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

Induction of DNA damage by UVB and UVA radiation may generate mutations and genomic instability leading to carcinogenesis.,Therefore, skin cells being repeatedly exposed to ultraviolet (UV) light have acquired multilayered protective mechanisms to avoid malignant transformation.,Besides extensive DNA repair mechanisms, the damaged skin cells can be eliminated by induction of apoptosis, which is mediated through the action of tumor suppressor p53.,In order to prevent the excessive loss of skin cells and to maintain the skin barrier function, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway.,However, AKT/mTOR not only prevents cell death, but is also active in cell cycle transition and hyper-proliferation, thereby also counteracting p53.,In turn, AKT/mTOR is tuned down by the negative regulators being controlled by the p53.,This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance.,Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence.,Conclusively, AKT/mTOR via an extensive cross talk with p53 influences the UV response in the skin with no black and white scenario deciding over death or survival.

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 major advances in targeted melanoma therapies, drug resistance limits their efficacy.,Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules.,LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored.,In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1.,Collectively, our results present MIRAT’s direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer.

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

Despite the extensive research approaches applied to characterise malignant melanoma, no specific molecular markers are available that are clearly related to the progression of this disease.,In this study, our aims were to define a gene expression signature associated with the clinical outcome of melanoma patients and to provide an integrative interpretation of the gene expression -, copy number alterations -, and promoter methylation patterns that contribute to clinically relevant molecular functional alterations.,Gene expression profiles were determined using the Affymetrix U133 Plus2.0 array.,The NimbleGen Human CGH Whole-Genome Tiling array was used to define CNAs, and the Illumina GoldenGate Methylation platform was applied to characterise the methylation patterns of overlapping genes.,We identified two subclasses of primary melanoma: one representing patients with better prognoses and the other being characteristic of patients with unfavourable outcomes.,We assigned 1,080 genes as being significantly correlated with ulceration, 987 genes were downregulated and significantly enriched in the p53, Nf-kappaB, and WNT/beta-catenin pathways.,Through integrated genome analysis, we defined 150 downregulated genes whose expression correlated with copy number losses in ulcerated samples.,These genes were significantly enriched on chromosome 6q and 10q, which contained a total of 36 genes.,Ten of these genes were downregulated and involved in cell-cell and cell-matrix adhesion or apoptosis.,The expression and methylation patterns of additional genes exhibited an inverse correlation, suggesting that transcriptional silencing of these genes is driven by epigenetic events.,Using an integrative genomic approach, we were able to identify functionally relevant molecular hotspots characterised by copy number losses and promoter hypermethylation in distinct molecular subtypes of melanoma that contribute to specific transcriptomic silencing and might indicate a poor clinical outcome of melanoma.

Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients.,Since most patients do not respond, predictive biomarkers to guide treatment selection are needed.,We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response.,In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous.,A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions.,Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of ‘PD-1 signalling', ‘allograft rejection' and ‘T-cell receptor signalling', among others.,In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4+ and CD8+ tumour infiltrate.,MHC-II+ tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.,Immunotherapy is used to treat melanoma, however patient responses vary widely highlighting the need for factors that can predict therapeutic success.,Here, the authors show that MHC-II molecules expressed by tumour cells are positively correlated with a good response to therapy and overall patient survival.

Cells release multiple, distinct, forms of extracellular vesicles including structures known as microvesicles which are known to alter the extracellular environment.,Despite growing understanding of microvesicle biogenesis, function, and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown.,Here we demonstrate that in amoeboid-like invasive tumor cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles.,MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion.,VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices.,Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients.,Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

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

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.

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.

Activating mutations in BRAF kinase are common in melanomas.,Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation.,However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials.,Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.

Approximately one‐third of Merkel cell carcinoma (MCC) patients eventually develop distant metastatic disease.,Little is known about whether the location of the primary lesion is predictive of initial distant metastatic site, or if survival likelihood differs depending on the metastatic site.,Such data could inform imaging/surveillance practices and improve prognostic accuracy.,Multivariate and competing‐risk analyses were performed on a cohort of 215 MCC patients with distant metastases, 31% of whom had two or more initial sites of distant metastasis.,At time of initial distant metastasis in the 215 patients, metastatic sites (n = 305) included non‐regional lymph nodes (present in 41% of patients), skin/body wall (25%), liver (23%), bone (21%), pancreas (8%), lung (7%), and brain (5%).,Among the 194 patients who presented with MCC limited to local or regional sites (stage I‐III) but who ultimately developed distant metastases, distant progression occurred in 49% by 1 year and in 80% by 2 years following initial diagnosis.,Primary MCC locations differed in how likely they were to metastasize to specific organs/sites (P < .001).,For example, liver metastases were far more likely from a head/neck primary (43% of 58 patients) versus a lower limb primary (5% of 39 patients; P < .0001).,Skin‐only distant metastasis was associated with lower MCC‐specific mortality as compared to metastases in multiple organs/sites (HR 2.7; P = .003), in the liver (HR 2.1; P = .05), or in distant lymph nodes (HR 2.0; P = .045).,These data reflect outcomes before PD1‐pathway inhibitor availability, which may positively impact survival.,In conclusion, primary MCC location is associated with a pattern of distant spread, which may assist in optimizing surveillance.,Because it is linked to survival, the site of initial distant metastasis should be considered when assessing prognosis.

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro.,To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSA sT), allowing Cre-mediated, conditional MCV sT expression.,Standard tamoxifen (TMX) administration to adult Ubc CreERT2; ROSA sT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days.,Conversely, most adult Ubc CreERT2; ROSA sT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis.,Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment.,Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth.,To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1 CreERT2/+; ROSA sT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally.,Tamoxifen administration to adult Atoh1 CreERT2/+; ROSA sT and Atoh1 CreERT2/+; ROSA sT; p53f lox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation.,Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Squamous cell carcinomas (SCC), including cutaneous SCCs, are by far the most frequent cancers in humans, accounting for 80% of all newly diagnosed malignancies worldwide.,The old dogma that SCC develops exclusively from stem cells (SC) has now changed to include progenitors, transit-amplifying and differentiated short-lived cells.,Accumulation of specific oncogenic mutations is required to induce SCC from each cell population.,Whilst as fewer as one genetic hit is sufficient to induce SCC from a SC, multiple events are additionally required in more differentiated cells.,Interestingly, the level of differentiation correlates with the number of transforming events required to induce a stem-like phenotype, a long-lived potential and a tumourigenic capacity in a progenitor, a transient amplifying or even in a terminally differentiated cell.,Furthermore, it is well described that SCCs originating from different cells of origin differ not only in their squamous differentiation status but also in their malignant characteristics.,This review summarises recent findings in cutaneous SCC and highlights transforming oncogenic events in specific cell populations.,It underlines oncogenes that are restricted either to stem or differentiated cells, which could provide therapeutic target selectivity against heterogeneous SCC.,This strategy may be applicable to SCC from different body locations, such as head and neck SCCs, which are currently still associated with poor survival outcomes.

Pro-apoptotic BCL2 associated X (BAX) is traditionally thought to be regulated by anti-apoptotic BCL-2 family members, like BCL2-like 1 (BCL-XL), at the protein level.,However, the posttranscriptional regulation of BAX is under explored.,In this study, we identified BAX as the novel downstream target of miR-365, which is supported by gain- and loss-of-function studies of onco-miR-365.,Loss of BAX by either RNA interference or highly-expressed miR-365 in cells of cutaneous squamous cell carcinoma (CSCC) enhanced the tumor resistance against apoptosis, while repressing cell proliferation, migration, and invasiveness.,In vivo experiment confirmed that BAX knockdown promotes the growth of CSCC xenografts.,Collectively, our results find a miR-365-BAX axis for alleviating the pro-apoptotic effects of BAX, which promotes CSCC development and may facilitate the generation of novel therapeutic regimens to the clinical treatment of CSCC.

Tumors that lack pre-existing immune infiltration respond poorly to T cell checkpoint blockade immunotherapy.,These cancers often surround themselves with high densities of suppressive myeloid stroma while excluding immunostimulatory dendritic cells.,Tumor-resident myeloid cells and selected lymphocyte populations retain expression of Toll-like Receptors (TLR) that sense common features of pathogens and activate innate immunity in response.,We explored whether agonists of TLR9 could augment innate immunity to promote tumor regression alone or in combination with T cell checkpoint blockade.,In the setting of the immunogenic B16-Ova (Ovalbumin) expressing melanoma model, local injection of the CpG oligonucleotide TLR9 agonist ODN1826 combined with systemic CTLA-4 blockade cured 45% of mice of both their treated and an untreated tumor on the opposite flank demonstrating the synergistic potential of this combination.,Next, in the non-immunogenic B16-F10 melanoma model, we showed that only intra-tumoral, but not systemic TLR9 activation augments the therapeutic potential of checkpoint blockade.,In this setting, intra-tumoral TLR9 activation cooperated equally with either CTLA-4 or PD-1 blockade co-administered locally or given systemically; however, the uninjected tumor rarely regressed.,Anti-CTLA-4 combinations were associated with improved intra-tumoral CD8 to regulatory T cell ratios, while anti-PD-1 combinations elicited improved ratios of CD8 T cells relative to suppressive myeloid stroma.,Using both a TLR9 agonist (MGN1703) and a CTLA-4 antibody (9D9-IgG2a) of increased potency cured 50% of bi-lateral B16-F10 melanoma.,These findings suggest that intra-tumoral TLR9 agonists can improve sensitivity of poorly immunogenic tumors to T cell checkpoint blockade, and that newer, higher potency TLR agonists and checkpoint antibodies can raise the therapeutic ceiling for this combination therapy.

