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Many studies have demonstrated that berberine inhibited the cell migration and invasion in human cancer cell lines.,However, the exact molecular mechanism of berberine inhibiting the cell migration and invasion of human melanoma A375.,S2 and A375.,S2/PLX (PLX4032 induced resistant A375.,S2) skin cancer cells remains unknown.,In this study, we investigated the anti-metastasis mechanisms of berberine in human melanoma cancer A375.,S2 cells and A375.,S2/PLX resistant cells in vitro.,Berberine at low concentrations (0, 1, 1.5 and 2 μM) induced cell morphological changes and reduced the viable cell number and inhibited the mobility, migration, and invasion of A375.,S2 cells that were assayed by wound healing and transwell filter.,The gelatin zymography assay showed that berberine slightly inhibited MMP-9 activity in A375.,S2 cells.,Results from western blotting indicated that berberine inhibited the expression of MMP-1, MMP-13, E-cadherin, N-cadherin, RhoA, ROCK1, SOS-1, GRB2, Ras, p-ERK1/2, p-c-Jun, p-FAK, p-AKT, NF-κB, and uPA after 24 h of treatment, but increased the PKC and PI3K in A375.,S2 cells.,PLX4032 is an inhibitor of the BRAFV600E mutation and used for the treatment of cancer cells harboring activated BRAF mutations.,Berberine decrease cell number and inhibited the cell mobility in the resistant A375.,S2 (A375.,S2/PLX, PLX4032 generated resistant A375.,S2 cells).,Based on these observations, we suggest that the potential of berberine as an anti-metastatic agent in melanoma that deserves to be investigated in more detail, including in vivo studies in future.

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.

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.

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

Merkel cell carcinoma (MCC) is a rare, aggressive, cutaneous neuroendocrine neoplasm with annual incidence rates of 0.13-1.6 cases/100,000/year worldwide as of 2018.,Chemotherapy for metastatic MCC (mMCC) has high objective response rates (ORRs), but responses are not durable and overall survival (OS) is poor.,Avelumab (anti-programmed death-ligand 1) has demonstrated meaningful survival benefit and durable responses in clinical trials for mMCC.,This study investigated real-world clinical outcomes in avelumab-treated patients with advanced (stage IIIB/IV) MCC in US academic medical centers.,We conducted a retrospective chart review of patients with advanced MCC who initiated avelumab between March 1, 2017, and July 31, 2019, at six US academic centers.,Data were requested for eligible patients from index date through December 31, 2020.,Descriptive analyses were conducted to assess demographic and clinical characteristics, real-world ORR (rwORR), real-world duration of response, real-world progression-free survival (rwPFS), and OS.,Ninety patients with advanced MCC (82%, stage IV; 18%, stage IIIB) received avelumab.,Median follow-up was 20.8 months (95% CI: 19.1 to 24.2).,Median age was 68 years (range, 48-83), and the majority of patients were men (58%) and white (93%).,The primary tumor was most commonly located on the lower limb (38%), with metastases mostly located in lymph nodes (68%), lung (52%), and viscera (52%).,Approximately 42% and 26% of patients had an Eastern Cooperative Oncology Group performance status of 2 and 3, respectively.,Seventy-three patients (81%) received avelumab as first-line treatment of advanced MCC, while 17 (19%) received avelumab as second-line or later treatment.,The median duration of avelumab treatment was 13.5 months (95% CI: 6.4 to 30.6), with 42% of patients still receiving avelumab by the end of follow-up.,Patients with avelumab treatment had an rwORR of 73% (95% CI: 64 to 83), median rwPFS of 24.4 months (95% CI: 8.31 to not estimable (NE)), and median OS of 30.7 months (95% CI: 11.2 to NE).,This real-world study of patients with advanced MCC demonstrated that avelumab treatment resulted in a high response rate with durable responses and prolonged survival.,The study findings validate the results demonstrated in prospective clinical trials and other observational studies.

Merkel cell carcinoma (MCC) is a rare but clinically aggressive cancer with a high mortality rate.,In recent years, antibodies blocking the interactions among PD-1 and its ligands have generated durable tumor regressions in patients with advanced MCC.,However, there is a paucity of data regarding effective therapy for patients whose disease is refractory to PD-1 pathway blockade.,This retrospective case series describes a heterogeneous group of patients treated with additional immune checkpoint blocking therapy after MCC progression through anti-PD-1.,Among 13 patients treated with anti-CTLA-4, alone or in combination with anti-PD-1, objective responses were seen in 4 (31%).,Additionally, one patient with MCC refractory to anti-PD-1 and anti-CTLA-4 experienced tumor regression with anti-PD-L1.,Our report - the largest case series to date describing this patient population - provides evidence that sequentially-administered salvage immune checkpoint blocking therapy can potentially activate anti-tumor immunity in patients with advanced anti-PD-1-refractory MCC and provides a strong rationale for formally testing these agents in multicenter clinical trials.,Additionally, to the best of our knowledge, our report is the first to demonstrate possible anti-tumor activity of second-line treatment with a PD-L1 antibody in a patient with anti-PD-1-refractory disease.,The online version of this article (10.1186/s40425-019-0661-6) contains supplementary material, which is available to authorized users.

The classification of melanoma into four histological subtypes has been questioned regarding its clinical validity in providing relevant information for treatment for metastatic tumors.,Specific genetic alterations are associated with particular clinical and histopathological features, suggesting that these could be helpful in refining existing melanoma classification schemes.,We analyzed BRAF V600E mutated melanomas to explore the Reflectance confocal microscopy (RCM) utility as a screening aid in the evaluation of the most appropriate patients for genetic testing.,Thus, 32 melanomas were assessed regarding their BRAF V600E mutational status.,Experts blinded to dermoscopic images and V600E immunohistochemistry results evaluated RCM images regarding previously described melanoma features.,BRAF positive melanomas were related to younger age (p = 0.035), invasive melanomas (p = 0.03) and to the presence of hiporreflective cells (p = 0.02), epidermal nests (p = 0.02), dermal-epidermal junction nests (p = 0.05), edged papillae (p = 0.05), and bright dots (p = 0.05), and to absence of junctional thickening due to isolated cells (p = 0.01) and meshwork (p = 0.02).,This study can not characterize other mutations in the BRAF, because the immunohistochemistry is specific to the type V600E.,The findings should encourage the genetic evaluation of BRAF mutation.,This study highlights the potential of RCM as a supplementary tool in the screening of BRAF-mutated melanomas.

Histopathologic interpretation of dermoscopic and reflectance confocal microscopy (RCM) features of cutaneous melanoma was timidly carried out using perpendicular histologic sections, which does not mimic the same plane of the image achieved at both techniques (horizontal plane).,The aim of this study was to describe the transverse histologic sections research technique and correlate main dermoscopic features characteristic of cutaneous melanoma (atypical network, irregular globules and pseudopods) with RCM and histopathology in perpendicular and transverse sections in order to offer a more precise interpretation of in vivo detectable features.,Four melanomas and 2 nevi with different dermoscopic clues have been studied.,Lesion areas that showed characteristic dermoscopic features were imaged by dermoscopy and confocal microscopy and directly correlated with histopathology in perpendicular and transverse sections.,We presented the possibility to perform transverse sections as a new approach to understand RCM features.,Atypical network showed different aspects in the 2 melanomas: in one case it was characterized by pleomorphic malignant melanocytes with tendency to form aggregates, whereas in the other elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembled the mitochondrial aspect at confocal and histopathology transversal sections.,Pigment globules in melanomas and nevi differed for the presence of large atypical cells in the former, and pseudopods showed up as elongated nests protruded toward the periphery of the lesion.,Transverse histologic research sections have a consistent dermoscopic and confocal correlate, and it may represent an help in confocal feature interpretation and an advance in improving melanoma diagnosis and knowledge of the biology of melanocytic lesions.

The roles of NK cells in human melanoma remain only partially understood.,We characterized NK cells from peripheral blood ex vivo by flow cytometry obtained from late stage (III/IV) melanoma patients.,Interestingly, we found that the abundance of CD56bright NK cells negatively correlate with overall patient survival, together with distant metastases, in a multivariate cox regression analysis.,The patients’ CD56bright NK cells showed upregulation of CD11a, CD38 and CD95 as compared to healthy controls, pointing to an activated phenotype as well as a possible immune regulatory role in melanoma patients.,After stimulation in vitro, CD56bright NK cells produced less TNFα and GMCSF in patients than controls.,Furthermore, IFNγ production by the CD56bright NK cells correlated inversely with overall survival.,Our results highlight that abundance and function of CD56bright NK cells are associated with melanoma patient survival, emphasizing the potential of NK cell subsets for biomarker discovery and future therapeutic targeting.

While immune checkpoint blockade has greatly improved clinical outcomes in diseases such as melanoma, there remains a need for predictive biomarkers to determine who will likely benefit most from which therapy.,To date, most biomarkers of response have been identified in the tumors themselves.,Biomarkers that could be assessed from peripheral blood would be even more desirable, because of ease of access and reproducibility of sampling.,We used mass cytometry (CyTOF) to comprehensively profile peripheral blood of melanoma patients, in order to find predictive biomarkers of response to anti-CTLA-4 or anti-PD-1 therapy.,Using a panel of ~ 40 surface and intracellular markers, we performed in-depth phenotypic and functional immune profiling to identify potential predictive biomarker candidates.,Immune profiling of baseline peripheral blood samples using CyTOF revealed that anti-CTLA-4 and anti-PD-1 therapies have distinct sets of candidate biomarkers.,The distribution of CD4+ and CD8+ memory/non-memory cells and other memory subsets was different between responders and non-responders to anti-CTLA-4 therapy.,In anti-PD-1 (but not anti-CTLA-4) treated patients, we discovered differences in CD69 and MIP-1β expressing NK cells between responders and non-responders.,Finally, multivariate analysis was used to develop a model for the prediction of response.,Our results indicate that anti-CTLA-4 and anti-PD-1 have distinct predictive biomarker candidates.,CD4+ and CD8+ memory T cell subsets play an important role in response to anti-CTLA-4, and are potential biomarker candidates.,For anti-PD-1 therapy, NK cell subsets (but not memory T cell subsets) correlated with clinical response to therapy.,These functionally active NK cell subsets likely play a critical role in the anti-tumor response triggered by anti-PD-1.,The online version of this article (10.1186/s40425-018-0328-8) 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.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes.,In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas.,Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells.,Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness.,Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity.,Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3′ untranslated region of MITF, BCL2 and cyclin D2.,Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression.,The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182.,Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells.,Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.

Cancer immunotherapy has generated significant excitement, mainly as a result of the development of immune checkpoint inhibitors.,The blockade of PD-1 or its ligand with antibodies has resulted in impressive clinical efficacy.,However, a subset of patients does not respond to biologic therapeutics, and another subset suffers from severe immune-related adverse events in certain cases.,The modulation of the immune system with small molecules might yield surprising benefits.,CD8+ cells were obtained through a magnetic cell sorting system (MACS), and their capabilities for IFN-γ release and PD-1 expression were analyzed.,The in vitro effects of drugs were studied in a coculture system of tumor cells and activated CD8+ cells.,We further isolated the primary tumor cells in tumor-bearing mice treated with CAI, DMF, 1-MT or a combination (CAI and DMF/CAI and 1-MT) and analyzed the percentages of CD8+ T cells and PD-1+CD8+ T cells among TILs.,The selective anti-tumor immune reactions of the two drug combinations were confirmed in a coculture system consisting of B16-OVA cells and OVA-specific CTLs derived from OT-1 transgenic mice.,The anti-tumor effects of the single drugs or combined therapies were assessed according to their capability to slow tumor growth and extend the life span of tumor-bearing mice, and they were compared with the effects of PD-1 antibody.,CAI increased IFN-γ release from activated T cells, which might strengthen the anti-proliferative and anti-metastatic effects on cancer cells.,However, CAI also stimulated IDO1-Kyn metabolic circuitry in the tumor microenvironment and facilitated tumor cell immune evasion.,Combining CAI with 1-MT or DMF disrupted PD-1 expression and promoted IFN-γ production in CD8+ T cells, and it also increased T lymphocyte infiltration in the tumor microenvironment, inhibited tumor growth and prolonged the life spans of tumor-bearing mice.,Inhibitors of the IDO1-Kyn-AhR pathway could abolish the negative effects of CAI on CD8+ T cells and result in complementary and beneficial anti-tumor immune effects.,The combination of CAI with 1-MT or DMF greatly augmented the ability of CD8+ T cells to kill malignant cells and showed a strong anti-cancer capability that was superior to that of either of the single agents was is comparable with that of anti-PD-1 antibody.,The combinations of small molecules utilized in this study may serve as valuable new immunotherapy strategies for cancer treatment.,The online version of this article (10.1186/s40425-019-0725-7) contains supplementary material, which is available to authorized users.

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

The overall 5-year survival for melanoma is 91%.,However, if distant metastasis occurs (stage IV), cure rates are < 15%.,Hence, melanoma detection in earlier stages (stages I-III) maximises the chances of patient survival.,We measured the expression of a panel of 17 microRNAs (miRNAs) (MELmiR-17) in melanoma tissues (stage III; n = 76 and IV; n = 10) and serum samples (collected from controls with no melanoma, n = 130; and patients with melanoma (stages I/II, n = 86; III, n = 50; and IV, n = 119)) obtained from biobanks in Australia and Germany.,In melanoma tissues, members of the ‘MELmiR-17’ panel were found to be predictors of stage, recurrence, and survival.,Additionally, in a minimally-invasive blood test, a seven-miRNA panel (MELmiR-7) detected the presence of melanoma (relative to controls) with high sensitivity (93%) and specificity (≥ 82%) when ≥ 4 miRNAs were expressed.,Moreover, the ‘MELmiR-7’ panel characterised overall survival of melanoma patients better than both serum LDH and S100B (delta log likelihood = 11, p < 0.001).,This panel was found to be superior to currently used serological markers for melanoma progression, recurrence, and survival; and would be ideally suited to monitor tumour progression in patients diagnosed with early metastatic disease (stages IIIa-c/IV M1a-b) to detect relapse following surgical or adjuvant treatment.,•A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%).,•In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases.,•The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.,A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%).,In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases.,The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.

Circulating cell-free(cf) microRNAs (miRNAs) have been reported to exist in plasma.,MicroRNA-210(miR-210) is known to play important roles in the tumor hypoxic state.,We hypothesized that the expression levels of cf-miR-210 in plasma would predict early clinical recurrence in melanoma patients.,A direct miRNA assay on plasma (RT-qPCR-DP) was developed to improve cf-miRNA assay logistics, eliminate RNA extraction, and reduce specimen amount required.,RNA was extracted from formalin-fixed paraffin-embedded (FFPE) melanoma tissues (n = 108) and assessed by RT-qPCR.,Plasma (10 μl; n = 264) was procured from AJCC Stage III/IV patients in phase III clinical trials.,A RT-qPCR-DP was performed to detect cf-miR-210.,MiR-210 was significantly higher in metastatic tumors compared to primary tumors.,Cf-miR-210 was significantly higher in melanoma patients versus healthy donor controls.,In serial bloods within individual patients, cf-miR-210 < 3 months prior to disease recurrence significantly increased compared to baseline levels (p = 0.012).,ROC curve analysis demonstrated that patients with elevated cf-miR-210 were more likely to have disease recurrence.,Moreover, cf-miR-210 increase significantly correlated with poorer prognosis (p < 0.001).,Lactate dehydrogenase (LDH) level was also assessed within patients, and the AIC values for proportional hazards regression models of cf-miR-210(120.01) and LDH (122.91) demonstrated that cf-miR-210 is a better recurrence indicator.,We concluded enhanced cf-miR-210 provides identification of early systemic melanoma recurrence.

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

Here, we showed that the secretome of senescent melanoma cells drives basal melanoma cells towards a mesenchymal phenotype, with characteristic of stems illustrated by increased level of the prototype genes FN1, SNAIL, OCT4 and NANOG.,This molecular reprogramming leads to an increase in the low-MITF and slow-growing cell population endowed with melanoma-initiating cell features.,The secretome of senescent melanoma cells induces a panel of 52 genes, involved in cell movement and cell/cell interaction, among which AXL and ALDH1A3 have been implicated in melanoma development.,We found that the secretome of senescent melanoma cells activates the STAT3 pathway and STAT3 inhibition prevents secretome effects, including the acquisition of tumorigenic properties.,Collectively, the findings provide insights into how the secretome of melanoma cells entering senescence upon chemotherapy treatments increases the tumorigenicity of naïve melanoma cells by inducing, through STAT3 activation, a melanoma-initiating cell phenotype that could favor chemotherapy resistance and relapse.

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest.,Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action.,EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells).,A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed.,At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown.,EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner.,A role of RyR as a direct target of EC was also suggested by molecular modelling studies.,In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

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

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

Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment1; however, optimal regimens have not been defined.,Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322).,RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed.,Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs).,Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy.,These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.

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

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research.,However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma.,In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS.,Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated.,We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol.,We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis.,Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.

The protein kinase V-Raf murine sarcoma viral oncogene homolog B (BRAF) is an oncogenic driver and therapeutic target in melanoma.,Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in melanoma patients bearing tumors that express BRAF Val600 mutations, but a vast majority of these patients develop drug resistance.,Here we show that loss of Stromal antigen 2 or 3 (STAG2 or STAG3), which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi.,We identified loss-of-function mutations in STAG2 as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines.,Knockdown of STAG2 or STAG3 decreased sensitivity of Val600Glu BRAF-mutant melanoma cells and xenograft tumors to BRAFi.,Loss of STAG2 inhibited CCCTC-binding factor (CTCF)-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of ERK signaling.,Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3.

