<4000/μl during treatment appear to be early markers associated with OS.,Nivolumab might have minimal efficacy in patients with a massive tumor burden.

Uveal melanoma (UM) represents the most frequent primary intraocular tumor, however, limited therapeutic options are still available.,We have previously shown that cluster of differentiation 47 (CD47) is significantly upregulated in UM cells following inflammatory stimuli and that it represents a predictor of disease progression.,Here, we aimed to better characterize the pathophysiological role of CD47 in UM.,We show that CD47 is not modulated at different cancer stages, although patients with the lowest expression of CD47 show significant better progression-free survival, after correcting for the presence of BAP1, GNAQ, and GNA11 mutations.,By stratifying patients based on the expression of CD47 in the tumor, we observed that patients with high levels of CD47 have a significant increase in immune score as compared to patients with low levels of CD47.,In particular, deconvolution analysis of infiltrating immune cell populations revealed that a significantly higher number of CD4+ and CD8+ T cells can be found in patients with high CD47 levels, with the most enriched populations being the Th2, Treg, and CD8+ Tcm cells.,We also show that a large number of transcripts are significantly modulated between the groups of patients with high and low levels of CD47, with a significant enrichment of interferon IFN-alpha regulated genes.,The results from this study may propel the development of anti-CD47 therapies for UM patients.

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

Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma.,Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D.,KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis.,KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates.,Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states.,Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells.,Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells.,We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.

In this study, Falletta et al. show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,Their results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.,The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance.,Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown.,In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance.,However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood.,Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors.,However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion.,Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance.,Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Deregulations or mutations of WNT/β-catenin signaling have been associated to both tumour formation and progression.,However, contradictory results concerning the role of β-catenin in human melanoma address an open question on its oncogenic nature and prognostic value in this tumour.,Changes in WNT signaling pathways have been linked to phenotype switching of melanoma cells between a highly proliferative/non-invasive and a slow proliferative/metastatic condition.,We used a novel panel of cell lines isolated from melanoma specimens, at initial passages, to investigate phenotype differences related to the levels and activity of WNT/β-catenin signaling pathway.,This in vitro cell system revealed a marked heterogeneity that comprises, in some cases, two distinct tumour-derived subpopulations of cells presenting a different activation level and cellular distribution of β-catenin.,In cells derived from the same tumor, we demonstrated that the prevalence of LEF1 (high β-catenin expressing cells) or TCF4 (low β-catenin expressing cells) as β-catenin partner for DNA binding, is associated to the expression of two distinct profiles of WNT-responsive genes.,Interestingly, melanoma cells expressing relative low level of β-catenin and an invasive markers signature were associated to the TNF-α-induced pro-inflammatory pathway and to the chemotherapy resistance, suggesting that the co-existence of melanoma subpopulations with distinct biological properties could influence the impact of chemo- and immunotherapy.

Melanoma is the most aggressive skin cancer; there is no cure in advanced stages.,Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools.,FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance.,We hypothesized a role for FKBP51 in melanoma invasive behaviour.,FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion.,In addition, FKBP51 induced some melanoma stem cell (MCSC) genes.,Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap.,Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains.,In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis.,The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program.

Histone lactylation, a metabolic stress-related histone modification, plays an important role in the regulation of gene expression during M1 macrophage polarization.,However, the role of histone lactylation in tumorigenesis remains unclear.,Here, we show histone lactylation is elevated in tumors and is associated with poor prognosis of ocular melanoma.,Target correction of aberrant histone lactylation triggers therapeutic efficacy both in vitro and in vivo.,Mechanistically, histone lactylation contributes to tumorigenesis by facilitating YTHDF2 expression.,Moreover, YTHDF2 recognizes the m6A modified PER1 and TP53 mRNAs and promotes their degradation, which accelerates tumorigenesis of ocular melanoma.,We reveal the oncogenic role of histone lactylation, thereby providing novel therapeutic targets for ocular melanoma therapy.,We also bridge histone modifications with RNA modifications, which provides novel understanding of epigenetic regulation in tumorigenesis.,The online version contains supplementary material available at 10.1186/s13059-021-02308-z.

Dynamic N6-methyladenosine (m6A) RNA modification generated and erased by N6-methyltransferases and demethylases regulates gene expression, alternative splicing and cell fate.,Ocular melanoma, comprising uveal melanoma (UM) and conjunctival melanoma (CM), is the most common primary eye tumor in adults and the 2nd most common melanoma.,However, the functional role of m6A modification in ocular melanoma remains unclear.,m6A assays and survival analysis were used to explore decreased global m6A levels, indicating a late stage of ocular melanoma and a poor prognosis.,Multiomic analysis of miCLIP-seq, RNA-seq and Label-free MS data revealed that m6A RNA modification posttranscriptionally promoted HINT2 expression.,RNA immunoprecipitation (RIP)-qPCR and dual luciferase assays revealed that HINT2 mRNA specifically interacted with YTHDF1.,Furthermore, polysome profiling analysis indicated a greater amount of HINT2 mRNA in the translation pool in ocular melanoma cells with higher m6A methylation.,Here, we show that RNA methylation significantly inhibits the progression of UM and CM.,Ocular melanoma samples showed decreased m6A levels, indicating a poor prognosis.,Changes in global m6A modification were highly associated with tumor progression in vitro and in vivo.,Mechanistically, YTHDF1 promoted the translation of methylated HINT2 mRNA, a tumor suppressor in ocular melanoma.,Our work uncovers a critical function for m6A methylation in ocular melanoma and provides additional insight into the understanding of m6A modification.

Recent advances in cancer treatment have emerged from new immunotherapies targeting T-cell inhibitory receptors, including cytotoxic T-lymphocyte associated antigen (CTLA)-4 and programmed cell death (PD)-1.,In this context, anti-CTLA-4 and anti-PD-1 monoclonal antibodies have demonstrated survival benefits in numerous cancers, including melanoma and non-small-cell lung carcinoma.,PD-1-expressing CD8+ T lymphocytes appear to play a major role in the response to these immune checkpoint inhibitors (ICI).,Cytotoxic T lymphocytes (CTL) eliminate malignant cells through recognition by the T-cell receptor (TCR) of specific antigenic peptides presented on the surface of cancer cells by major histocompatibility complex class I/beta-2-microglobulin complexes, and through killing of target cells, mainly by releasing the content of secretory lysosomes containing perforin and granzyme B.,T-cell adhesion molecules and, in particular, lymphocyte-function-associated antigen-1 and CD103 integrins, and their cognate ligands, respectively, intercellular adhesion molecule 1 and E-cadherin, on target cells, are involved in strengthening the interaction between CTL and tumor cells.,Tumor-specific CTL have been isolated from tumor-infiltrating lymphocytes and peripheral blood lymphocytes (PBL) of patients with varied cancers.,TCRβ-chain gene usage indicated that CTL identified in vitro selectively expanded in vivo at the tumor site compared to autologous PBL.,Moreover, functional studies indicated that these CTL mediate human leukocyte antigen class I-restricted cytotoxic activity toward autologous tumor cells.,Several of them recognize truly tumor-specific antigens encoded by mutated genes, also known as neoantigens, which likely play a key role in antitumor CD8 T-cell immunity.,Accordingly, it has been shown that the presence of T lymphocytes directed toward tumor neoantigens is associated with patient response to immunotherapies, including ICI, adoptive cell transfer, and dendritic cell-based vaccines.,These tumor-specific mutation-derived antigens open up new perspectives for development of effective second-generation therapeutic cancer vaccines.

In the recent past years, many discoveries in the tumor microenvironment have led to changes in the management of melanoma and it is rising up hopes, specially, to those in advanced stages.,FDA approved seven new drugs from 2011 to 2014.,They are: Vemurafenib, Dabrafenib and Trametinib, kinases inhibitors used for patients that have BRAFV600E mutation; Ipilimumab (anti-CTLA4), Pembrolizumab (anti-PD-1) and Nivolumab (anti-PD-1), monoclonal antibodies that stimulate the immune system; and Peginterferon alfa-2b, an anti-proliferative cytokine used as adjuvant therapy.,In this article, we will review the molecular bases for these new metastatic melanoma therapeutic agents cited above and also analyze new molecular discoveries in melanoma study, as Cancer-Testis antigens (CT).,They are capable of induce humoral and cellular immune responses in cancer patients and because of this immunogenicity and their restrict expression in normal tissues, they are considered an ideal candidate for vaccine development against cancer.,Among CT antigens, NY-ESO-1 is the best characterized in terms of expression patterns and immunogenicity.,It is expressed in 20-40% of all melanomas, more in metastatic lesions than in primary ones, and it is very heterogeneous inter and intratumoral.,Breslow index is associate with NY-ESO-1 expression in primary cutaneous melanomas, but its relation to patient survival remains controversial.

Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future.,This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells.,The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development.,In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research.,This model is particularly suitable for live cell imaging and high-throughput drug screening in a large-scale fashion.,Thanks to the recent advances in genome editing, such as the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) methodologies, the mechanisms associated with cancer development and progression, as well as drug resistance can be investigated and comprehended.,With these unique tools, the zebrafish represents a powerful platform for skin cancer research in the development of target therapies.,Here, we will review the advantages of using the zebrafish model for drug discovery and toxicological and phenotypical screening.,We will focus in detail on the most recent progress in the field of zebrafish model generation for the study of melanoma and squamous cell carcinoma (SCC), including cancer cell injection and transgenic animal development.,Moreover, we will report the latest compounds and small molecules under investigation in melanoma zebrafish models.

This review aims to summarize the current knowledge of molecular pathways and their clinical relevance in melanoma.,Metastatic melanoma was a grim diagnosis, but in recent years tremendous advances have been made in treatments.,Chemotherapy provided little benefit in these patients, but development of targeted and new immune approaches made radical changes in prognosis.,This would not have happened without remarkable advances in understanding the biology of disease and tremendous progress in the genomic (and other “omics”) scale analyses of tumors.,The big problems facing the field are no longer focused exclusively on the development of new treatment modalities, though this is a very busy area of clinical research.,The focus shifted now to understanding and overcoming resistance to targeted therapies, and understanding the underlying causes of the heterogeneous responses to immune therapy.