Melanoma is among the most aggressive tumors, and the occurrence of metastasis leads to a precipitous drop in the patients' survival.,Therefore, identification of metastasis-associated biomarkers and therapeutic targets will contribute a lot to improving melanoma theranostics.,Recently, microRNAs (miRNAs) have been implicated in modulating cancer invasion and metastasis, and are proved as potential non-invasive biomarkers in sera for various tumors.,Here, we reported miR-23a as a novel metastasis-associated miRNA that played a remarkable role in modulating melanoma invasive and metastatic capacity and was of great value in predicting melanoma metastasis and prognosis.,We found that serum miR-23a level was significantly down-regulated in metastatic melanoma patients and highly correlated with poor clinical outcomes.,In addition, miR-23a level was also remarkably decreased in metastatic melanoma tissues and cell lines.,Furthermore, overexpressed miR-23a suppressed the invasive and migratory property of melanoma cells by abrogating autophagy through directly targeting ATG12.,Specially, miR-23a-ATG12 axis attenuated melanoma invasion and migration through autophagy-mediated AMPK-RhoA pathway.,Finally, the overexpression of miR-23a prevented melanoma metastasis in vivo.,Taken together, our findings demonstrate that the metastasis-associated miR-23a is not only a potential biomarker, but also a valuable therapeutic target for melanoma.

In response to the dynamic intra‐tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia‐associated transcription factor (MITF).,The response to hypoxia is driven by hypoxia‐inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis.,HIF1α indirectly represses MITF that can activate HIF1α expression.,Although HIF and MITF share a highly related DNA‐binding specificity, it is unclear whether they co‐regulate subset of target genes.,Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown.,Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia.,Surprisingly, under hypoxia MITF is transiently up‐regulated by HIF1α and co‐regulates a subset of HIF targets including VEGFA.,Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo‐hypoxia.,Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in 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.

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

Cancer is a disease consisting of both genetic and epigenetic changes.,While increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear.,Here, we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma.,Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs1-4, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples.,Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo.,Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo.,We demonstrate that the tumour promoting function of mH2A loss is mediated, at least in part, through direct transcriptional up-regulation of CDK8.,Suppression of CDK8, a colorectal cancer oncogene5, 6, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss.,Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples.,Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

Cell-to-cell variability generates subpopulations of drug-tolerant cells that diminish the efficacy of cancer drugs.,Efficacious combination therapies are thus needed to block drug-tolerant cells via minimizing the impact of heterogeneity.,Probabilistic models such as Bliss independence have been developed to evaluate drug interactions and their combination efficacy based on probabilities of specific actions mediated by drugs individually and in combination.,In practice, however, these models are often applied to conventional dose-response curves in which a normalized parameter with a value between zero and one, generally referred to as fraction of cells affected (fa), is used to evaluate the efficacy of drugs and their combined interactions.,We use basic probability theory, computer simulations, time-lapse live cell microscopy, and single-cell analysis to show that fa metrics may bias our assessment of drug efficacy and combination effectiveness.,This bias may be corrected when dynamic probabilities of drug-induced phenotypic events, i.e. induction of cell death and inhibition of division, at a single-cell level are used as metrics to assess drug efficacy.,Probabilistic phenotype metrics offer the following three benefits.,First, in contrast to the commonly used fa metrics, they directly represent probabilities of drug action in a cell population.,Therefore, they deconvolve differential degrees of drug effect on tumor cell killing versus inhibition of cell division, which may not be correlated for many drugs.,Second, they increase the sensitivity of short-term drug response assays to cell-to-cell heterogeneities and the presence of drug-tolerant subpopulations.,Third, their probabilistic nature allows them to be used directly in unbiased evaluation of synergistic efficacy in drug combinations using probabilistic models such as Bliss independence.,Altogether, we envision that probabilistic analysis of single-cell phenotypes complements currently available assays via improving our understanding of heterogeneity in drug response, thereby facilitating the discovery of more efficacious combination therapies to block drug-tolerant cells.

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.

Preclinical studies have suggested that epigenetic therapy could enhance immunogenicity of cancer cells.,We report the results of the PEMDAC phase 2 clinical trial (n = 29; NCT02697630) where the HDAC inhibitor entinostat was combined with the PD-1 inhibitor pembrolizumab in patients with metastatic uveal melanoma (UM).,The primary endpoint was objective response rate (ORR), and was met with an ORR of 14%.,The clinical benefit rate at 18 weeks was 28%, median progression free survival was 2.1 months and the median overall survival was 13.4 months.,Toxicities were manageable, and there were no treatment-related deaths.,Objective responses and/or prolonged survival were seen in patients with BAP1 wildtype tumors, and in one patient with an iris melanoma that exhibited a UV signature.,Longer survival also correlated with low baseline ctDNA levels or LDH.,In conclusion, HDAC inhibition and anti-PD1 immunotherapy results in durable responses in a subset of patients with metastatic UM.,Trial registration ClinicalTrials.gov registration number: NCT02697630 (registered 3 March 2016).,EudraCT registration number: 2016-002114-50.,The authors report the results of the phase II PEMDAC clinical study testing the combination of the HDAC inhibitor entinostat with the anti- PD-1 antibody pembrolizumab in uveal melanoma.,Low tumor burden, a wildtype BAP1 gene in the tumor or iris melanoma correlates with response and longer survival.

Melanoma cells rely on developmental programs during tumor initiation and progression.,Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a−/− melanoma model and that its expression is necessary for primary melanoma formation.,Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model.,Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype.,We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes.,Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss.,Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.,Melanoma cells can switch between proliferative and invasive phenotypes.,Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes.

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

Tumor spreading is associated with the degradation of extracellular matrix proteins, mediated by the overexpression of matrix metalloproteinase 9 (MMP-9).,Although, such overexpression was linked to epigenetic promoter methylation, the role of intragenic methylation was not clarified yet.,Melanoma was used as tumor model to investigate the relationship between the DNA intragenic methylation of MMP9 gene and MMP-9 overexpression at transcriptional and protein levels.,Computational analysis revealed DNA hypermethylation within the intragenic CpG-2 region of MMP9 gene in melanoma samples with high MMP-9 transcript levels.,In vitro validation showed that CpG-2 hotspot region was hypermethylated in the A375 melanoma cell line with highest mRNA and protein levels of MMP-9, while low methylation levels were observed in the MEWO cell line where MMP-9 was undetectable.,Concordant results were demonstrated in both A2058 and M14 cell lines.,This correlation may give further insights on the role of MMP-9 upregulation in melanoma.

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

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

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

Melanoma tumors are highly heterogeneous, comprising of different cell types that vary in their potential for growth and invasion.,Heterogeneous expression of the Microphthalmia-associated Transcription Factor (MITF) and the POU domain transcription factor BRN2 (POU3F2) has been found in malignant melanoma.,Changing expression of these transcription factors as the disease progresses has been linked to the metastatic mechanism of phenotype switching.,We therefore investigated the effects of MITF and BRN2 expression in melanoma growth and metastasis.,Depletion of MITF resulted in a cell population that had a slowed cell cycle progression, was less invasive in vitro and had hindered tumor and metastasis forming ability in mouse xenograft studies.,BRN2 depletion left a cell population with intact proliferation and invasion in vitro; however metastatic growth was significantly reduced in the mouse xenograft model.,These results suggest that the proliferative population within melanoma tumors express MITF, and both MITF and BRN2 are important for metastatic growth in vivo.,This finding highlights the importance of BRN2 and MITF expression in development of melanoma metastasis.

The diversity of functional phenotypes observed within a tumor does not exclusively result from intratumoral genetic heterogeneity but also from the response of cancer cells to the microenvironment.,We have previously demonstrated that the morphological and functional phenotypes of melanoma can be dynamically altered upon external stimuli.,In the present study, transcriptome profiles were generated to explore the molecules governing phenotypes of melanospheres grown in the bFGF(+)EGF(+) serum-free cultures and monolayers maintained in the serum-containing medium.,Higher expression levels of MITF-dependent genes that are responsible for differentiation, e.g., TYR and MLANA, and stemness-related genes, e.g., ALDH1A1, were detected in melanospheres.,These results were supported by the observation that the melanospheres contained more pigmented cells and cells exerting the self-renewal capacity than the monolayers.,In addition, the expression of the anti-apoptotic, MITF-dependent genes e.g., BCL2A1 was also higher in the melanospheres.,The enhanced activity of MITF in melanospheres, as illustrated by the increased expression of 74 MITF-dependent genes, identified MITF as a central transcriptional regulator in melanospheres.,Importantly, several genes including MITF-dependent ones were expressed in melanospheres and original tumors at similar levels.,The reduced MITF level in monolayers might be partially explained by suppression of the Wnt/β-catenin pathway, and DKK1, a secreted inhibitor of this pathway, was highly up-regulated in monolayers in comparison to melanospheres and original tumors.,Furthermore, the silencing of DKK1 in monolayers increased the percentage of cells with self-renewing capacity.,Our study indicates that melanospheres can be used to unravel the molecular pathways that sustain intratumoral phenotypic heterogeneity.,Melanospheres directly derived from tumor specimens more accurately mirrored the morphology and gene expression profiles of the original tumors compared to monolayers.,Therefore, melanospheres represent a relevant preclinical tool to study new anticancer treatment strategies.

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.

Long non-coding RNAs (lncRNAs) are involved in tumorigenesis.,Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNAs, is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear.,In this study, the aberrant up-regulation of MALAT1 was detected in melanoma.,We determined that MALAT1 promotes melanoma cells proliferation, invasion and migration by sponging miR-22.,MiR-22 was decreased and acted as a tumor suppressor in melanoma, and MMP14 and Snail were the functional targets of miR-22.,Furthermore, MALAT1 could modulate MMP14 and Snail by operating as a competing endogenous RNA (ceRNA) for miR-22.,The effects of MALAT1 in malignant melanoma is verified using a xenograft model.,This finding elucidates a new mechanism for MALAT1 in melanoma development and provides a potential target for melanoma therapeutic intervention.

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

Melanoma is characterised by its ability to metastasise at early stages of tumour development.,Current clinico‐pathologic staging based on the American Joint Committee on Cancer criteria is used to guide surveillance and management in early‐stage disease, but its ability to predict clinical outcome has limitations.,Herein we review the genomics of melanoma subtypes including cutaneous, acral, uveal and mucosal, with a focus on the prognostic and predictive significance of key molecular aberrations.,© 2018 The Authors.,The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease.,Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency.,Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing.,In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin.,Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis.,These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.