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.

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

Matrix metalloproteinase-2 (MMP-2) is a key regulator in the migration of tumor cells. αvβ3 integrin has been reported to play a critical role in cell adhesion and regulate the migration of tumor cells by promoting MMP-2 activation.,However, little is known about the effects of MMP-2 on αvβ3 integrin activity and αvβ3 integrin-mediated adhesion and migration of tumor cells.,Human melanoma cells were seeded using an agarose drop model and/or subjected to in vitro analysis using immunofluorescence, adhesion, migration and invasion assays to investigate the relationship between active MMP-2 and αvβ3 integrin during the adhesion and migration of the tumor cells.,We found that MMP-2 was localized at the leading edge of spreading cells before αvβ3 integrin. αvβ3 integrin-mediated adhesion and migration of the tumor cells were inhibited by a MMP-2 inhibitor.,MMP-2 cleaved fibronectin into small fragments, which promoted the adhesion and migration of the tumor cells.,MMP-2 cleaves fibronectin into small fragments to enhance the adhesion and migration of human melanoma cells mediated by αvβ3 integrin.,These results indicate that MMP-2 may guide the direction of the tumor cell migration.

Mammalian target of rapamycin complex 2 (mTORC2) with its pivotal component rapamycin-insensitive companion of mTOR (RICTOR) is the major regulator of AKT phosphorylation and is increasingly implicated in tumor growth and progression.,In cutaneous melanoma, an extremely aggressive and highly metastatic disease, RICTOR overexpression is involved in tumor development and invasiveness.,Therefore, we investigated the impact of RICTOR inhibition in melanoma cells in vitro and in vivo with special emphasis on hepatic metastasis.,Moreover, our study focused on the interaction of tumor cells and hepatic stellate cells (HSC) which play a crucial role in the hepatic microenvironment.,In silico analysis revealed increased RICTOR expression in melanoma cells and tissues and indicated higher expression in advanced melanoma stages and metastases.,In vitro, transient RICTOR knock-down via siRNA caused a significant reduction of tumor cell motility.,Using a syngeneic murine splenic injection model, a significant decrease in liver metastasis burden was detected in vivo.,Moreover, stimulation of melanoma cells with conditioned medium (CM) from activated HSC or hepatocyte growth factor (HGF) led to a significant induction of AKT phosphorylation and tumor cell motility.,Blocking of RICTOR expression in cancer cells diminished constitutive and HGF-induced AKT phosphorylation as well as cell motility.,Interestingly, RICTOR blockade also led to an abrogation of CM-induced effects on AKT phosphorylation and motility in melanoma cells.,In conclusion, these results provide first evidence for a critical role of mTORC2/RICTOR in melanoma liver metastasis via cancer cell/HSC interactions.

Type XIX collagen is a minor collagen associated with basement membranes.,It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices).,Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion.,In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX).,Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway.,On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation.,Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed.,NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

Immune checkpoint inhibitors (ICIs) have changed the clinical management of melanoma.,However, not all patients respond, and current biomarkers including PD-L1 and mutational burden show incomplete predictive performance.,The clinical validity and utility of complex biomarkers have not been studied in melanoma.,Cutaneous metastatic melanoma patients at eight institutions were evaluated for PD-L1 expression, CD8+ T-cell infiltration pattern, mutational burden, and 394 immune transcript expression.,PD-L1 IHC and mutational burden were assessed for association with overall survival (OS) in 94 patients treated prior to ICI approval by the FDA (historical-controls), and in 137 patients treated with ICIs.,Unsupervised analysis revealed distinct immune-clusters with separate response rates.,This comprehensive immune profiling data were then integrated to generate a continuous Response Score (RS) based upon response criteria (RECIST v.1.1).,RS was developed using a single institution training cohort (n = 48) and subsequently tested in a separate eight institution validation cohort (n = 29) to mimic a real-world clinical scenario.,PD-L1 positivity ≥1% correlated with response and OS in ICI-treated patients, but demonstrated limited predictive performance.,High mutational burden was associated with response in ICI-treated patients, but not with OS.,Comprehensive immune profiling using RS demonstrated higher sensitivity (72.2%) compared to PD-L1 IHC (34.25%) and tumor mutational burden (32.5%), but with similar specificity.,In this study, the response score derived from comprehensive immune profiling in a limited melanoma cohort showed improved predictive performance as compared to PD-L1 IHC and tumor mutational burden.,The online version of this article (10.1186/s40425-018-0344-8) contains supplementary material, which is available to authorized users.

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

Resistance to immune checkpoint blockade and targeted therapy in melanoma patients is currently one of the major clinical challenges.,With the approval of talimogene laherparepvec (T-VEC), oncolytic viruses are now in clinical practice for locally advanced or non-resectable melanoma.,Here, we describe the usage of T-VEC in stage IVM1b-M1c melanoma patients, who achieved complete remission or stable disease upon systemic treatment but suffered from a loco-regional recurrence.,To our knowledge, there are no case reports so far describing T-VEC as a means to overcome acquired resistance to immune checkpoint blockade or targeted therapy.,All melanoma patients in our department treated with T-VEC in the period of 2016-2018 were evaluated retrospectively.,Data on clinicopathological characteristics, treatment response, and toxicity were analyzed.,Fourteen melanoma patients were treated with T-VEC in our center.,Six patients (43%) received T-VEC first-line.,In eight patients (57%), T-VEC followed a prior systemic therapy.,Three patients with M1b stage and one patient with M1c stage melanoma were treated with T-VEC.,These patients suffered from loco-regional progress, whilst distant metastases had regressed during prior systemic treatment. 64% of patients showed a benefit from therapy with T-VEC.,The durable response rate was 36%.,T-VEC represents an effective and tolerable treatment option.,This is true not only for loco-regionally advanced melanoma patients, but also for patients with stable or regressive systemic metastases who develop loco-regionally acquired resistance upon treatment with immune checkpoint blockade or targeted therapy.,A sensible selection of suitable patients seems to be crucial.

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity.,Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation.,Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients.,While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions.,Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival.,While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade.,This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma.,The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

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

We report a genome-wide association study of melanoma, conducted by GenoMEL, of 2,981 cases, of European ancestry, and 1,982 study-specific controls, plus a further 6,426 French and UK population controls, all genotyped for 317,000 or 610,000 SNPs.,The analysis confirmed previously known melanoma susceptibility loci.,The 7 novel regions with at least one SNP with p<10−5 and further local imputed or genotyped support were selected for replication using two other genome-wide studies (from Australia and Houston, Texas).,Additional replication came from UK and Dutch case-control series.,Three of the 7 regions replicated at p<10−3: an ATM missense polymorphism (rs1801516, overall p=3.4×10−9); a polymorphism within MX2 (rs45430, p=2.9×10−9) and a SNP adjacent to CASP8 (rs13016963, p=8.6×10−10).,A fourth region near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication.,Unlike the previously known regions, the novel loci showed no association with nevus or pigmentation phenotypes in a large UK case-control series.

Colonization is believed a rate-limiting step of metastasis cascade.,However, its underlying mechanism is not well understood.,Uveal melanoma (UM), which is featured with single organ liver metastasis, may provide a simplified model for realizing the complicated colonization process.,Because DDR1 was identified to be overexpressed in UM cell lines and specimens, and abundant pathological deposition of extracellular matrix collagen, a type of DDR1 ligand, was noted in the microenvironment of liver in metastatic patients with UM, we postulated the hypothesis that DDR1 and its ligand might ignite the interaction between UM cells and their surrounding niche of liver thereby conferring strengthened survival, proliferation, stemness and eventually promoting metastatic colonization in liver.,We tested this hypothesis and found that DDR1 promoted these malignant cellular phenotypes and facilitated metastatic colonization of UM in liver.,Mechanistically, UM cells secreted TGF-β1 which induced quiescent hepatic stellate cells (qHSCs) into activated HSCs (aHSCs) which secreted collagen type I.,Such a remodeling of extracellular matrix, in turn, activated DDR1, strengthening survival through upregulating STAT3-dependent Mcl-1 expression, enhancing stemness via upregulating STAT3-dependent SOX2, and promoting clonogenicity in cancer cells.,Targeting DDR1 by using 7rh, a specific inhibitor, repressed proliferation and survival in vitro and in vivo outgrowth.,More importantly, targeting cancer cells by pharmacological inactivation of DDR1 or targeting microenvironmental TGF-β1-collagen I loop exhibited a prominent anti-metastasis effect in mice.,In conclusion, targeting DDR1 signaling and TGF-β signaling may be a novel approach to diminish hepatic metastasis in UM.

Acidosis characterizes the microenvironment of most solid tumors and is considered a new hallmark of cancer.,It is mainly caused by both “aerobic” and “anaerobic” glycolysis of differently adapted cancer cells, with the final product lactic acid being responsible of the extracellular acidification.,Many evidences underline the role of extracellular acidosis in tumor progression.,Among the different findings, we demonstrated that acidosis-exposed cancer cells are characterized by an epithelial-to-mesenchymal transition phenotype with high invasive ability, high resistance to apoptosis, anchorage-independent growth, and drug therapy.,Acidic melanoma cells over-express SOX2, which is crucial for the maintenance of their oxidative metabolism, and carbonic anhydrase IX, that correlates with poor prognosis of cancer patients.,Considering these evidences, we realized that the profile outlined for acid cancer cells inevitably remind us the stemness profile.,Therefore, we wondered whether extracellular acidosis might induce in cancer cells the acquisition of stem-like properties and contribute to the expansion of the cancer stem cell sub-population.,We found that a chronic adaptation to acidosis stimulates in cancer cells the expression of stem-related markers, also providing a high in vitro/in vivo clonogenic and trans-differentiating ability.,Moreover, we observed that the acidosis-induced stem-like phenotype of melanoma cells was reversible and related to the EMT induction.,These findings help to characterize a further aspect of stem cell niche, contributing to the sustainment and expansion of cancer stem cell subpopulation.,Thus, the usage of agents controlling tumor extracellular acidosis might acquire great importance in the clinic for the treatment of aggressive solid tumor.,• Extracellular acidosis up-regulates EMT and stem-related markers in melanoma cells,• Acidic medium up-regulates in vitro self-renewal capacity of melanoma cells,• Chronic acidosis adaptation induces trans-differentiation ability in melanoma cells,• Melanoma cells adapted to acidosis show higher tumor-initiating potential than control cells,• Extracellular acidosis promotes a stem-like phenotype in prostate and colorectal carcinoma cells

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

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

We conducted a phase I trial evaluating pembrolizumab+hypofractionated radiotherapy (HFRT) for patients with metastatic cancers.,There were two strata (12 patients each): (i) NSCLC/melanoma progressing on prior anti-PD-1 therapy, (ii) other cancer types; anti-PD-1-naive.,Patients received 6 cycles of pembrolizumab, starting 1 week before HFRT.,Patients had ≥2 lesions; only one was irradiated (8 Gy × 3 for first half; 17 Gy × 1 for second half in each stratum) and the other(s) followed for response.,Of the 24 patients, 20 (83%) had treatment-related adverse events (AEs) (all grade 1 or 2).,There were eight grade 3 AEs, none treatment related.,There were no dose-limiting toxicities or grade 4/5 AEs.,Stratum 1: two patients (of 12) with progression on prior PD-1 blockade experienced prolonged responses (9.2 and 28.1 months).,Stratum 2: one patient experienced a complete response and two had prolonged stable disease (7.4 and 7.0 months).,Immune profiling demonstrated that anti-PD-1 therapy and radiation induced a consistent increase in the proliferation marker Ki67 in PD-1-expressing CD8 T cells.,HFRT was well tolerated with pembrolizumab, and in some patients with metastatic NSCLC or melanoma, it reinvigorated a systemic response despite previous progression on anti-PD-1 therapy.,Clinical Trial Registration: NCT02303990 (www.clinicaltrials.gov).

Immune checkpoint inhibitors1 result in impressive clinical responses2-5 but optimal results will require combination with each other6 and other therapies.,This raises fundamental questions about mechanisms of non-redundancy and resistance.,Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) and reproduced this effect in mouse models.,Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common.,Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T cell exhaustion.,Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1.,Anti-CTLA4 predominantly inhibits T regulatory cells (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio.,RT enhances the diversity of the T cell receptor (TCR) repertoire of intratumoral T cells.,Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones.,Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion.,Similar to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed.,Thus, PD-L1 on melanoma cells allows tumors to escape anti-CTLA4-based therapy, and the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms.

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

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.

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

Cutaneous melanoma is a metastatic disease characterized by high resistance to treatment, the incidence of which has alarmingly increased worldwide over the past years.,A thorough characterization of tumor onset, progression and metastasis is compulsory to overcome the gaps existent in melanoma biology.,The present study suggests a well-established protocol and a detailed histological description of human melanoma models in ovo and in vivo obtained by the inoculation of A375 cells to chick embryo chorioallantoic membrane (CAM) and Balb/c nude mice.,The inoculation of A375 cells on CAM led to the formation of compact primary and secondary tumors on day 4 post-inoculation, with mean surface area values of 2.2±0.4 mm2 and 1.5±0.3 mm2, respectively.,Moreover, the vessels around the tumors presented a spike wheel pattern, indicating a strong angiogenic reaction.,All the injected mice, apart from one, developed solid polypoid primary tumors with lobulated surfaces and intense vascularization, and achromic epithelioid malignant melanocytes with vesiculous nuclei and necrosis area were detected.,Metastasis was histologically confirmed in only 30% of the mice with the tumor xenografts.,These data indicate that the standardization protocols proposed are complex and reproducible, and can be further employed for the therapeutic surveillance of antiangiogenic and anticancer agents.

Here, we showed that the secretome of senescent melanoma cells drives basal melanoma cells towards a mesenchymal phenotype, with characteristic of stems illustrated by increased level of the prototype genes FN1, SNAIL, OCT4 and NANOG.,This molecular reprogramming leads to an increase in the low-MITF and slow-growing cell population endowed with melanoma-initiating cell features.,The secretome of senescent melanoma cells induces a panel of 52 genes, involved in cell movement and cell/cell interaction, among which AXL and ALDH1A3 have been implicated in melanoma development.,We found that the secretome of senescent melanoma cells activates the STAT3 pathway and STAT3 inhibition prevents secretome effects, including the acquisition of tumorigenic properties.,Collectively, the findings provide insights into how the secretome of melanoma cells entering senescence upon chemotherapy treatments increases the tumorigenicity of naïve melanoma cells by inducing, through STAT3 activation, a melanoma-initiating cell phenotype that could favor chemotherapy resistance and relapse.

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

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

Aurora kinase A (AurkA) is over-expressed in melanoma and its inhibition has been observed to limit tumor growth, suggesting a potential role in melanoma treatment.,A human melanoma cell line with the B-RAF (V600E) mutation (A375mel) was exposed to B-RAF inhibitor (GSK2118436), MEK inhibitor (GSK1120212) and AurkA inhibitor (MLN8054) as single agents or in various combinations (BRAF plus AurkA inhibitor, MEK plus AurkA inhibitor or triple combination BRAF plus MEK plus AurkA inhibitor).,Cell proliferation was assessed using xCELLigence technology.,Total protein extracts were examined for p53 and c-Myc protein expression by Western blot analysis.,Drug anti-tumor effects were further assessed using a 3D-human melanoma skin reconstruction model, in which tissues were incubated with serum-free medium containing control, B-RAF plus MEK inhibitor, MEK plus AurkA inhibitor or the triple combination.,AurkA inhibitor plus B-RAF inhibitor, AurkA inhibitor plus MEK inhibitor or triple combination had a markedly greater anti-proliferative effect on A375 (BRAFV600E) melanoma cells than single agents.,In the 3D human skin model, the triple combination had a greater anti-tumor effect at the epidermal/dermal junction than control or either double combination.,However, S-100 and Ki-67 positively stained spindle-shaped cells were detected in the dermal stratum, suggesting the presence of alive and proliferating melanoma cells.,These findings provide new prospects for melanoma research, including combined B-RAF/AurkA inhibition for B-RAF mutated melanomas and MEK/AurkA inhibitor combination for patients without B-RAF mutations.,Moreover, for the first time, we have shown that a B-RAF, MEK and AurkA inhibitor triple drug combination offers increased efficacy against melanoma cell growth and might be considered as a potential treatment strategy for enhancing clinical response in melanoma.,However, although this triple drug combination was more effective at the epidermal/dermal junction, the suggested presence of alive and proliferating melanoma cells in the dermal stratum could result in drug resistance and disease recurrence.,Molecular characterization of these dermal cells may be critical for the development of novel therapeutic strategies.

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

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

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.

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

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations.,In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin.,Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line.,High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively.,Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM.,Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation.,According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

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

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways.,MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations.,In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055.,While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only.,In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model.,We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells.,For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis.,Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis.,These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate.,Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment.,The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression.,Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients.,More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients.,Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors.,Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions.,A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases.,The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay.,Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src.,Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects.,These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

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

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.