Recreational sunbed use accounts for the main non-solar source of exposure to ultraviolet radiation in fair-skinned Western populations.,Indoor tanning is associated with increased risks for acute and chronic dermatological diseases.,The current community-based study assessed the one-year prevalence of sunbed use and associated skin health habits among a representative, gender-balanced sample of 1500 Austrian citizens.,Overall one-year prevalence of sunbed use was 8.9% (95% confidence interval (CI) 7.5%-10.4%), with slightly higher prevalence in females (9.2%, 95% CI 7.3%-11.2%) compared to males (8.6%, 95% CI 6.7%-10.6%).,Factors predicting sunbed use were younger age (by trend decreasing with older age), place of living, smoking, skin type (by trend increasing with darker skin), sun exposure, motives to tan, and use of UV-free tanning products.,Despite media campaigns on the harmful effects of excessive sunlight and sunbed exposure, we found a high prevalence of self-reported sunbed use among Austrian citizens.,From a Public (Skin) Health perspective, the current research extends the understanding of prevailing leisure time skin health habits in adding data on prevalence of sunbed use in the general Austrian population.

Despite educational and public health campaigns to convey the risks of indoor tanning, many individuals around the world continue to engage in this behavior.,Few descriptive studies of indoor tanning have collected information pertaining to the lifetime history of indoor tanning, thereby limiting our ability to understand indoor tanning patterns and potentially target interventions for individuals who not only initiate, but continue to persistently engage in indoor tanning.,In-person interviews elicited detailed retrospective information on lifetime history of indoor tanning among white individuals (n = 401) under age 40 seen by a dermatologist for a minor benign skin condition.,These individuals were controls in a case-control study of early-onset basal cell carcinoma.,Outcomes of interest included ever indoor tanning in both males and females, as well as persistent indoor tanning in females - defined as females over age 31 who tanned indoors at least once in the last three or all four of four specified age periods (ages 11-15, 16-20, 21-30 and 31 or older).,Multivariate logistic regression was used to identify sociodemographic and lifestyle correlates of ever and persistent indoor tanning in females.,Approximately three-quarters (73.3%) of females and 38.3% of males ever tanned indoors, with a median age of initiation of 17.0 and 21.5, respectively.,Among indoor tanners, 39.3% of females and 21.7% of males reported being burned while indoor tanning.,Female ever indoor tanners were younger, had darker color eyes, and sunbathed more frequently than females who never tanned indoors.,Using unique lifetime exposure data, 24.7% of female indoor tanners 31 and older persistently tanned indoors starting as teenagers.,Female persistent indoor tanners drank significantly more alcohol, were less educated, had skin that tanned with prolonged sun exposure, and sunbathed outdoors more frequently than non-persistent tanners.,Indoor tanning was strikingly common in this population, especially among females.,Persistent indoor tanners had other high-risk behaviors (alcohol, sunbathing), suggesting that multi-faceted behavioral interventions aimed at health promotion/disease prevention may be needed in this population.

Several distinct melanoma syndromes have been defined, and genetic tests are available for the associated causative genes.,Guidelines for melanoma genetic testing have been published as an informal “rule of twos and threes,” but these guidelines apply to CDKN2A testing and are not intended for the more recently described non-CDKN2A melanoma syndromes.,In order to develop an approach for the full spectrum of hereditary melanoma patients, we have separated melanoma syndromes into two types: “melanoma dominant” and “melanoma subordinate.”,Syndromes in which melanoma is a predominant cancer type are considered melanoma dominant, although other cancers, such as mesothelioma or pancreatic cancers, may also be observed.,These syndromes are associated with defects in CDKN2A, CDK4, BAP1, MITF, and POT1.,Melanoma-subordinate syndromes have an increased but lower risk of melanoma than that of other cancer(s) seen in the syndrome, such as breast and ovarian cancer or Cowden syndrome.,Many of these melanoma-subordinate syndromes are associated with well-established predisposition genes (e.g., BRCA1/2, PTEN).,It is likely that these predisposition genes are responsible for the increased susceptibility to melanoma as well but with lower penetrance than that observed for the dominant cancer(s) in those syndromes.,In this review, we describe our extension of the “rule of twos and threes” for melanoma genetic testing.,This algorithm incorporates an understanding of the spectrum of cancers and genes seen in association with melanoma to create a more comprehensive and tailored approach to genetic testing.

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

Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumours and is often adjusted during disease progression.,Our study aimed at extending our previous findings in skin metastases to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node metastases of stages III and IV melanoma patients.,TYRP1 mRNA expression in 104 lymph node metastases was quantified by real-time PCR and normalised to S100 calcium-binding protein B (S100B) mRNA expression to correct for tumour load.,TYRP1/S100B ratios were calculated and median was used as cutoff value.,TYRP1/S100B mRNA values were correlated to clinical follow-up and histopathological characteristics of the primary lesion.,A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free (DFS) and overall survival (OS; Cox regression analysis, P=0.005 and 0.01, respectively), increased Breslow thickness (Spearman's rho test, P<0.001) and the presence of ulceration (Mann-Whitney test, P=0.02) of the primaries.,Moreover, high TYRP1/S100B was of better prognostic value (lower P-value) for OS than Breslow thickness and ulceration.,Finally, it was well conserved during disease progression with respect to high/low TYRP1 groups.,High TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavourable clinical outcome.,Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients.

The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established.,Genome-wide alterations in DNA methylation are a common event in cancer.,This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients.,Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients.,The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis.,Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association.,Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis.,Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively.,The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences.,Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01).,LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC.,Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

YAP and its paralog protein TAZ are downstream effectors of the Hippo pathway.,Both are amplified in many human cancers and promote cell proliferation and epithelial-mesenchymal transition.,Little is known about the status of the Hippo pathway in cutaneous melanoma.,We profiled Hippo pathway component expression in a panel of human melanoma cell lines and melanocytic lesions, and characterized the capacity of YAP and TAZ to control melanoma cell behavior.,YAP and TAZ immuno-staining in human samples revealed mixed cytoplasmic and nuclear staining for both proteins in benign nevi and superficial spreading melanoma.,TAZ was expressed at higher levels than YAP1/2 in all cell lines and in those with high invasive potential.,Stable YAP or TAZ knockdown dramatically reduced the expression of the classical Hippo target CCN2/connective-tissue growth factor (CTGF), as well as anchorage-independent growth, capacity to invade Matrigel, and ability form lung metastases in mice following tail-vein injection.,YAP knockdown also reduced invasion in a model of skin reconstruct.,Inversely, YAP overexpression increased melanoma cell invasiveness, associated with increased TEA domain-dependent transcription and CCN2/CTGF expression.,Together, these results demonstrate that both YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells and may represent worthy targets for therapeutic intervention.

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality.,Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling.,Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity.,Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance.,To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications.,By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.,By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models.,Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination.,Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.

Resistance to BRAF/MEK inhibitor therapy in BRAFV600‐mutated advanced melanoma remains a major obstacle that limits patient benefit.,Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood.,Here, we investigated the process of matrix‐mediated drug resistance (MMDR) in response to BRAFV600 pathway inhibition in melanoma.,We demonstrate that physical and structural cues from fibroblast‐derived ECM abrogate anti‐proliferative responses to BRAF/MEK inhibition.,MMDR is mediated by drug‐induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors.,Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM‐mediated resistance to BRAF‐targeted therapy.,In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug‐induced collagen remodeling, and delays tumor relapse.,Mechanistically, DDR‐dependent MMDR fosters a targetable pro‐survival NIK/IKKα/NF‐κB2 pathway.,These findings reveal a novel role for a collagen‐rich matrix and DDR in tumor cell adaptation and resistance.,They also provide important insights into environment‐mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma.,Resistance to MAPK targeted therapy remains a major challenge in melanoma management.,This study shows that BRAF/MEK inhibitor combination induces collagen remodeling that fosters activation of Discoidin Domain Receptors (DDR) and turns on a drug tolerant pathway.,Targeting DDR overcomes this pathway.

It is widely accepted that chronic inflammation initiates and promotes carcinogenesis and tumor progression in various cell types.,However, this paradigm has not been comprehensively investigated in melanoma.,To investigate the effects of chronic inflammation on the progression of melanoma, we established a murine inflammatory skin model and investigated the relationship between skin inflammation and melanoma progression.,In a murine model, B16F10 melanoma cells in inflamed skin grew significantly more rapidly than cells in control skin.,The stromal expression of periostin was upregulated in inflamed skin, and significantly more CD163+ M2 macrophages were recruited to the melanomas in inflamed skin.,We then immunohistologically examined the expression of stromal periostin and the infiltration of CD163+ M2 macrophages in human acral lentiginous melanomas (n = 94) and analyzed the statistical associations with clinicopathological variables.,In human melanomas, high periostin expression and a large number of infiltrated M2 macrophages were significantly correlated with poor prognosis.,Furthermore, we confirmed that periostin promotes the proliferation of murine and human melanoma cells in vitro.,Our findings indicate that periostin and M2 macrophages play a critical role in melanoma progression and prognosis in both humans and mice, indicating that periostin is a potential target for treating progressive melanoma.

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

Mutations in GNAQ and GNA11, encoding the oncogenic G-protein alpha subunit q and 11, respectively, occur frequently in the majority of uveal melanomas.,Exons 4 and 5 from GNAQ and GNA11 were amplified and sequenced from 92 ciliary body and choroidal melanomas.,The mutation status was correlated with disease-free survival (DFS) and other parameters.,None of the tumours harboured a GNAQ exon 4 mutation.,A GNAQ mutation in exon 5 codon 209 was found in 46 out of 92 (50.0%) of the tumours.,Only 1 out of 92 (1.1%) melanomas showed a mutation in GNA11 exon 4 codon 183, whereas 39 out of 92 (42.4%) harboured a mutation in exon 5 of GNA11 codon 209.,Six tumours did not show any mutations in exons 4 and 5 of these genes.,Univariate analyses showed no correlation between DFS and the mutation status.,GNAQ and GNA11 mutations are, in equal matter, not associated with patient outcome.