Genes in the S100 family are abnormally expressed in a variety of tumor cells and are associated with clinical pathology, but their prognostic value in melanoma patients has not yet been fully elucidated.,In this study, we extracted and profiled S100 family mRNA expression data and corresponding clinical data from the Gene Expression Omnibus database to analyze how expression of these genes correlates with clinical pathology.,Compared with normal skin, S100A1, S100A13, and S100B were expressed at significantly higher levels in melanoma samples.,S100A2, S100A7, S100A8, S100A9, S100A10, S100A11, and S100P were all highly expressed in primary melanoma samples but were expressed at low levels in metastatic melanoma, and all of these genes were strongly correlated with each other (P<0.001).,We found the expression of these S100 family genes to be significantly correlated with both lymphatic and distant melanoma metastasis, as well as with American Joint Committee on Cancer grade but not with Clark’s grade, age, or sex.,This suggests that expression of these genes may be related to the degree of tumor invasion.,Although further validation through basic and clinical trials is needed, our results suggest that the S100 family genes have the potential to play an important role in the diagnosis of melanoma.,S100 expression may be related to tumor invasion and may facilitate the early diagnosis of melanoma, allowing for a more accurate prognosis.,Targeted S100 therapies are also potentially viable strategies in the context of melanoma.

BRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma.,We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors.,Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib.,245 plasma samples from 36 patients were analyzed.,In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib.,At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16).,BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment.,During treatment, disease progression (PD) was diagnosed in 27 of 36 patients.,An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %.,An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27).,Quantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors.,Its potential as an early predictor of acquired resistance deserves further evaluation.

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas.,SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear.,Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss.,Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3’ss-sequence context.,SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants.,Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage.,Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.,Mutations in the splicing factor SF3B1 are found in uveal melanoma.,Here, Alsafadi et al. use RNA-sequencing data from these cancers and experimental models, and show that mutant SF3B1 promotes alternative branchpoints in a specific gene subset and that the mutant protein gains a new function.

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

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance.,Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse.,We first characterized CSCs in melanoma cell lines.,We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells.,We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture.,Based on these data, we investigated whether δ-TT might target melanoma CSCs.,We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres.,In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker.,These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

Melanoma is the most dangerous and treatment-resistant skin cancer.,Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC).,Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior.,Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity.,However, their ability to target the CSC compartment in melanoma is not known.,Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models.,While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects.,Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids.,Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels.,Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

BRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma.,We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors.,Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib.,245 plasma samples from 36 patients were analyzed.,In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib.,At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16).,BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment.,During treatment, disease progression (PD) was diagnosed in 27 of 36 patients.,An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %.,An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27).,Quantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors.,Its potential as an early predictor of acquired resistance deserves further evaluation.

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

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

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance.,Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse.,We first characterized CSCs in melanoma cell lines.,We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells.,We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture.,Based on these data, we investigated whether δ-TT might target melanoma CSCs.,We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres.,In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker.,These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

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

Uveal melanoma (UM) is the most common cancer of the eye in adults.,Many UM patients develop metastases for which no curative treatment has been identified.,Novel therapeutic approaches are therefore urgently needed.,UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors.,Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors.,However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents.,Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071.,The most promising approaches were further investigated in vitro using our unique panel of UM cell lines.,When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs.,Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death.,While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease.,Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients.

In the present study, we analyzed the role of microRNA-194 circulating regulated human melanoma cell growth.,We found that microRNA-194 expression was markedly suppressed in human melanoma patients, compared with negative control group.,Next, disease-free survival (DFS) and overall survival (OS) of high expression in human melanoma patients was higher than those of low expression in human melanoma patients.,MicroRNA-194 overexpression inhibited cell proliferation, induced apoptosis, increased caspase-3/−9 activities and promoted Bax/Bcl-2 of human melanoma cells.,Furthermore, microRNA-194 overexpression also suppressed PI3K/AKT/FoxO3a signaling pathway and induced p53/p21 signaling pathway.,PI3K inhibitor, suppressed PI3K, phosphorylation-AKT, FoxO3a protein expression and increased the effects of microRNA-194 overexpression on cell growth, apoptosis, caspase-3/−9 activities and Bax/Bcl-2 protein expression of human melanoma cells through the induction of p53/p21 signaling pathway.,Taken together, these data indicate that circulating microRNA-194 regulated human melanoma cells via PI3K/AKT/FoxO3a and p53/p21 signaling pathway.

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

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.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases1, while rare variants in CDK4, BRCA2, BAP1, and the promoter of TERT, have also been linked to the disease2-5.,Here we set out to identify novel high-penetrance susceptibility genes in unexplained cases by sequencing 184 melanoma patients from 105 pedigrees recruited in the United Kingdom, the Netherlands, and Australia that were negative for variants in known predisposition genes.,We identify families where melanoma co-segregates with loss-of-function variants in the protection of telomeres 1 (POT1) gene, a proportion of members presenting with an early age of onset and multiple primaries.,We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide-/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding, leading to increased telomere length.,Thus, POT1 variants predispose to melanoma formation via a direct effect on telomeres.

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

Uveal melanoma (UM) is highly refractory to treatment with dismal prognosis in advanced stages.,The value of the combined checkpoint blockade with CTLA-4 and PD-1 inhibition in metastatic UM is currently unclear.,Patients with metastatic or unresectable UM treated with ipilimumab in combination with a PD-1 inhibitor were collected from 16 German skin cancer centers.,Patient records of 64 cases were analyzed for response, progression-free survival (PFS), overall survival (OS), and safety.,Clinical parameters and serum biomarkers associated with OS and treatment response were determined with Cox regression modelling and logistic regression.,The best overall response rate to combined checkpoint blockade was 15.6% with 3.1 and 12.5% complete and partial response, respectively.,The median duration of response was 25.5 months (range 9.0-65.0).,Stable disease was achieved in 21.9%, resulting in a disease control rate of 37.5% with a median duration of the clinical benefit of 28.0 months (range 7.0-65.0).,The median PFS was 3.0 months (95% CI 2.4-3.6).,The median OS was estimated to 16.1 months (95% CI 12.9-19.3).,Regarding safety, 39.1% of treated patients experienced a severe, treatment-related adverse event according to the CTCAE criteria (grade 3: 37.5%; grade 4: 1.6%).,The most common toxicities were colitis (20.3%), hepatitis (20.3%), thyreoiditis (15.6%), and hypophysitis (7.8%).,A poor ECOG performance status was an independent risk factor for decreased OS (p = 0.007).,The tolerability of the combined checkpoint blockade in UM may possibly be better than in trials on cutaneous melanoma.,This study implies that combined checkpoint blockade represents the hitherto most effective treatment option available for metastatic UM available outside of clinical trials.

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.

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).

Melanomas are tumors originating from melanocytes and tend to show early metastasis secondary to the loss of cellular adhesion in the primary tumor, resulting in high mortality rates.,Cancer-specific active immunotherapy is an experimental form of treatment that stimulates the immune system to recognize antigens on the surface of cancer cells.,Current experimental approaches in immunotherapy include vaccines, biochemotherapy, and the transfer of adoptive T cells and dendritic cells.,Several types of vaccines, including peptide, viral, and dendritic cell vaccines, are currently under investigation for the treatment of melanoma.,These treatments have the same goal as drugs that are already used to stimulate the proliferation of T lymphocytes in order to destroy tumor cells; however, immunotherapies aim to selectively attack the tumor cells of each patient.,In this comprehensive review, we describe recent advancements in the development of immunotherapies for melanoma, with a specific focus on the identification of neoantigens for the prediction of their elicited immune responses.,This review is expected to provide important insights into the future of immunotherapy for melanoma.

Brain metastases afflict approximately half of patients with metastatic melanoma (MM) and small cell lung cancer (SCLC) and represent the direct cause of death in 60 to 70% of those affected.,Standard of care remains ineffective in both types of cancer with the challenge of overcoming the blood brain barrier (BBB) exacerbating the clinical problem.,Our purpose is to determine and characterize the potential of albendazole (ABZ) as a cytotoxic and radiosensitizing agent against MM and SCLC cells.,Here, ABZ's mechanism of action as a DNA damaging and microtubule disrupting agent is assessed through analysis of histone H2AX phosphorylation and cell cyle progression.,The cytotoxicity of ABZ alone and in combination with radiation therapy is determined though clonogenic cell survival assays in a panel of MM and SCLC cell lines.,We further establish ABZ's ability to act synergistically as a radio-sensitizer through combination index calculations and apoptotic measurements of poly (ADP-ribose) polymerase (PARP) cleavage.,ABZ induces DNA damage as measured by increased H2AX phosphorylation.,ABZ inhibits the growth of MM and SCLC at clinically achievable plasma concentrations.,At these concentrations, ABZ arrests MM and SCLC cells in the G2/M phase of the cell cycle after 12 hours of treatment.,Exploiting the notion that cells in the G2/M phase are the most sensitive to radiation therapy, we show that treatment of MM and SCLC cells treated with ABZ renders them more sensitive to radiation in a synergistic fashion.,Additionally, MM and SCLC cells co-treated with ABZ and radiation exhibit increased apoptosis at 72 hours.,Our study suggests that the orally available antihelminthic ABZ acts as a potent radiosensitizer in MM and SCLC cell lines.,Further evaluation of ABZ in combination with radiation as a potential treatment for MM and SCLC brain metastases is warranted.

Long non-coding RNAs (lncRNAs) are a largely uncharacterized group of non-coding RNAs with diverse regulatory roles in various biological processes.,Recent observations have elucidated the functional roles of lncRNAs in cutaneous biology, e.g. in proliferation and differentiation of epidermal keratinocytes and in cutaneous wound repair.,Furthermore, the role of lncRNAs in keratinocyte-derived skin cancers is emerging, especially in cutaneous squamous cell carcinoma (cSCC), which presents a significant burden to health care services worldwide and causes high mortality as metastatic disease.,Elucidation of the functions of keratinocyte-specific lncRNAs will improve understanding of the molecular pathogenesis of epidermal disorders and skin cancers and can be exploited in development of new diagnostic and therapeutic applications for keratinocyte carcinomas.,In this review, we summarize the current evidence of functionally important lncRNAs in cutaneous biology and in keratinocyte carcinomas.

Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC), together known as keratinocyte cancers (KCs), are the commonest cancer in white ethnic populations.,Recent improvements to registry data collection in England has allowed more accurate analysis of the epidemiology of BCC and cSCC and for the first time we are able to provide an accurate (representative) tumour burden for KC in the U.K.,To estimate the incidence of BCC and cSCC in the U.K.,A cohort of patients with KCs between 2013 and 2015 were identified using linkage to diagnostic codes derived from pathology reports collected into the national cancer registry.,Data from England's cancer registry were combined with data from Scotland, Northern Ireland and Wales.,European age‐standardized incidence rates (EASRs) of the first BCC and cSCC per patient per annum (PPPA) were calculated.,In the U.K, the EASR of the first BCC and cSCC PPPA in 2013-15 were 285 and 77 per 100 000 person years, respectively (211 120 KCs total in 2015).,The mean annual percentage increase was 5% between 2013 and 2015 for both BCC and cSCC.,By counting the first KC PPPA, we include an additional 51% KCs compared with the previous reporting technique which counts only the first BCC and cSCC in a patient's lifetime, yet it represents a probable underestimation of 5-11% of the true tumour count.,Based on an improved methodology, a more representative incidence of KC is presented, which is essential to healthcare planning and will lead to improved understanding of the epidemiology of KC.,What's already known about this topic?,Keratinocyte cancers (KCs) are the most common cancers affecting white ethnic populations.The incidence of basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) is increasing worldwide including the U.K., most commonly in elderly male Caucasian patients.These cancers are traditionally substantially underreported and frequently excluded from national cancer statistics.,Keratinocyte cancers (KCs) are the most common cancers affecting white ethnic populations.,The incidence of basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) is increasing worldwide including the U.K., most commonly in elderly male Caucasian patients.,These cancers are traditionally substantially underreported and frequently excluded from national cancer statistics.,What does this study add?,Using improved data collection methods in England and validated tumour‐reporting techniques, we report the most accurate BCC and cSCC incidence data for the U.K. ever published.Identifying the first BCC and cSCC per patient per annum, the incidence of BCC and cSCC in the U.K. (excluding Wales) was 285 and 77 per 100 000 person years, respectively, between 2013 and 2015, with more than 210 000 KCs in the U.K. in 2015.,Using improved data collection methods in England and validated tumour‐reporting techniques, we report the most accurate BCC and cSCC incidence data for the U.K. ever published.,Identifying the first BCC and cSCC per patient per annum, the incidence of BCC and cSCC in the U.K. (excluding Wales) was 285 and 77 per 100 000 person years, respectively, between 2013 and 2015, with more than 210 000 KCs in the U.K. in 2015.,Linked Editorial: Whiteman.,Br J Dermatol 2019; 181:434-435.,Plain language summary available online,Respond to this article

The study aims to evaluate the effects of miR-136 on the proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of melanoma cells by targetting premelanosome protein (PMEL) through the Wnt signaling pathway.,After establishment of melanoma mouse models, melanoma (model group) and normal tissues (normal group) were collected.,Immunohistochemistry was performed to determine PMEL protein concentration.,Mouse melanoma cells were assigned into control, blank, negative control (NC), miR-136 mimics, miR-136 inhibitors, siRNA-PMEL, and miR-136 inhibitors + siRNA-PMEL, LiC1 (Wnt signaling pathway activator), and siRNA-PMEL+ LiCl groups.,MTT, Scratch test, Transwell assay, and flow cytometry were performed to measure cell proliferation, migration, invasion, and apoptosis.,Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to evaluate miR-136, PMEL, β-catenin, Wnt3a, Bcl-2, Bax, Caspase, E-cadherin, and N-cadherin expressions.,PMEL is highly expressed in melanoma tissues.,MiR-136, Bax, Caspase, and E-cadherin expressions decreased in the model group, whereas PMEL, β-catenin, Bcl-2, Wnt3a, and N-cadherin expressions increased.,Bax, Caspase, and E-cadherin expressions increased in the miR-136 mimics and siRNA-PMEL groups, whereas the expressions decreased in the miR-136 inhibitors group and LiC1 group.,PMEL, β-catenin, Bcl-2, Wnt3a, and N-cadherin expressions, cell proliferation, migration, and invasion decreased, and the apoptosis rate inceased in the miR-136 mimics and siRNA-PMEL groups; whereas the tendencies were opposite to those in the miR-136 inhibitors group and LiC1 group.,In the siRNA-PMEL+ LiCl group, PMEL expression decreased.,These findings indicated that overexpression of miR-136 inhibits melanoma cell EMT, proliferation, migration, invasion, and promotes apoptosis by targetting PMEL through down-regulation of the Wnt signaling pathway.

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

Histopathologic examination is sometimes inadequate for accurate and reproducible diagnosis of certain melanocytic neoplasms.,As a result, more sophisticated and objective methods have been sought.,The goal of this study was to identify a gene expression signature that reliably differentiated benign and malignant melanocytic lesions and evaluate its potential clinical applicability.,Herein, we describe the development of a gene expression signature and its clinical validation using multiple independent cohorts of melanocytic lesions representing a broad spectrum of histopathologic subtypes.,Using quantitative reverse‐transcription polymerase chain reaction (PCR) on a selected set of 23 differentially expressed genes, and by applying a threshold value and weighting algorithm, we developed a gene expression signature that produced a score that differentiated benign nevi from malignant melanomas.,The gene expression signature classified melanocytic lesions as benign or malignant with a sensitivity of 89% and a specificity of 93% in a training cohort of 464 samples.,The signature was validated in an independent clinical cohort of 437 samples, with a sensitivity of 90% and specificity of 91%.,The performance, objectivity, reliability and minimal tissue requirements of this test suggest that it could have clinical application as an adjunct to histopathology in the diagnosis of melanocytic neoplasms.

Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy.,Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1.,However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes.,IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both.,Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation.,We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment.,Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts.,Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis.,Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

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

Cathepsins represent a group of proteases involved in determining the metastatic potential of cancer cells.,Among these are cysteinyl- (e.g. cathepsin B and cathepsin L) and aspartyl-proteases (e.g. cathepsin D), normally present inside the lysosomes as inactive proenzymes.,Once released in the extracellular space, cathepsins contribute to metastatic potential by facilitating cell migration and invasiveness.,In the present work we first evaluated, by in vitro procedures, the role of cathepsins B, L and D, in the remodeling, spreading and invasiveness of eight different cell lines: four primary and four metastatic melanoma cell lines.,Among these, we considered two cell lines derived from a primary cutaneous melanoma and from a supraclavicular lymph node metastasis of the same patient.,To this purpose, the effects of specific chemical inhibitors of these proteases, i.e.,CA-074 and CA-074Me for cathepsin B, Cathepsin inhibitor II for cathepsin L, and Pepstatin A for cathepsin D, were evaluated.,In addition, we also analyzed the effects of the biological inhibitors of these cathepsins, i.e. specific antibodies, on cell invasiveness.,We found that i) cathepsin B, but not cathepsins L and D, was highly expressed at the surface of metastatic but not of primary melanoma cell lines and that ii) CA-074, or specific antibodies to cathepsin B, hindered metastatic cell spreading and dissemination, whereas neither chemical nor biological inhibitors of cathepsins D and L had significant effects.,Accordingly, in vivo studies, i.e. in murine xenografts, demonstrated that CA-074 significantly reduced human melanoma growth and the number of artificial lung metastases.,These results suggest a reappraisal of the use of cathepsin B inhibitors (either chemical or biological) as innovative strategy in the management of metastatic melanoma disease.

Melanoma affects about 6000 patients a year in Spain.,A group of medical oncologists from Spanish Society of Medical Oncology (SEOM) and Spanish Multidisciplinary Melanoma Group (GEM) has designed these guidelines to homogenize the management of these patients.,The diagnosis must be histological and determination of BRAF status has to be performed in patients with stage ≥ III.,Stage I-III resectable melanomas will be treated surgically.,In patients with stage III melanoma, adjuvant treatment with immunotherapy or targeted therapy is also recommended.,Patients with unresectable or metastatic melanoma will receive treatment with immunotherapy or targeted therapy, the optimal sequence of these treatments remains unclear.,Brain metastases require a separate consideration, since, in addition to systemic treatment, they may require local treatment.,Patients must be followed up closely to receive or change treatment as soon as their previous clinical condition changes, since multiple therapeutic options are available.

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

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

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

A major challenge for managing melanoma is its tumour heterogeneity based on individual co‐existing melanoma cell phenotypes.,These phenotypes display variable responses to standard therapies, and they drive individual steps of melanoma progression; hence, understanding their behaviour is imperative.,Melanoma phenotypes are defined by distinct transcriptional states, which relate to different melanocyte lineage development phases, ranging from a mesenchymal, neural crest‐like to a proliferative, melanocytic phenotype.,It is thought that adaptive phenotype plasticity based on transcriptional reprogramming drives melanoma progression, but at which stage individual phenotypes dominate and moreover, how they interact is poorly understood.,We monitored melanocytic and mesenchymal phenotypes throughout melanoma progression and detected transcriptional reprogramming at different stages, with a gain in mesenchymal traits in circulating melanoma cells (CTCs) and proliferative features in metastatic tumours.,Intriguingly, we found that distinct phenotype populations interact in a cooperative manner, which generates tumours of greater “fitness,” supports CTCs and expands organotropic cues in metastases.,Fibronectin, expressed in mesenchymal cells, acts as key player in cooperativity and promotes survival of melanocytic cells.,Our data reveal an important role for inter‐phenotype communications at various stages of disease progression, suggesting these communications could act as therapeutic target.