Acral melanoma is the most common type of melanoma in Asians, and usually results in a poor prognosis due to late diagnosis.,We applied a convolutional neural network to dermoscopy images of acral melanoma and benign nevi on the hands and feet and evaluated its usefulness for the early diagnosis of these conditions.,A total of 724 dermoscopy images comprising acral melanoma (350 images from 81 patients) and benign nevi (374 images from 194 patients), and confirmed by histopathological examination, were analyzed in this study.,To perform the 2-fold cross validation, we split them into two mutually exclusive subsets: half of the total image dataset was selected for training and the rest for testing, and we calculated the accuracy of diagnosis comparing it with the dermatologist’s and non-expert’s evaluation.,The accuracy (percentage of true positive and true negative from all images) of the convolutional neural network was 83.51% and 80.23%, which was higher than the non-expert’s evaluation (67.84%, 62.71%) and close to that of the expert (81.08%, 81.64%).,Moreover, the convolutional neural network showed area-under-the-curve values like 0.8, 0.84 and Youden’s index like 0.6795, 0.6073, which were similar score with the expert.,Although further data analysis is necessary to improve their accuracy, convolutional neural networks would be helpful to detect acral melanoma from dermoscopy images of the hands and feet.

Background.,One of the fatal disorders causing death is malignant melanoma, the deadliest form of skin cancer.,The aim of the modern dermatology is the early detection of skin cancer, which usually results in reducing the mortality rate and less extensive treatment.,This paper presents a study on classification of melanoma in the early stage of development using SVMs as a useful technique for data classification.,Method.,In this paper an automatic algorithm for the classification of melanomas in their early stage, with a diameter under 5 mm, has been presented.,The system contains the following steps: image enhancement, lesion segmentation, feature calculation and selection, and classification stage using SVMs.,Results.,The algorithm has been tested on 200 images including 70 melanomas and 130 benign lesions.,The SVM classifier achieved sensitivity of 90% and specificity of 96%.,The results indicate that the proposed approach captured most of the malignant cases and could provide reliable information for effective skin mole examination.,Conclusions.,Micro-melanomas due to the small size and low advancement of development create enormous difficulties during the diagnosis even for experts.,The use of advanced equipment and sophisticated computer systems can help in the early diagnosis of skin lesions.

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.

MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs.,Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes.,In the present study, we investigated the role of miRNA-34b/c in uveal melanoma.,Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples.,Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry.,Cell apoptosis was measured by caspase3/7 assay.,Cell migration was evaluated by transwell migration assay.,The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay.,In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting.,miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs.,The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G1 arrest rather than the induction of apoptosis.,Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells.,Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.,Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.

Melanomas can switch to a dedifferentiated cell state upon exposure to cytotoxic T cells.,However, it is unclear whether such tumor cells pre-exist in patients and whether they can be resensitized to immunotherapy.,Here, we chronically expose (patient-derived) melanoma cell lines to differentiation antigen-specific cytotoxic T cells and observe strong enrichment of a pre-existing NGFRhi population.,These fractions are refractory also to T cells recognizing non-differentiation antigens, as well as to BRAF + MEK inhibitors.,NGFRhi cells induce the neurotrophic factor BDNF, which contributes to T cell resistance, as does NGFR.,In melanoma patients, a tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance, and NGFRhi tumor fractions are associated with immune exclusion.,Lastly, pharmacologic NGFR inhibition restores tumor sensitivity to T cell attack in vitro and in melanoma xenografts.,These findings demonstrate the existence of a stable and pre-existing NGFRhi multitherapy-refractory melanoma subpopulation, which ought to be eliminated to revert intrinsic resistance to immunotherapeutic intervention.,Dedifferentiation state has been associated with therapy resistance in melanoma.,Here, the authors uncover a pre-existing NGFR-expressing, targetable subpopulation that is resistant to immunotherapy and other treatments in melanoma cells and preclinical models.

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

Malignant melanoma is a class of malignant tumors derived from melanocytes. lncRNAs have been considered as pro-/anti-tumor factors in progression of cancers.,The function of lncRNA TUG1 on growth of melanoma was investigated in this study.,The TUG1 and miR-129-5p expression were examined via qRT-PCR.,The protein expression was investigated by Western blotting assay.,Luciferase reporter assay was used to assess if lncRNA TUG1 can bind to miR-129-5p and if miR-129-5p can target AEG1 mRNA.,CCK-8 and apoptosis assay were used to detect cell growth and apoptosis.,The metastasis of melanoma cells was detected by wound-healing and Transwell assays.,The effects of TUG1 on growth of melanoma in vivo and cell chemoresistance were investigated via xenograft animal experiment and CCK-8 assay.,The expression of TUG1 and AEG1 was elevated and the miR-129-5p level was decreased in melanoma specimens and cell lines.,Downregulation of either TUG1 or AEG1 suppressed cell growth and metastasis. miR-129-5p can bind directly to AEG1 and TUG1 can directly sponge miR-129-5p.,Inhibition of TUG1 expression suppressed the expression of Bcl-2, MMP-9, and cyclin D1, and raised the level of cleaved caspase3 by modulating AEG1 level in melanoma cells.,Inhibition of TUG1 reduced the growth of tumors in vivo and improved the chemosensitivity of A375 cells to cisplatin and 5-FU.,Reduction of TUG1 level suppressed cell growth and metastasis by regulating AEG1 expression mediated by targeting miR-129-5p.,Suppression of lnc TUG1 may be a promising therapeutic strategy in the treatment of malignant melanoma.

Genomic locus at chromosome 9p21 that contains the CDKN2A and CDKN2B tumor suppressor genes is inactivated through mutations, deletions and promoter methylation in multiple human cancers.,Additionally, the locus encodes an anti-sense RNA (ANRIL).,Both hemizygous and homozygous deletions at the locus targeting multiple genes are fairly common in different cancers.,We in this study investigated breakpoints in five melanoma cell lines, derived from metastasized tumors, with previously identified homozygous deletions using array comparative genomic hybridization (aCGH).,For breakpoint mapping, we used primer approximation multiplex PCR (PAMP) and inverse PCR techniques.,Our results showed that three cell lines carried complex rearrangements.,In two other cell lines, with focal deletions of 141 kb and 181 kb, we identified fusion gene products, involving MTAP and ANRIL.,We also confirmed the complex rearrangements and focal deletions in DNA from tumor tissues corresponding to three cell lines.,The rapid amplification of 3′cDNA ends (3′RACE) carried out on transcripts resulted in identification of three isoforms of MTAP-ANRIL fusion gene.,Screening of cDNA from 64 melanoma cell lines resulted in detection of fusion transcripts in 13 (20%) cell lines that involved exons 4-7 of the MTAP and exon 2 or 5 of the ANRIL genes.,We also detected fusion transcripts involving MTAP and ANRIL in two of the seven primary melanoma tumors with focal deletion at the locus.,The results from the study, besides identifying complex rearrangements involving CDKN2A locus, show frequent occurrence of fusion transcripts involving MTAP and ANRIL genes.

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

The circadian clock controls the timing of the cell cycle in healthy tissues and clock disruption is known to increase tumourigenesis.,Melanoma is one of the most rapidly increasing forms of cancer and the precise molecular circadian changes that occur in a melanoma tumor are unknown.,Using a melanoma zebrafish model, we have explored the molecular changes that occur to the circadian clock within tumors.,We have found disruptions in melanoma clock gene expression due to a major impairment to the light input pathway, with a parallel loss of light-dependent activation of DNA repair genes.,Furthermore, the timing of mitosis in tumors is perturbed, as well as the regulation of certain key cell cycle regulators, such that cells divide arhythmically.,The inability to co-ordinate DNA damage repair and cell division is likely to promote further tumourigenesis and accelerate melanoma development.

Acral lentiginous melanoma (ALM), a relatively rare subtype of cutaneous melanoma, has been reported to have a worse prognosis than other melanomas.,We aimed to assess clinical findings in Caucasian ALM patients and compare the data with a matched cohort of superficial spreading melanoma (SSM) patients.,We studied 63 patients with ALM and 63 randomly stage- and limb-matched patients with SSM (non-ALM).,In both cohorts, guideline-adjusted diagnosis, treatment and follow-up were performed.,We did not observe differences in prognostic factors (e.g., tumor thickness, ulceration) between the two cohorts.,Both in ALM and non-ALM patients positive sentinel lymph node was a significant independent predictor for disease relapse and melanoma-specific death.,However, disease relapse and melanoma-specific death rates did not significantly differ between ALM and non-ALM patients.,An overall 5-year melanoma-specific survival of 82.5% and 81% was observed in ALM and non-ALM patients, respectively.,Our data confirm that patients with ALM have no worse outcome than non-ALM patients when correcting for significant prognostic factors.,Hence, the reportedly high rates of fatal ALM cases should not be ascribed to pathobiological differences between ALM and non-ALM but are most likely are a consequence of a delay in diagnosis and thus advanced stage of ALM.

Like many cancers, an early diagnosis of melanoma is fundamental to ensure a good prognosis, although an important proportion of stage I-II patients may still develop metastasis during follow‐up.,The aim of this work was to discover serum biomarkers in patients diagnosed with primary melanoma that identify those at a high risk of developing metastasis during the follow‐up period.,Proteomic and mass spectrophotometry analysis was performed on serum obtained from patients who developed metastasis during the first years after surgery for primary tumors and compared with that from patients who remained disease‐free for more than 10 years after surgery.,Five proteins were selected for validation as prognostic factors in 348 melanoma patients and 100 controls by ELISA: serum amyloid A and clusterin; immune system proteins; the cell adhesion molecules plakoglobin and vitronectin and the antimicrobial protein dermcidin.,Compared to healthy controls, melanoma patients have high serum levels of these proteins at the moment of melanoma diagnosis, although the specific values were not related to the histopathological stage of the tumors.,However, an analysis based on classification together with multivariate statistics showed that tumor stage, vitronectin and dermcidin levels were associated with the metastatic progression of patients with early‐stage melanoma.,Although melanoma patients have increased serum dermcidin levels, the REPTree classifier showed that levels of dermcidin <2.98 μg/ml predict metastasis in AJCC stage II patients.,These data suggest that vitronectin and dermcidin are potent biomarkers of prognosis, which may help to improve the personalized medical care of melanoma patients and their survival.,What's new?,The discovery of serum biomarkers capable of predicting metastatic risk during post‐operative follow‐up in early‐stage primary melanoma patients could significantly benefit patient prognosis and survival.,The present study identifies two promising biomarker candidates: vitronectin and dermcidin.,Analysis and comparison of serum proteins in melanoma patients who either remained disease‐free or developed metastases in the years after surgery to remove primary tumors revealed a strong association between metastatic progression of early‐stage melanomas (AJCC stage III) and vitronectin and dermcidin serum levels.,Further study of these proteins could open up new opportunities in the effort to improve long‐term survival among melanoma patients.

Objective: To determine the epidemiological profile of malignant melanoma cases treated at the National Institute for Neoplastic Diseases “Dr.,Eduardo Caceres Graziani” (INEN) over the period 1952 to 2008.,Study Design: All clinical records with complete data of patients presenting a histopathological diagnosis of malignant melanoma of the oral cavity were reviewed.,Data such as age, gender, location, tumor size, disease length, presence of metastasis, treatment received and year of admission were recorded.,Results: During the study period 97 cases were found.,The average age of patients was 52.85±1.6 years old mostly between 50 and 59 years old; the predominant gender was the female.,The most common location was the palate and there was 58.8% of cases with a tumor size bigger than or equal to 4 cm.,The length of the disease in 38.1% of the cases was longer than a year and in great part of the cases (69.1%) there was no metastasis.,The treatment of choice was the surgery plus radiotherapy in 38.1% of the cases.,According to the admission date it was also noted that the number of cases is increasing.,Conclusion: The results of this study demonstrate a late diagnosis and an increasing frequency of this neoplasia in the oral cavity.,Key words: Melanoma, oral cavity, epidemiology.

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.

Background.,The MC1R gene implicated in melanogenesis and skin pigmentation is highly polymorphic.,Several alleles are associated with red hair and fair skin phenotypes and contribute to melanoma risk.,Objective.,This work aims to assess the effect of different classes of MC1R variants, notably rare variants, on melanoma risk.,Methods.,MC1R coding region was sequenced in 1131 melanoma patients and 869 healthy controls.,MC1R variants were classified as RHC (R) and non-RHC (r).,Rare variants (frequency < 1%) were subdivided into two subgroups, predicted to be damaging (D) or not (nD).,Results.,Both R and r alleles were associated with melanoma (OR = 2.66 [2.20-3.23] and 1.51 [1.32-1.73]) and had similar population attributable risks (15.8% and 16.6%).,We also identified 69 rare variants, of which 25 were novel.,D variants were strongly associated with melanoma (OR = 2.38 [1.38-4.15]) and clustered in the same MC1R domains as R alleles (intracellular 2, transmembrane 2 and 7).,Conclusion.,This work confirms the role of R and r alleles in melanoma risk in the French population and proposes a novel class of rare D variants as important melanoma risk factors.,These findings may improve the definition of high-risk subjects that could be targeted for melanoma prevention and screening.

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.

The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress.,Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas.,The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation.,In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated.,NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation.,Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG.,Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation.,These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression.

Molecular chaperone heat shock protein 90 (Hsp90) inhibitors are promising targeted cancer therapeutic drugs, with the advantage that they deplete multiple oncogenic client proteins and modulate all the classical hallmarks of cancer.,They are now in clinical trial and show potential for activity in melanoma and other malignancies.,Here we explore the metabolic response to Hsp90 inhibition in human melanoma cells using magnetic resonance spectroscopy.,We show that, concomitant with growth inhibition and re-differentiation, Hsp90 inhibition in human melanoma cells is associated with increased glycerophosphocholine content.,This was seen with both the clinical geldanamycin-based Hsp90 drug 17-AAG and the structurally dissimilar Hsp90 inhibitor CCT018159.,The effect was noted in both BRAF mutant SKMEL28 and BRAF wildtype CHL-1 melanoma cells.,Elevated content of the -CH2+CH3 fatty acyl chains and cytoplasmic mobile lipid droplets was also observed in 17-AAG-treated SKMEL28 cells.,Importantly, the phospholipase A2 inhibitor bromoenol lactone prevented the rise in glycerophosphocholine seen with 17-AAG, suggesting a role for phospholipase A2 activation in the Hsp90 inhibitor-induced metabolic response.,Our findings provide a basis for using metabolic changes as non-invasive indicators of Hsp90 inhibition and potentially as biomarkers of anticancer activity with Hsp90 drugs in malignant melanoma and possibly in other cancers.

In contrast to other cancer types, melanoma incidence has been increasing over the last 50 years, and while it still represents less than 5% of all cutaneous malignancies, melanoma accounts for the majority of skin cancer deaths, due to its propensity to metastasise.,Whilst melanoma most commonly affects the skin, it can also arise in mucosal surfaces, the eye, and the brain.,For new therapies to be developed, a better understanding of the genetic landscape, signalling pathways, and tumour-microenvironmental interactions is needed.,This is where animal models are of critical importance.,The mouse is the foremost used model of human melanoma.,Arguably this is due to its plethora of benefits as a laboratory animal; however, it is important to note that unlike humans, melanocytes are not present at the dermal-epidermal junction in mice and mice do not develop melanoma without genetic manipulation.,In contrast, there are numerous reports of animals that spontaneously develop melanoma, ranging from sharks and parrots to hippos and monkeys.,In addition, several domesticated and laboratory‐bred animals spontaneously develop melanoma or UV‐induced melanoma, specifically, fish, opossums, pigs, horses, cats, and dogs.,In this review, we look at spontaneously occurring animal ‘models’ of melanoma and discuss their relevance to the different types of melanoma found in humans.,© 2020 The Authors.,The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..

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

Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients.,Until today most studies utilized autologous NK cells and yielded disappointing results.,Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC).,We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching.,Combination of the various strategies yielded improved effects.,In addition, we developed various clinically-compatible ex vivo expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors.,The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes.,This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.

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

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma.,However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown.,In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma.,We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ.,In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples.,We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation.,Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720.,Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma.

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

Targeted therapies directed at commonly overexpressed pathways in melanoma have clinical activity in numerous trials.,Little is known about how these therapies influence microRNA (miRNA) expression, particularly with combination regimens.,Knowledge of miRNAs altered with treatment may contribute to understanding mechanisms of therapeutic effects, as well as mechanisms of tumor escape from therapy.,We analyzed miRNA expression in metastatic melanoma tissue samples treated with a novel combination regimen of Temsirolimus and Bevacizumab.,Given the preliminary clinical activity observed with this combination regimen, we hypothesized that we would see significant changes in miRNA expression with combination treatment.,Using microarray analysis we analyzed miRNA expression levels in melanoma samples from a Cancer Therapy Evaluation Program-sponsored phase II trial of combination Temsirolimus and Bevacizumab in advanced melanoma, which elicited clinical benefit in a subset of patients.,Pre-treatment and post-treatment miRNA levels were compared using paired t-tests between sample groups (patients), using a p-value < 0.01 for significance.,microRNA expression remained unchanged with Temsirolimus alone; however, expression of 15 microRNAs was significantly upregulated (1.4 to 2.5-fold) with combination treatment, compared to pre-treatment levels.,Interestingly, twelve of these fifteen miRNAs possess tumor suppressor capabilities.,We identified 15 putative oncogenes as potential targets of the 12 tumor suppressor miRNAs, based on published experimental evidence.,For 15 of 25 miRNA-target mRNA pairings, changes in gene expression from pre-treatment to post-combination treatment samples were inversely correlated with changes in miRNA expression, supporting a functional effect of those miRNA changes.,Clustering analyses based on selected miRNAs suggest preliminary signatures characteristic of clinical response to combination treatment and of tumor BRAF mutational status.,To our knowledge, this is the first study analyzing miRNA expression in pre-treatment and post-treatment human metastatic melanoma tissue samples.,This preliminary investigation suggests miRNAs that may be involved in the mechanism of action of combination Temsirolimus and Bevacizumab in metastatic melanoma, possibly through inhibition of oncogenic pathways, and provides the preliminary basis for further functional studies of these miRNAs.