Recent studies have shown that immunotherapies and molecular targeted therapies are effective for advanced melanoma.,Non-antigen-specific immunotherapies such as immunocheckpoint blockades have been shown to be effective in the treatment of advanced melanoma.,However, the response rates remain low.,To improve their efficacy, they should be combined with antigen-specific immunotherapy.,Elevated expression of the transcription factor, Forkhead box M1 (FOXM1), has been reported in various human cancers, and it has been shown to have potential as a target for immunotherapy.,The purpose of this study was to investigate the FOXM1 expression in human melanoma samples and cell lines, to evaluate the relationship between the FOXM1 expression and the clinical features of melanoma patients and to investigate the association between the FOXM1 and MAPK and PI3K/AKT pathways in melanoma cell lines.,We conducted the quantitative reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines, and investigated melanoma and nevus tissue samples by qRT-PCR and immunohistochemistry.,We performed MEK siRNA and PI3K/AKT inhibitor studies and FOXM1 siRNA studies in melanoma cell lines.,We found that FOXM1 was expressed in all of the melanoma cell lines, and was expressed in 49% of primary melanomas, 67% of metastatic melanomas and 10% of nevi by performing immunohistochemical staining.,Metastatic melanoma samples exhibited significantly higher mRNA levels of FOXM1 (p = 0.004).,Primary melanomas thicker than 2 mm were also more likely to express FOXM1.,Patients whose primary melanoma expressed FOXM1 had a significantly poorer overall survival compared to patients without FOXM1 expression (p = 0.024).,Downregulation of FOXM1 by siRNA significantly inhibited the proliferation of melanoma cells, and blockade of the MAPK and PI3K/AKT pathways decreased the FOXM1 expression in melanoma cell lines.,In conclusion, FOXM1 is considered to be a new therapeutic target for melanoma.

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

Increasing evidence suggests that certain types of cancers are more common in people with diabetes mellitus (DM).,This study aimed to investigate the risk of skin cancer in patients with DM in Taiwan.,In this retrospective cohort study using data from the Taiwan Longitudinal Health Insurance Research Database, the risk of developing overall skin cancer, including nonmelanoma skin cancer (NMSC) and melanoma, was compared by Poisson regression analysis and Cox regression analysis between the DM and non-DM cohorts.,The DM cohort with newly diagnosed DM (n = 41,898) and a non-DM cohort were one-to-one matched by age, sex, index date, and comorbidities (coronary artery disease, hyperlipidemia, hypertension, chronic kidney disease, chronic obstructive pulmonary disease, and obesity).,Compared with non-DM cohort statistically, for the people with DM aged ≥60 years, the incidence rates of overall skin cancer and NMSC were significantly higher (overall: DM/non-DM: number [n] = 99/76, incidence rate ratio [IRR] = 1.44, P = 0.02; NMSC: DM/non-DM: n = 94/66, IRR = 1.57, P = 0.005).,By Cox regression analysis, the risk of developing overall skin cancer or NMSC was significantly higher after adjusting for sex, comorbidities, and overall diseases with immunosuppression status (overall: adjusted hazard ratio [AHR] = 1.46, P = 0.01; NMSC: AHR = 1.6, P = 0.003).,Other significant risk factors were older males for skin cancer (overall: AHR = 1.68, P = 0.001; NMSC: AHR = 1.59, P = 0.004; melanoma: AHR = 3.25, P = 0.04), chronic obstructive pulmonary disease for NMSC (AHR = 1.44, P = 0.04), and coronary artery disease for melanoma (AHR = 4.22, P = 0.01).,The risk of developing melanoma was lower in the DM cohort than in the non-DM cohort, but without significance (AHR = 0.56, P = 0.28; DM/non-DM: n = 5/10).,The incidence rate and risk of developing overall skin cancer, including NMSC, was significantly higher in older adults with DM.,Other significant risk factors for older adults with DM were males for NMSC and melanoma, chronic obstructive pulmonary disease for NMSC, and coronary artery disease for melanoma.

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma.,To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma.,Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2.,Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes.,To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4.,Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma.,Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS.,Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

Uveal melanoma is a clinically distinct and particularly lethal subtype of melanoma originating from melanocytes in the eye.,Here, we performed multi-region DNA sequencing of primary uveal melanomas and their matched metastases from 35 patients.,We observed novel driver mutations and established the order in which these and known driver mutations undergo selection.,Metastases had genomic alterations distinct from their primary tumors, and metastatic dissemination sometimes occurred early during the development of the primary tumor.,Our study offers new insights into the genetics and evolution of this melanoma subtype, providing potential biomarkers for progression and therapy.

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

The ETS family modulates immune response and drug efficiency to targeted therapies, but their role in melanoma is largely unclear.,In this study, the ETS family was systematically analyzed in multiple public data sets.,Bioinformatics tools were used to characterize the function of ETV7 in melanoma.,A prognostic model was constructed using the LASSO Cox regression method.,We found that ETV7 was the only differentially expressed gene with significant prognostic relevance in melanoma.,Enrichment analysis of seven independent data sets indicated ETV7 participation in various immune-related pathways.,ETV7 particularly showed a strong positive correlation with CD8+ T cell infiltration.,The prognostic model based on ETV7 and its hub genes showed a relatively good predictive value in training and testing data sets.,Thus, ETV7 can potentially regulate the immune microenvironment in melanoma.

The tumor microenvironment (TME) plays a critical role in tumorigenesis, development, and therapeutic efficacy.,Major advances have been achieved in the treatment of various cancers through immunotherapy.,Nevertheless, only a minority of patients have positive responses to immunotherapy, which is partly due to conditions of the immunosuppressive microenvironment.,Therefore, it is essential to identify prognostic biomarkers that reflect heterogeneous landscapes of the TME.,Based upon the ESTIMATE algorithm, we evaluated the infiltrating levels of immune and stromal components derived from patients afflicted by various types of cancer from The Cancer Genome Atlas database (TCGA).,According to respective patient immune and stromal scores, we categorized cases into high‐ and low‐scoring subgroups for each cancer type to explore associations between TME and patient prognosis.,Gene Set Enrichment Analyses (GSEA) were conducted and genes enriched in IFNγ response signaling pathway were selected to facilitate establishment of a risk model for predicting overall survival (OS).,Furthermore, we investigated the associations between the prognostic signature and tumor immune infiltration landscape by using CIBERSORT algorithm and TIMER database.,Among the cancers assessed, the immune scores for skin cutaneous melanoma (SKCM) were the most significantly correlated with patients' survival time (P < .0001).,We identified and validated a five‐IFNγ response‐related gene signature (UBE2L6, PARP14, IFIH1, IRF2, and GBP4), which was closely correlated with the prognosis for SKCM afflicted patients.,Multivariate Cox regression analysis indicated that this risk model was an independent prognostic factor for SKCM.,Tumor‐infiltrating lymphocytes and specific immune checkpoint molecules had notably differential levels of expression in high‐ compared to low‐risk samples.,In this study, we established a novel five‐IFNγ response‐related gene signature that provided a better and increasingly comprehensive understanding of tumor immune landscape, and which demonstrated good performance in predicting outcomes for patients afflicted by SKCM.,We established an effective five‐IFNγ response‐related gene‐based risk score, which has potential to be a novel prognostic signature and may provide insight into tumor immune microenvironment of cutaneous melanoma.

Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions.,Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth.,Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers.,In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis.,Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion.,We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2.,Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes.,Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes.,To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes.,These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT.,Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients.,High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas.,The HMW-MAA-positive cells were isolated using immunomagnetic beads.,After removing CD45+ cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA+, CD45−, MART-1/gp100+).,Single, isolated CTC were then subjected to BRAF and KIT mutational analysis.,CTC (HMW-MAA+, CD45−, MART-1/gp100+) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively.,The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients.,In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient.,Melanoma cells show clonal heterogeneity.,Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.

Melanoma is common; 15,906 people in the UK were diagnosed with melanoma in 2015 and incidence has increased fivefold in 30 years.,Melanoma affects old and young people, with poor prognosis once metastatic.,UK guidelines recommend people treated for cutaneous melanoma receive extended outpatient, hospital follow up to detect recurrence or new primaries.,Such follow up of the growing population of melanoma survivors is burdensome for both individuals and health services.,Follow up is important since approximately 20% of patients with early-stage melanoma experience a recurrence and 4-8% develop a new primary; the risk of either is highest in the first 5 years.,Achieving Self-directed Integrated Cancer Aftercare (ASICA) is a digital intervention to increase total-skin-self-examination (TSSE) by people treated for melanoma, with usual follow up.,We aim to recruit 240 adults with a previous first-stage 0-2C primary cutaneous melanoma, from secondary care in North-East Scotland and the East of England.,Participants will be randomised to receive the ASICA intervention (a tablet-based digital intervention to prompt and support TSSE) or control group (treatment as usual).,Patient-reported and clinical data will be collected at baseline, including the modified Melanoma Worry Scale (MWS), the Hospital Anxiety and Depression Scale (HADs), the EuroQoL 5-dimension 5-level questionnaire (EQ-5D-5 L), and questions about TSSE practice, intentions, self-efficacy and planning.,Participants will be followed up by postal questionnaire at 3, 6 and 12 months following randomization, along with a 12-month review of clinical data.,The primary timepoint for outcome analyses will be12 months after randomisation.,If the ASICA intervention improves the practice of TSSE in those affected by melanoma, this may lead to improved psychological well-being and earlier detection of recurrent and new primary melanoma.,This could impact both patients and National Health Service (NHS) resources.,This study will determine if a full-scale randomised controlled trial can be undertaken in the UK NHS to provide the high-quality evidence needed to determine the effectiveness of the intervention.,ASICA is a pilot study evaluating the effectiveness of the practice of digitally supported TSSE in those affected by melanoma.,Clinical Trials.gov, NCT03328247.,Registered on 1 November 2017.,The online version of this article (10.1186/s13063-019-3453-x) contains supplementary material, which is available to authorized users.

