Datasets:

GleghornLab

/

abstract_domain_skincancer

like 0

Objective To synthesise the literature on indoor tanning and non-melanoma skin cancer.,Design Systematic review and meta-analysis.,Data sources PubMed (1966 to present), Embase (1974 to present), and Web of Science (1898 to present).,Study selection All articles that reported an original effect statistic for indoor tanning and non-melanoma skin cancer were included.,Articles that presented no data, such as review articles and editorials, were excluded, as were articles in languages other than English.,Data extraction Two investigators independently extracted data.,Random effects meta-analysis was used to summarise the relative risk of ever use versus never use of indoor tanning.,Dose-response effects and exposure to indoor tanning during early life were also examined.,The population attributable risk fraction for the United States population was calculated.,Results 12 studies with 9328 cases of non-melanoma skin cancer were included.,Among people who reported ever using indoor tanning compared with those who never used indoor tanning, the summary relative risk for squamous cell carcinoma was 1.67 (95% confidence interval 1.29 to 2.17) and that for basal cell carcinoma was 1.29 (1.08 to 1.53).,No significant heterogeneity existed between studies.,The population attributable risk fraction for the United States was estimated to be 8.2% for squamous cell carcinoma and 3.7% for basal cell carcinoma.,This corresponds to more than 170 000 cases of non-melanoma skin cancer each year attributable to indoor tanning.,On the basis of data from three studies, use of indoor tanning before age 25 was more strongly associated with both squamous cell carcinoma (relative risk 2.02, 0.70 to 5.86) and basal cell carcinoma (1.40, 1.29 to 1.52).,Conclusions Indoor tanning is associated with a significantly increased risk of both basal and squamous cell skin cancer.,The risk is higher with use in early life (<25 years).,This modifiable risk factor may account for hundreds of thousands of cases of non-melanoma skin cancer each year in the United States alone and many more worldwide.,These findings contribute to the growing body of evidence on the harms of indoor tanning and support public health campaigns and regulation to reduce exposure to this carcinogen.

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.

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

Solid organ transplant recipients, who are medically immunosuppressed to prevent graft rejection, have increased melanoma risk, but risk factors and outcomes are incompletely documented.,We evaluated melanoma incidence among 139,991 non-Hispanic white transplants using linked U.S. transplant-cancer registry data (1987-2010).,We used standardized incidence ratios (SIRs) to compare incidence to the general population, and incidence rate ratios (IRRs) from multivariable Poisson models to assess risk factors.,Separately, we compared post-melanoma survival among transplant recipients (N=182) and non-recipients (N=131,358) using multivariable Cox models.,Among transplant recipients, risk of invasive melanoma (N=519) was elevated (SIR=2.20, 95%CI 2.01-2.39), especially for regional stage tumors (SIR=4.11, 95%CI 3.27-5.09).,Risk of localized tumors was stable over time after transplantation, but higher with azathioprine maintenance therapy (IRR=1.35, 95%CI 1.03-1.77).,Risk of regional/distant stage tumors peaked within 4 years following transplantation and increased with polyclonal antibody induction therapy (IRR=1.65, 95%CI 1.02-2.67).,Melanoma-specific mortality was higher among transplant recipients than non-recipients (HR 2.98, 95%CI 2.26-3.93).,Melanoma exhibits increased incidence and aggressive behavior under transplant-related immunosuppression.,Some localized melanomas may result from azathioprine, which acts synergistically with ultraviolet radiation, while T-cell depleting induction therapies may promote late stage tumors.,Our findings support sun safety practices and skin screening for transplant recipients.

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer.,Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood.,Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells.,Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell.,Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods.,By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro.,In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro.,These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

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

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

Clinical reports of limited and treatable cancer metastases, a disease state that exists in a transitional zone between localized and widespread systemic disease, were noted on occasion historically and are now termed oligometastasis.,The ramification of a diagnosis of oligometastasis is a change in treatment paradigm, i.e. if the primary cancer site (if still present) is controlled, or resected, and the metastatic sites are ablated (surgically or with radiation), a prolonged disease-free interval, and perhaps even cure, may be achieved.,Contemporary molecular diagnostics are edging closer to being able to determine where an individual metastatic deposit is within the continuum of malignancy.,Preclinical models are on the outset of laying the groundwork for understanding the oligometastatic state.,Meanwhile, in the clinic, patients are increasingly being designated as having oligometastatic disease and being treated owing to improved diagnostic imaging, novel treatment options with the potential to provide either direct or bridging therapy, and progressively broad definitions of oligometastasis.

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

The present study aimed to investigate the potential role of the long non-coding RNA (lncRNA) Pvt1 oncogene (non-protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms.,The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A-375 and sk-mel-5 cell lines, was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analyses.,The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively.,The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay.,The effect of PVT1 on microRNA-200c (miR-200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay.,PVT1 was highly expressed in the melanoma tissues and cells.,Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage.,Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells.,The expression of E-cadherin was significantly increased and the expression of N-cadherin and vimentin was significantly decreased in the PVT1-silenced group.,The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control.,The ChIP assay revealed that the expression of miR-200c was decreased significantly in the PVT1-silenced group compared with the controls.,Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR-200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.

Melanoma is the most aggressive and dangerous form of skin cancer that develops from transformed melanocytes.,It is crucial to identify melanoma at its early stages, in situ, as it is “curable” at this stage.,However, after metastasis, it is difficult to treat and the five-year survival is only 25%.,In recent years, a better understanding of the etiology of melanoma and its progression has made it possible for the development of targeted therapeutics, such as vemurafenib and immunotherapies, to treat advanced melanomas.,In this review, we focus on the molecular mechanisms that mediate melanoma development and progression, with a special focus on the immune evasion strategies utilized by melanomas, to evade host immune surveillances.,The proposed mechanism of action and the roles of immunotherapeutic agents, ipilimumab, nivolumab, pembrolizumab, and atezolizumab, adoptive T- cell therapy plus T-VEC in the treatment of advanced melanoma are discussed.,In this review, we implore that a better understanding of the steps that mediate melanoma onset and progression, immune evasion strategies exploited by these tumor cells, and the identification of biomarkers to predict treatment response are critical in the design of improved strategies to improve clinical outcomes for patients with this deadly disease.

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.

miRNAs are central players in cancer biology and they play a pivotal role in mediating the network communication between tumor cells and their microenvironment.,In melanoma, miRNAs can impair or facilitate a wide array of processes, and here we will focus on: the epithelial to mesenchymal transition (EMT), the immune milieu, and metabolism.,Multiple miRNAs can affect the EMT process, even at a distance, for example through exosome-mediated mechanisms. miRNAs also strongly act on some components of the immune system, regulating the activity of key elements such as antigen presenting cells, and can facilitate an immune evasive/suppressive phenotype. miRNAs are also involved in the regulation of metabolic processes, specifically in response to hypoxic stimuli where they can mediate the metabolic switch from an oxidative to a glycolytic metabolism.,Overall, this review discusses and summarizes recent findings on miRNA regulation in the melanoma tumor microenvironment, analyzing their potential diagnostic and therapeutic applications.

Epidermal stem cells (ESCs) are crucial for maintenance and self- renewal of skin epithelium and also for regular hair cycling.,Their role in wound healing is also indispensable.,ESCs reside in a defined outer root sheath portion of hair follicle-also known as the bulge region.,ECS are also found between basal cells of the interfollicular epidermis or mucous membranes.,The non-epithelial elements such as mesenchymal stem cell-like elements of dermis or surrounding adipose tissue can also contribute to this niche formation.,Cancer stem cells (CSCs) participate in formation of common epithelial malignant diseases such as basal cell or squamous cell carcinoma.,In this review article, we focus on the role of cancer microenvironment with emphasis on the effect of cancer-associated fibroblasts (CAFs).,This model reflects various biological aspects of interaction between cancer cell and CAFs with multiple parallels to interaction of normal epidermal stem cells and their niche.,The complexity of intercellular interactions within tumor stroma is depicted on example of malignant melanoma, where keratinocytes also contribute the microenvironmental landscape during early phase of tumor progression.,Interactions seen in normal bulge region can therefore be an important source of information for proper understanding to melanoma.,The therapeutic consequences of targeting of microenvironment in anticancer therapy and for improved wound healing are included to article.

While cancer immunotherapies including checkpoint blockade antibodies, adoptive T cell therapy, and even some vaccines have given rise to major clinical responses with durability in many cases, a subset of patients who initially respond subsequently develop secondary resistance to therapy.,Tumor-intrinsic mechanisms of acquired immunotherapy resistance are incompletely understood.,Baseline and treatment-resistant tumors underwent molecular analysis via transcriptional profiling or genomic sequencing for oncogenic alterations and histologic analysis for T cell infiltration to investigate mechanisms contributing to T cell exclusion and acquired resistance to immunotherapy.,We describe two patients with metastatic melanoma who initially showed a durable partial response to either a melanoma-peptide/interleukin-12 vaccine or combined anti-CTLA-4 + anti-PD-1 therapy, but subsequently developed new treatment-resistant metastases.,In the first case, the recurrent tumor showed new robust tumor expression of β-catenin, whereas in the second case genomic sequencing revealed acquired PTEN loss.,Both cases were associated with loss of T cell infiltration, and both pathways have been mechanistically linked to immune resistance preclinically.,Our results suggest that secondary resistance to immunotherapies can arise upon selection for new oncogenic variants that mediate T cell exclusion.,To identify the spectrum of underlying mechanisms of therapeutic resistance, similar evaluation for the emergence of tumor-intrinsic alterations in resistant lesions should be done prospectively at the time of relapse in a range of additional patients developing secondary resistance.

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

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

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.

Circulating melanoma cells (CMCs) are thought to be valuable in improving measures of prognosis in melanoma patients and may be a useful marker of residual disease to identify non-metastatic patients requiring adjuvant therapy.,We investigated whether immunomagnetic enrichment targeting multiple markers allows more efficient enrichment of CMCs from patient peripheral blood than targeting a single marker.,Furthermore, we aimed to determine whether the number of CMCs in patient blood was associated with disease stage.,We captured CMCs by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271, both individually and in combination, by immunomagnetic enrichment.,CMCs were enriched and quantified from the peripheral blood of 10 non-metastatic and 13 metastatic melanoma patients.,Targeting all markers in combination resulted in the enrichment of more CMCs than when any individual marker was targeted (p < 0.001-0.028).,Furthermore, when a combination of markers was targeted, a greater number of CMCs were enriched in metastatic patients compared with non-metastatic patients (p = 0.007).,Our results demonstrated that a combination of markers should be targeted for optimal isolation of CMCs.,In addition, there are significantly more CMCs in metastatic patients compared with non-metastatic patients and therefore quantification of CMCs may prove to be a useful marker of disease progression.

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

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

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.

Metastatic melanoma (MM) is a highly aggressive cancer with a median overall survival of 6-9 months, notwithstanding the numerous efforts in development of new therapeutic approaches.,To this aim we tested the clinical applicability of the Ion Torrent Personal Genome Machine to simultaneously screen MM patients in order to individuate new or already known SNPs and mutations able to predict the duration of response to BRAF inhibitors.,An Ampliseq Custom Panel, including 11 crucial full length genes involved in melanoma carcinogenesis and therapy response pathways, was created and used to analyze 25 MM patients.,We reported BRAFV600 and NRASQ61 mutations in 68% and 24% of samples, respectively.,Moreover, we more frequently identified the following alterations related to BRAF status: PIK3CAI391M (44%) and KITD737N (36%) mutations, CTLA4T17A (52%), MC1RV60L (32%) and MITFS473A (60%) polymorphisms.,Considering the progression free survival (PFS), statistical analyses showed that BRAFV600 patients without any of these more frequent alterations had a higher median PFS.,Protein structure changes seem to be due to these variants by in silico analysis.,In conclusion, a Next-Generation Sequencing approach with custom panel may provide new information to evaluate tumor-specific therapeutic susceptibility and individual prognosis to improve the care of MM patients.

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

Patients with melanoma brain metastases have a poor prognosis and historically have been excluded from clinical trials.,The Expanded Access Program (EAP) provided an opportunity to evaluate the feasibility of ipilimumab (3 mg/kg every 3 weeks for four doses) in patients with stage 3 (unresectable) or 4 melanoma and asymptomatic brain metastases, who had failed or did not tolerate previous treatments and had no other therapeutic option available.,Tumor assessments were conducted at baseline and week 12 using immune-related response criteria and patients were monitored for adverse events (AEs).,Of 855 patients participating in the EAP in Italy, 146 had asymptomatic brain metastases.,With a median follow-up of 4 months, the global disease control rate was 27 %, including 4 patients with a complete response and 13 with a partial response.,Median progression-free survival and overall survival were 2.8 and 4.3 months, respectively and approximately one-fifth of patients were alive 1 year after starting ipilimumab.,In total, 29 % of patients reported a treatment-related AE of any grade, which were grade 3/4 in 6 % of patients.,AEs were generally reversible with treatment as per protocol-specific guidelines.,Ipilimumab shows durable benefits in some patients with advanced melanoma metastatic to the brain, with safety results consistent with those previously reported in clinical trials.

Ipilimumab is a monoclonal antibody that antagonizes cytotoxic T lymphocyte antigen-4, a negative regulator of the immune system.,The authors report on advanced refractory melanoma patients treated in a compassionate use trial of ipilimumab at the Memorial Sloan-Kettering Cancer Center.,Patients with advanced refractory melanoma were treated in a compassionate use trial with ipilimumab 10 mg/kg every 3 weeks for 4 doses.,Those with evidence of clinical benefit at Week 24 (complete response [CR], partial response [PR], or stable disease [SD]) then received ipilimumab every 12 weeks.,A total of 53 patients were enrolled, with 51 evaluable.,Grade 3/4 immune-related adverse events were noted in 29% of patients, with the most common immune-related adverse events being pruritus (43%), rash (37%), and diarrhea (33%).,On the basis of immune-related response criteria, the response rate (CR + PR) was 12% (95% confidence interval [CI], 5%-25%), whereas 29% had SD (95% CI, 18%-44%).,The median progression-free survival was 2.6 months (95% CI, 2.3-5.2 months), whereas the median overall survival (OS) was 7.2 months (95% CI, 4.0-13.3 months).,Patients with an absolute lymphocyte count (ALC) ≥1000/μL after 2 ipilimumab treatments (Week 7) had a significantly improved clinical benefit rate (51% vs 0%; P = .01) and median OS (11.9 vs 1.4 months; P < .001) compared with those with an ALC <1000/μL.,The results confirm that ipilimumab is clinically active in patients with advanced refractory melanoma.,The ALC after 2 ipilimumab treatments appears to correlate with clinical benefit and OS, and should be prospectively validated.,Cancer 2010.,© 2010 American Cancer Society.,This description of 51 patients with advanced, treatment-refractory melanoma who were enrolled in a compassionate use trial of ipilimumab at Memorial Sloan-Kettering Cancer Center confirms that ipilimumab is active in this disease setting.,In addition, the results suggest that the absolute lymphocyte count after 2 ipilimumab treatments (at Week 7) highly correlates with the rate of clinical benefit at Week 24 and overall survival.

Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans and it can be locally invasive and metastatic to distant sites.,MicroRNAs (miRNAs or miRs) are endogenous, small, non-coding RNAs of 19-25 nucleotides in length, that are involved in regulating gene expression at a post-transcriptional level.,MicroRNAs have been implicated in diverse biological functions and diseases.,In cancer, miRNAs can proceed either as oncogenic miRNAs (onco-miRs) or as tumor suppressor miRNAs (oncosuppressor-miRs), depending on the pathway in which they are involved.,Dysregulation of miRNA expression has been shown in most of the tumors evaluated.,MiRNA dysregulation is known to be involved in the development of cutaneous squamous cell carcinoma (CSCC).,In this review, we focus on the recent evidence about the role of miRNAs in the development of CSCC and in the prognosis of this form of skin cancer.

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.

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

Uveal melanoma is the most common primary malignant tumor of the eye in adults, predominantly found in Caucasians.,Local tumor control of uveal melanoma is excellent, yet this malignancy is associated with relatively high mortality secondary to metastasis.,Various clinical, histopathological, cytogenetic features and gene expression features help in estimating the prognosis of uveal melanoma.,The clinical features associated with poor prognosis in patients with uveal melanoma include older age at presentation, male gender, larger tumor basal diameter and thickness, ciliary body location, diffuse tumor configuration, association with ocular/oculodermal melanocytosis, extraocular tumor extension, and advanced tumor staging by American Joint Committee on Cancer classification.,Histopathological features suggestive of poor prognosis include epithelioid cell type, high mitotic activity, higher values of mean diameter of ten largest nucleoli, higher microvascular density, extravascular matrix patterns, tumor-infiltrating lymphocytes, tumor-infiltrating macrophages, higher expression of insulin-like growth factor-1 receptor, and higher expression of human leukocyte antigen Class I and II.,Monosomy 3, 1p loss, 6q loss, and 8q and those classified as Class II by gene expression are predictive of poor prognosis of uveal melanoma.,In this review, we discuss the prognostic factors of uveal melanoma.,A database search was performed on PubMed, using the terms “uvea,” “iris,” “ciliary body,” “choroid,” “melanoma,” “uveal melanoma” and “prognosis,” “metastasis,” “genetic testing,” “gene expression profiling.”,Relevant English language articles were extracted, reviewed, and referenced appropriately.

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.

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.

Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation.,Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome.,We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity.,We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis.,Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism.,The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines.,Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics.,Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy.

Presently melanoma still lacks adequate treatment options for metastatic disease.,While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity.,Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity.,We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy).,This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16.,F10 melanoma growth in up to 85% of C57BL/6 mice.,Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation.,MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B- and T cells in its therapeutic effect.,Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clinic.

This is the first prospective study of a combination therapy involving a cardenolide and a MEK inhibitor for metastatic melanoma.,Whereas BRAF mutant melanomas can exhibit profound responses to treatment with BRAF and MEK inhibitors, there are fewer options for BRAF wild-type melanomas.,In preclinical studies, we discovered that cardenolides synergize with MEK inhibitor to promote the regression of patient-derived xenografts irrespective of BRAF mutation status.,We therefore conducted a phase 1B study of digoxin 0.25 mg and trametinib 2 mg given orally once daily in 20 patients with advanced, refractory, BRAF wild-type melanomas.,The most common adverse events were rash, diarrhea, nausea, and fatigue.,The response rate was 4/20 or 20% with response durations of 2, 4, 6, and 8 months.,The disease control rate (including partial responses and stable disease) was 13/20 or 65% of patients, including 5/6 or 83% of patients with NRAS mutant melanomas and 8/14 or 57% of NRAS wild-type melanomas.,Patients with stable disease had disease control for 2, 2, 2, 4, 5, 6, 7, 10, and 10 months.,Xenografts from four patients recapitulated the treatment responses observed in patients.,Based on these pilot results, an expansion arm of digoxin plus MEK inhibitor is warranted for NRAS mutant metastatic melanoma patients who are refractory or intolerant of immunotherapy.,Digoxin plus trametinib is well tolerated and achieves a high rate of disease control in BRAF wild-type metastatic melanoma patients.

In 2003, a skin cancer screening campaign based on total body skin examination was launched in the federal state of Schleswig-Holstein, Germany. 20% of adults aged 20 and over were screened.,In 2008, a 48% decline in melanoma mortality was reported.,In the same year, skin screening was extended to the rest of Germany.,We evaluated whether melanoma mortality trends decreased in Germany as compared with surrounding countries where skin screening is uncommon.,We also evaluated whether the initial decreasing mortality trend observed in Schleswig-Holstein was maintained with a longer follow-up.,Regional and national melanoma mortality data from 1995 to 2013 were extracted from the GEKID database and the Federal Statistical Office.,Mortality data for Germany and surrounding countries from 1980 to 2012 were extracted from the WHO mortality database.,Age-adjusted (European Standard Population) mortality rates were computed and joinpoint analysis performed for Schleswig-Holstein, Germany and surrounding countries.,In Schleswig-Holstein, melanoma mortality rates declined by 48% from 2003 to 2008, and from 2009 to 2013 returned to levels observed before screening initiation.,During the 5 years of the national programme (2008-2012), melanoma mortality rates increased by 2.6% (95% CI −0.1 to 5.2) in men and 0.02% (95% CI −1.8 to 1.8) in women.,No inflexion point in trends was identified after 2008 that could have suggested a decreasing melanoma mortality.,Trends of cutaneous melanoma mortality in Germany from 1980 to 2012 did not differ from those observed in surrounding countries.,The transient decrease mortality in Schleswig-Holstein followed by return to pre-screening levels could reflect a temporal modification in the reporting of death causes.,An in-depth evaluation of the screening programme is required.

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

Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma.,We tested a cohort of ocular melanoma samples for similar mutations.,The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.,Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas.,Mutations had a UV signature and were identical to those found in cutaneous melanoma.,Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas.,Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

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

Although recent studies have shown that adenosine-to-inosine (A-to-I) RNA editing occurs in microRNAs, its effects on tumor growth and metastasis are not well understood.,We present evidence of CREB-mediated low expression of ADAR1 in metastatic melanoma cell lines and tumor specimens.,Re-expression of ADAR1 resulted in the suppression of melanoma growth and metastasis in vivo.,Consequently, we identified 3 miRs undergoing A-to-I editing in the low-metastatic melanoma but not in highly metastatic cell lines.,One of these miRs, miR-455-5p has two A-to-I RNA editing sites.,The biological function of edited miR-455-5p is different from the unedited form as it recognizes different set of genes.,Indeed, w.t. miR-455-5p promotes melanoma metastasis via inhibition of the tumor suppressor gene CPEB1.,Moreover, w.t. miR-455 enhances melanoma growth and metastasis in vivo while the edited form inhibits these features.,These results demonstrate a previously unrecognized role of RNA editing in melanoma progression.

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

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

microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway.,While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma.,Here, we investigated the biological and clinical significance of miR-7-5p in melanoma.,We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest.,Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo.,We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis.,Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1β, IL-6 and IL-8.,Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown.,Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors.,Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling.,Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease.

T cell trafficking at vascular sites has emerged as a key step in antitumor immunity.,Chemokines are credited with guiding the multistep recruitment of CD8+ T cells across tumor vessels.,However, the multiplicity of chemokines within tumors has obscured the contributions of individual chemokine receptor/chemokine pairs to this process.,Moreover, recent studies have challenged whether T cells require chemokine receptor signaling at effector sites.,Here, we investigate the hierarchy of chemokine receptor requirements during T cell trafficking to murine and human melanoma.,These studies reveal a non-redundant role for GαI-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8+ effectors that is indispensable for therapeutic efficacy.,In contrast, functional CCR2 and CCR5 on CD8+ effectors fail to support trafficking despite the presence of intratumoral cognate chemokines.,Taken together, these studies identify CXCR3-mediated trafficking at the tumor vascular interface as a critical checkpoint to effective T cell-based cancer immunotherapy.

Interleukin-9 is a T cell cytokine that acts through a γC-family receptor on target cells.,We determined that T cells from mice deficient in the TH17 pathway genes ROR-γ and IL-23R produced abundant IL-9, and observed significant growth inhibition of B16F10 melanoma tumor in these mice.,IL-9 blocking antibodies reversed this tumor growth inhibition, and enhanced tumor growth in normal mice.,IL9R−/− mice showed accelerated tumor growth, while administration of rIL-9 to tumor bearing mice inhibited tumor growth.,Adoptive transfer of tumor antigen-specific TH9 cells blocked tumor growth; this was reversed by anti-IL-9.,Exogenous rIL-9 inhibited tumor growth in Rag1−/− mice, but not in mast cell deficient mice, suggesting a T cell independent process.,Finally, we found TH9 cells in normal human skin and blood, and low IL-9 production from melanoma tumor infiltrating lymphocytes.,These results suggest a role for IL-9 in tumor immunity, and suggest therapeutic strategies.

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

Tumor metastasis is still the leading cause of melanoma mortality.,Luteolin, a natural flavonoid, is found in fruits, vegetables, and medicinal herbs.,The pharmacological action and mechanism of luteolin on the metastasis of melanoma remain elusive.,In this study, we investigated the effect of luteolin on A375 and B16‐F10 cell viability, migration, invasion, adhesion, and tube formation of human umbilical vein endothelial cells.,Epithelial-mesenchymal transition (EMT) markers and pivotal molecules in HIF‐1α/VEGF signaling expression were analysed using western blot assays or quantitative real‐time polymerase chain reaction.,Results showed that luteolin inhibits cellular proliferation in A375 and B16‐F10 melanoma cells in a time‐dependent and concentration‐dependent manner.,Luteolin significantly inhibited the migratory, invasive, adhesive, and tube‐forming potential of highly metastatic A375 and B16‐F10 melanoma cells or human umbilical vein endothelial cells at sub‐IC50 concentrations, where no significant cytotoxicity was observed.,Luteolin effectively suppressed EMT by increased E‐cadherin and decreased N‐cadherin and vimentin expression both in mRNA and protein levels.,Further, luteolin exerted its anti‐metastasis activity through decreasing the p‐Akt, HIF‐1α, VEGF‐A, p‐VEGFR‐2, MMP‐2, and MMP‐9 proteins expression.,Overall, our findings first time suggests that HIF‐1α/VEGF signaling‐mediated EMT and angiogenesis is critically involved in anti‐metastasis effect of luteolin as a potential therapeutic candidate for melanoma.

This phase II study evaluated the safety and clinical activity of pazopanib, a potent and mutlitargeted tyrosine kinase inhibitor (TKI) of vascular endothelial growth factor receptors (VEGFRs)-1, -2 and -3, platelet-derived growth factor receptor (PDGFR)-α and β, and cKit, in combination with metronomic paclitaxel in patients with metastatic melanoma.,Sixty chemotherapy-naive patients received pazopanib at a starting dose of 800 mg daily in combination with metronomic dosing of paclitaxel 80 mg/m2 weekly thrice every 4 weeks.,The primary endpoint was 6-month progression-free survival (PFS) rate, while secondary endpoints included 1-year overall survival rate, RECIST response rates, progression-free survival rates and median overall survival.,Prior BRAF-targeted therapy or checkpoint inhibitors were permitted.,The 6-month PFS rate was 68%, with a 1-year OS rate of 48%.,Objective response rate was 37% comprising one complete and 20 partial responses.,Stable disease at 8 weeks was noted in 32 patients (55%) with an overall clinical benefit rate of 93%.,Six-month median progression-free survival was 8 months and median OS was 12.7 months.,The most frequently (> 15%) reported non-hematologic, treatment-related adverse events were fatigue, diarrhea, hypertension, transaminitis and peripheral neuropathy.,Treatment-related non-fatal bowel perforation, a known class effect, occurred in one patient.,No significant association was noted between plasma levels of pazopanib and response.,The combination of pazopanib and metronomic paclitaxel was well-tolerated, demonstrating significant activity in metastatic melanoma.,Further evaluation of this combination is warranted.

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

The incidence of malignant melanoma is increasing faster than that for any other cancer.,Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis.,Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis.,To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs).,Differential expression was validated by qRT-PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression.,In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas.,In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion.,We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.

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

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

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

The high mortality rate of melanoma is broadly associated with its metastatic potential.,Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis.,Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients.,Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay.,Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P‐value = 0.044) and the lymphangiogenic receptor VEGFR‐3 (P‐value = 0.002) were independent prognostic factors of overall survival in melanoma patients.,Enhanced reduced representation bisulfite sequencing‐based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells.,In patients, VEGFC (P‐value = 0.031), ANGPT2 (P‐value < 0.001), and SIX1 (P‐value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival.,Hence, our data suggest that these angio‐ and lymphangiogenesis factors are potential biomarkers of melanoma prognosis.,Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease.,Therapeutic options for metastatic UM are limited, with clinical trials having little impact.,Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris).,While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM.,All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX).,We identify three other significantly mutated genes (TP53, RPL5 and CENPE).,Uveal melanoma has a propensity to metastasise.,Here, the authors report the whole genome sequence of 103 uveal melanomas and find that the tumour mutational burden is variable and that two subsets of tumours are characterised by MBD4 mutations and a UV exposure signature.

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

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

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.

FENDRR (Fetal-lethal non-coding developmental regulatory RNA, LncRNA FOXF1-AS1) is a recently identified tumor suppressor long non-coding (LncRNA) RNA, and its expression has been linked with epigenetic modulation of the target genes involved in tumor immunity.,In this study, we aimed to understand the role of FENDRR in predicting immune-responsiveness and the inflammatory tumor environment.,Briefly, FENDRR expression and its relationship to immune activation signals were assessed in murine cell lines.,Data suggested that tumor cells (e.g., C26 colon, 4T1 breast) that typically upregulate immune activation genes and the MHC class I molecule exhibited high FENDRR expression levels.,Conversely, tumor cells with a generalized downregulation of immune-related gene expression (e.g., B16F10 melanoma) demonstrated low to undetectable FENDRR levels.,Mechanistically, the modulation of FENDRR expression enhanced the inflammatory and WNT signaling pathways in tumors.,Our early data suggest that FENDRR can play an important role in the development of immune-relevant phenotypes in tumors, and thereby improve cancer immunotherapy.

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

Mutations in the receptor tyrosine kinase c-KIT (KIT) are frequent oncogenic alterations in melanoma and are predominantly detected in tumors of acral, mucosal, and chronically sun-damaged skin.,Research indicates that melanocytes with aberrant KIT signaling can be found in the distant periphery of the primary tumor; However, it is hitherto unknown whether KIT might confer a migratory advantage, thereby enabling genetically abnormal cells to populate a distal area.,In this study, we investigated the role of mutant KIT in melanocyte- and melanoma cell migration using KIT mutant lines as well as genetically manipulated murine and primary human melanocytes.,Our results revealed that melanocytes, stably transduced with mutant KIT closed a gap inflicted on cell monolayers faster than wild-type controls.,Similarly, KIT mutant human melanoma lines were able to populate a larger area in a 3D in vitro skin model compared to KIT wild type and BRAF mutant lines.,Genomic profiling revealed that genes associated with increased cell-dispersal of KIT mutant variants were linked to a statistically significant up-regulation of 60 migratory genes (z-score 1.334; p=0.0001).,In addition, in vivo experiments harnessing a mouse xenograft model of early melanoma development demonstrated rapid lateral migration of KIT mutant cells compared to respective controls.,The specific kinase inhibitors imatinib and nilotinib, could abrogate this migratory advantage in vitro and in vivo.,Our work suggests that KIT inhibition might help to target migratory active, KIT mutant melanoma cells, thus representing a potential strategy to reduce spread and local recurrence.