The abnormal expression of several microRNAs has a causal role in tumorigenesis with either antineoplastic or oncogenic functions.,Here we demonstrated that miR-126 and miR-126* play a tumor suppressor role in human melanoma through the direct or indirect repression of several key oncogenic molecules.,The expression levels of miR-126&126* were elevated in normal melanocytes and primary melanoma cell lines, whereas they markedly declined in metastatic cells.,Indeed, the restored expression of miR-126&126* in two advanced melanoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro as well as of growth and dissemination in vivo.,In accordance, the reverse functional effects were obtained by knocking down miR-126&126* by transfecting antisense LNA oligonucleotides in melanoma cells.,Looking for the effectors of these antineoplastic functions, we identified ADAM9 and MMP7, two metalloproteases playing a pivotal role in melanoma progression, as direct targets of miR-126&126*.,In addition, as ADAM9 and MMP7 share a role in the proteolytic cleavage of the HB-EGF precursor, we looked for the effectiveness of this regulatory pathway in melanoma, confirming the decrease of HB-EGF activation as a consequence of miR-126&126*-dependent downmodulation of ADAM9 and MMP7.,Finally, gene profile analyses showed that miR-126&126* reexpression was sufficient to inactivate other key signaling pathways involved in the oncogenic transformation, as PI3K/AKT and MAPK, and to restore melanogenesis, as indicated by KIT/MITF/TYR induction.,In view of this miR-126&126* wide-ranging action, we believe that the replacement of these microRNAs might be considered a promising therapeutic approach.

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression.,The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages.,In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids.,In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts.,The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts.,Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts.,The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium.,An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity.,Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids.,Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts.,Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

The cancer-associated fibroblast (CAF) population is implicated in immune dysregulation.,Here, we test the hypothesis that CAF profiles in pretreatment tumor specimens are associated with response to immune checkpoint blockade of programmed cell death 1 (PD-1).,Pretreatment whole tissue sections from 117 melanoma patients treated with anti-PD-1 therapy were assessed by multiplex immunofluorescence to detect CAFs defined by Thy1, smooth muscle actin (SMA), and fibroblast activation protein (FAP).,Two independent image analysis technologies were used: inForm software (PerkinElmer) to quantify cell counts, and AQUA™ to measure protein by quantitative immunofluorescence (QIF).,CAF parameters by both methodologies were assessed for association with previously measured immune markers (CD3, CD4, CD8, CD20, CD68, PD-L1), best overall response, progression-free survival (PFS), and overall survival (OS).,CAF parameters, by cell counts or QIF, did not correlate with immune markers nor with best overall response.,However, both Thy1 and FAP cell counts had significant positive associations with PFS (all P < 0.05) and OS (all P < 0.003).,SMA cell counts showed negative associations with outcome in anti-PD-1 treated patients.,Similar associations were not observed in a control cohort of historical melanoma patients predating immunotherapy.,Instead, FAP was a negative prognostic biomarker (P = 0.01) in the absence of immunotherapy.,Multivariable analyses revealed significant PFS and OS associations with the CAF parameters were independent of baseline variables.,Pretreatment CAF profiles are associated with melanoma immunotherapy outcome.,Multiplex CAF analysis has potential as an objective companion diagnostic in immuno-oncology.,The online version of this article (10.1186/s40425-019-0675-0) contains supplementary material, which is available to authorized users.

Efficient treatments against metastatic melanoma dissemination are still lacking.,Here, we report that low-cytotoxic concentrations of 5-aza-2′-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo.,We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation.,Alone, this miR showed an anti-invasive and anti-metastatic effect.,Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2′-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes.,In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation.,Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma.,The online version of this article (10.1186/s13148-018-0600-2) contains supplementary material, which is available to authorized users.

Previous research indicates that microRNA-25 (miR-25) regulates carcinogenesis and the progression of various cancers, but the role of miR-25 in melanoma remains unclear.,We observed that miR-25 was significantly upregulated in melanoma cell lines and tissue samples.,Downregulation of miR-25 markedly suppressed invasion and proliferation of melanoma cells in vitro; however, overexpression of miR-25 markedly increased melanoma cell invasion and proliferation.,Moreover, we observed Dickkopf-related protein 3 (DKK3) as a direct target of miR-25 in vitro.,Upregulation of DKK3 partially attenuated the oncogenic effect of miR-25 on melanoma cells.,Ectopic expression of miR-25 in melanoma cells induced β-catenin accumulation in nuclear and inhibited TCF4 (T cell factor 4) activity, as well as the expression of c-Myc and Cyclin D1.,In a nude xenograft model, miR-25 upregulation significantly increased A375 melanoma growth.,In summary, miR-25 is upregulated in melanoma and promotes melanoma cell proliferation and invasion, partially by targeting DKK3.,These results were indicated that miR-25 may serve as a potential target for the treatment of melanoma in the future.

The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease.,Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy.,The object of this study is recognition of the possible role of mTOR kinase inhibitors-everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma.,Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors.,The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination.,The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor- everolimus with a B-RAF inhibitor-PLX-4032.,Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor-everolimus with ERK1/2 inhibitor-U126 or MEK inhibitor-AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor-LY294002 and AKT inhibitor-MK-2206.,The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment.,The online version of this article (10.1007/s13577-019-00270-4) contains supplementary material, which is available to authorized users.

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

The therapeutic landscape for advanced melanoma has expanded in recent years.,This expansion has largely been driven by investigational work in melanoma tumor biology and immunology that has been successfully translated to the clinical setting.,Molecular evidence generated through benchside experimentation identified BRAF and MEK as key molecular targets in melanoma.,This commentary will highlight this work and provide a rationale for the continued importance of translational work in the field of targeted melanoma therapies.

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.

Dogs have been considered as an excellent immunocompetent model for human melanoma due to the same tumor location and the common clinical and pathological features with human melanoma.,However, the differences in the melanoma transcriptome between the two species have not been yet fully determined.,Considering the role of oncogenes in melanoma development, in this study, we first characterized the transcriptome in canine oral melanoma and then compared the transcriptome with that of human melanoma.,The global transcriptome from 8 canine oral melanoma samples and 3 healthy oral tissues were compared by RNA-Seq followed by RT-qPCR validation.,The results revealed 2,555 annotated differentially expressed genes, as well as 364 novel differentially expressed genes.,Dog chromosomes 1 and 9 were enriched with downregulated and upregulated genes, respectively.,Along with 10 significant transcription site binding motifs; the NF-κB and ATF1 binding motifs were the most significant and 4 significant unknown motifs were indentified among the upregulated differentially expressed genes.,Moreover, it was found that canine oral melanoma shared >80% significant oncogenes (upregulated genes) with human melanoma, and JAK-STAT was the most common significant pathway between the species.,The results identified a 429 gene signature in melanoma, which was up-regulated in both species; these genes may be good candidates for therapeutic development.,Furthermore, this study demonstrates that as regards oncogene expression, human melanoma contains an oncogene group that bears similarities with dog oral melanoma, which supports the use of dogs as a model for the development of novel therapeutics and experimental trials before human application.

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

Melanoma is a severe and life-threatening malignancy derived from melanocytes.,The traditional treatment for melanoma could not sustain satisfactory outcomes long term; however, the recent immune checkpoint treatment has made a breakthrough in these problems.,Nivolumab is a representative immune checkpoint treatment, and this PD-1-targeted therapy has evolutionally developed and improved the clinical outcome in a recent decade.,On the other hand, the clinical application of immune checkpoint treatment presents clinicians with novel questions, especially how to obtain additional efficacy and overcome the disadvantage by using this treatment.,To answer these problems, we first investigated the distribution of PD-L1 in various organs to clarify the organs most affected by anti-PD-1 antibody treatment.,Among various organs, lung, placenta, spleen, heart, and thyroid highly expressed PD-L1, while skin, thalamus, hippocampus, ovary, stomach, testis, and prostate showed lower expressions of PD-L1.,Furthermore, the immune profiles were also examined in tumors and peripheral blood in patients with melanoma.,PD-1 was highly expressed in CD8 and CD4 cells, and B cells also highly expressed PD-1 compared with NK cells.,However, there was no significant difference in Th1/Th2/Th17 cytokines and inhibitory cytokine IL-10.,Although nevus showed a low expression of PD-L1 compared with healthy skin, PD-L1 expression was increased in growth-phase melanoma.,Finally, we analyzed the peripheral blood profiles in patients treated with nivolumab.,PD-1-bearing dendritic cells (DCs) were increased during nivolumab treatment and Lin-CD11c+HLA-DR+ cells were highly increased during nivolumab treatment.,These findings indicate a clue to answering the problems during nivolumab treatment and suggest to us the importance of multiple aspect observation during immune checkpoint treatment.

Previous studies have identified an inverse association between melanoma and smoking; however, data from population‐based studies are scarce.,To determine the association between smoking and socioeconomic (SES) on the risk of development of melanoma.,Furthermore, we sought to determine the implications of smoking and SES on survival.,We conducted a population‐based case-control study.,Cases were identified from the Welsh Cancer Intelligence and Surveillance Unit (WCISU) during 2000-2015 and controls from the general population.,Smoking and SES were obtained from data linkage with other national databases.,The association of smoking status and SES on the incidence of melanoma were assessed using binary logistic regression.,Multivariate survival analysis was performed on a melanoma cohort using a Cox proportional hazard model using survival as the outcome.,During 2000-2015, 9636 patients developed melanoma.,Smoking data were obtained for 7124 (73·9%) of these patients.,There were 26 408 controls identified from the general population.,Smoking was inversely associated with melanoma incidence [odds ratio (OR) 0·70, 95% confidence interval (CI) 0·65-0·76].,Smoking was associated with an increased overall mortality [hazard ratio (HR) 1·30, 95% CI 1·09-1·55], but not associated with melanoma‐specific mortality.,Patients with higher SES had an increased association with melanoma incidence (OR 1·58, 95% CI 1·44-1·73).,Higher SES was associated with an increased chance of both overall (HR 0·67, 95% CI 0·56-0·81) and disease‐specific survival (HR 0·69, 95% CI 0·53-0·90).,Our study has demonstrated that smoking appeared to be associated with reduced incidence of melanoma.,Although smoking increases overall mortality, no association was observed with melanoma‐specific mortality.,Further work is required to determine if there is a biological mechanism underlying this relationship or an alternative explanation, such as survival bias.,What's already known about this topic?,Previous studies have been contradictory with both negative and positive associations between smoking and the incidence of melanoma reported.Previous studies have either been limited by publication bias because of selective reporting or underpowered.,Previous studies have been contradictory with both negative and positive associations between smoking and the incidence of melanoma reported.,Previous studies have either been limited by publication bias because of selective reporting or underpowered.,What does this study add?,Our large study identified an inverse association between smoking status and melanoma incidence.Although smoking status was negatively associated with overall disease survival, no significant association was noted in melanoma‐specific survival.Socioeconomic status remains closely associated with melanoma.,Although higher socioeconomic populations are more likely to develop the disease, patients with lower socioeconomic status continue to have a worse prognosis.,Our large study identified an inverse association between smoking status and melanoma incidence.,Although smoking status was negatively associated with overall disease survival, no significant association was noted in melanoma‐specific survival.,Socioeconomic status remains closely associated with melanoma.,Although higher socioeconomic populations are more likely to develop the disease, patients with lower socioeconomic status continue to have a worse prognosis.,Linked Comment: Thompson and Friedman.,Br J Dermatol 2020; 182:1080.,Respond to this article,Plain language summary available online

Checkpoint inhibitors (CPIs) are thought to be effective against cutaneous melanoma in part because of the large burden of somatic mutations (neoantigens) generated from exposure to ultraviolet radiation.,However, rare melanoma subtypes arising from acral skin, mucosal surfaces, and the uveal tract are largely sun-shielded.,Genomic studies show these sun-shielded melanomas have a paucity of neoantigens and unique biology; they are thought to be largely resistant to immunotherapy.,It has not been definitively shown that CPI improves survival in metastatic sun-shielded melanoma.,We reviewed a single institutional experience using antibodies against CTLA-4, PD-1 and/or PD-L1 to treat patients with metastatic melanoma.,Primary tumor histology was categorized as cutaneous, unknown, acral, mucosal, or uveal.,We studied demographic data, treatment characteristics, and overall survival (OS) after CPI.,We treated 428 patients with metastatic melanoma from 2007 to 2019.,Primary tumors were cutaneous in 283 (66%), unknown in 55 (13%), acral in 22 (5%), mucosal in 38 (9%), and uveal in 30 (7%).,Patients with metastatic disease from cutaneous primary tumors had median OS after CPI of 45 months compared with 17 months for acral (p=0.047), 18 months for mucosal (p=0.003), and 12 months for uveal (p<0.001).,For all patients with sun-shielded melanoma (n=90), first treatment with anti-PD-1 or anti-PD-L1 was followed by a median OS of 9 months compared with 18 months after anti-CTLA-4 (p=0.010) and 20 months after combination therapy (p=0.003).,There were 21 patients who achieved actual 3-year survival; 20 received both anti-CTLA-4 and anti-PD-1, either sequentially or in combination.,Over 80% of 3-year survivors with progressive disease were treated with local therapy after CPI.,Long survival in patients with metastatic melanoma from acral, mucosal, and uveal primary tumors was associated with receipt of both anti-CTLA-4 and anti-PD-1 antibodies.,Complete responses were rare, and local therapy was frequently employed to control disease progression.,While sun-shielded melanomas exhibit worse outcomes after CPI than cutaneous melanomas, with an aggressive multidisciplinary approach, 5-year survival is still possible for 25%-32% of these patients.

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.

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.

Double positive (DP) CD4CD8 Tαβ cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated.,We recently reported the increased frequency of DP Tαβ cells in human breast pleural effusions.,This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas.,We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry.,Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples.,Interestingly, a high proportion of these cells produced TNF-α in response to autologous melanoma cell lines.,Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells.,In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node.,This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen.,However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells.,In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo.,Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of immunotherapies.

Aberrant expression of microRNAs plays vital roles in tumor development and progression.,As transcription factors (TFs) are the critical components of signaling cascades, specific targeting effects of microRNAs to specific TFs may determine the role of microRNAs in different cancers.,In this study, we identified Nuclear Factor I/B (NFIB) as one of the targets of miR-365 which was previously verified as an onco-miR in cutaneous squamous cell carcinoma (CSCC).,Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically up-regulated miR-365 expression levels.,The siRNA-based knockdown of NFIB mimic the carcinogenic transformation of normal cells by ectopically expression of miR-365 which indicates depletion of NFIB is necessary for miR-365 to exert its pro-carcinogenic function.,NFIB may represent a functional barrier targeted by miR-365 to the development of CSCC.,Further studies also discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development which may be potentially utilized as therapeutic target to improve the clinical CSCC treatment.

The expression levels of miR-365 vary in different malignancies.,Herein, we found that miR-365 was overexpressed in both cells and clinical specimens of cutaneous squamous cell carcinoma (SCC).,We demonstrated that the HaCaTpre-miR-365-2 cell line, which overexpressed miR-365, could induce subcutaneous tumors in vivo.,Antagomir-365, an anti-miR-365 oligonucleotide, inhibited cutaneous tumor formation in vivo, along with G1 phase arrest and apoptosis of cancer cells.,These findings suggest that miR-365 may act as an onco-miR in cutaneous SCC both in vitro and in vivo.,The present study provides valuable insight into the role of miR-365 in cutaneous SCC formation, which can help develop new drug and miR-365 target-based therapies for cutaneous SCC.