To develop a digital intervention to prompt, support, and respond to the outcomes of total skin self-examinations (TSSEs) at home by people treated for cutaneous melanoma.,A complex intervention development study.,Northeast Scotland.,Semistructured scoping interviews; people previously treated for cutaneous melanoma (n=21).,Pilot testing: people treated for melanoma stages 0-2C (n=20); general practitioners (n=6); and a nurse specialist in dermatology (n=1).,A tablet-based digital intervention designed to prompt and support TSSEs comprising instructional videos and electronic reporting (including photographs) to a clinical nurse specialist in dermatology, with subsequent clinical triage.,Qualitative assessment of intervention feasibility and acceptability, and quantitative assessment of intentions and confidence to perform TSSEs in pilot participants.,The majority of pilot participants were strongly positive and adhered well to the intervention (n=15), with 7 of these reporting symptoms of concern at some point during the 6-month pilot. 4 patients complied intermittently, 3 reporting skin problems at least once during the pilot, and 1 withdrew. 2 patients underwent skin surgery as a result of participating in the pilot, with 1 diagnosed as having a recurrent melanoma and the other, a benign lesion.,A number of practical issues to improve the usability of the intervention were identified.,The proportion of participants reporting intention to check their skin at least monthly increased during the intervention as did confidence to conduct a skin check.,People previously treated for cutaneous melanoma are prepared to use digital technology to support them in conducting TSSE.,An intervention has been developed which is practical, effective and safe, and after addressing minor practical issues, could now be evaluated for clinical outcomes in a randomised clinical trial.

Uveal melanoma (UM) is the most common intraocular malignancy in adults.,Despite improvements in surgical, radiation and chemotherapy treatments, the overall survival of UM and prognosis remain poor.,In the present study, we hypothesized that Sirtuin 1 and 2 (SIRT1/2), class III histone deacetylases (HDACs), were critical in controlling the destiny of bulk tumor cells and cancer stem cells (CSCs) of UM.,We testified this hypothesis in four lines of UM cells (92.1, Mel 270, Omm 1 and Omm 2.3).,Our results showed that inhibition of SIRT1/2 by Tenovin-6 induced apoptosis in UM cells by activating the expression of tumor suppressor genes such as p53 and elevating reactive oxygen species (ROS).,Tenovin-6 inhibited the growth of UM cells.,Tenovin-6 and vinblastine was synergistic in inducing apoptosis of UM cell line 92.1 and Mel 270.,Furthermore, Tenovin-6 eliminated cancer stem cells in 92.1 and Mel 270 cells.,In conclusion, our findings suggest that Tenovin-6 may be a promising agent to kill UM bulk tumor cells and CSCs.

Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies.,Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments.,Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2.,Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics.,In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines.,Four well characterized UM PDXs were used for in vivo experiments.,S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent.,Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC).,S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect.,However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models.,In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts.,Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration.,The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine.,These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

The failure in achieving a durable clinical immune response against cancer cells depends on the ability of cancer cells to establish a microenvironment that prevent cytotoxic immune cells to infiltrate tumors and kill cancer cells.,Therefore, the key approach to achieving successful antitumor immune response is to harness strategies allowing the reorientation of immune cells to the tumor.,Herein we reveal that inhibiting autophagy induces a massive infiltration of natural killer immune cells into the tumor bed, and a subsequent dramatic decrease in the tumor volume of melanomas.,These results highlight the role of targeting autophagy in breaking the immunosuppressive tumor microenvironment barrier, thus allowing the infiltration of natural killer cells into the tumor to kill cancer cells.,While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown.,Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas.,We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors.,We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5.,Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors.,Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK.,Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells.,Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients’ survival.,We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment.,We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines.,PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation.,Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM.,Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM.,There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines.,Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines.,However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines.,In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

Resistance to targeted cancer therapies is an important clinical problem.,The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms.,To address this challenge, we improved and applied the experimental-computational perturbation biology method.,Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations.,The models are computationally executed to predict the effects of thousands of untested perturbations.,In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK.,Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction.,In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.,DOI:http://dx.doi.org/10.7554/eLife.04640.001,Drugs that target the activity of specific genes could potentially form precise cancer therapies.,Some cancers, including the aggressive skin cancer called melanoma, initially respond well to such treatments.,However, resistance to drugs develops quickly, leading to the rapid regrowth of the tumors.,Resistance can develop in a number of ways.,For example, to prevent the drug from working or to compensate for the effects of a drug, cancer cells can adapt their signaling processes or acquire genetic mutations or other modifications that affect how genes are expressed.,A well-designed combination of drugs that targets multiple molecular pathways can make it harder for cells to resist treatment, as this limits the number of available ‘escape’ pathways that bypass the drug targets.,However, it is difficult to accurately predict how a cell will respond when treated with a particular drug, making it extremely challenging to design effective drug combinations.,In 2013, researchers developed a technique to build predictive models of cellular response pathways based on data collected from perturbation experiments followed by mathematical modeling.,Now, Korkut et al.-including several of the researchers involved in the 2013 work-have refined this technology and applied it to the problem of preventing drug resistance in cancer cells.,Computer simulations that used the mathematical models suggested a particular strategy of ‘upstream-downstream targeting’ in cells that were insensitive to the clinically successful drug vemurafenib (an inhibitor of RAF proteins, which are often mutated in cancers).,In the landscape of signaling pathways, the target of the upstream drug is on or near the mutated RAF protein. c-Myc, the indirect target of the downstream drug helps to express genes that trigger signals that cause the cells to grow.,Inhibiting both targets in parallel may have the dual advantage of blocking the activation of the tumor-specific growth pathway while reducing the cancer cells' attempts to bypass the activation block.,An initial test of the designed drug combination required moving from computer simulations to the laboratory using cell cultures originally derived from melanoma tumors.,When Korkut et al. applied the drug combination, the combined treatment successfully blocked cell growth.,The results suggest that the data-driven computer modeling strategy termed perturbation biology could be a useful tool for identifying effective cancer drug combinations for further preclinical research, possibly followed by clinical trials.,DOI:http://dx.doi.org/10.7554/eLife.04640.002

Recently, melanoma has become the most malignant and commonly occurring skin cancer.,Melanoma is not only the major source (75%) of deaths related to skin cancer, but also it is hard to be treated by the conventional drugs.,Recent research indicated that angiogenesis is an important factor for tumor initiation, expansion, and response to therapy.,Thus, we proposed a novel multi-scale agent-based computational model that integrates the angiogenesis into tumor growth to study the response of melanoma cancer under combined drug treatment.,Our multi-scale agent-based model can simulate the melanoma tumor growth with angiogenesis under combined drug treatment.,The significant synergistic effects between drug Dox and drug Sunitinib demonstrated the clinical potential to interrupt the communication between melanoma cells and its related vasculatures.,Also, the sensitivity analysis of the model revealed that diffusivity related to the micro-vasculatures around tumor tissues closely correlated with the spread, oscillation and destruction of the tumor.,Simulation results showed that the 3D model can represent key features of melanoma growth, angiogenesis, and its related micro-environment.,The model can help cancer researchers understand the melanoma developmental mechanism.,Drug synergism analysis suggested that interrupting the communications between melanoma cells and the related vasculatures can significantly increase the drug efficacy against tumor cells.

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.

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.

Over the past decades, melanoma-related mortality has remained nearly stable.,The main reason is treatment failure of metastatic disease and the inherently linked knowledge gap regarding metastasis formation.,In order to elicit invasion, melanoma cells manipulate the tumor microenvironment, gain motility, and adhere to the extracellular matrix and cancer-associated fibroblasts.,Melanoma cells thereby express different cell adhesion molecules like laminins, integrins, N-cadherin, and others.,Epithelial-mesenchymal transition (EMT) is physiological during embryologic development, but reactivated during malignancy.,Despite not being truly epithelial, neural crest-derived malignancies like melanoma share similar biological programs that enable tumorigenesis, invasion, and metastasis.,This complex phenomenon is termed phenotype switching and is intertwined with oncometabolism as well as dormancy escape.,Additionally, it has been shown that primary melanoma shed exosomes that create a favorable premetastatic niche in the microenvironment of secondary organs and lymph nodes.,Although the growing body of literature describes the aforementioned concepts separately, an integrative holistic approach is missing.,Using melanoma as a tumor model, this review will shed light on these complex biological principles in an attempt to clarify the mechanistic metastatic pathways that dictate tumor and patient fate.

Epacadostat is a potent inhibitor of the immunosuppressive indoleamine 2,3-dioxygenase 1 (IDO1) enzyme.,We present phase 1 results from a phase 1/2 clinical study of epacadostat in combination with ipilimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 antibody, in advanced melanoma (NCT01604889).,Only the phase 1, open-label portion of the study was conducted, per the sponsor’s decision to terminate the study early based on the changing melanoma treatment landscape favoring exploration of programmed cell death protein 1 (PD-1)/PD-ligand 1 inhibitor-based combination strategies.,Such decision was not related to the safety of epacadostat plus ipilimumab.,Patients received oral epacadostat (25, 50, 100, or 300 mg twice daily [BID]; 75 mg daily [50 mg am, 25 mg pm]; or 50 mg BID intermittent [2 weeks on/1 week off]) plus intravenous ipilimumab 3 mg/kg every 3 weeks.,Fifty patients received ≥1 dose of epacadostat.,As of January 20, 2017, 2 patients completed treatment and 48 discontinued, primarily because of adverse events (AEs) and disease progression (n = 20 each).,Dose-limiting toxicities occurred in 11 patients (n = 1 each with epacadostat 25 mg BID, 50 mg BID intermittent, 75 mg daily; n = 4 each with epacadostat 50 mg BID, 300 mg BID).,The most common immune-related treatment-emergent AEs included rash (50%), alanine aminotransferase elevation (28%), pruritus (28%), aspartate aminotransferase elevation (24%), and hypothyroidism (10%).,Among immunotherapy-naive patients (n = 39), the objective response rate was 26% by immune-related response criteria and 23% by Response Evaluation Criteria in Solid Tumors version 1.1.,No objective response was seen in the 11 patients who received prior immunotherapy.,Epacadostat exposure was dose proportional, with clinically significant IDO1 inhibition at doses ≥25 mg BID.,When combined with ipilimumab, epacadostat ≤50 mg BID demonstrated clinical and pharmacologic activity and was generally well tolerated in patients with advanced melanoma.,ClinicalTrials.gov identifier, NCT01604889.,Registration date, May 9, 2012, retrospectively registered.,The online version of this article (10.1186/s40425-019-0562-8) contains supplementary material, which is available to authorized users.