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

Around a fifth of melanomas exhibit an activating mutation in the oncogene NRas that confers constitutive signaling to proliferation and promotes tumor initiation.,NRas signals downstream of the major melanocyte tyrosine kinase receptor c-kit and activated NRas results in increased signaling via the extracellular signal-regulated kinase (ERK)/MAPK/ERK kinase/mitogen-activated protein kinase (MAPK) pathways to enhance proliferation.,The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling.,We tested the effects of activated NRasQ61K on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model.,We find an important role for Rac1 downstream of NRasQ61K in mediating dermal melanocyte survival in vivo in mouse, but surprisingly NRasQ61K does not appear to affect melanoblast motility or proliferation during mouse embryogenesis.,We also show that genetic deletion or pharmacological inhibition of Rac1 in NRasQ61K induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.,The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis.,Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.,Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth.,Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines.,Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib.,Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination.,The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.,The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines.,However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

Vascular pericytes are an important cellular component in the tumor microenvironment, however, their role in supporting cancer invasion is poorly understood.,We hypothesized that PDGF-BB could be involved in the transition of human retinal pericytes (HRPC) in cancer-activated fibroblasts (CAF), induced by the 92.1 uveal melanoma (UM) cell line.,In our model system, HRPC were conditioned by co-culturing with 92.1UM for 6 days (cHRPC), in the presence or absence of imatinib, to block PDGF receptor-β (PDGFRβ).,The effects of the treatments were tested by wound healing assay, proliferation assay, RT-PCR, high-content screening, Western blot analysis, and invasion assay.,Results showed profound changes in cHRPC shape, with increased proliferation and motility, reduction of NG2 and increase of TGF-β1, α-SMA, vimentin, and FSP-1 protein levels, modulation of PDGF isoform mRNA levels, phospho-PDGFRβ, and PDGFRβ, as well as phospho-STAT3 increases.,A reduction of IL-1β and IFNγ and an increase in TNFα, IL10, and TGF-β1, CXCL11, CCL18, and VEGF mRNA in cHRPC were found.,Imatinib was effective in preventing all the 92.1UM-induced changes.,Moreover, cHRPC elicited a significant increase of 92.1UM cell invasion and active MMP9 protein levels.,Our data suggest that retinal microvascular pericytes could promote 92.1UM growth through the acquisition of the CAF phenotype.

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.

Melanoma cells display distinct intrinsic phenotypic states.,Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines.,We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy.,Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states.,These findings have implications for cancer biology and the identification of new therapeutic strategies.,Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.,The regulation of the distinct intrinsic phenotypic states in melanoma remain poorly characterised.,Here, multi-omics analysis for a panel of 68 early passage melanoma cell lines reveals that cancer cell intrinsic transcriptomic programs are associated with distinct immune features.

Melanoma is the deadliest form of skin cancer, affecting men more frequently and severely than women.,Although recent studies suggest that differences in activity of the androgen receptor (AR) underlie the observed sex bias, little is known about AR activity in melanoma.,Here we show that AR and EGR1 bind to the long non-coding RNA SLNCR and increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes.,ChIP-seq reveals that ligand-free AR is enriched on SLNCR-regulated melanoma genes and that AR genomic occupancy significantly overlaps with EGR1 at consensus EGR1 binding sites.,We present a model in which SLNCR recruits AR to EGR1-bound genomic loci and switches EGR1-mediated transcriptional activation to repression of the tumor suppressor p21Waf1/Cip1.,Our data implicate the regulatory triad of SLNCR, AR, and EGR1 in promoting oncogenesis and may help explain why men have a higher incidence of and more rapidly progressive melanomas compared with women.

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

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

Cutaneous squamous cell carcinoma (cSCC) is prevalent in the world, accounting for a huge part of non-melanoma skin cancer.,Most cSCCs are associated with a distinct pre-cancerous lesion, the actinic keratosis (AK).,However, the progression trajectory from normal skin to AK and cSCC has not been fully demonstrated yet.,To identify genes involved in this progression trajectory and possible therapeutic targets for cSCC, here we constructed a UV-induced cSCC mouse model covering the progression from normal skin to AK to cSCC, which mimicked the solar UV radiation perfectly using the solar-like ratio of UVA and UVB, firstly.,Then, transcriptome analysis and a series of bioinformatics analyses and cell experiments proved that Rorα is a key transcript factor during cSCC progression.,Rorα could downregulate the expressions of S100a9 and Sprr2f in cSCC cells, which can inhibit the proliferation and migration in cSCC cells, but not the normal keratinocyte.,Finally, further animal experiments confirmed the inhibitory effect of cSCC growth by Rorα in vivo.,Our findings showed that Rorα would serve as a potential novel target for cSCC, which will facilitate the treatment of cSCC in the future.

Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers.,However, most DC vaccines so far have achieved only limited success in cancer treatment.,Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response.,In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT.,The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA.,The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model.,We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation).,Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells.,ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells.,Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines.

Supplemental Digital Content is available in the text.,Management of PD-1 blockade resistance in metastatic melanoma (MM) remains challenging.,Immunotherapy or chemotherapy alone provides limited benefit in this setting.,Chemo-immunotherapy (CIT) has demonstrated favorable efficacy and safety profiles in lung cancer.,Our pre-clinical study showed that in MM patients who have failed PD-1 blockade, the addition of chemotherapy increases CX3CR1+ therapy-responsive CD8+ T-cells with enhanced anti-tumor activity, resulting in improved clinical response.,Here, we examined the clinical outcomes of CIT in MM patients after PD-1 blockade failure and the treatment-related changes in CX3CR1+ therapy-responsive CD8+ T-cells.,We reviewed MM patients seen between January 2012 and June 2018 who failed anti-PD-1-based therapy and received subsequent CIT, immune checkpoint inhibitors (ICI) or chemotherapy alone.,Overall survival (OS), objective response rate (ORR), event-free survival (EFS), and toxicities were assessed.,Among 60 patients, 33 received CIT upon disease progression on PD-1 blockade.,At a median follow-up of 3.9 years, the CIT group had a median OS of 3.5 years [95% confidence interval (CI) 1.7-NR] vs.,1.8 years (95% CI 0.9-2; P = 0.002) for those who received subsequent ICI (n = 9) or chemotherapy alone (n = 18), with ORR of 59% vs.,15% (P = 0.0003), respectively.,The median EFS was 7.6 months (95% CI 6-10) following CIT vs.,3.4 months (95% CI 2.8-4.1; P = 0.0005) following ICI or chemotherapy alone.,Therapy-responsive CX3CR1+CD8+ T-cells showed dynamic increase with successful CIT.,CIT showed favorable clinical outcomes and acceptable safety profile in PD-1 blockade-resistant patients.,CX3CR1+CD8+ therapy-responsive T-cells can be potentially used for monitoring disease response to CIT.

Every biological experiment requires a choice of throughput balanced against physiological relevance.,Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput.,Here we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements.,We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput.,We demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability.,We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models.,The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts.,Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations.,In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.,The online version of this article (10.1186/s12885-019-5694-1) contains supplementary material, which is available to authorized users.

Rationale: Melanoma is an aggressive tumor of the skin and drug resistance is still a major problem in melanoma therapy.,Novel targets and effective agents to overcome drug resistant melanoma are urgently needed in clinical therapy.,Methods: Gene Expression Omnibus (GEO) database analysis, pathway enrichment analysis, and survival rate analysis were utilized to identify a candidate target.,An anchorage-independent cell growth assay, flow cytometry, Western blot, and a xenograft mouse model were used to study the function of Aurora kinase B (AURKB) in both drug-sensitive and drug-resistant melanoma.,Next, HI-511, a novel dual-target inhibitor targeting both AURKB and BRAF V600E, was designed and examined by an in vitro kinase assay.,Methods as indicated above in addition to a BRAF V600E/PTEN-loss melanoma mouse model were used to demonstrate the effect of HI-511 on melanoma development in vitro and in vivo.,Results: AURKB is highly expressed in melanoma and especially in vemurafenib-resistant melanoma and the expression was correlated with patient survival rate.,Knocking down AURKB inhibited cell growth and induced apoptosis in melanoma, which was associated with the BRAF/MEK/ERKs and PI3-K/AKT signaling pathways.,Importantly, we found that HI-511, a novel dual-target inhibitor against AURKB and BRAF V600E, suppresses both vemurafenib-sensitive and vemurafenib-resistant melanoma growth in vitro and in vivo by inducing apoptosis and mediating the inhibition of the BRAF/MEK/ERKs and PI3K/AKT signaling pathways.,Conclusion: AURKB is a potential target for melanoma treatment.,HI-511, a novel dual-target inhibitor against both AURKB and BRAF V600E, could achieve durable suppression of melanoma growth, even drug-resistant melanoma growth.

The optimal treatment sequence for patients with advanced BRAF V600 mutant melanoma is unknown.,BRAF/MEK inhibition (BRAF/MEKi), single agent anti‐PD‐1 (aPD‐1) antibodies and combination immune checkpoint inhibition with nivolumab and ipilimumab (niv/ipi) are all approved; however, they have not been prospectively compared.,Therefore, we sought to compare overall survival of patients with advanced BRAF mutant melanoma treated with either front‐line BRAF/MEKi, aPD‐1, or niv/ipi.,Patients with advanced BRAF mutant melanoma who had received BRAF/MEKi, niv/ipi, or aPD‐1 in the front‐line setting were identified from a nationwide database comprising de‐identified patient‐level structured and unstructured data derived from electronic health records.,Survival was compared using Kaplan‐Meier curves and log‐rank analysis.,Univariate and multivariate Cox regression models were used to measure the effect of front‐line treatment, age (>64 or not), LDH (elevated or not), and Eastern Cooperative Oncology Group (ECOG) performance status (>1 or not) on survival.,Five hundred and sixty seven patients with advanced disease and treated with front‐line aPD‐1 (n = 162), BRAF/MEKi (n = 297) or niv/ipi (n = 108) were identified.,With a median follow‐up of 22.4 months, median overall survival (OS) for patients treated with front‐line niv/ipi was not reached (NR) while median OS for patients treated with aPD‐1 or BRAF/MEKi was 39.5 months and 13.2 months, respectively.,Front‐line treatment with PD‐1 and niv/ipi were associated with statistically longer survival than BRAF/MEKi in multivariate analyses.,In our real‐world retrospective analysis, patients with advanced BRAF mutant melanoma treated with front‐line niv/ipi or aPD‐1 had longer survival compared to those treated with front‐line BRAF/MEKi.,Real‐world overall survival of patients with advanced BRAF mutant melanoma treated with front‐line BRAF/MEK inhibitors, anti‐PD‐1 antibodies, or nivolumab/ipilimumab.