Patient: Female, 56-year-old,Final Diagnosis: Malignant melanoma,Symptoms: Tumor,Medication:-,Clinical Procedure: -,Specialty: Oncology,Rare disease,Primary vaginal malignant melanoma is a rare and aggressive tumor with a high risk of local recurrence and distant metastasis.,Although there are several available treatment options, none are considered as standard.,Surgical resection is the first treatment choice because of its superior survival benefits.,The patient was a 56-year-old woman with a vaginal mass.,At the first visit to our institution, a 20×20 mm black and flat lesion on the lower third of the posterior vaginal wall and a polypoid mass near the vaginal fornix were detected by gynecologic examination.,Study of the tumor on the posterior vaginal wall suggested that it did not extend to the uterine cervix.,The preoperative diagnosis was vaginal malignant melanoma FIGO stage I (cT1, cN0, cM0).,The patient underwent a total vaginectomy, pelvic and inguinal lymphadenectomy, modified radical hysterectomy, and bilateral salpingo-oophorectomy.,The tumor cells were arranged in sheets and nests and exhibited nuclear pleomorphism, eosinophilic cytoplasm, brisk mitotic activity, and melanin production.,The overlying mucosa was ulcerated.,The tumor thickness was 2.5 mm and no residual lesion was found at the surgical margin.,No adjuvant therapies were performed.,The patient is alive without recurrence 15 months after the initial treatment.,This is a case of vaginal malignant melanoma for which complete response was achieved by radical tumor re-section, without severe adverse effects and with no observed recurrence 15 months after the surgery.

immunotherapy with immune checkpoint inhibitors has become one of the standard therapeutic modalities for patients with advanced melanoma.,Melanoma of the female lower genital tract is a rare and aggressive disease, with poor long-term clinical outcomes.,To date, no study evaluated the role of immunotherapy in metastatic melanoma of the lower genital tract.,Data of women with metastatic melanoma of the lower genital tract were prospectively collected.,Survival outcomes over time was assessed using Kaplan-Meier model.,Seven cases of metastatic melanoma of the lower genital tract (vulva [n=2], vagina [n=4], and uterine cervix [n=1]) treated with immune checkpoint inhibitors are reviewed.,Two patients had metastatic disease at diagnosis, while 5 patients developed metastatic disease at a mean (standard deviation) time of 9.9 (±3.0) months from primary diagnosis.,Four patients received an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) (ipilimumab) and 3 received an anti-programmed cell death 1 (PD-1) (pembrolizumab [n=2], nivolumab [n=1]) therapy.,The response rate to immunotherapy was 28.5%.,Patients receiving an anti-PD-1 experienced a better progression-free survival than patients treated with anti-CTLA4 (p=0.01, log-rank test).,Although not reaching statistical significance, overall survival was better in patients having an anti-PD-1 therapy in comparison to anti-CTLA4 (p=0.15, log-rank test).,Results from our series confirm the poor prognosis of women with metastatic melanoma of the lower genital tract, thus supporting the need of exploring new treatment modalities.,Further studies are warranted to improve knowledge on the role of immunotherapy in metastatic melanoma of the lower genital tract.

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

Melanoma is currently divided on a genetic level according to mutational status.,However, this classification does not optimally predict prognosis.,In prior studies, we have defined gene expression phenotypes (high-immune, pigmentation, proliferative and normal-like), which are predictive of survival outcome as well as informative of biology.,Herein, we employed a population-based metastatic melanoma cohort and external cohorts to determine the prognostic and predictive significance of the gene expression phenotypes.,We performed expression profiling on 214 cutaneous melanoma tumors and found an increased risk of developing distant metastases in the pigmentation (HR, 1.9; 95% CI, 1.05-3.28; P=0.03) and proliferative (HR, 2.8; 95% CI, 1.43-5.57; P=0.003) groups as compared to the high-immune response group.,Further genetic characterization of melanomas using targeted deep-sequencing revealed similar mutational patterns across these phenotypes.,We also used publicly available expression profiling data from melanoma patients treated with targeted or vaccine therapy in order to determine if our signatures predicted therapeutic response.,In patients receiving targeted therapy, melanomas resistant to targeted therapy were enriched in the MITF-low proliferative subtype as compared to pre-treatment biopsies (P=0.02).,In summary, the melanoma gene expression phenotypes are highly predictive of survival outcome and can further help to discriminate patients responding to targeted therapy.

The Melanoma Gene Database (MGDB) is a manually curated catalog of molecular genetic data relating to genes involved in melanoma.,The main purpose of this database is to establish a network of melanoma related genes and to facilitate the mechanistic study of melanoma tumorigenesis.,The entries describing the relationships between melanoma and genes in the current release were manually extracted from PubMed abstracts, which contains cumulative to date 527 human melanoma genes (422 protein-coding and 105 non-coding genes).,Each melanoma gene was annotated in seven different aspects (General Information, Expression, Methylation, Mutation, Interaction, Pathway and Drug).,In addition, manually curated literature references have also been provided to support the inclusion of the gene in MGDB and establish its association with melanoma.,MGDB has a user-friendly web interface with multiple browse and search functions.,We hoped MGDB will enrich our knowledge about melanoma genetics and serve as a useful complement to the existing public resources.,Database URL:http://bioinfo.ahu.edu.cn:8080/Melanoma/index.jsp

The acquisition of invasive properties in melanoma is associated with a high proclivity for metastasis, but the underlying pathways are poorly characterized.,The Hippo pathway plays an important role in organ size control and is dysregulated in some types of tumors.,The present study, “Pro-invasive activity of the Hippo pathway effectors YAP and TAZ in cutaneous melanoma” by Nallet-Staub et al., provides the first in-depth analysis of expression of the Hippo pathway effectors YAP (yes-associated protein) and TAZ (Tafazzin) in human melanocytic lesions.,Importantly, results from this study demonstrate a causal relationship between YAP/TAZ levels and melanoma cell tumorigenicity and invasiveness.

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.

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.

Circular RNAs (circRNAs) have been reported to have critical regulatory roles in tumor biology.,However, their contribution to melanoma remains largely unknown.,CircRNAs derived from oncogene CD151 were detected and verified by analyzing a large number of melanoma samples through quantitative real-time polymerase chain reaction (qRT-PCR).,Melanoma cells were stably transfected with lentiviruses using circ_0020710 interference or overexpression plasmid, and then CCK-8, colony formation, wound healing, transwell invasion assays, and mouse xenograft models were employed to assess the potential role of circ_0020710.,RNA immunoprecipitation, luciferase reporter assay and fluorescence in situ hybridization were used to evaluate the underlying mechanism of circ_0020710.,Our findings indicated that circ_0020710 was generally overexpressed in melanoma tissues, and high level of circ_0020710 was positively correlated with malignant phenotype and poor prognosis of melanoma patients.,Elevated circ_0020710 promoted melanoma cell proliferation, migration and invasion in vitro as well as tumor growth in vivo.,Mechanistically, we found that high level of circ_0020710 could upregulate the CXCL12 expression via sponging miR-370-3p.,CXCL12 downregulation could reverse the malignant behavior of melanoma cells conferred by circ_0020710 over expression.,Moreover, we also found that elevated circ_0020710 was correlated with cytotoxic lymphocyte exhaustion, and a combination of AMD3100 (the CXCL12/CXCR4 axis inhibitor) and anti-PD-1 significantly attenuated tumor growth.,Elevated circ_0020710 drives tumor progression via the miR-370-3p/CXCL12 axis, and circ_0020710 is a potential target for melanoma treatment.

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

Long intergenic noncoding RNA 00961 (Linc00961) has been identified as a tumor suppressor in various types of cancer.,However, the critical roles of Linc00961 in the carcinogenesis and progression of skin melanoma (SM) are yet to be fully elucidated.,The present study revealed via reverse transcription-quantitative PCR analysis that Linc00961 was downregulated in the tissues of patients with SM compared with benign nevi, and in A375, A2058 and SK-MEL-28 cell lines compared with human melanocytes.,Furthermore, overexpression of Linc00961 inhibited cell proliferation, and promoted the apoptosis of A375 and SK-MEL-28 cells in vitro and in vivo, as determined by Cell Counting Kit-8 and flow cytometry assays, and tumor xenograft studies, respectively.,Overexpression of Linc00961 also led to an attenuation of the migration and invasive capabilities of A375 and SK-MEL-28 cells, measured using Transwell assays.,Functionally, it was demonstrated that Linc00961 acted as a competing endogenous RNA (ceRNA) by competitively sponging microRNA-367 (miR-367) in A375 and SK-MEL-28 cells; restoration of miR-367 rescued the inhibitory effects of Linc00961 on A375 and SK-MEL-28 cells.,Finally, it was observed that phosphate and tension homology deleted on chromosome 10 (PTEN), an established target of miR-367 in A375 and SK-MEL-28 cells, was positively regulated by Linc00961, and its inhibition reversed the inhibitory effects of Linc00961 on the proliferation and invasion of A375 and SK-MEL-28 cells.,Collectively, the present study revealed that Linc00961 was downregulated inSM, and furthermore, Linc00961 was identified as a ceRNA that inhibits the proliferation and invasion of A375 and SK-MEL-28 cells by modulating the miR-367/PTEN axis.

Combined inhibition of BRAF and MEK1/2 (CIBM) improves therapeutic efficacy of BRAF-mutant melanoma.,However, drug resistance to CIBM is inevitable and the drug resistance mechanisms still remain to be elucidated.,Here, we show that BMK1 pathway contributes to the drug resistance to CIBM.,Considering that ERK1/2 pathway regulates cellular processes by phosphorylating, we first performed a SILAC phosphoproteomic profiling of CIBM.,Phosphorylation of 239 proteins was identified to be downregulated, while phosphorylation of 47 proteins was upregulated.,Following siRNA screening of 47 upregulated proteins indicated that the knockdown of BMK1 showed the most significant ability to inhibit the proliferation of CIBM resistant cells.,It was found that phosphorylation of BMK1 was enhanced in resistant cells, which suggested an association of BMK1 with drug resistance.,Further study indicated that phospho-activation of BMK1 by MEK5D enhanced the resistance to CIBM.,Conversely, inhibition of BMK1 by shRNAi or BMK1 inhibitor (XMD8-92) impaired not only the acquirement of resistance to CIBM, but also the proliferation of CIBM resistant cells.,Further kinome-scale siRNA screening demonstrated that SRC\MEK5 cascade promotes the phospho-activation of BMK1 in response to CIBM.,Our study not only provides a global phosphoproteomic view of CIBM in melanoma, but also demonstrates that inhibition of BMK1 has therapeutic potential for the treatment of melanoma.

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

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

Metastatic melanoma is a malignant cancer with generally poor prognosis, with no targeted chemotherapy.,To identify epigenetic changes related to melanoma, we have determined genome-wide methylated CpG island distributions by next-generation sequencing.,Melanoma chromosomes tend to be differentially methylated over short CpG island tracts.,CpG islands in the upstream regulatory regions of many coding and noncoding RNA genes, including, for example, TERC, which encodes the telomerase RNA, exhibit extensive hypermethylation, whereas several repeated elements, such as LINE 2, and several LTR elements, are hypomethylated in advanced stage melanoma cell lines.,By using CpG island demethylation profiles, and by integrating these data with RNA-seq data obtained from melanoma cells, we have identified a co-expression network of differentially methylated genes with significance for cancer related functions.,Focused assays of melanoma patient tissue samples for CpG island methylation near the noncoding RNA gene SNORD-10 demonstrated high specificity.

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.

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.

To assess clinical characteristics, treatment and survival of patients with uveal melanoma in China.,The retrospective study included all patients with malignant uveal melanoma who were consecutively examined in the study period from January 2005 and June 2015 in the Beijing Tongren hospital.,The mean age of the 582 patients (295(50.7%) women) was 44.6±12.6 years (range:5-77 years).,The tumors were located most often in the superior temporal region (in 117(21.5%) patients) and least common in the inferior region (in 31(5.7%) patients).,In 548(94.2%) patients, the tumors were located in the choroid, in 33(5.7%) patients in the ciliary body, and in one (0.2%) patient in the iris.,Treatment included episcleral brachytherapy (415(71.3%) patients), local tumor resection (48(8.2%) patients) and primary enucleation (119(20.4%) patients).,In 53 individuals out of the 415 patients with primary brachytherapy, episcleral brachytherapy was followed by enucleation, due to an increasing tumor size or due to uncontrolled neovascular glaucoma.,Median follow-up time was of 30 months (range: 1-124 months; mean: 34.8 ± 24.4 months).,Overall survival rate at 5 and 10 years was of 92.7% and 85.1%.,Younger age (P = 0.017), tumor location in the nasal meridian(P = 0.004), smaller tumor size (P<0.001), hemispheric tumor shape (P = 0.025), histological tumor cell type (spindle-cell type versus epitheloid cell type;P = 0.014), and type of treatment (episcleral brachytherapy versus local tumor resection and versus primary enucleation; P<0.001) were significantly associated with the overall survival in univariate analysis, while in multivariate analysis only smaller tumor size was significantly (P<0.001; RR: 4.75; 95% confidence interval:2.11,10.7) associated with better overall survival.,In this study on clinical characteristics of uveal melanoma of a larger group of patients from China, the onset age was considerably younger and survival rate better than in studies from Western countries.,Tumor size was the most significant factor for survival.

Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases.,To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs.,We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1).,In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above.,In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2).,TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278.,Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036).,Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs.,We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses.,Collectively, these data support the use of immunotherapies for virus-negative MCCs.

Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor frequently associated with clonal integration of a polyomavirus, MCPyV, and MCC tumor cells express putative polyomavirus oncoproteins small T antigen (sTAg) and truncated large T antigen (tLTAg).,Here, we show robust transforming activity of sTAg in vivo in a panel of transgenic mouse models.,Epithelia of pre-term sTAg-expressing embryos exhibited hyperplasia, impaired differentiation, increased proliferation and apoptosis, and activation of a DNA damage response.,Epithelial transformation did not require sTAg interaction with the PP2A protein complex, a tumor suppressor in some other polyomavirus transformation models, but was strictly dependent on a recently-described sTAg domain that binds Fbxw7, the substrate-binding component of the SCF ubiquitin ligase complex.,Postnatal induction of sTAg using a Cre-inducible transgene also led to epithelial transformation with development of lesions resembling squamous cell carcinoma in situ and elevated expression of Fbxw7 target proteins.,Our data establish that expression of MCPyV sTAg alone is sufficient for rapid neoplastic transformation in vivo, implicating sTAg as an oncogenic driver in MCC and perhaps other human malignancies.,Moreover, the loss of transforming activity following mutation of the sTAg Fbxw7 binding domain identifies this domain as crucial for in vivo transformation.

Over half of cutaneous melanoma tumors have BRAFV600E/K mutations.,Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses.,In this study we demonstrate that approximately 50-60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases.,In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes.,Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anti-cancer compounds.,We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors.,Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHigh BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms.,Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

Previous analysis of COMBI-d (NCT01584648) demonstrated improved progression-free survival (PFS) and overall survival (OS) with combination dabrafenib and trametinib versus dabrafenib monotherapy in BRAF V600E/K-mutant metastatic melanoma.,This study was continued to assess 3-year landmark efficacy and safety after ≥36-month follow-up for all living patients.,This double-blind, phase 3 study enrolled previously untreated patients with BRAF V600E/K-mutant unresectable stage IIIC or stage IV melanoma.,Patients were randomized to receive dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or dabrafenib plus placebo.,The primary endpoint was PFS; secondary endpoints were OS, overall response, duration of response, safety, and pharmacokinetics.,Between 4 May and 30 November 2012, a total of 423 of 947 screened patients were randomly assigned to receive dabrafenib plus trametinib (n = 211) or dabrafenib monotherapy (n = 212).,At data cut-off (15 February 2016), outcomes remained superior with the combination: 3-year PFS was 22% with dabrafenib plus trametinib versus 12% with monotherapy, and 3-year OS was 44% versus 32%, respectively.,Twenty-five patients receiving monotherapy crossed over to combination therapy, with continued follow-up under the monotherapy arm (per intent-to-treat principle).,Of combination-arm patients alive at 3 years, 58% remained on dabrafenib plus trametinib.,Three-year OS with the combination reached 62% in the most favourable subgroup (normal lactate dehydrogenase and <3 organ sites with metastasis) versus only 25% in the unfavourable subgroup (elevated lactate dehydrogenase).,The dabrafenib plus trametinib safety profile was consistent with previous clinical trial observations, and no new safety signals were detected with long-term use.,These data demonstrate that durable (≥3 years) survival is achievable with dabrafenib plus trametinib in patients with BRAF V600-mutant metastatic melanoma and support long-term first-line use of the combination in this setting.

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

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity.,Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III-IV) and 16 early-stage (I-II) melanoma patients.,CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed.,Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation.,Pairwise comparison of CTC subpopulations longitudinally before and 6-13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01).,Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82-41.75, P<0.01).,Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

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

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

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

The abnormal expression of long noncoding RNA- (lncRNA-) MEG3 was clearly identified in a number of malignant tumors, but the specific function of MEG3 remains unknown in malignant melanoma until now.,The research attempts to explore the effects of MEG3 on the growth and metastasis of malignant melanoma.,MEG3 and miR-499-5p expression were determined by qRT-PCR method.,Western blotting assay was applied to detect protein expression.,Luciferase reporter assay was used to assess the correlation between MEG3 and miR-499-5p and between CYLD and miR-499-5p.,Cell growth, cell cycle, and cell apoptosis were examined by CCK-8 assay, EdU assay, and flow cytometry assay, respectively.,The invasion ability of melanoma cells was investigated by wound-healing and Transwell assays.,The effect of MEG3 on growth of melanoma in vivo and cell chemosensitivity was detected by xenograft animal model and CCK-8 assay.,As a result, the expression of MEG3 was decreased in melanoma tissues and cell lines.,The level of MEG3 was significantly associated with poor prognosis.,MEG3 could bind to miR-499-5p and CYLD mRNA contained a binding site of miR-499-5p.,The expression of CYLD was reduced and the level of miR-499-5p was elevated in melanoma tissues and cell lines.,Luciferase reporter assay and western blot assay confirmed that MEG3 regulated the expression of CYLD by sponging miR-499-5p.,Functionally, upregulation of MEG3 inhibited melanoma cell proliferation, invasion, and migration, enhanced melanoma cell apoptosis, arrested melanoma cell cycle, and regulated the expression of E-cadherin, N-cadherin, and cyclin D1 by regulating CYLD expression mediated by sponging miR-499-5p.,Importantly, overexpression of MEG3 suppressed the growth of xenograft tumor and improved chemotherapy sensitivity of A375 cells to cisplatin and 5-FU treatment.,In conclusion, MEG3 has a crucial function in the tumorigenesis of melanoma, and MEG3 may be a potential therapeutic target in the treatment of melanoma.