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

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5).,Mouse models have been broadly utilized to study tissue morphogenesis in vivo.,However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans.,Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis.,The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types.,The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis.,Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction.,The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin.,As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes.,Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo.,Recently, dermal stem cells have been identified in the human dermis (6).,These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression.,Notably, melanoma can disseminate using amoeboid invasive strategies.,We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells.,Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation.,Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures.,As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive.,This pattern is further enhanced in metastatic lesions.,We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy.,Amoeboid cells are associated with melanoma invasive capacity.,Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours.

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

Clinical diagnosis of early melanoma (Breslow thickness less than 0.8 mm) is crucial to disease-free survival.,However, it is subjective and can be exceedingly difficult, leading to missed melanomas, or unnecessary excision of benign pigmented skin lesions.,An objective technique is needed to improve the diagnosis of early melanoma.,We have developed a method to improve diagnosis of (thin) melanoma, based on Raman spectroscopy.,In an ex vivo study in a tertiary referral (pigmented lesions) centre, high-wavenumber Raman spectra were collected from 174 freshly excised melanocytic lesions suspicious for melanoma.,Measurements were performed on multiple locations within the lesions.,A diagnostic model was developed and validated on an independent data set of 96 lesions.,Approximately 60% of the melanomas included in this study were melanomas in situ.,The invasive melanomas had an average Breslow thickness of 0.89 mm.,The diagnostic model correctly classified all melanomas (including in situ) with a specificity of 43.8%, and showed a potential improvement of the number needed to treat from 6.0 to 2.7, at a sensitivity of 100%.,This work signifies an important step towards accurate and objective clinical diagnosis of melanoma and in particular melanoma with Breslow thickness <0.8 mm.

The present study was conducted to identify possible dermoscopic patterns, associated with mitotic rate > 1/mm2, histological ulceration in melanoma and metastatic disease.,For this retrospective data analysis all clinical and dermoscopic digital images of primary malignant melanomas between 2008 and 2013 documented at the Department of Dermatology Graz were included, using the internal image data-base. 550 patients with 559 melanomas were included.,While clinical or dermoscopic analysis considered ulceration to be present in 120 (21.5%) and 117 (20.9%) of all lesions, respectively, histopathology reported ulceration in only 96 cases (17.2%).,The presence of milky-red areas, shiny-white streaks, a blue-white veil and blue-grey areas in dermoscopy is highly correlated with histological ulceration and a mitotic rate > 1/mm2.,The dermoscopic patterns shiny-white streaks, milky-red areas and blue-white veil were also significantly associated with development of distant metastases.,Our study proves a significant correlation between the dermoscopic patterns "blue white veil“, "milky-red areas”and "shiny-white streaks”and the histological findings "ulceration”and "mitotic rate > 1/mm2“.,Furthermore these dermoscopic patterns are highly related to distant metastases.,Thus, dermoscopy renders earlier prognostic statements possible.

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.

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

The hypoxic environment within solid tumors impedes the efficacy of chemotherapeutic treatments.,Here, we demonstrate that hypoxia augments the capacity of melanoma cells to withstand cisplatin and doxorubicin cytotoxicity.,We show that B16F10 cells derived from spontaneously formed melanoma and YUMM1.7 cells, engineered to recapitulate human‐relevant melanoma driver mutations, profoundly differ in their vulnerabilities to cisplatin and doxorubicin.,The differences are manifested in magnitude of proliferative arrest and cell death rates, extent of mtDNA depletion, and impairment of mitochondrial respiration.,In both models, cytotoxicity is mitigated by hypoxia, which augments glycolytic metabolism.,Collectively, the findings implicate metabolic reprogramming in drug evasion and suggest that melanoma tumors with distinct genetic makeup may have differential drug vulnerabilities, highlighting the importance of precision anticancer treatments.,Cancer cells have robust drug evasion capabilities.,We show that the genetically distinct B16F10 and YUMM1.7 melanoma models have differential drug vulnerabilities, suggesting that tumors arising from the same tissue may acquire unique weaknesses and differentially respond to therapy.,Notwithstanding, in hypoxia, both cell models exploit metabolic reprogramming for drug evasion that could explain chemotherapy failures in arresting tumor growth.

Recent advances in cancer treatment have emerged from new immunotherapies targeting T-cell inhibitory receptors, including cytotoxic T-lymphocyte associated antigen (CTLA)-4 and programmed cell death (PD)-1.,In this context, anti-CTLA-4 and anti-PD-1 monoclonal antibodies have demonstrated survival benefits in numerous cancers, including melanoma and non-small-cell lung carcinoma.,PD-1-expressing CD8+ T lymphocytes appear to play a major role in the response to these immune checkpoint inhibitors (ICI).,Cytotoxic T lymphocytes (CTL) eliminate malignant cells through recognition by the T-cell receptor (TCR) of specific antigenic peptides presented on the surface of cancer cells by major histocompatibility complex class I/beta-2-microglobulin complexes, and through killing of target cells, mainly by releasing the content of secretory lysosomes containing perforin and granzyme B.,T-cell adhesion molecules and, in particular, lymphocyte-function-associated antigen-1 and CD103 integrins, and their cognate ligands, respectively, intercellular adhesion molecule 1 and E-cadherin, on target cells, are involved in strengthening the interaction between CTL and tumor cells.,Tumor-specific CTL have been isolated from tumor-infiltrating lymphocytes and peripheral blood lymphocytes (PBL) of patients with varied cancers.,TCRβ-chain gene usage indicated that CTL identified in vitro selectively expanded in vivo at the tumor site compared to autologous PBL.,Moreover, functional studies indicated that these CTL mediate human leukocyte antigen class I-restricted cytotoxic activity toward autologous tumor cells.,Several of them recognize truly tumor-specific antigens encoded by mutated genes, also known as neoantigens, which likely play a key role in antitumor CD8 T-cell immunity.,Accordingly, it has been shown that the presence of T lymphocytes directed toward tumor neoantigens is associated with patient response to immunotherapies, including ICI, adoptive cell transfer, and dendritic cell-based vaccines.,These tumor-specific mutation-derived antigens open up new perspectives for development of effective second-generation therapeutic cancer vaccines.

Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit.,We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug.,Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly.,This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal.,Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors.,Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing.,Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.

Thousands of putative enhancers are characterized in the human genome, yet few have been shown to have a functional role in cancer progression.,Inhibiting oncokinases, such as EGFR, ALK, ERBB2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by up-regulation of the HGF receptor, MET.,The mechanisms mediating such genomic responses to targeted therapy are unknown.,Here, we identify lineage-specific enhancers at the MET locus for multiple common tumor types, including a melanoma lineage-specific enhancer 63 kb downstream from the MET TSS.,This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition.,Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function.,Targeted genomic deletion (<7 bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation.,Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of a dominant transcription factor and block innate resistance to oncokinase therapy.

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

Some genetic melanocortin-1 receptor (MC1R) variants responsible for human red hair color (RHC-variants) are consequently associated with increased melanoma risk.,Although MC1R signaling is critically dependent on its palmitoylation primarily mediated by the ZDHHC13 protein-acyl transferase, whether increasing MC1R palmitoylation represents a viable therapeutic target to limit melanomagenesis in redheads is unknown.,Here we identify a specific and efficient in vivo strategy to induce MC1R palmitoylation for therapeutic benefit.,We validate the importance of ZDHHC13 to MC1R signaling in vivo by targeted expression of ZDHHC13 in C57BL/6J-MC1RRHC mice and subsequently inhibit melanomagenesis.,By identifying APT2 as the MC1R depalmitoylation enzyme, we are able to demonstrate that administration of the selective APT2 inhibitor ML349 treatment efficiently increases MC1R signaling and represses UVB-induced melanomagenesis in vitro and in vivo.,Targeting APT2, therefore, represents a preventive/therapeutic strategy to reduce melanoma risk, especially in individuals with red hair.,Melanocortin-1 receptor is a palmitoylated protein and variants of the receptor are associated with red hair colour and susceptibility to melanoma.,Here, the authors describe a method to enhance the palmitoylation of the receptor, which can inhibit melanomagenesis in mice.

MicroRNAs refer to small RNA molecules that destroy the messenger RNA by binding on them inhibiting the production of protein.,However, the role of miR-155 in uveal melanoma metastasis remains largely unknown.,In this study, we found that miR-155 was upregulated in both uveal melanoma cells and tissues.,Transfection of miR-155 mimic into uveal melanoma cells led to an increase in cell growth and invasion; in contrast, inhibition of miR-155 resulted in opposite effects.,Also, we identified Nedd4-family interacting protein 1 as a direct target of miR-155, and the expression of Nedd4-family interacting protein 1 was inhibited by miR-155.,Furthermore, ectopic expression of Nedd4-family interacting protein 1 restored the effects of miR-155 on cell proliferation and invasion of uveal melanoma cells.,In conclusion, miR-155 acts as a tumor promotor in uveal melanoma through increasing cell proliferation and invasion.,Thus, miR-155 might serve as a potential therapeutic target in patients with uveal melanoma.

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

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.

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.

Combining PD-L1 blockade with inhibition of oncogenic mitogen-activated protein kinase (MAPK) signaling may result in long-lasting responses in patients with advanced melanoma.,This phase 1, open-label, dose-escalation and -expansion study (NCT02027961) investigated safety, tolerability and preliminary efficacy of durvalumab (anti-PD-L1) combined with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for patients with BRAF-mutated melanoma (cohort A, n = 26), or durvalumab and trametinib given concomitantly (cohort B, n = 20) or sequentially (cohort C, n = 22) for patients with BRAF-wild type melanoma.,Adverse events and treatment discontinuation rates were more common than previously reported for these agents given as monotherapy.,Objective responses were observed in 69.2% (cohort A), 20.0% (cohort B) and 31.8% (cohort C) of patients, with evidence of improved tumor immune infiltration and durable responses in a subset of patients with available biopsy samples.,In conclusion, combined MAPK inhibition and anti-PD-L1 therapy may provide treatment options for patients with advanced melanoma.,Immune checkpoints inhibitors or MAPK inhibitors are currently used as standard of care therapies for patients with advanced melanoma.,Here the authors report a phase 1 clinical trial testing the anti-PD-L1 antibody durvalumab in combination with the BRAF inhibitor dafrafenib and the MEK inhibitor trametinib in patients with BRAFV600-mutant melanoma.