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

Long non-coding RNAs (lncRNAs) are involved in tumorigenesis.,Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNAs, is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear.,In this study, the aberrant up-regulation of MALAT1 was detected in melanoma.,We determined that MALAT1 promotes melanoma cells proliferation, invasion and migration by sponging miR-22.,MiR-22 was decreased and acted as a tumor suppressor in melanoma, and MMP14 and Snail were the functional targets of miR-22.,Furthermore, MALAT1 could modulate MMP14 and Snail by operating as a competing endogenous RNA (ceRNA) for miR-22.,The effects of MALAT1 in malignant melanoma is verified using a xenograft model.,This finding elucidates a new mechanism for MALAT1 in melanoma development and provides a potential target for melanoma therapeutic intervention.

BRAF mutations are frequent in cutaneous melanomas and BRAF inhibitors(BRAFi) have shown remarkable clinical efficacy in BRAF mutant melanoma patients.,However, acquired drug resistance can occur rapidly and tumor(s) often progress thereafter.,Various mechanisms of BRAFi resistance have recently been described; however, the mechanism of resistance remains controversial.,In this study we developed BRAFi resistant melanoma cell lines and found that metastasis related EMT properties of BRAFi resistant cells were enhanced significantly.,Upregulation of EGFR was observed in BRAFi resistant cell lines and patient tumors due to demethylation of EGFR regulatory DNA elements.,EGFR induced PI3K/AKT pathway activation in BRAFi resistant cells through epigenetic regulation.,Treatment of EGFR inhibitor was effective in BRAFi resistant melanoma cell lines.,The study demonstrates that EGFR epigenetic activation has important implications in BRAFi 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.

Melanoma is a highly lethal cutaneous cancer with the tendency for early invasion and metastasis.,Integrated miRNA transcriptome sequence analysis of human melanoma tumors and adjacent control tissues identified 17 miRNAs differentially expressed in melanoma tissues: let-7a-5p, let-7b-5p, let-7c, miR-374a-3p, miR-100-5p, miR-7, miR-195, miR-1908, miR-214, miR-221, miR-199a-5p, miR-21, miR-18, miR-34a, miR-199a-3p, miR-92a and miR-106b.,Among these, miR-34a was most significantly down-regulated in melanoma tissues, and its expression correlated with TNM melanoma stage. miR-34a overexpression inhibited expression and activity of the transcription factor ZEB1, resulting in decreased proliferation and migration of melanoma cells.,Moreover, miR-34a overexpression inhibited ZEB1 expression and melanoma tumor growth in vivo, in a melanoma nude mouse model.,Together, these findings demonstrate that miR-34a inhibits melanoma growth by targeting the proto-oncogene ZEB1 and suggest the miR-34a -ZEB1 axis may serve as a novel target for melanoma treatment.

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

To explore the effect of VEGF (vascular endothelial growth factor) on the vasculogenic mimicry (VM) formation of Choroidal Melanoma (CM) through PI3k signal pathway, to find novel targets for CM therapy.,This research investigated the molecular mechanism of VEGF promoting VM formation of CM.,First, we evaluated the expressions of VEGF in 20 CM specimens by immunohistochemical determination.,Then we detected expressions of VEGF, AKT, MT1-MMP, MMP2, and MMP9 of OCM-1 in hypoxia. siRNA was used to inhibit the expression of VEGF, to realize the control of the VM formation.,The VM formation was evaluated through wound healing assay, transwell assay, and apoptosis.,And then we testify the correlation of the VM and the factors in protein and mRNA level preliminarily.,VEGF protein was expressed in CM in all 20 cases of CM, especially along the VM.,In hypoxia, the expression of VEGF in OCM-1 increased significantly.,VEGF gene deletion reduced the proliferation, migration, and invasion of OCM-1.,VEGF gene deletion impaired the expression of invasive associated genes like VEGF, p-AKT, AKT, MT1-MMP, MMP2, and MMP9.,These results indicate that VEGF induce VM formation in CM by activating PI3K/AKT signaling pathway.,VEGF promoted VM formation by the PI3K signal transduction pathway, indicating a molecular mechanism which may be used to develop new therapeutic targets for the clinical treatment of CM.

Melanoma cells can switch their phenotypes in response to microenvironmental insults.,Heterogeneous melanoma populations characterized by long-term growth and a high self-renewal capacity can be obtained in vitro in EGF(+)bFGF(+) medium whilst invasive potential of melanoma cells is increased in serum-containing cultures.,In the present study, we have shown that originally these patient-derived melanoma populations exhibit variable expression of pro-survival genes from the BCL-2 family and inhibitors of apoptosis (IAPs), and differ in the baseline MCL-1 transcript stability as well.,While being transferred to serum-containing medium, melanoma cells are well protected from death.,Immediate adaptive response of melanoma cells selectively involves a temporary MCL-1 increase, both at mRNA and protein levels, and BCL-XL can complement MCL-1, especially in MITFlow populations.,Thus, the extent of MCL-1 and BCL-XL contributions seems to be cell context-dependent.,An increase in MCL-1 level results from a transiently enhanced stability of its transcript, but not from altered protein turnover.,Inhibition of MCL-1 preceding transfer to serum-containing medium caused the induction of cell death in a subset of melanoma cells, which confirms the involvement of MCL-1 in melanoma cell survival during the rapid alteration of growth conditions.,Additionally, immediate response to serum involves the transient increase in MITF expression and inhibition of ERK-1/2 activity.,Uncovering the mechanisms of adaptive response to rapid changes in microenvironment may extend our knowledge on melanoma biology, especially at the stage of dissemination.

The effect of apatinib on the formation of vasculogenic mimicry (VM) was studied in a malignant melanoma cell line.,MUM-2B cells cultured in three-dimensional Matrigel were treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.5 μmol/L) of apatinib to test its effect on VM in vitro, followed by MTT proliferation and transwell invasion assays to determine the effect of apatinib on cell proliferation and invasion of MUM-2B cells.,In vivo, we used a melanoma cancer model to test the effect of short-term apatinib (100, 200, 300 mg/kg) treatment on VM.,Western blotting, immunohistochemistry staining, and CD31-PAS dual staining were performed to assess the expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2, and formation of VM.,The results showed apatinib-treated groups formed a lesser number of VM in 3D matrigel, while the cell viability in MTT proliferation assay and the number of migration cells in transwell invasion assay were significantly lower in apatinib-treated groups.,In addition, short-term apatinib treatment inhibited angiogenesis, VM formation, and tumor growth in models of melanoma cancer.,Mice in apatinib-treated groups showed a markedly reduced expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2.,In summary, apatinib could inhibit the expression of VEGFR-2, and downregulate the ERK1/2/PI3K/MMP-2 signaling cascade, which may be one of the underlying mechanisms by which apatinib inhibits angiogenesis and the development of VM in models of melanoma cancer, and restrains the formation of VM by MUM-2B cells.,Apatinib shows inhibitory effects on cell proliferation and invasion of MUM-2B cells, which is a close relationship with the VM.

Glycolytic reprogramming is a typical feature of many cancers; however, key regulators of glucose metabolism reengineering are poorly understood, especially in cutaneous squamous cell carcinoma (cSCC).,Here, Homeobox A9 (HOXA9), a direct target of onco-miR-365, is identified to be significantly downregulated in cSCC tumors and cell lines.,HOXA9 acts as a tumor suppressor and inhibits glycolysis in cSCC in vitro and in vivo by negatively regulating HIF-1α and its downstream glycolytic regulators, HK2, GLUT1 and PDK1.,Mechanistic studies show that HOXA9-CRIP2 interaction at glycolytic gene promoters impeds HIF-1α binding, repressing gene expression in trans.,Our results reveal a miR-365-HOXA9-HIF-1α regulatory axis that contributes to the enhanced glycolysis in cSCC development and may represent an intervention target for cSCC therapy.,Hypoxia-inducible transcription factor HIF-1α promotes glycolysis allowing cell survival under stress.,Here the authors show, using both cell lines and animal models, that in cutaneous squamous cell carcinoma HOXA9 acts as a tumor suppressor and inhibits glycolysis by associating with CRIP2 to repress HIF-1α binding to target genes.

Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis.,The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells.,A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness.,Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions.,Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells.,Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p.,Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively.,Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.

Skin cancer, including melanoma, basal cell carcinoma and cutaneous squamous cell carcinoma, has one of the highest global incidences of any form of cancer.,In 2016 more than 16,000 people were diagnosed with melanoma in the UK.,Over the last decade the incidence of melanoma has increased by 50% in the UK, and about one in ten melanomas are diagnosed at a late stage.,Among the keratinocyte carcinomas (previously known as non-melanoma skin cancers), basal cell carcinoma is the most common cancer amongst Caucasian populations.,The main risk factor for all skin cancer is exposure to ultraviolet radiation-more than 80% are considered preventable.,Primary care clinicians have a vital role to play in detecting and managing patients with skin lesions suspected to be skin cancer, as timely diagnosis and treatment can improve patient outcomes, particularly for melanoma.,However, detecting skin cancer can be challenging, as common non-malignant skin lesions such as seborrhoeic keratoses share features with less common skin cancers.,Given that more than 80% of skin cancers are attributed to ultraviolet (UV) exposure, primary care clinicians can also play an important role in skin cancer prevention.,This article is one of a series discussing cancer prevention and detection in primary care.,Here we focus on the most common types of skin cancer: melanoma, squamous cell carcinoma and basal cell carcinoma.,We describe the main risk factors and prevention advice.,We summarise key guidance on the symptoms and signs of skin cancers and their management, including their initial assessment and referral.,In addition, we review emerging technologies and diagnostic aids which may become available for use in primary care in the near future, to aid the triage of suspicious skin lesions.