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

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

Melanoma is one of the most aggressive skin cancers.,The 5-year survival rate of stage III melanoma patients ranges from 93% (IIIA) to 32% (IIID) with a high risk of recurrence after complete surgery.,The introduction of target and immune therapies has dramatically improved the overall survival, but the identification of patients with a high risk of relapse who will benefit from adjuvant therapy and the determination of the best treatment choice remain crucial.,Currently, patient prognosis is based on clinico-pathological features, highlighting the urgent need of predictive and prognostic markers to improve patient management.,In recent years, many groups have focused their attention on identifying molecular biomarkers with prognostic and predictive potential.,In this review, we examined the main candidate biomarkers reported in the literature.

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

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

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.

Here, we report that the long noncoding RNA (lncRNA) ovarian adenocarcinoma-amplified lncRNA (OVAAL) is a mediator of cancer cell resistance, counteracting the effects of apoptosis-inducing agents acting through both the extrinsic and intrinsic pathways.,Building upon previous reports associating OVAAL amplification with ovarian and endometrial cancers, we now show that OVAAL overexpression occurs during the pathogenesis of colorectal cancer and melanoma.,Mechanistically, our findings also establish that OVAAL expression more generally contributes a prosurvival role to cancer cells under steady-state conditions.,OVAAL accomplishes these actions utilizing distinct functional modalities: one promoting activation of RAF/MEK/ERK signaling and the other blocking cell entry into senescence.,Our study demonstrates that expression of a single OVAAL in cancer cells drives two distinct but coordinated actions contributing to cancer pathology.,Long noncoding RNAs (lncRNAs) function through a diverse array of mechanisms that are not presently fully understood.,Here, we sought to find lncRNAs differentially regulated in cancer cells resistant to either TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77, agents that act through the extrinsic and intrinsic apoptotic pathways, respectively.,This work identified a commonly up-regulated lncRNA, ovarian adenocarcinoma-amplified lncRNA (OVAAL), that conferred apoptotic resistance in multiple cancer types.,Analysis of clinical samples revealed OVAAL expression was significantly increased in colorectal cancers and melanoma in comparison to the corresponding normal tissues.,Functional investigations showed that OVAAL depletion significantly inhibited cancer cell proliferation and retarded tumor xenograft growth.,Mechanically, OVAAL physically interacted with serine/threonine-protein kinase 3 (STK3), which, in turn, enhanced the binding between STK3 and Raf-1.,The ternary complex OVAAL/STK3/Raf-1 enhanced the activation of the RAF protooncogene serine/threonine-protein kinase (RAF)/mitogen-activated protein kinase kinase 1 (MEK)/ERK signaling cascade, thus promoting c-Myc-mediated cell proliferation and Mcl-1-mediated cell survival.,On the other hand, depletion of OVAAL triggered cellular senescence through polypyrimidine tract-binding protein 1 (PTBP1)-mediated p27 expression, which was regulated by competitive binding between OVAAL and p27 mRNA to PTBP1.,Additionally, c-Myc was demonstrated to drive OVAAL transcription, indicating a positive feedback loop between c-Myc and OVAAL in controlling tumor growth.,Taken together, these results reveal that OVAAL contributes to the survival of cancer cells through dual mechanisms controlling RAF/MEK/ERK signaling and p27-mediated cell senescence.

Rationale: Malignant melanoma (MM) is the cutaneous neoplasia with the greatest mortality rates and one of the malignancies with the highest potential of dissemination.,The prognosis of patients with metastatic MM is grim, with a 5-years survival rate between 5-19%, and is dictated by the location and the number of metastases.,Objective: We aimed to estimate the survival of patients with metastatic MM from our study and find out if the metastasis’ location influences survival.,Methods and results: Between 2008 and 2013, 155 patients with cutaneous MM were diagnosed in our clinic.,All the patients were staged according to 2009 AJCC staging system.,The median follow-up period was of 24 months.,Survival was calculated by using the Kaplan-Meier method with a confidence level of 95%.,40.5% of the patients developed metastases in different organs, especially the brain.,80.6% of those with metastases died during the study.,The median overall survival, estimated for the entire group of patients who developed metastases, was of 5.3 months.,Discussion: The influence of metastases distribution on the overall survival was examined and it was noticed that there were statistically significant differences between the risks of death of various groups of patients, depending on metastasis topography.,Thus, the death probability of a patient with brain metastases is twice that of a patient with digestive metastasis, about 7 times higher than that of a patient with lung metastasis (p = 0.0004) and 12 times higher than the death risk of a patient with extra-regional lymph nodes or subcutaneous metastasis (p = 0.0000).

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation.,Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC.,Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin.,MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse.,Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development.,These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis.,Cutaneous squamous cell carcinomas is a growing problem but the driver genes causing this remain poorly defined.,Here, the authors demonstrate that inactivating driver mutations in TGFBR1 and TGFBR2 occur in vemurafenib-induced and sporadic cutaneous squamous cell carcinomas.

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.

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

Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate.,Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration.,As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses.,We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data.,We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry.,We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation).,In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration.,Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways.,Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways.,Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility.,The online version of this article (doi:10.1186/s12916-017-0851-3) contains supplementary material, which is available to authorized users.

We used a combination of whole-genome sequencing and in vitro validation to show that mutations that activated at least two pro-growth/survival pathways mediated intrinsic resistance to BRAF inhibition in a melanoma patient.,These data demonstrate how in-depth analysis can reveal intrinsic resistance to standard of care, providing an opportunity for alternative therapeutic strategies for patients who are likely to fail first-line treat-575 ment.,BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAFV600 mutations.,Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond.,Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance.,We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient.,We validated mechanisms of resistance in a cell line derived from the patient's tumour.,We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites.,We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours.,A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway.,Inhibition of both pathways synergised to block the growth of the cells.,Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination.,We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient.,These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered.,Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth.,In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale.,Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression (‘SCNA-genes’) in seven human metastatic melanoma cell lines.,We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis.,Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples.,From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules.,Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data.,Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis.,Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma.,Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

Supplemental Digital Content is available in the text,Pembrolizumab has been approved in the United States for treating advanced melanoma for >4 years.,We examined real-world pembrolizumab use and associated outcomes in US oncology clinical practices, including patients who would not be eligible for clinical trials.,Flatiron Health longitudinal database was used to identify adult patients with advanced melanoma initiating ≥1 dose of pembrolizumab from September 4, 2014, through December 31, 2016, with follow-up through December 31, 2017.,Patients in any clinical trial during the study period were excluded.,Overall survival (OS) and time on treatment from pembrolizumab initiation were analyzed using the Kaplan-Meier (KM) method.,Subgroup analyses were conducted to examine OS for several patient characteristics including Eastern Cooperative Oncology Group (ECOG) performance status >1, brain metastases, and corticosteroids before pembrolizumab initiation.,Pembrolizumab was administered to 315 (59%), 152 (29%), and 65 (12%) patients as first-, second-, and third-line/later therapy.,Median age at pembrolizumab initiation was 68 years (range, 18-84); most patients were male (66%) and white (94%).,Of those with available data, 38% had BRAF-mutant melanoma, 21% had elevated lactate dehydrogenase (LDH) level, and 23% had ECOG >1.,Overall, 18% had brain metastases, and 23% were prescribed corticosteroids <3 months before initiating pembrolizumab.,Median study follow-up was 12.9 months (range, 0.03-39.6).,Median OS was 21.8 months (95% confidence interval [CI] 16.8-29.1); KM 1-year and 2-year survival rates were 61% and 48%, respectively; and median time on pembrolizumab treatment was 4.9 months (95% CI 3.7-5.5).,Median OS for first-line pembrolizumab was not reached, and for second-line and third-line/later was 13.9 and 12.5 months, respectively (log-rank P = .0095).,Significantly better OS (all P ≤.0014, log-rank test) was evident for patients with ECOG performance status (PS) of 0 to 1 (vs >1), normal (vs elevated) LDH level, and no (vs yes) corticosteroid prescription <3 months before.,No difference was recorded in OS by brain metastases (log-rank P = .22) or BRAF mutation status (log-rank P = .90).,These findings support effectiveness of pembrolizumab in the real-world clinical setting and provide important insights into patient characteristics and outcomes associated with pembrolizumab therapy for a heterogeneous patient population with advanced melanoma, including patients who would not be eligible for clinical trials.

Immunotherapy with PD-1 antibodies has greatly increased prognosis of patients with advanced melanoma.,Identifying biomarkers that predict overall survival (OS) and response to immunotherapy is important.,OS and best overall response according to RECIST version 1.1 were analysed, and S100B and lactate dehydrogenase (LDH) serum levels were assessed retrospectively in 152 patients treated with anti-PD-1, and in 86 patients treated with anti-PD-1 plus anti-CTLA-4 antibodies at University Hospital Tuebingen, Germany.,In the pembrolizumab group, patients with elevated baseline S100B or LDH exhibited significantly impaired OS compared with patients with normal S100B (1-year OS: 51.1% vs 83.1%, log-rank P < .0001) and normal LDH (1-year OS: 44.4% vs 80.8%, P = .00022), respectively.,LDH increases of >25% and S100B increases of >145% compared to baseline were significantly associated with impaired OS (both P < .0001).,In patients treated with ipilimumab and nivolumab, baseline S100B and increasing S100B levels of >145% as well as baseline LDH were associated with impaired OS (P < .0001, P = .00060, and P = .0050, respectively), whereas increasing LDH of >25% was not (P = .64).,S100B could serve as a strong baseline marker for OS in melanoma patients receiving anti-PD-1 therapy.,Rising S100B levels during the first weeks of therapy could help guide treatment decisions.

In vitro studies and mathematical models are now being widely used to study the underlying mechanisms driving the expansion of cell colonies.,This can improve our understanding of cancer formation and progression.,Although much progress has been made in terms of developing and analysing mathematical models, far less progress has been made in terms of understanding how to estimate model parameters using experimental in vitro image-based data.,To address this issue, a new approximate Bayesian computation (ABC) algorithm is proposed to estimate key parameters governing the expansion of melanoma cell (MM127) colonies, including cell diffusivity, D, cell proliferation rate, λ, and cell-to-cell adhesion, q, in two experimental scenarios, namely with and without a chemical treatment to suppress cell proliferation.,Even when little prior biological knowledge about the parameters is assumed, all parameters are precisely inferred with a small posterior coefficient of variation, approximately 2-12%.,The ABC analyses reveal that the posterior distributions of D and q depend on the experimental elapsed time, whereas the posterior distribution of λ does not.,The posterior mean values of D and q are in the ranges 226-268 µm2h−1, 311-351 µm2h−1 and 0.23-0.39, 0.32-0.61 for the experimental periods of 0-24 h and 24-48 h, respectively.,Furthermore, we found that the posterior distribution of q also depends on the initial cell density, whereas the posterior distributions of D and λ do not.,The ABC approach also enables information from the two experiments to be combined, resulting in greater precision for all estimates of D and λ.

The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity.,Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties.,Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells.,We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium.,Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers.,They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts.,Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells.,In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities.,In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts.,Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity.,The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures.,While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells.,Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

Animal models serve as powerful tools for investigating the pathobiology of cancer, identifying relevant pathways, and developing novel therapeutic agents.,They have facilitated rapid scientific progress in many tumor entities.,However, for establishing a powerful animal model of uveal melanoma fundamental challenges remain.,To date, no animal model offers specific genetic attributes as well as histologic, immunologic, and metastatic features of uveal melanoma.,Syngeneic models with intraocular injection of cutaneous melanoma cells may suit best for investigating immunologic/tumor biology aspects.,However, differences between cutaneous and uveal melanoma regarding genetics and metastasis remain problematic.,Human xenograft models are widely used for evaluating novel therapeutics but require immunosuppression to allow tumor growth.,New approaches aim to establish transgenic mouse models of spontaneous uveal melanoma which recently provided preliminary promising results.,Each model provides certain benefits and may render them suitable for answering a respective scientific question.,However, all existing models also exhibit relevant limitations which may have led to delayed research progress.,Despite refined therapeutic options for the primary ocular tumor, patients' prognosis has not improved since the 1970s.,Basic research needs to further focus on a refinement of a potent animal model which mimics uveal melanoma specific mechanisms of progression and metastasis.,This review will summarise and interpret existing animal models of uveal melanoma including recent advances in the field.

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.

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.

Cutaneous T-cell lymphomas (CTCLs) are non-Hodgkin’s lymphomas resulting from clonal expansion and localization of malignant T-lymphocytes to the skin.,CTCL cells have defective apoptosis.,Signal transducers and activators of transcription (STAT) are a family of transcription factors known to play important roles in the development and progression of several human cancers by promoting cell proliferation and protecting against apoptosis.,In this study, we investigated the specific role of STAT3, a major component of the STAT family, in growth and survival of human CTCL cell line Hut78.,Western immunoblot analysis showed elevated expression of STAT3 and phospho-STAT3(Y705) in human CTCL cells as compared to freshly isolated peripheral blood lymphocytes (PBLs).,Specific knockdown of STAT3 expression in Hut78 cells by RNA interference induced morphological and biochemical changes indicating apoptotic cell death.,Moreover, STAT3 inhibition downregulated the expression of Bcl2 family of anti-apoptotic gene Bcl-xL.,These observations suggest that STAT3 is required for the survival of CTCL cells and strongly indicate that targeting STAT3 using siRNA techniques may serve a novel therapeutic strategy for the treatment of CTCL.

Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion.,In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined.,The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells.,Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells.,Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells.,In particular, cell adhesion of GD2+ cells was markedly strong.,As for cell migration velocity, GD2+ cells were slower than all other cells.,The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia.,Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells.,While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion.,On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites.,These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.

In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids).,Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor.,On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients.,Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients.,Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-3H]sphingosine and by FACS.,N-glycolyl GM3 was identified employing the 14 F7 antibody.,Gene expression was assayed by Real Time PCR.,Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [3H] thymidine incorporation.,Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis.,Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3.,Moreover, our data demonstrated that: a) a correlation could be traced between patients’ survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients’ survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins.,Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness.,Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool.,The online version of this article (doi:10.1186/1471-2407-14-560) contains supplementary material, which is available to authorized users.

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.

Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423-7).,Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.,We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen.,We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAFV600E mutation, as well as by cloning and sequencing of separated alleles.,Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients.,Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells.,Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas.,Selection of BRAF mutant alleles during progression was demonstrated in all the three patients.,Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.

Worldwide, metastatic melanoma of the skin has an aggressive course with high morbidity and mortality.,Therefore, an increased understanding of the pathogenesis of metastatic melanoma has gained increasing attention, including the role of epigenetic modification and competing endogenous RNA (ceRNA).,This study aimed to used bioinformatics data to undertake an integrative analysis of long noncoding RNA (lncRNA), microRNA (miRNA) and mRNA expression to construct a ceRNA network in metastatic melanoma.,Data from the Cancer Genome Atlas (TCGA), the Gene Ontology (GO) database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed.,There were 471 cases that included 103 primary solid tumors and 368 cases of metastatic melanoma that included transcriptome sequencing data (including lncRNA and mRNA); 452 cases had miRNA sequencing data.,Analysis of chip data identified 85 6 mRNAs, 67 miRNAs, and 250 lncRNAs that were differentially expressed in cases of metastatic melanoma, of which 25 miRNAs, 18 lncRNAs, and 18 mRNAs participated in the formation of ceRNAs.,Survival analysis identified seven differentially expressed mRNAs, five differentially expressed miRNAs (miRNA-29c, miRNA-100, miR-142-3p, miR-150, miR-516a-2), and six differentially expressed lncRNAs (AC068594.1, C7orf71, FAM41C, GPC5-AS1, MUC19, LINC00402) that were correlated with survival time in patients with metastatic melanoma.,Bioinformatics data and integrative analysis identified lncRNA, miRNA, and mRNA expression to construct a ceRNA and patient survival network in metastatic melanoma.,These findings support the need for further studies on the mechanisms involved in the regulation of metastatic melanoma by ceRNAs.

The abnormal expression of long noncoding RNA- (lncRNA-) MEG3 was clearly identified in a number of malignant tumors, but the specific function of MEG3 remains unknown in malignant melanoma until now.,The research attempts to explore the effects of MEG3 on the growth and metastasis of malignant melanoma.,MEG3 and miR-499-5p expression were determined by qRT-PCR method.,Western blotting assay was applied to detect protein expression.,Luciferase reporter assay was used to assess the correlation between MEG3 and miR-499-5p and between CYLD and miR-499-5p.,Cell growth, cell cycle, and cell apoptosis were examined by CCK-8 assay, EdU assay, and flow cytometry assay, respectively.,The invasion ability of melanoma cells was investigated by wound-healing and Transwell assays.,The effect of MEG3 on growth of melanoma in vivo and cell chemosensitivity was detected by xenograft animal model and CCK-8 assay.,As a result, the expression of MEG3 was decreased in melanoma tissues and cell lines.,The level of MEG3 was significantly associated with poor prognosis.,MEG3 could bind to miR-499-5p and CYLD mRNA contained a binding site of miR-499-5p.,The expression of CYLD was reduced and the level of miR-499-5p was elevated in melanoma tissues and cell lines.,Luciferase reporter assay and western blot assay confirmed that MEG3 regulated the expression of CYLD by sponging miR-499-5p.,Functionally, upregulation of MEG3 inhibited melanoma cell proliferation, invasion, and migration, enhanced melanoma cell apoptosis, arrested melanoma cell cycle, and regulated the expression of E-cadherin, N-cadherin, and cyclin D1 by regulating CYLD expression mediated by sponging miR-499-5p.,Importantly, overexpression of MEG3 suppressed the growth of xenograft tumor and improved chemotherapy sensitivity of A375 cells to cisplatin and 5-FU treatment.,In conclusion, MEG3 has a crucial function in the tumorigenesis of melanoma, and MEG3 may be a potential therapeutic target in the treatment of melanoma.