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.

Eye salvaging therapy of malignant melanomas of the uvea can preserve the eye in most cases, but still about half of patients die from metastatic disease.,Previous analyses of cell‐free DNA from plasma had shown detectable levels of tumor‐specific GNAQ/GNA11 mutations in patients with the clinical diagnosis of progressive disease.,However, data on the time span that elapses from the detection of ctDNA in plasma to the clinical detection of metastases (diagnostic lead time) are missing.,We examined 135 patients with uveal melanoma.,Cell‐free DNA was isolated from a total of 807 blood samples which were taken over a period of up to 41 months and analyzed for the presence of GNAQ/GNA11 mutations by deep amplicon sequencing.,Twenty‐one of the 135 patients developed metastases or recurrence.,A ctDNA signal was identified in the plasma of 17 of the 21 patients.,In 10 patients, this ctDNA signal preceded the clinical diagnosis of metastasis by 2-10 months.,In 10 other patients, a ctDNA signal was only detected in samples obtained shortly before or after radiotherapy.,The presence of a ctDNA signal in 16 of the remaining 125 patients was linked to clinical manifestation of metastases (n = 14) or tumor recurrence (n = 2) with a sensitivity and specificity of 80% and 96%, respectively.,Detection of ctDNA in plasma can provide a diagnostic lead time over the clinical diagnosis of metastases or tumor recurrence.,Longer lead times are to be expected if intervals between sampling are shortened.,In metastasized uveal melanoma (UM) patients, circulating tumor DNA (ctDNA) can be detected in blood.,Here we explored if cfDNA is a suitable biomarker for the early detection of metastatic disease in UM patients.,Our data show that this biomarker fulfills the expectation as, overall, about half of the patients who developed metastases showed a positive ctDNA signal prior to the clinical diagnosis of metastatic disease with a lead time ranging between 2 and 10 months.,Moreover, it is reasonable that, with more frequent sampling time points, diagnostic lead times will be even longer.

The outcomes of patients with stage III cutaneous melanoma who undergo complete surgical resection can be highly variable, and estimation of individual risk of disease recurrence and mortality remains imprecise.,With recent demonstrations of effective adjuvant targeted and immune checkpoint inhibitor therapy, more precise stratification of patients for costly and potentially toxic adjuvant therapy is needed.,We report the utility of pre-operative circulating tumour DNA (ctDNA) in patients with high-risk stage III melanoma.,ctDNA was analysed in blood specimens that were collected pre-operatively from 174 patients with stage III melanoma undergoing complete lymph node (LN) dissection.,Cox regression analyses were used to evaluate the prognostic significance of ctDNA for distant metastasis recurrence-free survival and melanoma-specific survival (MSS).,The detection of ctDNA in the discovery and validation cohort was 34% and 33%, respectively, and was associated with larger nodal melanoma deposit, higher number of melanoma involved LNs, more advanced stage and high lactate dehydrogenase (LDH) levels.,Detectable ctDNA was significantly associated with worse MSS in the discovery [hazard ratio (HR) 2.11 P < 0.01] and validation cohort (HR 2.29, P = 0.04) and remained significant in a multivariable analysis (HR 1.85, P = 0.04). ctDNA further sub-stratified patients with AJCC stage III substage, with increasing significance observed in more advanced stage melanoma.,Pre-operative ctDNA predicts MSS in high-risk stage III melanoma patients undergoing complete LN dissection, independent of stage III substage.,This biomarker may have an important role in determining prognosis and stratifying patients for adjuvant treatment.

The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity.,Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity.,In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma.,Patients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma.,Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I).,Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label.,Between July 2014 and July 2017, 131 patients were enrolled.,(P) was used more frequently (n=114, 87%) per investigator’s choice.,The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).,The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%.,Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9).,The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients.,The combination was well tolerated, and side effects were similar to what was expected from single agent (P).,In this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy.,Delineating mechanisms of secondary resistance to this strategy is increasingly important.,Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb).,Sustained IFNβ transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC).,Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation.,Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature.,Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy.

Long noncoding RNAs (lncRNAs) play pivotal roles in various malignant tumors.,Epidermal growth factor receptor (EGFR) signaling is associated with the pathogenesis of cutaneous squamous cell carcinoma (cSCC).,This study aimed to explore the role of LINC00520 in the development of cSCC via EGFR and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways.,A microarray analysis was applied to screen differentially expressed lncRNAs in cSCC samples.,The A431 cSCC cell line was transfected and assigned different groups.,The expression patterns of LINC00520, EGFR, and intermediates in the PI3K/Akt pathway were characterized using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis.,Cell proliferation, migration, and invasion were detected using the MTT assay, scratch test, and Transwell assay, respectively.,Cell-based experiments and a tumorigenicity assay were conducted to assess the effect of LINC00520 on cSCC progression.,This study was ended in September 2017.,Comparisons between two groups were analyzed with t-test and comparisons among multiple groups were analyzed using one-way analysis of variance.,The nonparametric Wilcoxon rank sum test was used to analyze skewed data.,The enumerated data were analyzed using the chi-square test or Fisher exact test.,Data from chip GSE66359 revealed depletion of LINC00520 in cSCC.,Cells transfected with LINC00520 vector and LINC00520 vector + si-EGFR showed elevated LINC00520 level but decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the si-LINC00520 group showed opposite trends (all P < 0.05).,Compared with the LINC00520 vector group, the LINC00520 vector + si-EGFR group showed decreased levels of the EGFR, PI3K, AKT, VEGF, MMP-2 and MMP-9 mRNAs and proteins, and inhibition of the growth, migration and adhesion of cSCC cells, while the LINC00520 vector + EGFR vector group showed opposite results (all P < 0.05).,Based on our results, LINC00520-targeted EGFR inhibition might result in the inactivation of the PI3K/Akt pathway, thus inhibiting cSCC development.

Basal cell carcinoma (BCC) is the most common human cancer and represents a growing public health care problem.,Several tumor suppressor genes and proto-oncogenes have been implicated in BCC pathogenesis, including the key components of the Hedgehog pathway, PTCH1 and SMO, the TP53 tumor suppressor, and members of the RAS proto-oncogene family.,Aberrant activation of the Hedgehog pathway represents the molecular driver in basal cell carcinoma pathogenesis, with the majority of BCCs carrying somatic point mutations, mainly ultraviolet (UV)-induced, and/or copy-loss of heterozygosis in the PTCH1 gene.,Recent advances in sequencing technology allowed genome-scale approaches to mutation discovery, identifying new genes and pathways potentially involved in BCC carcinogenesis.,Mutational and functional analysis suggested PTPN14 and LATS1, both effectors of the Hippo-YAP pathway, and MYCN as new BCC-associated genes.,In addition, emerging reports identified frequent non-coding mutations within the regulatory promoter sequences of the TERT and DPH3-OXNAD1 genes.,Thus, it is clear that a more complex genetic network of cancer-associated genes than previously hypothesized is involved in BCC carcinogenesis, with a potential impact on the development of new molecular targeted therapies.,This article reviews established knowledge and new hypotheses regarding the molecular genetics of BCC pathogenesis.

The incidence of malignant melanoma is rapidly increasing and current medicine is offering only limited options for treatment of the advanced disease.,For B-Raf mutated melanomas, treatment with mutation-specific drug inhibitors may be used.,Unfortunately, tumors frequently acquire resistance to the treatment.,Tumor microenvironment, namely cancer-associated fibroblasts, largely influence this acquired resistance.,In the present study, fibroblasts were isolated from a patient suffering from acrolentiginous melanoma (Breslow, 4.0 mm; Clark, IV; B-Raf V600E mutated).,The present study focused on the expression of structural and functional markers of fibroblast activation in melanoma-associated fibroblasts (MAFs; isolated prior to therapy initiation) as well as in autologous control fibroblasts (ACFs) of the same patient isolated during B-Raf inhibitor therapy, yet before clinical progression of the disease.,Analysis of gene transcription was also performed, as well as DNA methylation status analysis at the genomic scale of both isolates.,MAFs were positive for smooth muscle actin (SMA), which is a marker of myofibroblasts and the hallmark of cancer stoma.,Surprisingly, ACF isolated from the distant uninvolved skin of the same patient also exhibited strong SMA expression.,A similar phenotype was also observed in control dermal fibroblasts (CDFs; from different donors) exclusively following stimulation by transforming growth factor (TGF)-β1.,Immunohistochemistry confirmed that melanoma cells potently produce TGF-β1.,Significant differences were also identified in gene transcription and in DNA methylation status at the genomic scale.,Upregulation of SMA was observed in ACF cells at the protein and transcriptional levels.,The present results support recent experimental findings that tumor microenvironment is driving resistance to B-Raf inhibition in patients with melanoma.,Such an activated microenvironment may be viable for the growth of circulating melanoma cells.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo.,Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression.,The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells.,Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells.,In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development.,A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses.,NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance.,We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival.,Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy.,Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis.,Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types.

Melanoma cells driven by mutant B-RAF are highly resistant to chemotherapeutic treatments.,Recent Phase 1 results with PLX4032/RG7204/Vemurafenib, which selectively inhibits B-RAF/MEK/ERK1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field.,Nearly all patients in the phase 1-3 studies saw at least some response and the overall response rates were in between 81 and 48%.,However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time.,These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032.,It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond.,We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is up-regulated following inhibition of B-RAF-MEK signaling in mutant B-RAF melanoma cells.,Here, we show that up-regulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death.,Small interfering RNA (siRNA)-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines.,Additionally, up-regulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death.,Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment.,Together, these data indicate that up-regulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance.