Long non-coding RNAs (lncRNAs) have been shown to be implicated in the complex network of cancer including malignant melanoma and play important roles in tumorigenesis and progression.,However, their functions and downstream mechanisms are largely unknown.,This study aimed to investigate whether BRAF-activated non-coding RNA (BANCR), a novel and potential regulator of melanoma cell, participates in the proliferation of malignant melanoma and elucidate the underlying mechanism in this process.,We found that BANCR was abnormally overexpressed in human malignant melanoma cell lines and tissues, and increased with tumor stages by quantitative PCR.,BANCR knockdown induced by shRNA transfection significantly inhibited proliferation of tumor cells and inactivated MAPK pathway, especially by silencing the ERK1/2 and JNK component.,Moreover, combination treatment of BANCR knockdown and suppression ERK1/2 or JNK (induced by specific inhibitors U0126 or SP600125 respectively) produced synergistic inhibitory effects in vitro.,And the inhibitory effects induced by ERK1/2 or JNK could be rescued by BANCR overexpression.,By tumorigenicity assay in BALB/c nude mice, we further found that BANCR knockdown inhibited tumor growth in vivo.,In addition, patients with high expression of BANCR had a lower survival rate.,Taken together, we confirmed the abnormal upregulation of a novel lncRNA, BANCR, in human malignant melanoma.,BANCR was involved in melanoma cell proliferation both in vitro and in vivo.,The linkage between BANCR and MAPK pathway may provide a novel interpretation for the mechanism of proliferation regulation in malignant melanoma.

Vendramin et al. demonstrate that the integrated stress response in melanoma promotes cell plasticity and drives therapy resistance by boosting mitochondrial translation.,Therefore, repurposing of mitoribosome-targeting antibiotics offers a salvage strategy for the treatment of patients with limited therapeutic options.,The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance.,In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation.,We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics.,Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models.,Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy.,In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines.,Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion.,Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.

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

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.

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples.,Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively.,Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma.,Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data.,We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression.,Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors.,Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs).,Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters.,Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy.

Oral malignant melanoma (OMM) is the most common canine melanocytic neoplasm.,Overlap between the somatic mutation profiles of canine OMM and human mucosal melanomas suggest a shared UV-independent molecular aetiology.,In common with human mucosal melanomas, most canine OMM metastasise.,There is no reliable means of predicting canine OMM metastasis, and systemic therapies for metastatic disease are largely palliative.,Herein, we employed exon microarrays for comparative expression profiling of FFPE biopsies of 18 primary canine OMM that metastasised and 10 primary OMM that did not metastasise.,Genes displaying metastasis-associated expression may be targets for anti-metastasis treatments, and biomarkers of OMM metastasis.,Reduced expression of CXCL12 in the metastasising OMMs implies that the CXCR4/CXCL12 axis may be involved in OMM metastasis.,Increased expression of APOBEC3A in the metastasising OMMs may indicate APOBEC3A-induced double-strand DNA breaks and pro-metastatic hypermutation.,DNA double strand breakage triggers the DNA damage response network and two Fanconi anaemia DNA repair pathway members showed elevated expression in the metastasising OMMs.,Cross-validation was employed to test a Linear Discriminant Analysis classifier based upon the RT-qPCR-measured expression levels of CXCL12, APOBEC3A and RPL29.,Classification accuracies of 94% (metastasising OMMs) and 86% (non-metastasising OMMs) were estimated.

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

The search for melanoma biomarkers is crucial, as the incidence of melanoma continues to rise.,We have previously demonstrated that serum CEACAM1 (sCEACAM1) is secreted from melanoma cells and correlates with disease progression in metastatic melanoma patients.,Here, we have used a different cohort of melanoma patients with regional or metastatic disease (N = 49), treated with autologous vaccination.,By monitoring sCEACAM1 in serum samples obtained prior to and after vaccination, we show that sCEACAM1 correlates with disease state, overall survival, and S100B.,The trend of change in sCEACAM1 following vaccination (increase/decrease) inversely correlates with overall survival.,DTH skin test is used to evaluate patients' anti-melanoma immune response and to predict response to vaccination.,Importantly, sCEACAM1 had a stronger prognostic value than that of DTH, and when sCEACAM1 decreased following treatment, this was the dominant predictor of increased survival.,Collectively, our results point out the relevance of sCEACAM1 in monitoring melanoma patients.

Past studies have shown that upregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 is a major adaptive mechanism of melanoma cells to endoplasmic reticulum (ER) stress, and has an important role in resistance of the cells to apoptosis.,In this study, we show that the increase in transcription of Mcl-1 in melanoma cells triggered by pharmacological ER stress inducers is mediated by the transcription factor Ets-1.,By incremental deletion analysis of the Mcl-1 promoter, we identified a DNA fragment containing an Ets-1 binding site that is transcriptionally responsive to ER stress.,Mutations in the Ets-1 binding site or knockdown of Ets-1 inhibited the increase in Mcl-1, indicating that Ets-1 has a critical role in transcriptional upregulation of Mcl-1.,Similar to Mcl-1, Ets-1 was transcriptionally upregulated by ER stress.,This was mediated by the IRE1α/XBP-1 branch of the unfolded protein response, as upregulation of Ets-1 was inhibited in melanoma cell lines deficient in IRE1α or XBP-1 established by short hairpin RNA knockdown.,Activation of the PI3k/Akt pathway downstream of XBP-1 was also involved, in that inhibition of the pathway blocked upregulation of Ets-1.,Inhibition of Ets-1 enhanced ER stress-induced apoptosis in melanoma cell lines and in fresh melanoma isolates, recapitulating the effect of inhibition of Mcl-1.,These results reveal a key mechanism by which Mcl-1 is transcriptionally upregulated in melanoma cells by ER stress, and identify Ets-1 as a potential target for inhibition to sensitize melanoma cells to apoptosis.

The discovery of the BRAFV600E mutation led to the development of vemurafenib (PLX4032), a selective BRAF inhibitor specific to the kinase, for the treatment of metastatic melanomas.,However, initial success of the drug was dampened by the development of acquired resistance.,Melanoma was shown to relapse in patients following treatment with vemurafenib which eventually led to patients’ deaths.,It has been proposed that mechanisms of resistance can be due to (1) reactivation of the mitogen-activated protein kinase (MAPK) signalling pathway via secondary mutations, amplification or activation of target kinase(s), (2) the bypass of oncogenic pathway via activation of alternative signalling pathways, (3) other uncharacterized mechanisms.,Studies showed that receptor tyrosine kinases (RTK) such as PDGFRβ, IGF1R, EGFR and c-Met were overexpressed in melanoma cells.,Along with increased secretion of growth factors such as HGF and TGF-α, this will trigger intracellular signalling cascades.,This review discusses the role MAPK and Phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) pathways play in the mechanism of resistance of melanomas.

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.

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

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.

Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance.,To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution.,We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development.,Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines.,SATB2 binds and activates neural crest-regulators, including pdgfab and snai2.,The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo.,In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.

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.

Autophagy maintains homeostasis and is induced upon stress.,Yet, its mechanistic interaction with oncogenic signaling remains elusive.,Here, we show that in BRAFV600E-melanoma, autophagy is induced by BRAF inhibitor (BRAFi), as part of a transcriptional program coordinating lysosome biogenesis/function, mediated by the TFEB transcription factor.,TFEB is phosphorylated and thus inactivated by BRAFV600E via its downstream ERK independently of mTORC1.,BRAFi disrupts TFEB phosphorylation, allowing its nuclear translocation, which is synergized by increased phosphorylation/inactivation of the ZKSCAN3 transcriptional repressor by JNK2/p38-MAPK.,Blockade of BRAFi-induced transcriptional activation of autophagy-lysosomal function in melanoma xenografts causes enhanced tumor progression, EMT-transdifferentiation, metastatic dissemination, and chemoresistance, which is associated with elevated TGF-β levels and enhanced TGF-β signaling.,Inhibition of TGF-β signaling restores tumor differentiation and drug responsiveness in melanoma cells.,Thus, the “BRAF-TFEB-autophagy-lysosome” axis represents an intrinsic regulatory pathway in BRAF-mutant melanoma, coupling BRAF signaling with TGF-β signaling to drive tumor progression and chemoresistance.,The relationship between autophagy and BRAF signalling is unclear.,Here, the authors describe that BRAF inhibition induces the autophagy-lysosomal function in BRAF-mutant melanomas via modulation of the TFEB and ZKSCAN3 transcriptome, which downregulates TGF-β and suppresses melanoma progression.

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

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.

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

Anti-PD-1 therapy has shown significant clinical activity in advanced melanoma.,We developed and validated a clinical prediction scale for response to anti- PD-1 monotherapy.,A total of 315 patients with advanced melanoma treated with pembrolizumab (2 or 10 mg kg−1 Q2W or Q3W) or nivolumab (3 mg kg−1 Q2W) at four cancer centres between 2011 to 2013 served as the setting for the present cohort study.,Variables with significant association to response on a univariate analysis were entered into a forward stepwise logistic regression model and were given a score based on ORs to calculate a clinical prediction scale.,The developed clinical prediction scale included elevated LDH (1 point), age <65 years (1 point), female sex (1 point), history of ipilimumab treatment (2 points) and the presence of liver metastasis (2 points).,The scale had an area under the receiver-operating curve (AUC) of 0.73 (95% CI 0.67, 0.80) in predicting response to therapy.,The predictive performance of the score was maintained in the validation cohort (AUC 0.70 (95% CI 0.58, 0.81)) and the goodness-to-fit model demonstrated good calibration.,Based on a large cohort of patients, we developed and validated a simple five-factor prediction scale for the clinical activity of PD-1 antibodies in advanced melanoma patients.,This scale can be used to stratify patients participating in clinical trials.

Increased level of serum S100B can serve as a marker of metastatic spread in patients with cutaneous melanoma (CM).,In patients with elevated S100 B and/or clinical signs of disease progression PET-CT scan is a valuable tool for discovering metastases and planning treatment.,The aims of this study were to determine whether regular measurements of serum S100B are a useful tool for discovering patients with CM metastases and to evaluate the diagnostic value of PET-CT during the follow-up.,From September 2007 to February 2010, 115 CM patients included in regular follow up at the Institute of Oncology Ljubljana were appointed to PET-CT.,There were 82 (71.3%) patients with clinical signs of disease progression and 33 (28.7%) asymptomatic patients with two subsequent elevated values of S100B.,Sensitivity, specificity, positive and negative predictive value (PPV, NPV) of S100B and PET-CT were calculated using standard procedures.,Disease progression was confirmed in 81.7% of patients (in 86.5% of patients with clinical signs of disease progression and in 69.7% of asymptomatic patients with elevated S100B).,Sensitivity, specificity, PPV and NPV of S100B was 33.8%, 90.9%, 96.0% and 17.5% in patients with clinical signs of disease progression.,In 20.0% of patients increased serum S100B was the only sign of disease progression.,Sensitivity and PPV of S100 in this group of patients were 100.0% and 69.7%.,With PET-CT disease progression was diagnosed in 84.2% of symptomatic patients and in 72.7% of asymptomatic patients with elevated S100B.,The sensitivity, specificity, PPV and NPV of PET-CT for symptomatic patients was 98.5%, 90.9%, 98.5% and 90.9% and 100%, 90.0%, 95.8% and 100% for asymptomatic patients with elevated S100.,Measurements of serum S100B during regular follow-up of patients with CM are a useful tool for discovering disease progression in asymptomatic patients.,The value of its use increases if measurements are followed by extended whole body PET-CT.

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

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.

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.

In melanoma, the adaptative cell response to BRAF inhibitors includes altered patterns of cytokine production contributing to tumor progression and drug resistance.,Among the factors produced by PLX4032-resistant melanoma cell lines, CCL2 was higher compared to the sensitive parental cell lines and increased upon drug treatment.,CCL2 acted as an autocrine growth factor for melanoma cells, stimulating the proliferation and resistance to apoptosis.,In patients, CCL2 is detected in melanoma cells in tumors and in plasma at levels that correlate with tumor burden and lactate dehydrogenase.,Vemurafenib treatment increased the CCL2 levels in plasma, whereas the long-term clinical response was associated with low CCL2 levels.,Increased CCL2 production was associated with miRNA deregulation in the resistant cells. miR-34a, miR-100 and miR-125b showed high expression in both resistant cells and in tumor biopsies that were obtained from treated patients, and they were involved in the control of cell proliferation and apoptosis.,Inhibition of CCL2 and of the selected miRNAs restored both the cell apoptosis and the drug efficacy in resistant melanoma cells.,Therefore, CCL2 and miRNAs are potential prognostic factors and attractive targets for counteracting treatment resistance in metastatic melanoma.

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.

Carrying the cyclin-dependent kinase inhibitor 2A (CDKN2A) germline mutations is associated with a high risk for melanoma.,Penetrance of CDKN2A mutations is modified by pigmentation characteristics, nevus phenotypes, and some variants of the melanocortin-1 receptor gene (MC1R), which is known to have a role in the pigmentation process.,However, investigation of the associations of both MC1R variants and host phenotypes with melanoma risk has been limited.,We included 815 CDKN2A mutation carriers (473 affected, and 342 unaffected, with melanoma) from 186 families from 15 centers in Europe, North America, and Australia who participated in the Melanoma Genetics Consortium.,In this family-based study, we assessed the associations of the four most frequent MC1R variants (V60L, V92M, R151C, and R160W) and the number of variants (1, ≥2 variants), alone or jointly with the host phenotypes (hair color, propensity to sunburn, and number of nevi), with melanoma risk in CDKN2A mutation carriers.,These associations were estimated and tested using generalized estimating equations.,All statistical tests were two-sided.,Carrying any one of the four most frequent MC1R variants (V60L, V92M, R151C, R160W) in CDKN2A mutation carriers was associated with a statistically significantly increased risk for melanoma across all continents (1.24 × 10−6 ≤ P ≤ .0007).,A consistent pattern of increase in melanoma risk was also associated with increase in number of MC1R variants.,The risk of melanoma associated with at least two MC1R variants was 2.6-fold higher than the risk associated with only one variant (odds ratio = 5.83 [95% confidence interval = 3.60 to 9.46] vs 2.25 [95% confidence interval = 1.44 to 3.52]; Ptrend = 1.86 × 10−8).,The joint analysis of MC1R variants and host phenotypes showed statistically significant associations of melanoma risk, together with MC1R variants (.0001 ≤ P ≤ .04), hair color (.006 ≤ P ≤ .06), and number of nevi (6.9 × 10−6 ≤ P ≤ .02).,Results show that MC1R variants, hair color, and number of nevi were jointly associated with melanoma risk in CDKN2A mutation carriers.,This joint association may have important consequences for risk assessments in familial settings.

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.

Malignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy.,Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression.,This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets.,In the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models.,Our in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X) - and premature mitosis of S-phase cells.,Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts.,These data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.,The online version of this article (doi:10.1186/s12885-015-1474-8) contains supplementary material, which is available to authorized users.