Immunotherapy with checkpoint inhibitors has greatly prolonged the overall survival of cancer patients in melanoma and many other cancer types.,However, only a subset of patients shows clinical responses from these interventions, which was predicated by the T cell-inflamed tumor microenvironment.,T cell-inflamed phenotype is characterized by the infiltration of CD8+ T cells, CD8α/CD103-lineage dendritic cells (DCs), as well as high density of forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) that are associated with the efficacy of immune checkpoint blockade.,A number of regulators has been associated with T cell-inflammation in the tumor microenvironment, and WNT/β-catenin signaling is one of the best characterized.,The tumor-intrinsic WNT/β-catenin signaling activation is frequently associated with poor spontaneous T cell infiltration across most human cancers.,In this article, we review the essential roles of WNT/β-catenin signaling in the T cell-inflamed and non-T cell-inflamed tumor microenvironment, including the development and function of immune cells, activation of immune exclusion of tumor cells, and cancer immunosurveillance.,We also discuss the impact of this pathway in driving the non-T cell-inflamed tumor microenvironment in other tumor types.,To improve immunotherapy efficacy, we argue that targeting Wnt/β-catenin signaling should be a high priority for combinational cancer therapy to restore T cell infiltration.

A deepened understanding of the cellular and molecular processes in the tumor microenvironment is necessary for the development of precision immunotherapy (IT).,We simultaneously investigated CD3, PDL1, and IDO by immunohistochemistry in paired biopsies from various organs of 43 metastatic melanoma patients treated with IT and targeted therapy (TT).,Intraindividual biopsies taken after a period of weeks to months demonstrate discordant results in 30% of the cases.,Overlap of IDO and PDL1 increased after therapy.,IT only marginally impacted PDL1 expression over time in contrast to TT.,Standardized repeated assessments of multiple immune markers in repeated biopsies will generate detailed insights in melanoma's immune evolution and adaption during therapies and might be used to support treatment decisions.

To provide pooled longer term data from three groups of a phase 2 study of cemiplimab in patients with advanced cutaneous squamous cell carcinoma (CSCC), and to determine duration of response (DOR) and impact on quality of life (QoL).,Patients received cemiplimab 3 mg/kg every 2 weeks (group 1, metastatic CSCC [mCSCC], n=59; group 2, locally advanced CSCC, n=78) or cemiplimab 350 mg every 3 weeks (group 3, mCSCC, n=56).,Primary endpoint was objective response rate (ORR) per independent central review (ICR).,QoL was repeatedly measured at day 1 of each treatment cycle (groups 1 and 2: 8 weeks; group 3: 9 weeks).,Median duration of follow-up was 15.7 months.,Overall, ORR per ICR was 46.1% (95% CI: 38.9% to 53.4%).,Complete response (CR) rates were 20.3%, 12.8%, and 16.1% for groups 1, 2, and 3, respectively.,Median time to CR was 11.2 months.,Among patients with partial response or CR, the estimated proportion of patients with ongoing response at 12 months from the first objective response was 87.8% (95% CI: 78.5% to 93.3%), with median DOR not reached.,Kaplan-Meier estimated probability of overall survival (OS) was 73.3% (95% CI: 66.1% to 79.2%) at 24 months, with median OS not reached.,Global Health Status (GHS)/QoL improvements were observed as early as cycle 2 and were significantly improved and durable until last assessment.,Kaplan-Meier estimate of median time to first clinically meaningful improvement for pain was 2.1 (95% CI: 2.0 to 3.7) months and was significantly improved in responders versus non-responders (p<0.0001).,This is the largest (n=193) clinical dataset for a programmed cell death-1 inhibitor against advanced CSCC, confirming the sustained substantial clinical activity of cemiplimab in these patients, including new findings of improved CR rates over time, increasing DOR, and durable pain control and GHS/QoL improvement.,ClinicalTrials.gov Registry (NCT02760498), https://clinicaltrialsgov/ct2/show/NCT02760498.

Merkel cell carcinoma (MCC) is a rare but clinically aggressive cancer with a high mortality rate.,In recent years, antibodies blocking the interactions among PD-1 and its ligands have generated durable tumor regressions in patients with advanced MCC.,However, there is a paucity of data regarding effective therapy for patients whose disease is refractory to PD-1 pathway blockade.,This retrospective case series describes a heterogeneous group of patients treated with additional immune checkpoint blocking therapy after MCC progression through anti-PD-1.,Among 13 patients treated with anti-CTLA-4, alone or in combination with anti-PD-1, objective responses were seen in 4 (31%).,Additionally, one patient with MCC refractory to anti-PD-1 and anti-CTLA-4 experienced tumor regression with anti-PD-L1.,Our report - the largest case series to date describing this patient population - provides evidence that sequentially-administered salvage immune checkpoint blocking therapy can potentially activate anti-tumor immunity in patients with advanced anti-PD-1-refractory MCC and provides a strong rationale for formally testing these agents in multicenter clinical trials.,Additionally, to the best of our knowledge, our report is the first to demonstrate possible anti-tumor activity of second-line treatment with a PD-L1 antibody in a patient with anti-PD-1-refractory disease.,The online version of this article (10.1186/s40425-019-0661-6) contains supplementary material, which is available to authorized users.

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer1,2.,Here we show that targeted therapy with BRAF, ALK, or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed melanoma and lung adenocarcinoma cells.,This therapy-induced secretome (TIS) stimulates the outgrowth, dissemination, and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression.,The vemurafenib reactive secretome in melanoma is driven by down-regulation of the transcription factor FRA1.,In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of multiple signalling pathways, most prominently the AKT pathway.,Dual inhibition of RAF and PI3K/AKT/mTOR pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant melanoma tumours, suggesting this combination therapy as a strategy against tumour relapse.,Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

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

Analysis of melanomas for actionable mutations has become the standard of care.,Recently, a classification scheme has been proposed that categorizes BRAF mutations based on their mechanisms for activation of the MAPK pathway.,In this analysis BRAF, KIT, NRAS, and PIK3CA mutations were examined by next generation sequencing (NGS) in 446 melanomas in a clinical diagnostic setting.,KRAS and HRAS were also analyzed to elucidate coexisting BRAF and RAS mutations.,BRAF mutations were categorized into class-1 (kinase-activated, codon 600), class-2 (kinase-activated, non-codon 600) and class-3 (kinase-impaired), based on the newly proposed classification scheme.,NGS demonstrated high analytic sensitivity.,Among 355 mutations detected, variant allele frequencies were 2-5% in 21 (5.9%) mutations and 2-10% in 47 (13%) mutations.,Mutations were detected in BRAF (42%), NRAS (25%), KIT (4.9%) and PIK3CA (2.7%).,The incidence of class-1, class-2 and class-3 mutations were 33% (26% p.V600E and 6.1% p.V600K), 3.1 and 4.9% respectively.,With a broader reportable range of NGS, class-1, class-2 and class-3 mutations accounted for 77, 7.4 and 12% of all BRAF mutations.,Class-3 mutations, commonly affecting codons 594, 466 and 467, showed a higher incidence of coexisting RAS mutations, consistent with their RAS-dependent signaling.,Significant association with old age and primary tumors of head/neck/upper back suggest chronic solar damage as a contributing factor for melanomas harboring BRAF p.V600K or class-3 mutations.,This study categorizes the range, frequency, coexisting driver mutations and clinical characteristics of the three classes of BRAF mutations in a large cohort of melanomas in a clinical diagnostic setting.,Further prospective studies are warranted to elucidate the clinical outcomes and benefits of newly developed targeted therapy in melanoma patients carrying each class of BRAF mutation.,The online version of this article (10.1186/s12885-019-5864-1) contains supplementary material, which is available to authorized users.

BRAF mutations are present in 40 % of human skin melanomas.,Mutated tumors with an increased percentage of BRAF mutant alleles (BRAF-M%) may have a better response to RAF/MEK inhibitors.,We evaluated the BRAF-M% in melanomas, and the genetic causes of its variation.,BRAF-M% was quantified by pyrosequencing, real-time PCR (rtPCR) and/or picoliter-droplet PCR (dPCR).,BRAF mutant expression was detected by immunohistochemistry.,Chromosomal alterations were analyzed with fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) arrays.,BRAF-M% quantification obtained with pyrosequencing was highly correlated (R = 0.94) with rtPCR, and with dPCR.,BRAF-M% quantified from DNA and RNA were also highly correlated (R = 0.98).,Among 368 samples with >80 % tumor cells, 38.6 % had a BRAFV600E mutation.,Only 66.2 % cases were heterozygous (BRAF-M% 30 to 60 %).,Increased BRAF-M% (>60 %) was observed in 19 % of cases.,FISH showed a polysomy of chromosome 7 in 13.6 %, 35.3 % and 54.5 % of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005).,Amplification (5.6 %) and loss (3.2 %) of BRAF locus were rare.,By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi.,BRAF-M% is heterogeneous and frequently increased in BRAF-mutant melanomas.,Aneuploidy of chromosome 7 is more frequent in BRAF mutant melanomas, specifically in those with high BRAF-M%.,The online version of this article (doi:10.1186/s12885-015-1515-3) contains supplementary material, which is available to authorized users.

Despite the increasing number of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance.,While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug.,Here we track the responses of thousands of single melanoma cells to BRAF inhibitors and show that a subset of cells escapes drug via non-genetic mechanisms within the first three days of treatment.,Cells that escape drug rely on ATF4 stress signalling to cycle periodically in drug, experience DNA replication defects leading to DNA damage, and yet out-proliferate other cells over extended treatment.,Together, our work reveals just how rapidly melanoma cells can adapt to drug treatment, generating a mutagenesis-prone subpopulation that expands over time.,BRAF inhibitors are used to treat late-stage melanoma patients harbouring BRAF mutations.,Here the authors track the responses of single melanoma cells to BRAF inhibitors and show that a subset of cells rapidly escapes drug via non-genetic mechanisms and incurs DNA damage.

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.

Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches.,Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways.,Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention.,According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease.,Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development.,Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents.,The source code of the various versions of the model are available as S1 Archive.

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

This study aimed to investigate the prognostic value of HRAS mRNA expression in cutaneous melanoma.,Cutaneous melanoma is an aggressive cancer with an increasing incidence.,Few studies have focused on the transcriptional level of RAS isoforms (KRAS, NRAS, and HRAS) in cutaneous melanoma.,To gain further insight into RAS isoforms at transcriptional level, we obtained the cutaneous melanoma data from cBioPortal and investigated the RAS mRNA expression levels in different stages of melanoma and evaluated their correlation with clinical characteristics and patients' survival.,Furthermore, we retrieved and analyzed the coexpression data and performed pathway enrichment analysis.,Totally, 452 cutaneous melanoma cases were included in this study.,We found that lower HRAS expression level was associated with longer patient survival. 206 genes that negatively correlated with HRAS expression were positively correlated with KRAS and NRAS expression.,In contrast, no gene that positively correlated with HRAS expression was positively correlated with KRAS and NRAS expression.,In conclusion, our data showed that transcriptional regulation was different for the three RAS isoforms in cutaneous melanoma.,This study highlighted the prognostic value of HRAS mRNA expression and revealed that HRAS greatly differs from KRAS and NRAS at the transcriptional level.

PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome.,Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi).,However, the function and regulation of expression of PD-L1 remain incompletely understood.,After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression.,These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement.,Consistently, in vitro they showed significantly increased invasive properties.,This phenotype was controlled in part by PD-L1, as determined after silencing the molecule.,Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA.,Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays.,In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis.,Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi.,Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions.,In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells.,We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.

Malignant eccrine porocarcinoma is a rare skin adnexal cancer arising from the sweat glands.,Little is known about the epidemiology and incidence of eccrine porocarcinoma.,This registry-based study examined the epidemiology and incidence data for eccrine porocarcinoma from the Finnish Cancer Registry.,The study included all persons diagnosed with eccrine porocarcinoma in 2007 to 2017.,There were 69 cases in the study period; 34 (49%) male and 35 (51%) female patients.,Mean age at diagnosis was 75.5 years.,Incidence for men was 0.06 per 100,000 person-years and for women 0.04 per 100,000 person-years adjusted for age according to the World Standard Population.,Incidence increased with age.,There was one eccrine porocarcinoma-specific death among the 69 patients.,The incidence of eccrine porocarcinoma in Finland is therefore low.,The mean age at time of diagnosis and the location of eccrine porocarcinoma are consistent with previous reports.,The survival of patients with eccrine porocarcinoma is high.

Recent breakthroughs in tumor immunotherapy such as immune checkpoint blockade (ICB) antibodies, have demonstrated the capacity of the immune system to fight cancer in a number of malignancies such as melanoma and lung cancer.,The numbers, localization and phenotypes of tumor-infiltrating lymphocytes (TIL) are not only predictive of response to immunotherapy but also key modulators of disease progression.,In this review, we focus on TIL profiling in cutaneous melanoma using histopathological approaches and highlight the observed prognostic value of the primary TIL subsets.,The quantification of TIL in formalin-fixed tumor samples ranges from visual scoring of lymphocytic infiltrates in H&E to multiplex immunohistochemistry and immunofluorescence followed by enumeration using image analysis software.,Nevertheless, TIL enumeration in the current literature primarily relies upon single marker immunohistochemistry analyses of major lymphocyte subsets such as conventional T cells (CD3, CD4, CD8), regulatory T cells (FOXP3) and B cells (CD20).,We review key studies in the literature on associations between TIL subsets and patient survival.,We also cover recent findings with respect to the existence of ectopic lymphoid aggregates found in the TME which are termed tertiary lymphoid structures (TLS) and are generally a positive prognostic feature.,In addition to their prognostic significance, the existence of various TIL sub-populations has also been reported to predict a patient’s response to ICB.,Thus, the literature on the predictive potential of TIL subsets in melanoma patients receiving ICB has also been discussed.,Finally, we describe recently developed state-of-the-art profiling approaches for tumor infiltrating immune cells such as digital pathology scoring algorithms (e.g., Immunoscore) and multiplex proteomics-based immunophenotyping platforms (e.g., imaging mass cytometry).,Translating these novel technologies have the potential to revolutionize tumor immunopathology leading to altering our current understanding of cancer immunology and dramatically improving outcomes for patients.

Cutaneous melanoma can metastasise haematogenously and/or lymphogenously to form satellite/in-transit, lymph node or distant metastasis.,This study aimed to determine if BRAF and NRAS mutant and wild-type tumours differ in their site of first tumour metastasis and anatomical metastatic pathway.,Prospective cohort of patients with a histologically confirmed primary cutaneous melanoma at three tertiary referral centres in Melbourne, Australia from 2010 to 2015.,Multinomial regression determined clinical, histological and mutational factors associated with the site of first metastasis and metastatic pathway.,Of 1048 patients, 306 (29%) developed metastasis over a median 4.7 year follow-up period. 73 (24%), 192 (63%) and 41 (13%) developed distant, regional lymph node and satellite/in-transit metastasis as the first site of metastasis, respectively.,BRAF mutation was associated with lymph node metastasis (adjusted RRR 2.46 95% CI 1.07-5.69, P=0.04) and sentinel lymph node positivity (adjusted odds ratio [aOR] OR 1.55, 95% CI 1.14-2.10, P=0.005).,BRAF mutation and NRAS mutation were associated with increased odds of developing liver metastasis (aOR 3.09, 95% CI 1.49-6.42, P=0.003; aOR 3.17, 95% CI 1.32-7.58, P=0.01) and central nervous system (CNS) metastasis (aOR 4.65, 95% CI 2.23-9.69, P<0.001; aOR 4.03, 95% CI 1.72-9.44, P=0.001).,NRAS mutation was associated with lung metastasis (aOR 2.44, 95% CI 1.21-4.93, P=0.01).,BRAF mutation was found to be associated with lymph node metastasis as first metastasis and sentinel lymph node positivity.,BRAF and NRAS mutations were associated with CNS and liver metastasis and NRAS mutation with lung metastasis.,If these findings are validated in additional prospective studies, a role for heightened visceral organ surveillance may be warranted in patients with tumours harbouring these somatic mutations.