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas and targeting MEK as central kinase of this pathway is currently tested in clinical trials.,However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response.,Apart from dose-limitations, a reason for the low response to MEK targeting drugs is thought to be the up-regulation of counteracting signalling cascades as a direct response to MEK inhibition.,Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of drug being used.,We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352 while efficiently suppressing proliferation stimulates increased invasiveness.,Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion.,Most importantly, and surprisingly MEK inhibition results in a significant increase in MMP-2 and MT1-MMP expression.,All together MEK inhibition in melanoma cells induces a ‘mesenchymal’ phenotype that is characterised by protease driven invasion.,This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are main regulators of this process, the SRC kinase inhibitor saracatinib (AZD0530) completely abolished the MEK inhibitor induced invasion.,Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.

Cell-type specific signalling determines cell fate under physiological conditions, but it is increasingly apparent that also in cancer development the impact of any given oncogenic pathway on the individual cancer pathology is dependent on cell-lineage specific molecular traits.,For instance in colon and liver cancer canonical Wnt signalling produces increased cytoplasmic and nuclear localised beta-catenin, which correlates with invasion and poor prognosis.,In contrast, in melanoma increased cytoplasmic and nuclear beta-catenin is currently emerging as a marker for good prognosis and thus appears to have a different function compared to other cancer types; however this function is unknown.,We discovered that in contrast to its function in other cancers, in melanoma, beta-catenin blocks invasion.,We demonstrate that this opposing role of nuclear beta-catenin in melanoma is mediated through MITF, a melanoma-specific protein that defines the lineage background of this cancer type.,Downstream of beta-catenin MITF not only suppresses the Rho-GTPase regulated cell-morphology of invading melanoma cells, but also interferes with beta-catenin induced expression of the essential collagenase MT1-MMP, thus affecting all aspects of an invasive phenotype.,Importantly, overexpression of MITF in invasive colon cancer cells modifies beta-catenin directed signalling and induces a ‘melanoma-phenotype’.,In summary, the cell type specific presence of MITF in melanoma affects beta-catenin’s pro-invasive properties otherwise active in colon or liver cancer.,Thus our study reveals the general importance of considering cell-type specific signalling for the accurate interpretation of tumour markers and ultimately for the design of rational therapies.

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-negative regulator of receptor tyrosine kinase (RTK) signaling and a tumor suppressor in several cancers, but its involvement in melanoma is largely unexplored.,Here, we aim to determine the role of LRIG1 in melanoma tumorigenesis, RTK signaling, and BRAF inhibitor resistance.,We find that LRIG1 is downregulated during early tumorigenesis and that LRIG1 affects activation of the epidermal growth factor receptor (EGFR) in melanoma cells.,LRIG1-dependent regulation of EGFR signaling is evolutionary conserved to the roundworm C. elegans, where negative regulation of the EGFR-Ras-Raf pathway by sma-10/LRIG completely depends on presence of the receptor let-23/EGFR.,In a cohort of metastatic melanoma patients, we observe an association between LRIG1 and survival in the triple wild-type subtype and in tumors with high EGFR expression.,During in vitro development of BRAF inhibitor resistance, LRIG1 expression decreases; and mimics LRIG1 knockout cells for increased EGFR expression.,Treating resistant cells with recombinant LRIG1 suppresses AKT activation and proliferation.,Together, our results show that sma-10/LRIG is a conserved regulator of RTK signaling, add to our understanding of LRIG1 in melanoma and identifies recombinant LRIG1 as a potential therapeutic against BRAF inhibitor-resistant melanoma.

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.

Uveal melanoma (UM) is the most common intraocular malignancy in adults.,In contrast to cutaneous melanoma (CM), there is no standard therapy, and the efficacy and safety of dual checkpoint blockade with nivolumab and ipilimumab is not well defined.,We conducted a retrospective analysis of patients with metastatic UM (mUM) who received treatment with ipilimumab plus nivolumab across 14 academic medical centers.,Toxicity was graded using National Cancer Institute Common Terminology Criteria for Adverse Events V.5.0.,Progression-free survival (PFS) and overall survival (OS) were calculated using Kaplan-Meier methodology.,89 eligible patients were identified. 45% had received prior therapy, which included liver directed therapy (29%), immunotherapy (21%), targeted therapy (10%) and radiation (16%).,Patients received a median 3 cycles of ipilimumab plus nivolumab.,The median follow-up time was 9.2 months.,Overall response rate was 11.6%.,One patient achieved complete response (1%), 9 patients had partial response (10%), 21 patients had stable disease (24%) and 55 patients had progressive disease (62%).,Median OS from treatment initiation was 15 months and median PFS was 2.7 months.,Overall, 82 (92%) of patients discontinued treatment, 34 due to toxicity and 27 due to progressive disease.,Common immune-related adverse events were colitis/diarrhea (32%), fatigue (23%), rash (21%) and transaminitis (21%).,Dual checkpoint inhibition yielded higher response rates than previous reports of single-agent immunotherapy in patients with mUM, but the efficacy is lower than in metastatic CM.,The median OS of 15 months suggests that the rate of clinical benefit may be larger than the modest response rate.

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

The incidence of epidermal keratinocyte‐derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide.,To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC.,The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real‐time polymerase chain reaction.,The production of C1r and C1s was determined with Western blotting.,The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s.,Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes.,The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin.,Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa‐associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin.,Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal‐related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo.,These results provide evidence for the role of tumour‐cell‐derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC.,What's already known about this topic?,The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally.Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC.,The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally.,Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC.,What does this study add?,Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC.,Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC.,What is the translational message?,Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.,Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.,Linked Comment: https://doi.org/10.1111/bjd.18419.,https://doi.org/10.1111/bjd.18821 available online

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.

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.

Melanocyte development provides an excellent model for studying more complex developmental processes.,Melanocytes have an apparently simple aetiology, differentiating from the neural crest and migrating through the developing embryo to specific locations within the skin and hair follicles, and to other sites in the body.,The study of pigmentation mutations in the mouse provided the initial key to identifying the genes and proteins involved in melanocyte development.,In addition, work on chicken has provided important embryological and molecular insights, whereas studies in zebrafish have allowed live imaging as well as genetic and transgenic approaches.,This cross-species approach is powerful and, as we review here, has resulted in a detailed understanding of melanocyte development and differentiation, melanocyte stem cells and the role of the melanocyte lineage in diseases such as melanoma.,Summary: This Review discusses melanocyte development and differentiation, melanocyte stem cells, and the role of the melanocyte lineage in diseases such as melanoma.

Recently introduced systemic therapies for locally advanced and metastatic non-melanoma skin cancers (NMSCs) are paving the way for neoadjuvant approach.,Although none of the therapeutic options has currently gained indication in this setting, neoadjuvant approach for NMSCs is an open field and we are likely to see huge developments in the near future.,Targeted therapy with sonic hedgehog pathway inhibitors is very effective in locally advanced or multiple basal cell carcinomas while immunotherapy with immune checkpoint inhibitors appears to be promising for advanced cutaneous squamous cell carcinoma and Merkel cell carcinoma.,To date, targeted therapy and immunotherapy represent the frontiers in NMSC therapeutic management and, according to recent studies, good results can be achieved.

Non-melanoma skin cancers (NMSCs) include basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and Merkel cell carcinoma (MCC).,These neoplasms are highly diverse in their clinical presentation, as well as in their biological evolution.,While the deregulation of the Hedgehog pathway is commonly observed in BCC, SCC and MCC are characterized by a strikingly elevated mutational and neoantigen burden.,As result of our improved understanding of the biology of non-melanoma skin cancers, innovative treatment options including inhibitors of the Hedgehog pathway and immunotherapeutic agents have been recently investigated against these malignancies, leading to their approval by regulatory authorities.,Herein, we review the most relevant biological and clinical features of NMSC, focusing on innovative treatment approaches.

Metastatic melanoma is a devastating disease with limited therapeutic options.,MicroRNAs (miRNAs) are small non coding RNA molecules with important roles in post-transcriptional gene expression regulation, whose aberrant expression has been implicated in cancer.,We show that the expression of miRNAs from a large cluster on human chromosome 14q32 is significantly down-regulated in melanoma cell lines, benign nevi and melanoma samples relative to normal melanocytes.,This miRNA cluster resides within a parentally imprinted chromosomal region known to be important in development and differentiation.,In some melanoma cell lines, a chromosomal deletion or loss-of-heterozygosity was observed in the cis-acting regulatory region of this cluster.,In several cell lines we were able to re-express two maternally-induced genes and several miRNAs from the cluster with a combination of de-methylating agents and histone de-acetylase inhibitors, suggesting that epigenetic modifications take part in their silencing.,Stable over-expression of mir-376a and mir-376c, two miRNAs from this cluster that could be re-expressed following epigenetic manipulation, led to modest growth retardation and to a significant decrease in migration in-vitro.,Bioinformatic analysis predicted that both miRNAs could potentially target the 3'UTR of IGF1R.,Indeed, stable expression of mir-376a and mir-376c in melanoma cells led to a decrease in IGF1R mRNA and protein, and a luciferase reporter assay indicated that the 3'UTR of IGF1R is a target of both mir-376a and mir-376c.,Our work is the first to show that the large miRNA cluster on chromosome 14q32 is silenced in melanoma.,Our results suggest that down-regulation of mir-376a and mir-376c may contribute to IGF1R over-expression and to aberrant negative regulation of this signaling pathway in melanoma, thus promoting tumorigenesis and metastasis.

MicroRNAs (miRNAs) are 18-23 nucleotide non-coding RNAs that regulate gene expression in a sequence specific manner.,Little is known about the repertoire and function of miRNAs in melanoma or the melanocytic lineage.,We therefore undertook a comprehensive analysis of the miRNAome in a diverse range of pigment cells including: melanoblasts, melanocytes, congenital nevocytes, acral, mucosal, cutaneous and uveal melanoma cells.,We sequenced 12 small RNA libraries using Illumina's Genome Analyzer II platform.,This massively parallel sequencing approach of a diverse set of melanoma and pigment cell libraries revealed a total of 539 known mature and mature-star sequences, along with the prediction of 279 novel miRNA candidates, of which 109 were common to 2 or more libraries and 3 were present in all libraries.,Some of the novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers to assist in the early detection of distant metastases by measuring the circulating levels in blood.,Follow up studies of the functional roles of these pigment cell miRNAs and the identification of the targets should shed further light on the development and progression of melanoma.