Immunotherapy is a revolutionary strategy in cancer therapy, but the resistance of which is one of the important challenges.,Detecting the regulation of immune cells and biomarkers concerning immune checkpoint blockade (ICB) therapy is of great significance.,Here, we firstly constructed regulation networks for 11 immune cell clusters by integrating biological pathway data and single cell sequencing data in metastatic melanoma with or without ICB therapy.,We then dissected these regulation networks and identified differently expressed genes between responders and non-responders.,Finally, we trained and validated a logistic regression model based on ligands and receptors in the regulation network to predict ICB therapy response.,We discovered the regulation of genes across eleven immune cell stats.,Functional analysis indicated that these stat-specific networks consensually enriched in immune response corrected pathways and highlighted antigen processing and presentation as a core pathway in immune cell regulation.,Furthermore, some famous ligands like SIRPA, ITGAM, CD247and receptors like CD14, IL2 and HLA-G were differently expressed between cells of responders and non-responders.,A predictive model of gene sets containing ligands and receptors performed accuracy prediction with AUCs above 0.7 in a validation dataset suggesting that they may be server as biomarkers for predicting immunotherapy response.,In summary, our study presented the gene-gene regulation landscape across 11 immune cell clusters and analysis of these networks revealed several important aspects and immunotherapy response biomarkers, which may provide novel insights into immune related mechanisms and immunotherapy response prediction.,The online version contains supplementary material available at 10.1186/s12967-021-02962-8.

Supplemental Digital Content is available in the text.,The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) immune checkpoints are negative regulators of T-cell immune function.,Inhibition of these targets, resulting in increased activation of the immune system, has led to new immunotherapies for melanoma, non-small cell lung cancer, and other cancers.,Ipilimumab, an inhibitor of CTLA-4, is approved for the treatment of advanced or unresectable melanoma.,Nivolumab and pembrolizumab, both PD-1 inhibitors, are approved to treat patients with advanced or metastatic melanoma and patients with metastatic, refractory non-small cell lung cancer.,In addition the combination of ipilimumab and nivolumab has been approved in patients with BRAF WT metastatic or unresectable melanoma.,The roles of CTLA-4 and PD-1 in inhibiting immune responses, including antitumor responses, are largely distinct.,CTLA-4 is thought to regulate T-cell proliferation early in an immune response, primarily in lymph nodes, whereas PD-1 suppresses T cells later in an immune response, primarily in peripheral tissues.,The clinical profiles of immuno-oncology agents inhibiting these 2 checkpoints may vary based on their mechanistic differences.,This article provides an overview of the CTLA-4 and PD-1 pathways and implications of their inhibition in cancer therapy.

Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity.,The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy.,Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab).,Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS).,Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type.,Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies.,We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy.,Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease.,Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission.,However, most patients eventually relapse due to drug resistance.,Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition.,We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3.,Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase.,For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment.,By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma.,ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment.,Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro.,These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.

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.

Nivolumab 1 mg/kg plus ipilimumab 3 mg/kg (NIVO1+IPI3) is approved for first-line treatment of patients with advanced melanoma in several countries.,We conducted a phase IIIb/IV study (CheckMate 511) to determine if nivolumab 3 mg/kg plus ipilimumab 1 mg/kg (NIVO3+IPI1) improves the safety profile of the combination.,Patients (N = 360) age 18 years or older with previously untreated, unresectable stage III or IV melanoma were randomly assigned 1:1 to NIVO3+IPI1 or NIVO1+IPI3 once every 3 weeks for four doses.,After 6 weeks, all patients received NIVO 480 mg once every 4 weeks until disease progression or unacceptable toxicity.,The primary end point was a comparison of the incidence of treatment-related grade 3 to 5 adverse events (AEs) between groups.,Secondary end points included descriptive analyses of objective response rate, progression-free survival, and overall survival.,The study was not designed to formally demonstrate noninferiority of NIVO3+IPI1 to NIVO1+IPI3 for efficacy end points.,At a minimum follow-up of 12 months, incidence of treatment-related grade 3 to 5 AEs was 34% with NIVO3+IPI1 versus 48% with NIVO1+IPI3 (P = .006).,In descriptive analyses, objective response rate was 45.6% in the NIVO3+IPI1 group and 50.6% in the NIVO1+IPI3 group, with complete responses in 15.0% and 13.5% of patients, respectively.,Median progression-free survival was 9.9 months in the NIVO3+IPI1 group and 8.9 months in the NIVO1+IPI3 group.,Median overall survival was not reached in either group.,The CheckMate 511 study met its primary end point, demonstrating a significantly lower incidence of treatment-related grade 3-5 AEs with NIVO3+IPI1 versus NIVO1+IPI3.,Descriptive analyses showed that there were no meaningful differences between the groups for any efficacy end point, although longer follow up may help to better characterize efficacy outcomes.

Uveal melanoma (UM) is a global disease which especially occurs in elderly people.,Its incidence varies widely between populations, with the highest incidence among Caucasians, and a South-to-North increase in Europe.,As northern Europeans often have blond hair and light eyes, we wondered whether iris colour may be a predisposing factor for UM and if so, why.,We compared the distribution of iris colour between Dutch UM patients and healthy Dutch controls, using data from the Rotterdam Study (RS), and reviewed the literature regarding iris colour.,We describe molecular mechanisms that might explain the observed associations.,When comparing a group of Dutch UM patients with controls, we observed that individuals from Caucasian ancestry with a green/hazel iris colour (Odds Ratio (OR) = 3.64, 95% Confidence Interval (CI) 2.57-5.14) and individuals with a blue/grey iris colour (OR = 1.38, 95% CI 1.04-1.82) had a significantly higher crude risk of UM than those with brown eyes.,According to the literature, this may be due to a difference in the function of pheomelanin (associated with a light iris colour) and eumelanin (associated with a brown iris colour).,The combination of light-induced stress and aging may affect pheomelanin-carrying melanocytes in a different way than eumelanin-carrying melanocytes, increasing the risk of developing a malignancy.

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

MicroRNAs (miRs) play a key role in cancer etiology by coordinately repressing numerous target genes involved in cell proliferation, migration and invasion.,The genomic region in chromosome 9p21 that encompasses miR-31 is frequently deleted in solid cancers including melanoma; however the expression and functional role of miR-31 has not been previously studied in melanoma.,Here, we queried the expression status and performed functional characterization of miR-31 in melanoma tissues and cell lines.,We found that down-regulation of miR-31 was a common event in melanoma tumors and cell lines and was associated with genomic loss in a subset of samples.,Down-regulation of miR-31 gene expression was also a result of epigenetic silencing by DNA methylation, and via EZH2-mediated histone methylation.,Ectopic overexpression of miR-31 in various melanoma cell lines inhibited cell migration and invasion. miR-31 targets include oncogenic kinases such as SRC, MET, NIK (MAP3K14) and the melanoma specific oncogene RAB27a.,Furthermore, miR-31 overexpression resulted in down-regulation of EZH2 and a de-repression of its target gene rap1GAP; increased expression of EZH2 was associated with melanoma progression and overall patient survival.,Taken together, our study supports a tumor suppressor role for miR-31 in melanoma and identifies novel therapeutic targets.

Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells.,MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics.,SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression.,We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death.,BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP).,Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation.,BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter.,The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells.,Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.

Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common.,A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway).,IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival.,In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs.,We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs.,The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model.,The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status.,Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo.,Importantly, this combination therapy had minimal therapy-related toxicity in mice.,Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination.,Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells.,In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

Numerous tyrosine kinase inhibitors (TKIs) targeting c-Met are currently in clinical trials for several cancers.,Their efficacy is limited due to the development of resistance.,The present study aims to elucidate this mechanism of c-Met TKI resistance by investigating key mTOR and Wnt signaling proteins in melanoma cell lines resistant to SU11274, a c-Met TKI.,Xenografts from RU melanoma cells treated with c-Met TKIs SU11274 and JNJ38877605 showed a 7- and 6-fold reduction in tumor size, respectively.,Resistant cells displayed upregulation of phosphorylated c-Met, mTOR, p70S6Kinase, 4E-BP1, ERK, LRP6, and active β-catenin.,In addition, GATA-6, a Wnt signaling regulator, was upregulated, and Axin, a negative regulator of the Wnt pathway, was downregulated in resistant cells.,Modulation of these mTOR and Wnt pathway proteins was also prevented by combination treatment with SU11274, everolimus, an mTOR inhibitor, and XAV939, a Wnt inhibitor.,Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells.,The V600E BRAF mutation was found to be positive only in MU cells.,Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability.,These studies indicate activation of mTOR and Wnt signaling pathways in c-Met TKI resistant melanoma cells and suggest that concurrent targeting of c-Met, mTOR, and Wnt pathways and BRAF may improve efficacy over traditional TKI monotherapy in melanoma patients.

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

Patients with high-risk stage II/III resected melanoma commonly develop distant metastases.,At present, we cannot differentiate between patients who will recur or those who are cured by surgery.,We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma.,We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial.,Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples.,Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79-5.47; P < 0.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80-5.79; P < 0.0001) versus those with undetectable ctDNA.,Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83-5.83, P < 0.0001; DMFI: HR 3.45, 95% CI 1.88-6.34, P < 0.0001).,Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%-55%) versus 65% (95% CI 56%-72%) for those with undetectable ctDNA.,Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40-4.96); P = 0.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32-4.74, P = 0.005).,ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy.,ISRCTN 81261306

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.

Methylation of a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene suppresses that gene's transcription.,The expression of G0/G1 switch gene 2 (G0S2) is potentially associated with tumorigenesis.,The aim of this study is to elucidate the methylation status of the CGI located in the 5' region of G0S2 (hereinafter called 5' G0S2 CGI) in cutaneous squamous cell carcinoma (SCC).,Quantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to evaluate the methylation statuses of cutaneous SCC and normal epithelial cell samples.,Quantitative real-time reverse transcription-PCR was performed to evaluate RNA expression levels.,Immunohistochemical analysis was performed to detect protein expression.,G0S2 was suppressed in the five SCC cell lines with 5' G0S2 CGI methylation levels of nearly 100.0% and was expressed in the two normal cultured keratinocytes with methylation levels of almost 0.0%.,G0S2 was re-expressed in SCC cell lines treated with a demethylating agent.,The in vivo methylation levels of 5' G0S2 CGI as determined by RT-MSP varied widely (0.0% to 77.7%) in 17 cutaneous SCC samples and narrowly (0.1% to 7.3%) in 6 normal epidermis samples.,Nine cutaneous SCC samples exhibited higher methylation levels than the highest methylation level (7.3%) of the 6 normal epidermis samples.,Bisulfite sequencing showed dense methylated CpG sites within 5' G0S2 CGI in these highly methylated cutaneous SCC samples.,The methylation levels of the cutaneous SCC samples did not correlate with any clinical parameters investigated or with histopathological grading.,G0S2 is silenced by aberrant DNA methylation in a subset of cutaneous SCCs.

An impressive clinical success has been observed in treating a variety of cancers using immunotherapy with programmed cell death‐1 (PD‐1) checkpoint blockade.,However, limited response in most patients treated with anti‐PD‐1 antibodies remains a challenge, requiring better understanding of molecular mechanisms limiting immunotherapy.,In colorectal cancer (CRC) resistant to immunotherapy, mismatch‐repair‐proficient or microsatellite instability‐low (pMMR‐MSI‐L) tumors have low mutation burden and constitute ~85% of patients.,Here, we show that inhibition of N 6‐methyladenosine (m6A) mRNA modification by depletion of methyltransferases, Mettl3 and Mettl14, enhanced response to anti‐PD‐1 treatment in pMMR‐MSI‐L CRC and melanoma.,Mettl3‐ or Mettl14‐deficient tumors increased cytotoxic tumor‐infiltrating CD8+ T cells and elevated secretion of IFN‐γ, Cxcl9, and Cxcl10 in tumor microenvironment in vivo.,Mechanistically, Mettl3 or Mettl14 loss promoted IFN‐γ‐Stat1‐Irf1 signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2.,Finally, we found a negative correlation between METTL3 or METTL14 and STAT1 in 59 patients with pMMR‐MSI‐L CRC tumors.,Altogether, our findings uncover a new awareness of the function of RNA methylation in adaptive immunity and provide METTL3 and METTL14 as potential therapeutic targets in anticancer immunotherapy.,Disruption of m6A methyltransferases leads to enhanced immunotherapy response in colorectal cancer and melanoma cells due to enhanced IFN‐γ‐Stat1‐Irf1 signaling and modulation of the tumor microenvironment.

While recent years have seen a revolution in the treatment of metastatic cutaneous melanoma, no treatment has yet been able to demonstrate any prolonged survival in metastatic uveal melanoma.,Thus, metastatic uveal melanoma remains a disease with an urgent unmet medical need.,Reports of treatment with immune checkpoint inhibitors have thus far been disappointing.,Based on animal experiments, it is reasonable to hypothesize that the effect of immunotherapy may be augmented by epigenetic therapy.,Proposed mechanisms include enhanced expression of HLA class I and cancer antigens on cancer cells, as well as suppression of myeloid suppressor cells.,The PEMDAC study is a multicenter, open label phase II study assessing the efficacy of concomitant use of the PD1 inhibitor pembrolizumab and the class I HDAC inhibitor entinostat in adult patients with metastatic uveal melanoma.,Primary endpoint is objective response rate.,Eligible patients have histologically confirmed metastatic uveal melanoma, ECOG performance status 0-1, measurable disease as per RECIST 1.1 and may have received any number of prior therapies, with the exception of anticancer immunotherapy.,Twenty nine patients will be enrolled.,Patients receive pembrolizumab 200 mg intravenously every third week in combination with entinostat 5 mg orally once weekly.,Treatment will continue until progression of disease or intolerable toxicity or for a maximum of 24 months.,The PEMDAC study is the first trial to assess whether the addition of an HDAC inhibitor to anti-PD1 therapy can yield objective anti-tumoral responses in metastatic UM.,ClinicalTrials.gov registration number: NCT02697630.,(Registered 3 March 2016).,EudraCT registration number: 2016-002114-50.

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

Melanoma is an aggressive tumor with increasing incidence.,To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood.,In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis.,Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis.,This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.,Here, we analyzed Xmrk-induced gene expression using a microarray approach.,Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors.,The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA.,Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.,Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6).,Interestingly, most genes were blocked in presence of a SRC kinase inhibitor.,Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes.,Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration.,Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines.,The identified molecules constitute new possible molecular players in melanoma development.,Specifically, a role of FOSL1 in melanomagenic processes is demonstrated.,These data are the basis for future detailed analyses of the investigated target genes.

Epithelial cancer constitutes a major clinical challenge and molecular mechanisms underlying the process of tumour initiation are not well understood.,Here we demonstrate that hair follicle bulge stem cells (SCs) give rise to well-differentiated sebaceous tumours and show that SCs are not only crucial in tumour initiation, but are also involved in tumour plasticity and heterogeneity.,Our findings reveal that SC-specific expression of mutant Lef1, which mimics mutations found in human sebaceous tumours, drives sebaceous tumour formation.,Mechanistically, we demonstrate that mutant Lef1 abolishes p53 activity in SCs.,Intriguingly, mutant Lef1 induces DNA damage and interferes with SC-specific gatekeeper functions normally protecting against accumulations of DNA lesions and cell loss.,Thus, normal control of SC proliferation is disrupted by mutant Lef1, thereby allowing uncontrolled propagation of tumour-initiating SCs.,Collectively, these findings identify underlying molecular and cellular mechanisms of tumour-initiating events in tissue SCs providing a potential target for future therapeutic strategies.,The presence of multiple stem and progenitor cells in the skin has a major impact on the formation of different epidermal tumours.,Here the authors identify bulge stem cells as the cells of origin of sebaceous tumours through genetic lineage tracing and molecular studies in a mouse model.

Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients.,Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65-100 fold higher risk than in the normal population1.,By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent 1.,Here we report that genetic and pharmacological suppression of calcineurin/NFAT function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-rasV12 expressing primary human keratinocytes or keratinocyte-derived SCC cells.,Calcineurin/NFAT inhibition counteracts p53-dependent cancer cell senescence thereby increasing tumourigenic potential.,ATF3, a member of the “enlarged” AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumourigenic potential.,Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Originally described as interpatient variability, tumour heterogeneity has now been demonstrated to occur intrapatiently, within the same lesion, or in different lesions of the same patient.,Tumour heterogeneity involves both genetic and epigenetic changes.,Intrapatient heterogeneity is responsible for generating subpopulations of cancer cells which undergo clonal evolution with time.,Tumour heterogeneity develops also as a consequence of the selective pressure imposed by the immune system.,It has been demonstrated that tumour heterogeneity and different spatiotemporal interactions between all the cellular compontents within the tumour microenvironment lead to cancer adaptation and to therapeutic pressure.,In this context, the recent advent of single cell analysis approaches which are able to better study tumour heterogeneity from the genomic, transcriptomic and proteomic standpoint represent a major technological breakthrough.,In this review, using metastatic melanoma as a prototypical example, we will focus on applying single cell analyses to the study of clonal trajectories which guide the evolution of drug resistance to targeted therapy.

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.

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

We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS.,We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling.,This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS.,Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice.,Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression.,They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.,► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice

Malignant melanoma has the highest increase of incidence of malignancies in the western world.,In early stages, front line therapy is surgical excision of the primary tumor.,Metastatic disease has very limited possibilities for cure.,Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem.,The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high.,The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers.,Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation).,Fractionation produced higher numbers of protein identifications (4284).,Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping).,Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma “genomic subtypes”, (“pigmentation” and “high immune”) revealed several proteins showing differential levels of expression.,In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications.,The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.

Diversity between metastatic melanoma tumours in individual patients is known; however, the molecular and genetic differences remain unclear.,To examine the molecular and genetic differences between metastatic tumours, we performed gene-expression profiling of 63 melanoma tumours obtained from 28 patients (two or three tumours/patient), followed by analysis of their mutational landscape, using targeted deep sequencing of 1697 cancer genes and DNA copy number analysis.,Gene-expression signatures revealed discordant phenotypes between tumour lesions within a patient in 50% of the cases.,In 18 of 22 patients (where matched normal tissue was available), we found that the multiple lesions within a patient were genetically divergent, with one or more melanoma tumours harbouring 'private' somatic mutations.,In one case, the distant subcutaneous metastasis of one patient occurring 3 months after an earlier regional lymph node metastasis had acquired 37 new coding sequence mutations, including mutations in PTEN and CDH1.,However, BRAF and NRAS mutations, when present in the first metastasis, were always preserved in subsequent metastases.,The patterns of nucleotide substitutions found in this study indicate an influence of UV radiation but possibly also DNA alkylating agents.,Our results clearly demonstrate that metastatic melanoma is a molecularly highly heterogeneous disease that continues to progress throughout its clinical course.,The private aberrations observed on a background of shared aberrations within a patient provide evidence of continued evolution of individual tumours following divergence from a common parental clone, and might have implications for personalized medicine strategies in melanoma treatment.,Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize.,We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology.,Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival.,We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis.,In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters.,We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation.,Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.,Metastatic cells can mimic many of the phenotypic behaviors of embryonic cells.,Here, the authors generate a melanoblast-specific transcriptome using a genetically engineered mouse model and identify KDELR3 as a pro-metastasis gene in melanoma.

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.

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.

PTEN gene is considered one of the most mutated tumor suppressor genes in human cancer, and it's likely to become the first one in the near future.,Since 1997, its involvement in tumor suppression has smoothly increased, up to the current importance.,Germline mutations of PTEN cause the PTEN hamartoma tumor syndrome (PHTS), which include the past-called Cowden, Bannayan-Riley-Ruvalcaba, Proteus, Proteus-like, and Lhermitte-Duclos syndromes.,Somatic mutations of PTEN have been observed in glioblastoma, prostate cancer, and brest cancer cell lines, quoting only the first tissues where the involvement has been proven.,The negative regulation of cell interactions with the extracellular matrix could be the way PTEN phosphatase acts as a tumor suppressor.,PTEN gene plays an essential role in human development.,A recent model sees PTEN function as a stepwise gradation, which can be impaired not only by heterozygous mutations and homozygous losses, but also by other molecular mechanisms, such as transcriptional regression, epigenetic silencing, regulation by microRNAs, posttranslational modification, and aberrant localization.,The involvement of PTEN function in melanoma and multistage skin carcinogenesis, with its implication in cancer treatment, and the role of front office in diagnosing PHTS are the main reasons why the dermatologist should know about PTEN.

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.

Activated PI3K-AKT pathway may contribute to decrease sensitivity to inhibitors of key pathogenetic effectors (mutated BRAF, active NRAS or MEK) in melanoma.,Functional alterations are deeply involved in PI3K-AKT activation, with a minimal role reported for mutations in PIK3CA, the catalytic subunit of the PI3K gene.,We here assessed the prevalence of the coexistence of BRAF/NRAS and PIK3CA mutations in a series of melanoma samples.,A total of 245 tumor specimens (212 primary melanomas and 33 melanoma cell lines) was screened for mutations in BRAF, NRAS, and PIK3CA genes by automated direct sequencing.,Overall, 110 (44.9%) samples carried mutations in BRAF, 26 (10.6%) in NRAS, and 24 (9.8%) in PIK3CA.,All identified PIK3CA mutations have been reported to induce PI3K activation; those detected in cultured melanomas were investigated for their interference with the antiproliferative activity of the BRAF-mutant inhibitor vemurafenib.,A reduced suppression in cell growth was observed in treated cells carrying both BRAF and PIK3CA mutations as compared with those presenting a mutated BRAF only.,Among the analysed melanomas, 12/245 (4.9%) samples presented the coexistence of PIK3CA and BRAF/NRAS mutations.,Our study further suggests that PIK3CA mutations account for a small fraction of PI3K pathway activation and have a limited impact in interfering with the BRAF/NRAS-driven growth in melanoma.

Malignant melanoma is a rare disease in Asia, and knowledge on its characteristics and clinical outcome in Asian patients is limited.,The purpose of this observational study was to determine the clinical presentation and outcome of patients with melanoma in China.,A database was prospectively established for the purpose of this analysis.,The elements of the database included basic demographic data of patients and prognosticators previously reported in literature, as well as follow-up data including clinical outcome after treatment.,Medical record of all patients with pathologically diagnosed malignant melanoma consulted in our center since 2006 were retrieved and reviewed.,No patient was excluded in this study.,Statistical analyses including survival and multivariate analyses of factors associated with survival were respectively performed by Kaplan-Meier method and Cox proportional hazard model.,A total of 522 consecutive and nonselected cases were evaluated.,There were 218 cases (41.8%) of acral lentiginous melanoma (ALM), 118 (22.6%) of mucosal melanoma (MCM), 103 (19.7%) of nodular melanoma (NM), 33 (6.3%) of superficial spreading melanoma (SSM), and others were Lentigo maligna melanoma or unclassifiable disease.,The proportion of patients with clinical stage I, II, III, and IV diseases were 6.1%, 55.9%, 25.1%, and 12.8%, respectively.,Among the 357 cases of cutaneous melanoma, 234 patients (65.5%) had ulceration.,The 5-year overall survival rate of all 522 patients was 41.6%, and the median survival time was 3.92 years (95% CI, 3.282 to 4.558).,Five-year survival rates of patients with stage I, II, III, and IV diseases were 94.1%, 44.0%, 38.4% and 4.6% respectively (P < 0.001).,Multivariate analysis revealed that clinical stage and the ulceration were two significant prognosticators for OS.,In addition, extent of surgery and use of adjuvant therapy were significant prognosticators for DFS in patients with non-metastatic disease after definitive treatment.,Pathological subtype was not a significant prognostic factor to predict wither OS or DFS.,Prognoses of patients with malignant melanoma diagnosed in China were suboptimal, and most patients were diagnosed with locally advanced disease (i.e., stage II or above).,ALM and MCM are the two most commonly diagnosed pathological subtypes.,Clinical staging and presence of ulceration was significantly associated with clinical outcome in terms of OS, while treatment strategy including extent of surgery and use of adjuvant therapy were significant predictors of DFS.

S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation.,Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival.,In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial.,To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas.,The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry.,The Kaplan-Meier method was used to evaluate the patient survival.,The univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up.,Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas.,Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II-IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (P = 0.005).,Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) but not metastatic melanoma (P>0.05).,This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival.,Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease.

Dabrafenib plus trametinib improved relapse-free survival (RFS) versus placebo (hazard ratio [HR], 0.47; P < .001) in patients with resected BRAF V600-mutant stage III melanoma (BRF115532; COMBI-AD; ClinicalTrials.gov identifier: NCT01682083).,We present an updated RFS analysis on the basis of extended study follow-up and a cure-rate model analysis to estimate the fraction of patients expected to remain relapse free long term.,In this phase III trial, patients with resected BRAF V600-mutant stage III melanoma were randomly assigned to 12 months of adjuvant dabrafenib plus trametinib versus placebo.,We report updated RFS (primary end point) and distant metastasis-free survival.,RFS was also analyzed by subgroups defined by baseline disease stage (American Joint Committee on Cancer 7th and 8th editions), nodal metastatic burden, and ulceration status.,The fraction of patients who remained relapse free long term was estimated using a Weibull mixture cure-rate model.,At median follow-up of 44 months (dabrafenib plus trametinib) and 42 months (placebo), 3- and 4-year RFS rates were 59% (95% CI, 55% to 64%) and 54% (95% CI, 49% to 59%) in the dabrafenib plus trametinib arm and 40% (95% CI, 35% to 45%) and 38% (95% CI, 34% to 44%) in the placebo arm, respectively (HR, 0.49; 95% CI, 0.40 to 0.59).,Distant metastasis-free survival also favored dabrafenib plus trametinib (HR, 0.53; 95% CI, 0.42 to 0.67).,The estimated cure rate was 54% (95% CI, 49% to 59%) in the dabrafenib plus trametinib arm compared with 37% (95% CI, 32% to 42%) in the placebo arm.,Subgroup analysis of RFS demonstrated similar treatment benefit regardless of baseline factors, including disease stage, nodal metastatic burden, and ulceration.,Longer follow-up confirmed RFS benefit with dabrafenib plus trametinib.,Subgroup analysis suggested that dabrafenib plus trametinib benefited patients regardless of baseline factors.

Melanoma is a molecularly heterogeneous disease with many genetic mutations and altered signaling pathways.,Activating mutations in the BRAF oncogene are observed in approximately 50% of cutaneous melanomas and the use of BRAF inhibitor (BRAFi) compounds has been reported to improve the outcome of patients with BRAF-mutated metastatic melanoma.,However, the majority of these patients develop resistance within 6-8 months following the initiation of BRAFi treatment.,In this study, we examined the possible use of the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, ABT-888 (veliparib), as a novel molecule that may be successfully employed in the treatment of BRAFi-resistant melanoma cells.,Sensitive and resistant to BRAFi dabrafenib A375 cells were exposed to increasing concentrations of ABT-888.,Cell viability and apoptosis were assessed by MTT assay and Annexin V-FITC analysis, respectively.,The cell migratory and invasive ability was investigated using the xCELLigence technology and Boyden chamber assays, respectively.,ABT-888 was found to reduce cell viability and exhibited pro-apoptotic activity in melanoma cell lines, independently from the BRAF/NRAS mutation status, in a dose-dependent manner, with the maximal effect being reached in the 25-50 µM concentration range.,Moreover, ABT-888 promoted apoptosis in both the sensitive and resistant A375 cells, suggesting that ABT-888 may be useful in the treatment of BRAFi-resistant subsets of melanoma cells.,Finally, in accordance with the involvement of PARP1 in actin cytoskeletal machinery, we found that the cytoskeletal organization, motility and invasive capability of both the A375 and A375R cells decreased upon exposure to 5 µM ABT-888 for 24 h.,On the whole, the findings of this study highlight the pivotal role of PARP1 in the migration and invasion of melanoma cells, suggesting that ABT-888 may indeed be effective, not only as a pro-apoptotic drug for use in the treatment of BRAFi-resistant melanoma cells, but also in suppressing their migratory and invasive activities.

Skin cancer is one of the most common cancers in the world but is largely preventable through protective behavior.,The aim of this study was to evaluate the impact of an educational intervention based on the BASNEF model on skin cancer prevention and protective behavior in midwifery students in Urmia.,In this quasi-experimental study, the subject population comprised 100 midwifery students in two groups, study and control (n = 50 in each).,The sample was collected using a stratified random sampling method.,The data collection tool was a multi-section questionnaire which included demographic questions, knowledge and structures (attitude, enabling factors, subjective norms, behavior and attitude).,The educational intervention was carried out in three 45-minute sessions.,Data were collected during face-to-face discussions before the educational intervention and three months thereafter and analyzed using paired t-test and independent t-test statistics.,The results showed that after intervention, mean score of knowledge was significantly higher in the study group compared to the control group.,Significant improvement in mean scores for attitude, enabling factors, mental norms, and intent of behavior was limited to the study group Also, behavior for prevention of skin cancer was significantly better in the intervention group.,The results of this study showed that the BASNEF model is effective for promotion of skin cancer prevention behavior.

This experiment examined the impact of adding upward and/or downward social comparison information on the efficacy of an appearance-based sun protection intervention (UV photos and photoaging information).,Southern California college students (N = 126) were randomly assigned to one of four conditions: control, intervention, intervention plus upward social comparison, intervention plus downward social comparison.,The results demonstrated that all those who received the basic UV photo/photoaging intervention reported greater perceived susceptibility to photoaging (d = .74), less favorable tanning cognitions (d = .44), and greater intentions to sun protect (d = 1.32) relative to controls.,Of more interest, while the basic intervention increased sun protective behavior during the subsequent 5 weeks relative to controls (d = .44), the addition of downward comparison information completely negated this benefit.,Upward comparison information produced sun protection levels that were only slightly (and nonsignificantly) greater than in the basic intervention condition and, as such, does not appear to be a cost-effective addition.,Possible mechanisms that may have reduced the benefits of upward comparison information and contributed to the undermining effects of downward comparison information are discussed.

Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.