Since patients diagnosed with BRAF V600E and V600K mutated advanced melanoma show response to treatment with MAP kinase inhibitors, several sensitive methods have been developed to determine the V600 allele status of melanoma patients.,Vemurafenib (Zelboraf) and dabrafenib (Tafinlar) are specific BRAF V600 inhibitors recently approved by the US FDA as single agent treatments for unresectable or metastatic melanoma in patients with the BRAF V600 mutation.,We assessed the new CE THxID™-BRAF diagnostic test, which is also FDA-approved as a companion diagnostic test in the US under a specific reference and compared the results of this assay with both High Resolution Melting (HRM) and Sanger sequencing in 113 melanoma FFPE samples.,Invalid results were observed in 0/113 specimen with HRM, 5/113 (4.4%) with Sanger sequencing, and 1/113 (0.9%) with the THxID™-BRAF test.,Positive percentage agreement (PPA) was 93.5% (95% CI 82.5 - 97.8) for V600E and V600K mutations combined for the THxID™-BRAF test and HRM, and negative percentage agreement (NPA) was 100.0% (95% CI 94.5 - 100.0).,For the THxID™-BRAF test and Sanger, PPA was 100.0% (95% CI 92.1 - 100.0) and NPA 100.0% (95% CI 94.2 - 100.0).,One V600E sample identified by THxID™-BRAF test was detected as wild-type by HRM and uninterpretable by Sanger.,All V600K (n = 3) were detected using the 3 different approaches.,Finally, percent agreement values were not significantly different when using punches (n = 77) vs. slides (n = 36) or depending on samples characteristics such as pigmentation, necrosis, and tumor content.,This study demonstrated the high agreement between the FDA approved THxID™-BRAF assay, HRM, and Sanger sequencing.,It has also highlighted the potential of THxID™-BRAF to be applied to a broader range of sample types than claimed in the current “instructions for use”, an extension that would require the ad hoc validation and approval.

Cancer immunotherapy restores and/or enhances effector function of CD8+ T cells in the tumor microenvironment1,2.,CD8+ T cells activated by cancer immunotherapy execute tumor clearance mainly by inducing cell death through perforin-granzyme- and Fas/Fas ligand-pathways3,4.,Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent lipid peroxide accumulation5,6.,Although it was mechanistically illuminated in vitro7,8, emerging evidence has shown that ferroptosis may be implicated in a variety of pathological scenarios9,10.,However, the involvement of ferroptosis in T cell immunity and cancer immunotherapy is unknown.,Here, we find that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumor cells, and in turn, increased ferroptosis contributes to the anti-tumor efficacy of immunotherapy.,Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system xc-, restrains tumor cell cystine uptake, and as a consequence, promotes tumor cell lipid peroxidation and ferroptosis.,In preclinical models, depletion of cyst(e)ine by cyst(e)inase in combination with checkpoint blockade synergistically enhances T cell-mediated anti-tumor immunity and induces tumor cell ferroptosis.,Expression of system xc- is negatively associated with CD8+ T cell signature, IFNγ expression, and cancer patient outcome.,Transcriptome analyses before and during nivolumab therapy reveal that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8.,Thus, T cell-promoted tumor ferroptosis is a novel anti-tumor mechanism.,Targeting tumor ferroptosis pathway constitutes a therapeutic approach in combination with checkpoint blockade.

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

The efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration.,Oncogenic alterations of tumor cells have been associated to T cell exclusion.,Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy.,The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies.,We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy.,We evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses.,Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8+, CD4+ T cells number and their activation state (PD-1, Ki67).,ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses.,Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes.,Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated.,Combined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8+ T cell infiltration, independently of β-catenin pathway activation.,ZEB1 ectopic expression in melanoma cells impairs CD8+ T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade.,Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10.,Finally, Zeb1 knock-out, by promoting CD8+ T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression.,We identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma.,NCT02828202.

Developmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion.,This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic.,SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth.,In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1).,Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models.,Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth.,Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells.,Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways.,These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma.

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

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

In early 2011, we reviewed the initial success of the RAF inhibitor, vemurafenib, in mutant V600 BRAF melanoma patients.,It was soon evident that the response to RAF inhibitor is heterogeneous and that the short-term benefits are burdened by the development of resistance.,The field has progressed rapidly with the FDA-approval of vemurafenib and the development of other RAF and MEK inhibitors.,Despite these advances, the issue of RAF inhibitor resistance remains.,Here, we review recent clinical advances in the field, the growing number of resistance mechanisms, preclinical evidence for combinatorial trials using RAF inhibitors as the building blocks, and the new challenges that are arising.

The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive.,Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways.,Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression.,The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns.,Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced.,Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here.,Characteristically, production of ROS was seen early in response to wortmannin and MK-2206.,Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation.,This correlated with reduced Bax phosphorylation at threonine-167.,The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation.,The pathway may be considered for proapoptotic, anticancer strategies.

Recent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments.,In this study, silymarin nanoemulsion (SN) was used along with air CAP as therapeutic agent to counter human melanoma.,In this study, we examined the combined treatment of CAP and SN on G-361 human melanoma cells by evaluating cellular toxicity levels, reactive oxygen and nitrogen species (RONS) levels, DNA damage, melanoma-specific markers, apoptosis, caspases and poly ADP-ribose polymerase-1 (PARP-1) levels using flow cytometer.,Dual-treatment effects on the epithelial-mesenchymal transition (EMT), Hepatocyte growth factor (HGF/c-MET) pathway, sphere formation and the reversal of EMT were also assessed using western blotting and microscopy respectively.,SN and plasma-activated medium (PAM) were applied on tumor growth and body weight and melanoma-specific markers and the mesenchymal markers in the tumor xenograft nude mice model were checked.,Co-treatment of SN and air CAP increased the cellular toxicity in a time-dependent manner and shows maximum toxicity at 200 nM in 24 h.,Intracellular RONS showed significant generation of ROS (< 3 times) and RNS (< 2.5 times) in dual-treated samples compared to control.,DNA damage studies were assessed by estimating the level of γ-H2AX (1.8 times), PD-1 (> 2 times) and DNMT and showed damage in G-361 cells.,Increase in Caspase 8,9,3/7 (> 1.5 times), PARP level (2.5 times) and apoptotic genes level were also observed in dual treated group and hence blocking HGF/c-MET pathway.,Decrease in EMT markers (E-cadherin, YKL-40, N-cadherin, SNAI1) were seen with simultaneously decline in melanoma cells (BRAF, NAMPT) and stem cells (CD133, ABCB5) markers.,In vivo results showed significant reduction in SN with PAM with reduction in tumor weight and size.,The use of air CAP using μ-DBD and the SN can minimize the malignancy effects of melanoma cells by describing HGF/c-MET molecular mechanism of acting on G-361 human melanoma cells and in mice xenografts, possibly leading to suitable targets for innovative anti-melanoma approaches in the future.,The online version of this article (10.1186/s12964-019-0360-4) contains supplementary material, which is available to authorized users.

Cold atmospheric plasma (CAP) is a promising approach in anti-cancer therapy, eliminating cancer cells with high selectivity.,However, the molecular mechanisms of CAP action are poorly understood.,In this study, we investigated CAP effects on calcium homeostasis in melanoma cells.,We observed increased cytoplasmic calcium after CAP treatment, which also occurred in the absence of extracellular calcium, indicating the majority of the calcium increase originates from intracellular stores.,Application of previously CAP-exposed extracellular solutions also induced cytoplasmic calcium elevations.,A substantial fraction of this effect remained when the application was delayed for one hour, indicating the chemical stability of the activating agent(s).,Addition of ryanodine and cyclosporin A indicate the involvement of the endoplasmatic reticulum and the mitochondria.,Inhibition of the cytoplasmic calcium elevation by the intracellular chelator BAPTA blocked CAP-induced senescence.,This finding helps to understand the molecular influence and the mode of action of CAP on tumor cells.

To improve immunotherapy efficacy for melanoma, a coexpression network and key genes of M2 macrophages in melanoma were explored.,A prognostic risk assessment model was established for M2-related coexpressed genes, and the role of M2 macrophages in the immune microenvironment of melanoma was elucidated.,We obtained mRNA data from melanoma and peritumor tissue samples from The Cancer Genome Atlas-skin cutaneous melanoma (TCGA-SKCM).,Then, we used CIBERSORT to calculate the proportion of M2 macrophage cells.,A coexpression module most related to M2 macrophages in TCGA-SKCM was determined by analyzing the weighted gene coexpression network, and a coexpression network was established.,After survival analysis, factors with significant results were incorporated into a Cox regression analysis to establish a model.,The model's essential genes were analyzed using functional enrichment, GSEA, and subgroup and total carcinoma.,Finally, external datasets GSE65904 and GSE78220 were used to verify the prognostic risk model.,The yellow-green module was the coexpression module most related to M2 macrophages in TCGA-SKCM; NOTCH3, DBN1, KDELC2, and STAB1 were identified as the essential genes that promoted the infiltration of M2 macrophages in melanoma.,These genes are concentrated in antigen treatment and presentation, chemokine, cytokine, the T cell receptor pathway, and the IFN-γ pathway.,These factors were analyzed for survival, and factors with significant results were included in a Cox regression analysis.,According to the methods, a model related to M2-TAM coexpressed gene was established, and the formula was risk score = 0.25∗NOTCH3 + 0.008∗ DBN1 − 0.031∗KDELC2 − 0.032∗STAB1.,The new model was used to perform subgroup evaluation and external queue validation.,The results showed good prognostic ability.,We proposed a Cox proportional hazards regression model associated with coexpression genes of melanoma M2 macrophages that may provide a measurement method for generating prognosis scores in patients with melanoma.,Four genes coexpressed with M2 macrophages were associated with high levels of infiltration of M2 macrophages.,Our findings may provide significant candidate biomarkers for the treatment and monitoring of melanoma.

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

Resisting cell death is a hallmark of cancer.,Disturbances in the execution of cell death programs promote carcinogenesis and survival of cancer cells under unfavorable conditions, including exposition to anti-cancer therapies.,Specific modalities of regulated cell death (RCD) have been classified based on different criteria, including morphological features, biochemical alterations and immunological consequences.,Although melanoma cells are broadly equipped with the anti-apoptotic machinery and recurrent genetic alterations in the components of the RAS/RAF/MEK/ERK signaling markedly contribute to the pro-survival phenotype of melanoma, the roles of autophagy-dependent cell death, necroptosis, ferroptosis, pyroptosis, and parthanatos have recently gained great interest.,These signaling cascades are involved in melanoma cell response and resistance to the therapeutics used in the clinic, including inhibitors of BRAFmut and MEK1/2, and immunotherapy.,In addition, the relationships between sensitivity to non-apoptotic cell death routes and specific cell phenotypes have been demonstrated, suggesting that plasticity of melanoma cells can be exploited to modulate response of these cells to different cell death stimuli.,In this review, the current knowledge on the non-apoptotic cell death signaling pathways in melanoma cell biology and response to anti-cancer drugs has been discussed.

Nuclear factor-κB (NF-κB) inducing kinase (NIK) is a MAP3K that regulates the activation of NF-κB.,NIK is often highly expressed in tumor cells, including melanoma, but the significance of this in melanoma progression has been unclear.,Tissue microarray analysis of NIK expression reveals that dysplastic nevi (n=22), primary (n=15) and metastatic melanoma (n=13) lesions showed a statistically significant elevation in NIK expression when compared to benign nevi (n=30).,Moreover, when shRNA techniques were used to knock-down NIK, the resultant NIK-depleted melanoma cell lines exhibited decreased proliferation, increased apoptosis, and reduced tumor growth in a mouse xenograft model.,As expected, when NIK was depleted there was decreased activation of the non-canonical NF-κB pathway, while canonical NF-κB activation remained intact.,NIK depletion also resulted in reduced expression of genes that contribute to tumor growth, including CXCR4, c-MYC and c-MET, and pro-survival factors such as BCL2 and survivin.,These changes in gene expression are not fully explained by the attenuation of the non-canonical NF-κB pathway.,Shown here for the first time is the demonstration that NIK modulates β-catenin mediated transcription to promote expression of survivin.,NIK-depleted melanoma cells exhibited down-regulation of survivin as well as other β-catenin regulated genes including c-MYC, c-MET and CCND2.,These data indicate that NIK mediates both β-catenin and NF-κB regulated transcription to modulate melanoma survival and growth.,Thus, NIK may be a promising therapeutic target for melanoma.

Tissue-resident memory CD8+ T (Trm) cells mediate potent local innate and adaptive immune responses and play a central role against solid tumors.,However, whether Trm cells cross-talk with dendritic cells (DCs) to support anti-tumor immunity remains unclear.,Here we show that antigen-specific activation of skin Trm cells leads to maturation and migration to draining lymph nodes of cross-presenting dermal DCs.,Tumor rejection mediated by Trm cells triggers the spread of cytotoxic CD8+ T cell responses against tumor-derived neo- and self-antigens via dermal DCs.,These responses suppress the growth of intradermal tumors and disseminated melanoma lacking the Trm cell-targeted epitope.,Moreover, analysis of RNA sequencing data from human melanoma tumors reveals that enrichment of a Trm cell gene signature associates with DC activation and improved survival.,This work unveils the ability of Trm cells to amplify the breath of cytotoxic CD8+ T cell responses through DCs, thereby strengthening anti-tumor immunity.,Immunotherapy can induce antigen spreading of antitumor T cell response, which correlates with better outcomes.,Here the authors show that tissue-resident memory CD8 T cells promote antigen spreading via lysing tumor cells and promoting their uptake and cross-presentation by dendritic cells, thereby eliciting de novo T cell responses.

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

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.

Up to 50% of patients with uveal melanoma develop metastatic disease with poor prognosis.,Regional, mainly liver-directed, therapies may induce limited tumor responses but do not improve overall survival.,Response rates of metastatic uveal melanoma (MUM) to systemic chemotherapy are poor.,Insights into the molecular biology of MUM recently led to investigation of new drugs.,In this study, to compare response rates of systemic treatment for MUM we searched Pubmed/Web of Knowledge databases and ASCO website (1980-2013) for “metastatic/uveal/melanoma” and “melanoma/eye.”,Forty studies (one case series, three phase I, five pilot, 22 nonrandomized, and two randomized phase II, one randomized phase III study, data of three expanded access programs, three retrospective studies) with 841 evaluable patients were included in the numeric outcome analysis.,Complete or partial remissions were observed in 39/841 patients (overall response rate [ORR] 4.6%; 95% confidence intervals [CI] 3.3-6.3%), no responses were observed in 22/40 studies.,Progression-free survival ranged from 1.8 to 7.2, median overall survival from 5.2 to 19.0 months as reported in 21/40 and 26/40 studies, respectively.,Best responses were seen for chemoimmunotherapy (ORR 10.3%; 95% CI 4.8-18.7%) though mainly in first-line patients.,Immunotherapy with ipilimumab, antiangiogenetic approaches, and kinase inhibitors have not yet proven to be superior to chemotherapy.,MEK inhibitors are currently investigated in a phase II trial with promising preliminary data.,Despite new insights into genetic and molecular background of MUM, satisfying systemic treatment approaches are currently lacking.,Study results of innovative treatment strategies are urgently awaited.,Forty clinical studies on metastatic uveal melanoma were reviewed regarding responses to systemic treatments.,New insights into genetic and molecular background led to investigation of new substances but promising in vitro data have not yet been translated into satisfying treatment responses; however, preliminary results of ongoing studies are highly encouraging.

de novo fatty acid biosynthesis (DNFA) is a hallmark adaptation of many cancers that supports survival, proliferation, and metastasis.,Here we elucidate previously unexplored aspects of transcription regulation and clinical relevance of DNFA in cancers.,We show that elevated expression of DNFA genes is characteristic of many tumor types and correlates with poor prognosis, especially in melanomas.,Elevated DNFA gene expression depends on the SREBP1 transcription factor in multiple melanoma cell lines.,SREBP1 predominantly binds to the transcription start sites of DNFA genes, regulating their expression by recruiting RNA polymerase II to promoters for productive transcription elongation.,We find that SREBP1-regulated DNFA represents a survival trait in melanoma cells, regardless of proliferative state and oncogenic mutation status.,Indeed, malignant melanoma cells exhibit elevated DNFA gene expression after the BRAF/MEK signaling pathway is blocked (e.g. by BRAF inhibitors), and DNFA expression remains higher in melanoma cells resistant to vemurafenib treatment than in untreated cells.,Accordingly, DNFA pathway inhibition, whether by direct targeting of SREBP1 with antisense oligonucleotides, or through combinatorial effects of multiple DNFA enzyme inhibitors, exerts potent cytotoxic effects on both BRAFi-sensitive and -resistant melanoma cells.,Altogether, these results implicate SREBP1 and DNFA enzymes as enticing therapeutic targets in melanomas.

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

Metastatic melanoma is a recalcitrant tumor.,Although “targeted” and immune therapies have been highly touted, only relatively few patients have had durable responses.,To overcome this problem, our laboratory has established the melanoma patient-derived orthotopic xenograft (PDOX) model with the use of surgical orthotopic implantation (SOI).,Promising results have been obtained with regard to identifying effective approved agents and experimental therapeutics, as well as combinations of the two using the melanoma PDOX model.

Betulinic acid, a very promising anti-melanoma agent, has very low water solubility that causes low bioavailability.,To overcome this inconvenience, a highly water-soluble cyclodextrin was used (octakis-[6-deoxy-6-(2-sulfanyl ethanesulfonic acid)]-γ-cyclodextrin).,The complex was physico-chemically analyzed using differential scanning calorimetry (DSC), X-ray and scanning electron microscopy (SEM) methods and then in vitro tested for its antiproliferative activity by the MTT assay and by cell cycle analysis.,Finally, the complex was tested in vivo using an animal model of murine melanoma developed in C57BL/6J mice, where it caused a reduction in tumor volume and weight.,The study revealed the beneficial influence of betulinic acid inclusion into the cyclodextrin in terms of antiproliferative activity and in vivo tumor development.