Skin lesions are a severe disease globally.,Early detection of melanoma in dermoscopy images significantly increases the survival rate.,However, the accurate recognition of melanoma is extremely challenging due to the following reasons: low contrast between lesions and skin, visual similarity between melanoma and non-melanoma lesions, etc.,Hence, reliable automatic detection of skin tumors is very useful to increase the accuracy and efficiency of pathologists.,In this paper, we proposed two deep learning methods to address three main tasks emerging in the area of skin lesion image processing, i.e., lesion segmentation (task 1), lesion dermoscopic feature extraction (task 2) and lesion classification (task 3).,A deep learning framework consisting of two fully convolutional residual networks (FCRN) is proposed to simultaneously produce the segmentation result and the coarse classification result.,A lesion index calculation unit (LICU) is developed to refine the coarse classification results by calculating the distance heat-map.,A straight-forward CNN is proposed for the dermoscopic feature extraction task.,The proposed deep learning frameworks were evaluated on the ISIC 2017 dataset.,Experimental results show the promising accuracies of our frameworks, i.e., 0.753 for task 1, 0.848 for task 2 and 0.912 for task 3 were achieved.

An infrared (IR) absorbance sensor has been designed, realized and tested with the aim of detecting malignant melanomas in human skin biopsies.,The sensor has been designed to obtain fast measurements (80 s) of a biopsy using a small light spot (0.5 mm in diameter, typically five to 10 times smaller than the biopsy size) to investigate different biopsy areas.,The sensor has been equipped with a monochromator to record the whole IR spectrum in the 3330-3570 nm wavelength range (where methylene and methyl stretching vibrations occur) for a qualitative spectral investigation.,From the collected spectra, the CH2 stretch ratio values (ratio of the absorption intensities of the symmetric to asymmetric CH2 stretching peaks) are determined and studied as a cancer indicator.,Melanoma areas exhibit different spectral shapes and significantly higher CH2 stretch ratios when compared to healthy skin.,The results of the infrared investigation are compared with standard histology.,This study shows that the IR sensor is a promising supportive tool to improve the diagnosis of melanoma during histopathological analysis, decreasing the risk of misdiagnosis.

Although transformation of melanocytes to melanoma is rare, the rapid growth, systemic spread, as well as the chemoresistance of melanoma present significant challenges for patient care.,Here we review animal models of melanoma, including murine, canine, equine, and zebrafish models, and detail the immense contribution these models have made to our knowledge of human melanoma development, and to melanocyte biology.,We also highlight the opportunities for cross‐species comparative genomic studies of melanoma to identify the key molecular events that drive this complex disease.,© 2015 The Authors.,The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

E1697 was a phase III trial of adjuvant interferon (IFN)-α2b for one month (Arm B) versus observation (Arm A) in patients with resected melanoma at intermediate risk.,We evaluated the levels of candidate serum cytokines, the HLA genotype, polymorphisms of CTLA4 and FOXP3 genes and the development of autoantibodies for their association with relapse free survival (RFS) in Arm A and Arm B among 268 patients with banked biospecimens.,ELISA was used to test 5 autoantibodies.,Luminex/One Lambda LABTypeRSSO was used for HLA Genotyping.,Selected CTLA4 and FOXP3 Single nucleotide polymorphisms (SNPs) and microsatellites were tested for by polymerase chain reaction (PCR).,Sixteen serum cytokines were tested at baseline and one month by Luminex xMAP multiplex technology.,Cox Proportional Hazards model was applied and the Wald test was used to test the marginal association of each individual marker and RFS.,We used the Lasso approach to select the markers to be included in a multi-marker Cox Proportional Hazards model.,The ability of the resulting models to predict one year RFS was evaluated by the time-dependent ROC curve.,The leave-one-out method of cross validation (LOOCV) was used to avoid over-fitting of the data.,In the multi-marker modeling analysis conducted in Arm B, one month serum IL2Rα, IL-12p40 and IFNα levels predicted one year RFS with LOOCV AUC = 82%.,Among the three markers selected, IL2Rα and IFNα were the most stable (selected in all the cross validation cycles).,The risk score (linear combination of the 3 markers) separated the RFS curves of low and high risk groups well (p = 0.05).,This model did not hold for Arm A, indicating a differential marker profile in Arm B linked to the intervention (adjuvant therapy).,Early on-treatment proinflammatory serum markers (IL2Rα, IL-12p40, IFNα) significantly predict RFS in our cohort of patients treated with adjuvant IFN-α2b and warrant further study.

Mutations in GNAQ and GNA11, encoding the oncogenic G-protein alpha subunit q and 11, respectively, occur frequently in the majority of uveal melanomas.,Exons 4 and 5 from GNAQ and GNA11 were amplified and sequenced from 92 ciliary body and choroidal melanomas.,The mutation status was correlated with disease-free survival (DFS) and other parameters.,None of the tumours harboured a GNAQ exon 4 mutation.,A GNAQ mutation in exon 5 codon 209 was found in 46 out of 92 (50.0%) of the tumours.,Only 1 out of 92 (1.1%) melanomas showed a mutation in GNA11 exon 4 codon 183, whereas 39 out of 92 (42.4%) harboured a mutation in exon 5 of GNA11 codon 209.,Six tumours did not show any mutations in exons 4 and 5 of these genes.,Univariate analyses showed no correlation between DFS and the mutation status.,GNAQ and GNA11 mutations are, in equal matter, not associated with patient outcome.

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.

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

Melanoma represents a significant malignancy in humans and dogs.,Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model.,Canine melanomas rarely arise in sun-exposed sites.,Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma.,The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis.,Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs.,Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation.,We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas.,This represents opportunity to explore canine oral cavity melanoma as a preclinical model.

Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit.,We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug.,Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly.,This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal.,Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors.,Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing.,Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.

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.

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

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer.,BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma.,In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436).,Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death.,In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition.,However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors.,In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models.,Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab).,Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG.,While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB.,All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB+ cells.,This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance.,As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.,Agonistic 4-1BB antibodies developed for cancer immunotherapy have suffered from either hepatotoxicity or insufficient anti-cancer activity.,Here the authors determine the contribution of FcγR binding and agonistic strength to these outcomes, and engineer a 4-1BB antibody with potent anti-tumor effect and no liver toxicity in mice.

Whereas significant anti-tumor responses are observed in most BRAFV600E-mutant melanoma patients exposed to MAPK-targeting agents, resistance almost invariably develops.,Here, we show that in therapy-responsive cells BRAF inhibition induces downregulation of the processing of Sterol Regulator Element Binding (SREBP-1) and thereby lipogenesis.,Irrespective of the escape mechanism, therapy-resistant cells invariably restore this process to promote lipid saturation and protect melanoma from ROS-induced damage and lipid peroxidation.,Importantly, pharmacological SREBP-1 inhibition sensitizes BRAFV600E-mutant therapy-resistant melanoma to BRAFV600E inhibitors both in vitro and in a pre-clinical PDX in vivo model.,Together, these data indicate that targeting SREBP-1-induced lipogenesis may offer a new avenue to overcome acquisition of resistance to BRAF-targeted therapy.,This work also provides evidence that targeting vulnerabilities downstream of oncogenic signaling offers new possibilities in overcoming resistance to targeted therapies.,Melanoma patients harbouring BRAFV600E mutation generally develop resistance to targeted therapy.,In this study, the authors demonstrate that SREBP-1-mediated induction of lipid biosynthesis contributes to therapy resistance in BRAF mutant melanoma.

Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome.,Similarly to other tumors, permissive microenvironment is essential for melanoma progression.,Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue.,Here, we study the effect of melanoma cells on human primary keratinocytes (HPK).,Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).,Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed.,Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively.,Differentially expressed candidate genes were verified by RT-qPCR.,Biological activity of candidate proteins was assessed on cultured HPK.,Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases.,This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10.,We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK.,This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies.,We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.,We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro.,This interaction further highlights the role of intercellular interactions in melanoma.,The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.,The online version of this article (doi:10.1186/1476-4598-14-1) contains supplementary material, which is available to authorized users.

During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype.,Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis.,However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated.,Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes.,Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively.,SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells.,Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells.,We also observed that pharmacological inhibition of oncogenic BRAFV600E using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAFV600E.,Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration.,Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene.,In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found.,These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.

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

Cutaneous head and neck melanoma has poor outcomes and limited treatment options.,In OPTiM, a phase 3 study in patients with unresectable stage IIIB/IIIC/IV melanoma, intralesional administration of the oncolytic virus talimogene laherparepvec improved durable response rate (DRR; continuous response ≥6 months) compared with subcutaneous granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).,Retrospective review of OPTiM identified patients with cutaneous head and neck melanoma given talimogene laherparepvec (n = 61) or GM‐CSF (n = 26).,Outcomes were compared between talimogene laherparepvec and GM‐CSF treated patients with cutaneous head and neck melanoma.,DRR was higher for talimogene laherparepvec-treated patients than for GM‐CSF treated patients (36.1% vs 3.8%; p = .001).,A total of 29.5% of patients had a complete response with talimogene laherparepvec versus 0% with GM‐CSF.,Among talimogene laherparepvec-treated patients with a response, the probability of still being in response after 12 months was 73%.,Median overall survival (OS) was 25.2 months for GM‐CSF and had not been reached with talimogene laherparepvec.,Treatment with talimogene laherparepvec was associated with improved response and survival compared with GM‐CSF in patients with cutaneous head and neck melanoma.,© 2016 Wiley Periodicals, Inc.,Head Neck 38: 1752-1758, 2016

T-cell-based immunotherapies are promising treatments for cancer patients.,Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies.,From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy.,We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo.,Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells.,Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.,Many patients fail to respond to T cell based immunotherapies.,Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma.,However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8+ T cells for infusion.,One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8+ effector-memory T-cell expansion.,In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8+ T cells, on the yield, phenotype, and functional activity of expanded CD8+ T cells during the REP.,We found that CD8+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand.,However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity.,Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8+ post-REP TIL when re-cultured in the absence or presence of cytokines.,Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival.,This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.