In uveal melanoma (UM), the most frequent primary intraocular tumour in adults, loss of one entire chromosome 3 (monosomy 3 (M3)) is observed in ∼50% of tumours and is significantly associated with metastatic disease.,The strong association of metastatic disease with M3 offers the opportunity for molecular prognostic testing of UM patients.,To re-evaluate M3 as prognostic marker in our clinical and laboratory setting and to determine the metastatic potential of rare tumours with partial M3, we performed a comprehensive study on 374 UM patients treated by enucleation in our clinic within 10 consecutive years, starting in 1998.,Genotyping of all tumours was performed by microsatellite analysis.,Median follow-up time was 5.2 years.,The disease-specific mortality rates (death by UM metastases) for tumours with disomy 3 (D3) and M3 were 13.2% and 75.1%, respectively.,The disease-specific survival was worse when M3 was observed together with chromosome 8 alterations (P=0.020).,Death of UM metastases was also observed in 12 patients (9%) with D3 tumours.,The metastasising D3 tumours showed a larger basal tumour diameter (P=0.007), and were more frequently of mixed or epitheloid cell type (P<0.0001) than D3 tumours that did not metastasise.,Mortality rate of tumours showing partial M3 (8.3%) was as low as that for tumours with D3.,This shows that large tumours with disomy 3 have an increased risk to develop metastases.,On the basis of these results, our clinic offers routine prognostic testing of UM patients by chromosome 3 typing.

MicroRNAs (miRNAs) have been shown to function in many different cellular processes, including proliferation, apoptosis, differentiation and development. miR-181a, -181b, -181c and -181d are miR-181 members of the family, which has been rarely studied, especially uveal melanoma.,The expression level of miR-181 family in human uveal melanoma cell lines was measured via real-time PCR (RT-PCR).,The function of miR-181 on cell cycle was detected through Flow Cytometry assay.,Microarray assay and Bioinformatics analysis were used to find the potential target of miR-181b, and dual-luciferase reporter assays further identified the target gene.,MiR-181 family members were found to be highly homologous across different species and their upregulation significantly induces UM cell cycle progression.,Of the family members, miR-181b was significantly overexpressed in UM tissues and most UM cells.,Bioinformatics and dual luciferase reporter assay confirmed CTDSPL as a target of miR-181b. miR-181b over-expression inhibited CTDSPL expression, which in turn led to the phosphorylation of RB and an accumulation of the downstream cell cycle effector E2F1, promoting cell cycle progression in UM cells.,Knockdown CTDSPL using siRNAs showing the same effect, including increase of E2F1 and the progression of cell cycle.,MiR-181 family members are key negative regulators of CTDSPL-mediated cell cycle progression.,These results highlight that miR-181 family members, especially miR-181b, may be useful in the development of miRNA-based therapies and may serve as novel diagnostic and therapeutic candidate for UM.,The online version of this article (10.1186/s13046-018-0679-5) contains supplementary material, which is available to authorized users.

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.

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.

Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility.,How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood.,Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development.,Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion.,Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature.,We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces.,Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis.,CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients.,Its expression is also enriched in the invasive fronts of lesions.,Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth.,We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.,•TGF-β-SMAD promotes amoeboid migration in melanoma•Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility•Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients•TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,TGF-β-SMAD promotes amoeboid migration in melanoma,Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility,Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients,TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,Cantelli et al. find that, in melanoma, TGF-β-SMAD-CITED1 controls amoeboid behavior through activation of a transcriptional program.,As a result, melanoma cells detach from keratinocytes, increase their invasive potential, and efficiently colonize the lung.,This is a new function of TGF-β, independent of epithelial-to-mesenchymal transition.

Nicotinamide (NAM) is an amide form of vitamin B3 and the precursor of nicotinamide adenine dinucleotide (NAD+), an essential co-enzyme of redox reactions for adenosine triphosphate (ATP) production and for other metabolic processes.,As NAD+ status is critical in maintaining cellular energy, vitamin B3 deficiency mainly affects tissues that need high cellular energy causing pellagra and skin sun sensitivity.,In animal models, NAD+ deficiency leads to UV sensitivity of the skin, impairs DNA damage response, and increases genomic instability and cancer incidence.,Furthermore, NAD+ depletion is associated with human skin aging and cancer.,NAM prevents the UV-induced ATP depletion boosting cellular energy and enhances DNA repair activity in vitro and in vivo.,Moreover, NAM reduces skin cancer incidence and prevents the immune-suppressive effects of UV in mice.,Thus, NAM is involved in the maintenance of genomic stability and may have beneficial effects against skin aging changes and tumor development.,Clinical studies showed that topical use of NAM reduces cutaneous aging.,Furthermore, oral NAM administration reduces the level of UV-mediated immunosuppression and lowers the rate of non-melanoma skin cancers in high-risk patients.,Therefore, NAM replenishment strategy may be a promising approach for skin cancer chemoprevention.

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

Isolated limb perfusion combined with melphalan is an accepted treatment for obtaining locoregional control in advanced melanoma of the extremities and other malignant neoplasias restricted to the limb.,This study aims to examine the factors associated with toxicity caused by the regional method.,We considered the technical aspects of severe complications associated with the procedure in an attempt to diminish the patient morbidity that occurs during the learning curve.,We conducted a retrospective analysis of the records of patients who underwent perfusion at the AC Camargo Hospital in São Paulo, Brazil between January 2000 and January 2009.,The Wieberdink scale was applied to classify local toxicity and its relation to clinical and laboratory variables.,Fifty-eight perfusions were performed in 55 patients.,Most patients (86.2%) presented a toxicity level between I and III.,Grade V toxicity was seen in five cases (8.6%), four of which occurred in the first 2 years.,Creatine phosphokinase, an important predictive factor for toxicity, had an average value of 231.8 for toxicity grades I-III and 1286.2 for toxicity grades IV-V (p = 0.001).,There was a relationship between the melphalan dose and toxicity, which was 77 mg (25 to 130 mg) for toxicity grades I-II and 93.5 mg (45 to 120 mg) for toxicity grades IV-V (p = 0.0204).,It is possible to prevent the toxicity associated with melphalan by adjusting the dose according to the patient's body weight (especially for women and obese patients) and the creatine phosphokinase values in the postoperative period.

Approximately 5-8% of melanoma patients will develop in-transit metastases (IT-mets).,Tumor necrosis factor-α (TNF) and melphalan-based isolated limb perfusion (TM-ILP) is an attractive treatment modality in melanoma patients with multiple IT-mets.,This study reports on a 20 years experience and outlines the evolution and major changes since the introduction of TNF in ILP.,A total of 167 TM-ILPs were performed in 148 patients, between 1991 and 2009.,TM-ILPs were performed at high doses of TNF (3-4 mg) from 1991 to 2004 (n = 99) and at low doses of TNF (1-2 mg) from 2004 to 2009 (n = 68) under mild hyperthermic conditions (38°C-39.5°C.).,Melphalan doses were unchanged at 10-13 mg/l (leg and arm, respectively).,Characteristics for the 167 ILPs were: 81 stage IIIB, 65 stage IIIC, and 21 stage IV disease.,The overall response rate was 89% (n = 148).,(Complete response [CR] = 61%; partial response [PR] = 28%).,CR rates correlated with stage (P = .001) and with high-dose vs. low-dose TNF (70% vs.,49%; P < .006).,High-dose TNF prolonged local control (median 16 months vs. 11 months; P = .076).,Survival was not influenced by TNF dose.,CR after ILP and number of lesions also correlated with local progression-free interval.,Overall survival did correlate with stage of disease (P < .001), size of the lesions (P = .001), and a CR (P < .001).,This 2-decade single-center experience demonstrates that TM-ILP is a safe and effective treatment modality for melanoma patients with multiple IT-mets.,Higher dose of TNF was associated with significantly higher CR rates and prolonged local control without an effect on overall survival.

An important component of research using animal models is ensuring rigor and reproducibility.,This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy.,Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC).,Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied.,The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation.,We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank.,When tumors reached 190-230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine).,Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia.,Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors.,SC tumors reached the target tumor volume significantly faster compared with ID tumors.,Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not.,Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth.,These findings were then extended to additional immunotherapy regimens in four separate tumor models.,These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment.,Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

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

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.

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.

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.

For the first time new treatments in melanoma have produced significant responses in advanced diseases, but 30-90% of melanoma patients do not respond or eventually relapse after the initial response to the current treatments.,The resistance of these melanomas is likely due to tumor heterogeneity, which may be explained by models such as the stochastic, hierarchical, and phenotype-switching models.,This review will discuss the recent advancements in targeting BCL-2 family members for cancer treatments, and how this approach can be applied as an alternative option to combat melanoma, and overcome melanoma relapse or resistance in current treatment regimens.

To examine whether GNAQ and GNA11 somatic mutations previously identified in uveal melanomas of Caucasians are associated with uveal melanomas in Chinese patients.,Uveal melanomas treated by primary enucleation in Chinese patients underwent a mutation analysis of GNAQ and GNA11 with sequencing of exon 5 and exon 4.,The study included 50 patients with uveal melanoma and with a mean age of 47.6±13.0 years.,During the follow-up of at least 3 years, 20 (40%) patients developed extraocular metastases.,The frequencies of GNAQ and GNA11 somatic mutations in uveal melanoma were 18% (9/50) and 20% (10/50), respectively.,The mutations occurred exclusively in codon 209 of exon 5.,No mutations were detected in exon 4.,Mutations affecting codon 209 in GNAQ were c.626A>C(Q209P) (78%) and c.626A>T(Q209L) (22%).,Mutations affecting codon 209 in GNA11 were exclusively c.626A>T(Q209L) (100%).,In none of the tumors, mutations of BRAF and NRAS were detected.,GNAQ/11 mutations were marginally (P = 0.045) associated with optic disc involvement.,In Kaplan-Meier analysis, metastasis-free survival was not significantly (P = 0.94) associated with GNAQ/11 mutations.,Mutations of GNAQ and GNA11 can be found in Chinese patients as in Caucasian patients with uveal melanoma, with a higher frequency reported for Caucasian patients.

There is a need for more effective treatments for uveal melanoma.,The recombinant oncolytic adenovirus H101 replicates specifically in p53-depleted tumor cells, and has been approved for use by the Chinese State Food and Drug Administration.,However, this treatment is associated with subsequent remission.,Transfection of uveal melanoma cells with a small interfering RNA against Notch1 (siNotch1) effectively suppressed Notch1 expression, resulting in significant cell growth inhibition when combined with H101 treatment.,Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) greatly enhanced apoptosis and cell cycle arrest in vitro as compared to treatment with H101 or siNotch1 alone.,For in vivo treatments, the combined treatment of siNotch1 and H101 showed remarkable tumor growth inhibition and prolonged mouse survival in the OCM1 xenograft model.,We predict that Notch pathway deregulation could be a feature of uveal melanoma, and could be a therapeutic target, especially if p53 is concurrently targeted.

Dysregulation of the microRNA (miRNA) network is a typical feature of many cancers.,However, the key specific miRNAs involved in uveal melanoma carcinogenesis are largely unknown.,RT-qPCR was used to detected miR-652 expression in uveal melanoma tissues and cell lines. miR-652 inhibitor was transfected into uveal melanoma cells to decrease miR-652 expression and determine the biological role of miR-652 by CCK-8 and wound healing assays.,Bioinformatic analysis and dual luciferase reporter assay were used to predict and validate the target gene of miR-652.,HOXA9 siRNA was transfected into cells to confirm that miR-652 relies on regulation of HOXA9 to regulate cell proliferation and migration.,RT-qPCR showed that miR-652 was overexpressed in uveal melanoma cell lines (MUM-2B, MEL270) compared with melanocyte cells (ARPE-19).,Overexpression of miR-652 was also observed in uveal melanoma compared to paired non-tumor tissues.,Downregulation of miR-652 inhibited the cell proliferation ability and migration ability of uveal melanoma cells.,Using bioinformatic analysis, HOXA9 was found to be a potential target gene of miR-652.,The direct regulation of HOXA9 by miR-652 was experimentally validated in uveal melanoma cells by dual luciferase assay and Western blotting.,We also observed that miR-652 promoted HIF-1α signaling via repression of HOXA9 in uveal melanoma cells.,Silencing of HOXA9 attenuated the miR-652 inhibitor decreased cell growth rate and decreased migration ability in uveal melanoma cells.,Our data demonstrate an oncogenic role of miR-652 in uveal melanoma, showing that miR-652 may be a useful biomarker for prediction of prognosis for patients with uveal melanoma.

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

The present study aimed to investigate the potential role of the long non-coding RNA (lncRNA) Pvt1 oncogene (non-protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms.,The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A-375 and sk-mel-5 cell lines, was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analyses.,The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively.,The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay.,The effect of PVT1 on microRNA-200c (miR-200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay.,PVT1 was highly expressed in the melanoma tissues and cells.,Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage.,Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells.,The expression of E-cadherin was significantly increased and the expression of N-cadherin and vimentin was significantly decreased in the PVT1-silenced group.,The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control.,The ChIP assay revealed that the expression of miR-200c was decreased significantly in the PVT1-silenced group compared with the controls.,Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR-200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.

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

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

Rationale: Mutations in KIT, a major cancer driver gene, are now considered as important drug targets for the treatment of melanomas arising from mucosal and acral tissues and from chronically sun-damaged sites.,At present, imatinib is the only targeted drug for KIT-mutation-bearing melanomas that is recommended by the National Comprehensive Cancer Network (NCCN) Clinical Practice guidelines.,Patients with KIT mutations, however, are either insensitive or rapidly progress to imatinib insensitivity, which restricts its clinical use.,Thus, effective inhibitors of KIT-mutation-bearing melanomas are urgently needed.,Methods: A cohort of patient-derived tumor xenograft (PDX) models and corresponding PDX-derived cells (PDCs) from patients with melanomas harboring KIT mutations (KITV560D, KITK642E and KITD816V) were established, characterized, and then used to test the in vitro and, subsequently, in vivo inhibitory effects of a panel of known KIT inhibitors.,Results: Ponatinib was more potent than imatinib against cells bearing KIT mutations.,In vivo drug efficacy evaluation experiments showed that ponatinib treatment caused much stronger inhibition of KIT-mutation-bearing melanomas than did imatinib.,Mechanistically, molecular dynamics (MD) simulations revealed a plausible atomic-level explanation for the observation that ponatinib has a higher affinity for the KITD816V mutant protein than does imatinib.,Conclusions: Our study of KIT-mutation-and KITWT-bearing melanomas demonstrates that ponatinib is a far more potent inhibitor than is imatinib for KIT-mutation-bearing melanomas and thus underscores that ponatinib should be given priority consideration for the design of precision treatments for melanoma patients triaged to have KIT mutations.,Moreover, our work provides a rationale for undertaking clinical trials to examine the repurposing of ponatinib, which is already approved for use in leukemia, for use in treating a large subset of melanoma patients.

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.

Circulating melanoma cells (CMCs) are thought to be valuable in improving measures of prognosis in melanoma patients and may be a useful marker of residual disease to identify non-metastatic patients requiring adjuvant therapy.,We investigated whether immunomagnetic enrichment targeting multiple markers allows more efficient enrichment of CMCs from patient peripheral blood than targeting a single marker.,Furthermore, we aimed to determine whether the number of CMCs in patient blood was associated with disease stage.,We captured CMCs by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271, both individually and in combination, by immunomagnetic enrichment.,CMCs were enriched and quantified from the peripheral blood of 10 non-metastatic and 13 metastatic melanoma patients.,Targeting all markers in combination resulted in the enrichment of more CMCs than when any individual marker was targeted (p < 0.001-0.028).,Furthermore, when a combination of markers was targeted, a greater number of CMCs were enriched in metastatic patients compared with non-metastatic patients (p = 0.007).,Our results demonstrated that a combination of markers should be targeted for optimal isolation of CMCs.,In addition, there are significantly more CMCs in metastatic patients compared with non-metastatic patients and therefore quantification of CMCs may prove to be a useful marker of disease progression.

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

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

Conjunctival melanoma (CjM) is a rare, primary cancer of the ocular region.,Genetic and epigenetic characteristics of conjunctival melanoma have not been completely elucidated yet.,Conjunctival melanoma presents similarities with cutaneous melanoma, with substantial differences in the biological behavior.,We reviewed the genetic and epigenetic insights of CjM involved in invasion and metastatic spread.,CjM is commonly characterized by mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF), neurofibromin 1 (NF1) and telomerase reverse transcriptase (TERT), high expression of mammalian target of rapamycin (mTOR) and heat shock protein 90 (HSP90), frequent phosphatase and tensin homolog (PTEN) loss and upregulation of specific miRNAs.,These features should identify CjM as a distinct subset of melanoma with its own profile, which is more similar to cutaneous melanoma than mucosal melanoma and remarkably different from uveal melanoma.

We report phase 1b data from patients enrolled in the JAVELIN Solid Tumor clinical trial (NCT01772004) with unresectable stage IIIC or IV melanoma that had progressed after ≥1 line of therapy for metastatic disease.,Patients received avelumab (10 mg/kg)-a human anti-PD-L1 antibody.,Assessments included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety.,As of December 31, 2016, 51 patients were treated and followed for a median of 24.2 months (range, 16.1-31.5).,Most patients had cutaneous (n = 28 [54.9%]) or ocular (n = 16 [31.4%]) melanoma and had received a median of 2 prior lines of therapy (range, 0-4), including ipilimumab (n = 26 [51.0%]).,The confirmed ORR was 21.6% (95% CI, 11.3-35.3; complete response, 7.8%; partial response, 13.7%).,The median duration of response was not estimable (95% CI, 2.6 months-not estimable).,Median PFS and OS were 3.1 months (95% CI, 1.4-6.3) and 17.2 months (95% CI, 6.6-not estimable), respectively.,Subgroup analyses suggested meaningful clinical activity (ORR [95% CI]) in patients with non-ocular melanoma (31.4% [16.9-49.3]), PD-L1-positive tumors (42.1% [20.3-66.5]), or prior ipilimumab therapy (30.8% [14.3-51.8]).,Thirty-nine patients (76.5%) had a treatment-related adverse event (TRAE), most commonly infusion-related reaction (29.4%), fatigue (17.6%), and chills (11.8%); 4 patients (7.8%) had a grade 3 TRAE.,Five patients (9.8%) had an immune-related TRAE (all were grade 1/2).,No grade 4 TRAEs or treatment-related deaths were reported.,Avelumab showed durable responses, promising survival outcomes, and an acceptable safety profile in patients with previously treated metastatic melanoma.,ClinicalTrials.gov identifier: NCT01772004.,The online version of this article (10.1186/s40425-018-0459-y) contains supplementary material, which is available to authorized users.

Over two-thirds of melanomas have activating mutations in B-Raf, leading to constitutive activation of the B-Raf/MKK/ERK signaling pathway.,The most prevalent mutation, B-RafV600E, promotes cancer cell behavior through mechanisms that are still incompletely defined.,Here, we used a sensitive microarray profiling platform to compare microRNA (miRNA) expression levels between primary melanocytes and B-RafV600E-positive melanoma cell lines, and between melanoma cells treated in the presence and absence of an MKK1/2 inhibitor.,We identified a network of >20 miRNAs deregulated by B-Raf/MKK/ERK in melanoma cells, the majority of which modulate the expression of key cancer regulatory genes and functions.,Importantly, miRNAs within the network converge on protein regulation and cancer phenotypes, suggesting that these miRNAs might function combinatorially.,We show that miRNAs augment effects on protein repression and cell invasion when co-expressed, and gene-specific latency and interference effects between miRNAs were also observed.,Thus, B-Raf/MKK/ERK controls key aspects of cancer cell behavior and gene expression by modulating a network of miRNAs with cross-regulatory functions.,The findings highlight the potential for complex interactions between coordinately regulated miRNAs within a network.

The incidence of malignant melanoma is increasing faster than that for any other cancer.,Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis.,Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis.,To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs).,Differential expression was validated by qRT-PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression.,In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas.,In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion.,We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.

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.

Wu et al. report a novel IL-17-mediated cascade via the IL-17R-TRAF4-ERK5 axis that directly stimulates keratinocyte proliferation and skin tumor formation in mice.,Although IL-17 is emerging as an important cytokine in cancer promotion and progression, the underlining molecular mechanism remains unclear.,Previous studies suggest that IL-17 (IL-17A) sustains a chronic inflammatory microenvironment that favors tumor formation.,Here we report a novel IL-17-mediated cascade via the IL-17R-Act1-TRAF4-MEKK3-ERK5 positive circuit that directly stimulates keratinocyte proliferation and tumor formation.,Although this axis dictates the expression of target genes Steap4 (a metalloreductase for cell metabolism and proliferation) and p63 (a transcription factor for epidermal stem cell proliferation), Steap4 is required for the IL-17-induced sustained expansion of p63+ basal cells in the epidermis.,P63 (a positive transcription factor for the Traf4 promoter) induces TRAF4 expression in keratinocytes.,Thus, IL-17-induced Steap4-p63 expression forms a positive feedback loop through p63-mediated TRAF4 expression, driving IL-17-dependent sustained activation of the TRAF4-ERK5 axis for keratinocyte proliferation and tumor formation.

Uveal melanoma (UM) is the most common intraocular tumor in adults.,Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal.,UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy.,Hence, alternative diagnostic tools are needed.,Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring.,The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations.,We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients.,Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws.,Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15).,The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant.,In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth.,Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor.,In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients.,We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy.,Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors.,We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression.,These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.,The online version contains supplementary material available at 10.1186/s13046-021-01984-w.

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

Fibroblast growth factor (FGF)/Fibroblast growth factor receptor (FGFR) signaling regulates various cellular processes during the embryonic development and in the adult organism.,In the skin, fibroblasts and keratinocytes control proliferation and survival of melanocytes in a paracrine manner via several signaling molecules, including FGFs.,FGF/FGFR signaling contributes to the skin surface expansion in childhood or during wound healing, and skin protection from UV light damage.,Aberrant FGF/FGFR signaling has been implicated in many disorders, including cancer.,In melanoma cells, the FGFR expression is low, probably because of the strong endogenous mutation-driven constitutive activation of the downstream mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling pathway.,FGFR1 is exceptional as it is expressed in the majority of melanomas at a high level.,Melanoma cells that acquired the capacity to synthesize FGFs can influence the neighboring cells in the tumor niche, such as endothelial cells, fibroblasts, or other melanoma cells.,In this way, FGF/FGFR signaling contributes to intratumoral angiogenesis, melanoma cell survival, and development of resistance to therapeutics.,Therefore, inhibitors of aberrant FGF/FGFR signaling are considered as drugs in combination treatment.,The ongoing LOGIC-2 phase II clinical trial aims to find out whether targeting the FGF/FGFR signaling pathway with BGJ398 may be a good therapeutic strategy in melanoma patients who develop resistance to v-Raf murine sarcoma viral oncogene homolog B (BRAF)/MEK inhibitors.

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

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

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma.,Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease.,Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients.,We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis.,Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence.,Targeting these alterations blunts the development of drug resistance.

miR-150 has been demonstrated to inhibit tumor progression in various human cancers, including colorectal cancer, ovarian cancer, and thyroid cancer.,However, the role of miR-150 in melanoma remains to be determined.,In this study, we found that miR-150 was underexpressed in melanoma tissues and cell lines.,Through transfection of miR-150 mimics, we found that miR-150 significantly inhibited the proliferation, migration, and invasion of melanoma cells.,In mechanism, we found that MYB was a target of miR-150 in melanoma cells.,Overexpression of miR-150 significantly inhibited mRNA and protein levels of MYB in melanoma cells.,Moreover, there was an inverse correlation between the expression of miR-150 and MYB in melanoma tissues.,We also showed that MYB was upregulated in melanoma tissues and cell lines.,Through functional experiments, we found that restoration of MYB in miR-150-overexpressed melanoma cells rescued the proliferation, migration, and invasion.,Therefore, our findings demonstrated that miR-150 suppressed the proliferation, migration, and invasion of melanoma cell by downregulating MYB.

Skin cutaneous melanoma (SKCM) is characterized by both epigenetic DNA methylation (MET) abnormalities and genomic copy number variations (CNVs).,The resulting transcriptome dysregulation promotes progression of many cancers.,In this study, DNA copy numbers and MET, as well as mRNA expression, were examined in 466 SKCM samples from The Cancer Genome Atlas.,Our results indicate that CNVs-correlated (CNVcor) genes and MET-correlated (METcor) genes are coregulated to a remarkable degree.,In addition, integrative multi-omics analysis of both METcor and CNVcor genes revealed four SKCM subtypes with differing prognoses; these subtypes were validated with independent data.,Immune cell scores were markedly elevated in the iC1 subtype, which had the best prognosis.,Immune cell infiltration correlated with DNA MET or CNV level in SKCM.,In the iC3 subtype, which was associated with the most aggressive SKCM cases, FAM135B gene mutation frequencies were increased, while CD8A, GBP5, KIAA0040, and SAMHD1 expression were downregulated, suggesting that these genes play important roles in cancer development and immune responses.,Taken together, the results of our epigenetic and genomic transcriptome modulation analysis improve our understanding of SKCM pathobiology and may aid in the development of more effective therapies.

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.

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

A dynamic phenotypic change contributes to the metastatic progression and drug resistance in malignant melanoma.,Nevertheless, mechanisms for a phenotypic change have remained to be addressed.,Here, we show that Met receptor expression changes in a cell-autonomous manner and can distinguish phenotypical differences in growth, as well as in metastatic and drug-resistant characteristics.,In metastatic melanoma, the cells are composed of Met-low and Met-high populations.,Met-low populations have stem-like gene expression profiles, are resistant to chemotherapeutic agents, and have shown abundant angiogenesis and rapid tumor growth in subcutaneous inoculation.,Met-high populations have a differentiated phenotype, are relatively resistant to B-RAF inhibitor, and are highly metastatic to the lungs.,Met plays a definitive role in lung metastasis because the lung metastasis of Met-high cells requires Met, and treatment of mice with the Met-containing exosomes from Met-high cells facilitates lung metastasis by Met-low cells.,Clonal cell fate analysis showed the hierarchical phenotypical changes from Met-low to Met-high populations.,Met-low cells either showed self-renewal or changed into Met-high cells, whereas Met-high cells remained Met-high.,Clonal transition from Met-low to Met-high cells accompanied changes in the gene expression profile, in tumor growth, and in metastasis that were similar to those in Met-high cells.,These findings indicate that malignant melanoma has the ability to undergo phenotypic change by a cell-intrinsic/autonomous mechanism that can be characterized by Met expression.

Of more than 150,000 published studies evaluating new biomarkers, fewer than 100 biomarkers have been implemented for patient care[1].,One reason for this is lack of rigorous testing by the medical community to validate claims for biomarker clinical relevance, and potential reluctance to publish negative results when confirmation is not obtained.,Here we sought to determine the utility and reproducibility of immunohistochemical detection of hepatocyte growth factor (HGF) in melanoma tissue, an approach of potential assistance in defining patients with innate resistance to BRAF inhibitor therapy[2].,To this end, a published and a revised method that retained sensitivity but with greater specificity for HGF detection, were evaluated in cells known to endogenously express HGF, models where HGF is upregulated via cytokine induction, and via overexpression by gene transfection.,Consequent patient evaluation in collaboration with the Melanoma Institute Australia of a cohort of 41 melanoma specimens with extensive clinical annotation failed to validate HGF immunohistochemistry as a predictor of response to BRAF inhibitors.,Targeted therapies for advanced melanoma[3-5] and other cancers show great promise, and rigorous validation studies are thus indicated for approaches that seek to personalize such therapies in order to maximize therapeutic efficacy.

Skin melanoma is one of the most aggressive and difficult-to-treat human malignancies, characterized by poor survival rates, thus requiring urgent novel therapeutic approaches.,Although metabolic reprogramming has represented so far, a cancer hallmark, accumulating data indicate a high plasticity of cancer cells in modulating cellular metabolism to adapt to a heterogeneous and continuously changing microenvironment, suggesting a novel therapeutic approach for dietary manipulation in cancer therapy.,To this aim, we exposed melanoma cells to combined nutrient-restriction/sorafenib.,Results indicate that cell death was efficiently induced, with apoptosis representing the prominent feature.,In contrast, autophagy was blocked in the final stage by this treatment, similarly to chloroquine, which also enhanced melanoma cell sensitization to combined treatment.,Energy stress was evidenced by associated treatment with mitochondrial dysfunction and glycolysis impairment, suggesting metabolic stress determining melanoma cell death.,A reduction of tumor growth after cycles of intermittent fasting together with sorafenib treatment was also observed in vivo, reinforcing that the nutrient shortage can potentiate anti-melanoma therapy.,Our findings showed that the restriction of nutrients by intermittent fasting potentiates the effects of sorafenib due to the modulation of cellular metabolism, suggesting that it is possible to harness the energy of cancer cells for the treatment of melanoma.

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma.,Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms.,In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma.,Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors.,Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3.,Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion.,Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling.,Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present.,Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis.,Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.

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

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

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

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success.,Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes.,Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a−/−Pten−/− YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1).,We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy.,Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma.,The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

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

Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood.,Here we explore the oncogenic effects of BPTF in melanoma.,The consequences of differential expression of BPTF were explored using shRNA-mediated knockdown in several melanoma cell lines.,Immunoblotting was used to assess the expression of various proteins regulated by BPTF.,The functional role of BPTF in melanoma progression was investigated using assays of colony formation, invasion, cell cycle, sensitivity to selective BRAF inhibitors, and in xenograft models of melanoma progression (n = 12 mice per group).,The biomarker role of BPTF in melanoma progression was assessed using fluorescence in situ hybridization and immunohistochemical analyses.,All statistical tests were two-sided.,shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells.,Elevated BPTF copy number (mean ≥ 3) was observed in 28 of 77 (36.4%) melanomas.,BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival P = .03, and disease-specific survival P = .008), and promoted resistance to BRAF inhibitors in melanoma cell lines.,Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell subclones, indicating the continued presence of drug-responsive subclones within tumors demonstrating overall resistance to anti-BRAF agents.,These studies demonstrate multiple protumorigenic functions for BPTF and identify it as a novel target for anticancer therapy.,They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF.

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

Angiogenesis is an important mediator in tumor progression.,Vascular endothelial growth factor (VEGF) is one of the major cytokines that can influence angiogenesis.,However, the potential mechanism of tumor growth inhibition through anti-VEGF agents is still unclear.,This study was performed to examine whether ranibizumab could inhibit malignant melanoma growth in vitro and to determine the safety of ranibizumab on human adult retinal pigment epithelium cell line (ARPE-19 cells).,Malignant melanoma cells obtained from a clinic were cultured in vitro.,VEGF concentrations secreted by malignant melanoma cells and the ARPE-19 cells were examined by enzyme-linked immunosorbent assay (ELISA).,The two kinds of cells were both treated with VEGF and its antagonist, ranibizumab.,The dynamic changes of the two types of cells were monitored by real-time cell electronic sensing (RT-CES) assay.,The effect of ranibizumab on both types of cells was verified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay.,The expression of VEGF receptor 1 (VEGFR1) RNA in uveal melanoma was further investigated through the PCR technique.,The levels of VEGF secreted by malignant melanoma cells were much higher than those of ARPE-19 cells, and were markedly decreased in the action of 0.1 mg/ml ranibizumab.,However, there was no obvious reduction of VEGF in the presence of ranibizumab for ARPE-19 (p>0.05).,Meanwhile, RT-CES showed that the viability of malignant melanoma cells increased greatly in the presence of VEGF.,When VEGF was 20 ng/ml, viability of the malignant melanoma cells increased by 40% compared with the negative control.,There was no evident effect on proliferation of ARPE-19 (p>0.05).,Furthermore, the growth of malignant melanoma cells was obviously inhibited after ranibizumab intervention.,When ranibizumab was administered at 0.25 mg/ml, the survival rate of the malignant melanoma cells decreased to 57.5%.,Nevertheless, low-dose exposure to ranibizumab had only a slight effect on the growth of ARPE-19, and PCR result demonstrated that VEGFR1 plays a role in this tumor tissue rather than VEGFR2.,Ranibizumab can selectively inhibit malignant melanoma cell proliferation by decreasing the expression of VEGF; the possible mechanism of the inhibitory effect may involve VEGFR1 antagonism.

To determine if mosaic tuberous sclerosis complex (TSC) can be stratified into subtypes that correspond with prognosis and extent of disease.,Next-generation sequencing of skin tumor and other samples was used to identify patients with mosaic pathogenic variants in TSC1 or TSC2.,Extent of disease, onset age, and family history of TSC were determined through retrospective analysis of patient records.,The median number of disease findings and age at penetrance differed between mosaic patients with asymmetrically distributed facial angiofibromas (4 findings, 24y, n=7), mosaic patients with bilaterally symmetric facial angiofibromas (8 findings, 10y, n=12), and germline TSC patients (10 findings, 4y, n=29).,Cutaneous and internal organ involvement positively correlated in mosaic (R=0.62, p=0.005), but not germline (R=−0.24, p=0.24) TSC.,Variant allele fraction (VAF) in the blood (range: 0-19%) positively correlated with the number of major features (R=0.55, p=0.028).,Five had a TSC2 variant identified in the skin that was below detection in the blood.,One of 12 children from a mosaic parent had TSC.,The phenotype of mosaic TSC ranged from mild to indistinguishable from germline disease.,Patients with mosaicism and asymmetric facial angiofibromas exhibited fewer findings, later onset, and lower VAF in the blood.

Cancer treatment often tends to involve direct targeting enzymes essential for the growth and proliferation of cancer cells.,The aim of this study was the recognition of the possible role of selected protein kinases: PI3K, ERK1/2, and mTOR in cell proliferation and cell cycle in malignant melanoma.,We investigated the role of protein kinase inhibitors: U0126 (ERK1/2), LY294002 (PI3K), rapamycin (mTOR), everolimus (mTOR), GDC-0879 (B-RAF), and CHIR-99021 (GSK3beta) in cell proliferation and expression of crucial regulatory cell cycle proteins in human melanoma cells: WM793 (VGP) and Lu1205 (metastatic).,They were used either individually or in various combinations.,The study on the effect of signaling kinases inhibitors on proliferation-BrdU ELISA test after 48-72 h.,Their effect on the expression of cell cycle regulatory proteins: cyclin D1 and D3, cyclin-dependent kinase CDK4 and CDK6, and cell cycle inhibitors: p16, p21, and p27, was studied at the protein level (western blot).,Treatment of melanoma cells with protein kinase inhibitors led to significantly decreased cell proliferation except the use of a GSK-3β kinase inhibitors-CHIR-99021.,The significant decrease in the expression of selected cyclins and cyclin-dependent kinases (CDKs) with parallel increase in the expression of some of cyclin-dependent kinases inhibitors and in consequence meaningful reduction in melanoma cell proliferation by the combinations of inhibitors of signaling kinases clearly showed the crucial role of AKT, ERK 1/2, and mTOR signal transduction in melanoma progression.,The results unanimously indicate those pathways as an important target for treatment of melanoma.

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

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

Arsenic, a naturally occurring element, contaminates the drinking water of over 200 million people globally.,Chronic arsenic exposure causes multiple cancers including those originating from skin, lung and bladder, and is associated with liver, kidney, and prostate cancers.,Skin is the primary target organ for arsenic toxicity; chronic toxicity initially manifests as non-malignant hyperkeratoses (HK) and subsequently advances to malignant lesions, including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC).,In this study, we evaluate the miRNA expression profiles of premalignant (3 HK) and malignant (3 BCC and 3 SCC) skin lesions from individuals chronically exposed to high levels of arsenic (59-172 ppb) in their drinking water in West Bengal, India.,The lesions were histologically complex requiring histopathologic identification of keratinocytes to be isolated for RNA analyses.,Keratinocytes were harvested using Laser Capture Microdissection and miRNA expression profiles were determined using TaqMan® Array Human MiRNA A Card v2.0.,Thirty-five miRNAs were differentially expressed among the three lesion types analyzed.,Two miRNAs (miR-425-5p and miR-433) were induced in both BCC and SCC relative to HK indicating their association with malignancy.,Two other miRNAs (miR-184 and miR-576-3p) were induced in SCC relative to both BCC and HK suggesting selective induction in tumors capable of metastasis.,Six miRNAs (miR-29c, miR-381, miR-452, miR-487b, miR-494 and miR-590-5p) were selectively suppressed in BCC relative to both SCC and HK.,In conclusion, the differential miRNA expression was both phenotype- and stage-related.,These miRNAs are potential biomarkers and may serve as therapy targets for arsenic-induced internal tumors.

Vascular endothelial growth factor A (VEGFA) plays a key role in the angiogenesis of human skin.,Elevated levels of VEGFA are associated with several pathological conditions, including chronic inflammatory skin diseases and several types of skin cancer.,In particular, squamous cell carcinoma (SCC) of the skin, the second most common skin cancer in the general population, is characterized by invasive growth, pronounced angiogenesis and elevated levels of VEGFA.,The processing, turnover and production of VEGFA are extensively regulated at the post-transcriptional level, both by RNA-binding proteins and microRNAs (miRNAs).,In the present study, we identified a new miRNA recognition element in a downstream conserved region of the VEGFA 3′-UTR.,We confirmed the repressive effect of miR-361-5p on this element in vitro, identifying the first target for this miRNA.,Importantly, we found that miR-361-5p levels are inversely correlated with VEGFA expression in SCC and in healthy skin, indicating that miR-361-5p could play a role in cancers.

Preclinical studies suggest that BRAF inhibitors enhance anti-tumor immunity and antigen presentation.,Combination BRAF inhibition with immunotherapy is an appealing therapeutic approach.,We sequenced vemurafenib with HD IL-2 in patients with BRAF-mutated metastatic melanoma to improve long term outcomes.,Eligible patients were HD IL-2 eligible with metastatic BRAF V600 mutated melanoma.,Cohort 1 was treatment naïve and received vemurafenib 960 mg BID for 6 weeks before HD IL-2.,Cohort 2 received vemurafenib for 7-18 weeks before enrollment.,Both cohorts received HD IL-2 at 600,000 IU/kg every 8 h days 1-5 and days 15-19.,The primary objective was to assess complete responses (CR) at 10 weeks ±3 (assessment 1) and 26 weeks ±3 (assessment 2) from the start of HD IL-2.,Fifty-three patients were enrolled, (cohort 1, n = 38; cohort 2, n = 15).,Of these, 39 underwent assessment 1 and 15 assessment 2.,The CR rate at assessment 1 was 10% (95% CI 3-24) for both cohorts combined, and 27% (95% CI 8-55) at assessment 2.,Three-year survival was 30 and 27% for cohort 1 and cohort 2, respectively.,No unexpected toxicities occurred.,A shift in the melanoma treatment landscape during this trial adversely affected accrual, leading to early trial closure.,Vemurafenib in sequence with HD IL-2 did not change the known toxicity profile for either agent.,Lower than expected response rates to vemurafenib were observed.,Overall response rates and durability of responses appear similar to that observed with HD IL-2 alone.,NCTN, NCT01683188.,Registered 11 September 2012, http://www.clinicaltrials.gov/NCT01683188

MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated.,Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance.,To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression.,A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels.,Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance.,This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2).,A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death.,These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors.,These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.

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

Identification of melanoma patients at high risk for recurrence and monitoring for recurrence are critical for informed management decisions.,We hypothesized that serum microRNAs (miRNAs) could provide prognostic information at the time of diagnosis unaccounted for by the current staging system and could be useful in detecting recurrence after resection.,We screened 355 miRNAs in sera from 80 melanoma patients at primary diagnosis (discovery cohort) using a unique quantitative reverse transcription-PCR (qRT-PCR) panel.,Cox proportional hazard models and Kaplan-Meier recurrence-free survival (RFS) curves were used to identify a miRNA signature with prognostic potential adjusting for stage.,We then tested the miRNA signature in an independent cohort of 50 primary melanoma patients (validation cohort).,Logistic regression analysis was performed to determine if the miRNA signature can determine risk of recurrence in both cohorts.,Selected miRNAs were measured longitudinally in subsets of patients pre-/post-operatively and pre-/post-recurrence.,A signature of 5 miRNAs successfully classified melanoma patients into high and low recurrence risk groups with significant separation of RFS in both discovery and validation cohorts (p = 0.0036, p = 0.0093, respectively).,Significant separation of RFS was maintained when a logistic model containing the same signature set was used to predict recurrence risk in both discovery and validation cohorts (p < 0.0001, p = 0.033, respectively).,Longitudinal expression of 4 miRNAs in a subset of patients was dynamic, suggesting miRNAs can be associated with tumor burden.,Our data demonstrate that serum miRNAs can improve accuracy in identifying primary melanoma patients with high recurrence risk and in monitoring melanoma tumor burden over time.

Dermal neurofibromas (DNFs) are benign peripheral nerve sheath tumors thought to originate from Schwann cell progenitors.,These tumors represent one of the hallmarks of the neurofibromatosis type 1 (NF1) tumor predisposition syndrome, where they can number in the thousands.,While NF1-DNFs arise due to mutations in the NF1 gene, the vast majority of DNFs occur sporadically (sp-DNFs), where the genetic etiology is currently unknown.,Herein, we employed whole-exome sequencing of sp-DNFs to identify a recurrent mutation in the KIR2DL5 gene, which codes for a protein suppressor of natural killer (NK) cell activity.,While the function of KIR2DL5 outside of the immune system is unknown, we identified a KIR2DL5N173D mutation in three of nine sp-DNFs, resulting in loss of KIR2DL5 protein expression.,In contrast, two of these subjects had unrelated tumors, which retained KIR2DL5 protein expression.,Moreover, loss of KIR2DL5 expression was demonstrated in 15 of 45 independently-identified sp-DNFs.,Consistent with its potential role as a negative growth regulator important for neurofibroma maintenance, ectopic KIR2DL5N173D expression in normal human Schwann cells resulted in reduced KIR2DL5 expression and increased cell proliferation.,Similarly, KIR2DL5 short hairpin RNA knockdown (KD) decreased KIR2DL5 protein levels and increased cell proliferation, as well as correlated with PDGFRβ and downstream RAS/AKT/mTOR hyperactivation.,Importantly, inhibition of PDGFRβ or AKT/mTOR activity in KIR2DL5-KD human Schwann cells reduced proliferation to control levels.,Collectively, these findings establish KIR2DL5 as a new Schwann cell growth regulator relevant to sp-DNF pathogenesis, which links sporadic and NF1-associated DNFs through RAS pathway hyperactivation.

As surgical and/or ablative modalities, radiofrequency (RF) has been known to produce good clinical outcomes in dermatology.,Recently, 27.12 MHz RF has been introduced and has several advantages over conventional 4 or 6 MHz in terms of the precise ablation and lesser pain perception.,We aimed to evaluate the clinical efficacy and safety of 27.12 MHz RF for the treatment of benign cutaneous lesions.,Twenty female patient subjects were enrolled.,Digital photography and a USB microscope camera were used to monitor the clinical results before one session of treatment with 27.12 MHz RF and after 1 and 3 weeks.,Treated lesions included telangiectasias, cherry and spider angiomas, skin tags, seborrheic keratoses, lentigo, milium, dilated pore, acne, piercing hole, and one case of neurofibroma.,For vascular lesions, clinical results were excellent for 33.3%, good for 44.4%, moderate for 11.1%, and poor for 11.1%.,For nonvascular lesions (epidermal lesions and other benign cutaneous lesions), clinical results were excellent for 48.3%, good for 45.2%, moderate for 3.2%, and poor for 3.2%.,No serious adverse events were observed.,Mild adverse events reported were slight erythema, scale, and crust.,The 27.12 MHz RF treatment of benign vascular and nonvascular lesions appears safe and effective after 3 weeks of follow-up.

Tumor cells are subjected to evolutionary selection pressures during progression from initiation to metastasis.,We analyzed the clonal evolution of squamous skin carcinomas induced by DMBA/TPA treatment using the K5CreER-Confetti mouse and stage-specific lineage tracing.,We show that benign tumors are polyclonal, but only one population contains the Hras driver mutation.,Benign papillomas are therefore monoclonal in origin, but recruit neighboring epithelial cells during growth.,Papillomas that never progress to malignancy retain several distinct clones, whereas progression to carcinoma is associated with a clonal sweep.,Newly generated clones within carcinomas demonstrate intratumoral invasion and clonal intermixing, often giving rise to metastases containing two or more distinct clones derived from the matched primary tumor.,These data demonstrate that late stage tumor progression and dissemination are governed by evolutionary selection pressures that operate at a multicellular level, and thus differ from the clonal events that drive initiation and the benign-malignant transition.

Human papillomaviruses are causally associated with 5% of human cancers.,The recent discovery of a papillomavirus (MmuPV1) that infects laboratory mice provides unique opportunities to study the life cycle and pathogenesis of papillomaviruses in the context of a genetically manipulatable host organism.,To date, MmuPV1-induced disease has been found largely to be restricted to severely immunodeficient strains of mice.,In this study, we report that ultraviolet radiation (UVR), specifically UVB spectra, causes wild-type strains of mice to become highly susceptible to MmuPV1-induced disease.,MmuPV1-infected mice treated with UVB develop warts that progress to squamous cell carcinoma.,Our studies further indicate that UVB induces systemic immunosuppression in mice that correlates with susceptibility to MmuPV1-associated disease.,These findings provide new insight into how MmuPV1 can be used to study the life cycle of papillomaviruses and their role in carcinogenesis, the role of host immunity in controlling papillomavirus-associated pathogenesis, and a basis for understanding in part the role of UVR in promoting HPV infection in humans.