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.

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

Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance.,Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment.,In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression.,We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels.,In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis.,Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo.,Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice.,Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis.,Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation.,Inhibition of these two augments synthetic lethality in melanoma.

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

BCL2 19 kD protein-interacting protein 3 (BNIP3) is a BH3-containing protein of the BCL-2 family; it can regulate cell death, autophagy, and cytoprotection.,The upregulation of BNIP3 has been reported to relate to progression and poor prognosis in different cancer types.,However, the clinical significance of BNIP3 in uveal melanoma (UM) is still unknown.,In our study, 47 patients with UM were enrolled; the expression of BNIP3 was detected with immunohistochemistry.,According to BNIP3 immunohistochemical scores, the patients were divided into BNIP3 high- and low-expression subgroups.,The correlation between the expression of BNIP3 and clinicopathological factors was evaluated with Fisher’s test; the associations with survival rates were analyzed with log-rank test.,The independent prognostic factors were identified with the Cox-regression model.,BNIP3 was mainly localized in the cytoplasm, and high expression of BNIP3 accounted for 31.9% (15/47) of the patients in our study.,High expression of BNIP3 was demonstrated to be significantly associated with more pigment (P=0.018) and deeper scleral invasion (P=0.013).,High expression of BNIP3 was also correlated with lower overall survival rate (P=0.006).,Multivariate analysis confirmed positive ciliary body involvement and lymphatic infiltration as independent prognostic factors.,High expression of BNIP3 was significantly associated with poor prognosis of patients with UM, indicating that BNIP3 detection could help stratify high-risk patients and identify new therapies targeting BNIP3 as a promising approach to treat UM.

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.

Due to the critical impact of active AP-1 transcription factors in melanoma, it is important to define their target genes and to identify and ultimately inhibit oncogenic signals.,Here we mapped the genome-wide occupancy of the AP-1 family member c-Jun in different melanoma cells and correlated AP-1 binding with transcriptome data to detect genes in melanoma regulated by c-Jun.,Our analysis shows that c-Jun supports the malignant phenotype by deregulating genes in cancer-relevant signaling pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways.,Moreover, we demonstrate that the importance of c-Jun depends on melanoma stage and mutation status of the tumor suppressor PTEN.,Our study reveals that activation of c-Jun overrules the tumor suppressive effect of PTEN in early melanoma development.,These findings help to understand the relevance of c-Jun within cancer pathways in different melanoma cell types, especially in relation to MAPK and PI3K pathways, which are commonly deregulated in melanomas.,Consequently, targeting c-Jun in PTEN+ melanoma cells may represent a promising therapeutic strategy to inhibit survival of melanoma cells to prevent the development of a metastatic phenotype.

Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer.,The latter is due to its viral or UV-associated carcinogenesis.,For tumor progression MCC has to escape the host’s immuno-surveillance, e.g. by loss of HLA class-I expression.,Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines.,This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2.,Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells.,Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components.,Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model.,In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins.,In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.

The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies.,However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment.,We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines.,We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ.,Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118).,Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines.,In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature.,Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort.,We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine.,Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma.,Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma.,In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage.,A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

UV radiation (UV) is classified as a “complete carcinogen” because it is both a mutagen and a non-specific damaging agent and has properties of both a tumor initiator and a tumor promoter.,In environmental abundance, UV is the most important modifiable risk factor for skin cancer and many other environmentally-influenced skin disorders.,However, UV also benefits human health by mediating natural synthesis of vitamin D and endorphins in the skin, therefore UV has complex and mixed effects on human health.,Nonetheless, excessive exposure to UV carries profound health risks, including atrophy, pigmentary changes, wrinkling and malignancy.,UV is epidemiologically and molecularly linked to the three most common types of skin cancer, basal cell carcinoma, squamous cell carcinoma and malignant melanoma, which together affect more than a million Americans annually.,Genetic factors also influence risk of UV-mediated skin disease.,Polymorphisms of the melanocortin 1 receptor (MC1R) gene, in particular, correlate with fairness of skin, UV sensitivity, and enhanced cancer risk.,We are interested in developing UV-protective approaches based on a detailed understanding of molecular events that occur after UV exposure, focusing particularly on epidermal melanization and the role of the MC1R in genome maintenance.

The prognostic significance of BRAF and NRAS mutations in metastatic melanoma patients remains uncertain, with several studies reporting conflicting results, often biased by the inclusion of patients treated with BRAF and MEK (MAPK) inhibitors.,We therefore interrogated a historical cohort of patients free of the confounding influence of MAPK inhibitor therapy.,Patients with available archival tissue first diagnosed with metastatic melanoma between 2002 and 2006 were analysed.,Mutational analysis was performed using the OncoCarta Panel.,Patient characteristics, treatment outcome and survival were correlated with BRAF/NRAS mutation status.,In 193 patients, 92 (48%) melanomas were BRAF-mutant, 39 (20%) were NRAS-mutant and 62 (32%) were wild-type for BRAF/NRAS mutations (wt).,There was no difference in response to chemotherapy based on mutation status (35-37%).,The distant disease-free interval (DDFI) was significantly shorter in patients with wt melanoma (27.9 months vs 35.1 for BRAF and 49.1 for NRAS) although this was not significant in multivariate analysis.,Survival from stage IV melanoma diagnosis was not significantly different based on mutation status.,The DDFI was significantly shorter in patients with BRAFV600K/R versus BRAFV600E melanoma in univariate and multivariate analyses.,BRAF and NRAS mutation status does not influence survival in metastatic melanoma.

The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance.,In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.,Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot.,Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.,More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM).,The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib.,Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi.,In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance.,Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.,AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma.,Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration.,We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes.,In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs.,In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas.,Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.,•Prl3a prevents premature MSC differentiation in zebrafish regeneration•PRL3 binds DDX21 to restrain transcription of MITF-regulated endolysosomal genes•V-ATPase inhibitors rescue prl3a−/− premature MSC differentiation in regeneration•PRL3-regulated transcription controls MSC fate and characterizes PRL3-high melanomas,Prl3a prevents premature MSC differentiation in zebrafish regeneration,PRL3 binds DDX21 to restrain transcription of MITF-regulated endolysosomal genes,V-ATPase inhibitors rescue prl3a−/− premature MSC differentiation in regeneration,PRL3-regulated transcription controls MSC fate and characterizes PRL3-high melanomas,Activated melanocyte stem cells produce differentiated progenitors in regeneration.,Johansson et al. reveal that the oncogene PRL3 regulates this process.,PRL3 restricts productive transcription of MITF endolysosomal target genes bound by DDX21 in the melanocyte stem cell lineage to prevent premature melanoblast expansion during regeneration and in melanoma.

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

Recent reports suggested frequent occurrence of cancer associated somatic mutations within regulatory elements of the genome.,Based on initial exome sequencing of 21 melanomas, we report frequent somatic mutations in skin cancers in a bidirectional promoter of diphthamide biosynthesis 3 (DPH3) and oxidoreductase NAD-binding domain containing 1 (OXNAD1) genes.,The UV-signature mutations occurred at sites adjacent and within a binding motif for E-twenty six/ternary complex factors (Ets/TCF), at −8 and −9 bp from DPH3 transcription start site.,Follow up screening of 586 different skin lesions showed that the DPH3 promoter mutations were present in melanocytic nevi (2/114; 2%), melanoma (30/304; 10%), basal cell carcinoma of skin (BCC; 57/137; 42%) and squamous cell carcinoma of skin (SCC; 12/31; 39%).,Reporter assays carried out in one melanoma cell line for DPH3 and OXNAD1 orientations showed statistically significant increased promoter activity due to −8/−9CC > TT tandem mutations; although, no effect of the mutations on DPH3 and OXNAD1 transcription in tumors was observed.,The results from this study show occurrence of frequent somatic non-coding mutations adjacent to a pre-existing binding site for Ets transcription factors within the directional promoter of DPH3 and OXNAD1 genes in three major skin cancers.,The detected mutations displayed typical UV signature; however, the functionality of the mutations remains to be determined.

Ipilimumab improves the survival of metastatic melanoma patients.,Despite documented, durable objective responses, a significant number of patients fails to benefit from treatment.,The aim of this study was to identify an upfront marker for treatment benefit.,A total of 187 metastatic melanoma patients treated in three Italian Institutions with 3 mg kg−1 ipilimumab, and 27 patients treated with 10 mg kg−1 ipilimumab, were evaluated.,Neutrophil-to-lymphocyte ratio (NLR) was calculated from pre-therapy full blood counts.,Progression-free survival (PFS) and overall survival (OS) were assessed using the Kaplan-Meier method, and multivariate Cox models were applied, adjusting for confounders and other prognostic factors.,In the training cohort of 69 patients treated at European Institute of Oncology, pre-therapy NLR was identified as the strongest and independent marker for treatment benefit in multivariate analyses.,Patients with baseline NLR<5 had a significantly improved PFS (HR=0.38; 95% CI: 0.22-0.66; P=0.0006) and OS (HR=0.24; 95% CI: 0.13-0.46; P<0.0001) compared with those with a NLR⩾5.,Associations of low NLR with improved survival were confirmed in three validation cohorts of patients.,Our findings show that baseline NLR is strongly and independently associated with outcome of patients treated with ipilimumab, and may serve to identify patients most likely to benefit from this therapy.

Uveal melanoma is the most common primary intraocular malignancy in adults.,Despite successful control of the primary tumor, metastatic disease will ultimately develop in approximately 50% of patients, with the liver being the most common site for metastases.,The median survival for patients with liver metastases is between 6 and 12 months, and no treatment has in randomized trials ever been shown to prolong survival.,A previous phase II trial using isolated hepatic perfusion (IHP) has suggested a 14-month increase in overall survival compared with a historic control group consisting of the longest surviving patients in Sweden during the same time period (26 versus 12 months).,This is the protocol for a multicenter phase III trial randomizing patients with isolated liver metastases of uveal melanoma to IHP or best alternative care (BAC).,Inclusion criteria include liver metastases (verified by biopsy) and no evidence of extra-hepatic tumor manifestations by positron emission tomography-computed tomography (PET-CT).,The primary endpoint is overall survival at 24 months, with secondary endpoints including response rate, progression-free survival, and quality of life.,The planned sample size is 78 patients throughout five years.,Patients with isolated liver metastases of uveal melanoma origin have a short expected survival and no standard treatment option exists.,This is the first randomized clinical trial to evaluate IHP as a treatment option with overall survival being the primary endpoint.,ClinicalTrials.gov registration number: NCT01785316 (registered 1 February 2013).,EudraCT registration number: 2013-000564-29.

TAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.,The growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations.,Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.,Fourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM.,The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733.,The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733.,TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733.,MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines.,Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.,The MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

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

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.

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.

Melanocytic nevi are benign proliferations that sometimes turn into malignant melanoma in a way that is still unclear from the biochemical and genetic point of view.,Diagnostic and prognostic tools are then mostly based on dermoscopic examination and morphological analysis of histological tissues.,To investigate the role of mechanics and geometry in the morpholgical dynamics of melanocytic nevi, we study a computation model for cell proliferation in a layered non-linear elastic tissue.,Numerical simulations suggest that the morphology of the nevus is correlated to the initial location of the proliferating cell starting the growth process and to the mechanical properties of the tissue.,Our results also support that melanocytes are subject to compressive stresses that fluctuate widely in the nevus and depend on the growth stage.,Numerical simulations of cells in the epidermis releasing matrix metalloproteinases display an accelerated invasion of the dermis by destroying the basal membrane.,Moreover, we suggest experimentally that osmotic stress and collagen inhibit growth in primary melanoma cells while the effect is much weaker in metastatic cells.,Knowing that morphological features of nevi might also reflect geometry and mechanics rather than malignancy could be relevant for diagnostic purposes.

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.

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

Metastatic melanoma is the most aggressive form of skin cancer with a median overall survival of less than one year.,Advancements in our understanding of how melanoma evades the immune system as well as the recognition that melanoma is a molecularly heterogeneous disease have led to major improvements in the treatment of patients with metastatic melanoma.,In 2011, the US Food and Drug Administration (FDA) approved two novel therapies for advanced melanoma: a BRAF inhibitor, vemurafenib, and an immune stimulatory agent, ipilimumab.,The success of these agents has injected excitement and hope into patients and clinicians and, while these therapies have their limitations, they will likely provide excellent building blocks for the next generation of therapies.,In this review we will discuss the advantages and limitations of the two new approved agents, current clinical trials designed to overcome these limitations, and future clinical trials that we feel hold the most promise.

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

In Australia, more money is spent on skin cancer than any other malignancy.,Despite this, the mortality rate of melanoma, the deadliest form, has steadily increased over the past 50 years.,Diagnostic imprecision and a lack of complimentary molecular biomarkers are partially responsible for this lack of progress.,Whole-microRNAome profiling was performed on plasma samples from 32 patients with histologically confirmed melanoma and 16 normal controls.,A classification algorithm was trained on these data and independently validated on multiple previously published microRNA data sets, representing (i) melanoma patient- and normal-blood, (ii) melanoma and nevi biopsy tissue, and (iii) cell lines and purified exosomes.,38 circulating microRNAs had biologically and statistically significant differences between melanoma and normal plasma samples (MEL38).,A support vector machine algorithm, trained on these markers, showed strong independent classification accuracy (AUC 0.79-0.94).,A majority of MEL38 genes have been previously associated with melanoma and are known regulators of angiogenesis, metastasis, tumour suppression, and treatment resistance.,MEL38 exhibits disease state specificity and robustness to platform and specimen-type variation.,It has potential to become an objective diagnostic biomarker and improve the precision and accuracy of melanoma detection and monitoring.

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

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

Melanoma is the most highly malignant skin cancer, and nucleotide excision repair (NER) is involved in melanoma susceptibility.,In this analysis of 1042 melanoma patients, we evaluated whether genetic variants of NER genes may predict survival outcome of melanoma patients.,We used genotyping data of 74 tagging single nucleotide polymorphisms (tagSNPs) in eight core NER genes from our genome-wide association study (including 2 in XPA, 14 in XPC, 3 in XPE, 4 in ERCC1, 10 in ERCC2, 8 in ERCC3, 14 in ERCC4, and 19 in ERCC5) and evaluated their associations with prognosis of melanoma patients.,Using the Cox proportional hazards model and Kaplan-Meier analysis, we found a predictive role of XPE rs28720291, ERCC5 rs4150314, XPC rs2470458 and ERCC2 rs50871 SNPs in prognosis of melanoma patients (rs28720291: AG vs.,GG, adjusted hazard ratio [adjHR] = 11.2, 95% confidence interval [CI] 3.04-40.9, P = 0.0003; rs4150314: AG vs.,GG, adjHR = 4.76, 95% CI 1.09-20.8, P = 0.038; rs2470458: AA vs.,AG/GG, adjHR = 2.11, 95% CI 1.03-4.33, P = 0.040; and rs50871: AA vs.,AC/CC adjHR =2.27, 95% CI 1.18-4.35, P = 0.015).,Patients with an increasing number of unfavorable genotypes had dramatically increased death risk.,Genetic variants of NER genes, particularly XPE rs28720291, ERCC5 rs4150314, XPC rs2470458 and ERCC2 rs50871, may independently or jointly modulate survival outcome of melanoma patients.,Because our results were based on a median follow-up of 3 years without multiple test corrections, additional large prospective studies are needed to confirm our findings.

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

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

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

Melanoma is an aggressive malignancy of melanocytes and most commonly arises in the skin.,In 2002, BRAF gene mutations were identified in melanoma, and this finding resulted in the development of several small-molecule molecular inhibitors that specifically targeted the BRAF V600E mutation.,The development of targeted therapies for advanced-stage melanoma, including tyrosine kinase inhibitors (TKIs) of the BRAF (V600E) kinase, vemurafenib and dabrafenib, have been approved for the treatment of advanced melanoma leading to improved clinical outcomes.,However, the development of BRAF inhibitor (BRAFi) resistance has significantly reduced the therapeutic efficacy after prolonged treatment.,Recent studies have identified the molecular mechanisms for BRAFi resistance.,This review aims to describe the impact of BRAFi resistance on the pathogenesis of melanoma, the current status of molecular pathways involved in BRAFi resistance, including intrinsic resistance, adaptive resistance, and acquired resistance.,This review will discuss how an understanding of the mechanisms associated with BRAFi resistance may aid the identification of useful strategies for overcoming the resistance to BRAF-targeted therapy in patients with advanced-stage melanoma.

Vitamin D is a lipid soluble steroid hormone with pleiotropic biological properties, including regulation of cell proliferation, differentiation and apoptosis.,As to these desirable anticancer actions, 1,25-dihydroxyvitamins D and analogs have been reported to inhibit the proliferation and to induce differentiation of a wide variety of cancer cell types, including human malignant melanoma.,However, there is a need for novel and more efficacious vitamin D analogs, and how best to design such is still an open issue.,A series of double point modified (DPM) analogs of 1,25-dihydroxyvitamin D2 (1,25(OH)2D2) induced differentiation of the vitamin D receptor (VDR) positive A375 and VDR negative SK-MEL 188b human malignant melanoma cell lines.,Surprisingly, the dose of 1,25(OH)2D2 required to inhibit the proliferation of the A375 melanoma cell line by was several fold lower than that required in the case of 1,25(OH)2D3.,To evaluate the impact of the modification in the side chain (additional 22-hydroxyl) and in the A-ring (5,6-trans modification), the regular side-chain of vitamin D2 or D3 was retained in the structure of our analogs.,As expected, 5,6-trans modification was advantageous to enhancing the anti-proliferative activity of analogs, but not as a single point modification (SPM).,Very unexpectedly, the additional 22-hydroxyl in the side-chain reduced significantly the anti-proliferative activity of both the natural and 5,6-trans series analogs.,Finally, an induction of pigmentation in melanoma SK-MEL 188b cells was observed to sensitized cells to the effect of vitamin D analogs.

Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease.,The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease.,In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms.,Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis.,Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma.,This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents.,Mechanisms of resistance to therapy are also discussed.

The cellular defense system known as global-genome nucleotide excision repair (GG-NER) safeguards genome stability by eliminating a plethora of structurally unrelated DNA adducts inflicted by chemical carcinogens, ultraviolet (UV) radiation or endogenous metabolic by-products.,Xeroderma pigmentosum group C (XPC) protein provides the promiscuous damage sensor that initiates this versatile NER reaction through the sequential recruitment of DNA helicases and endonucleases, which in turn recognize and excise insulting base adducts.,As a DNA damage sensor, XPC protein is very unique in that it (a) displays an extremely wide substrate range, (b) localizes DNA lesions by an entirely indirect readout strategy, (c) recruits not only NER factors but also multiple repair players, (d) interacts avidly with undamaged DNA, (e) also interrogates nucleosome-wrapped DNA irrespective of chromatin compaction and (f) additionally functions beyond repair as a co-activator of RNA polymerase II-mediated transcription.,Many recent reports highlighted the complexity of a post-translational circuit that uses polypeptide modifiers to regulate the spatiotemporal activity of this multiuse sensor during the UV damage response in human skin.,A newly emerging concept is that stringent regulation of the diverse XPC functions is needed to prioritize DNA repair while avoiding the futile processing of undamaged genes or silent genomic sequences.

The melanocortin-1-receptor (MC1R) gene regulates human pigmentation and is highly polymorphic in populations of European origins.,The aims of this study were to evaluate the association between MC1R variants and the risk of non-melanoma skin cancer (NMSC), and to investigate whether risk estimates differed by phenotypic characteristics.,Data on 3527 NMSC cases and 9391 controls were gathered through the M-SKIP Project, an international pooled-analysis on MC1R, skin cancer and phenotypic characteristics.,We calculated summary odds ratios (SOR) with random-effect models, and performed stratified analyses.,Subjects carrying at least one MC1R variant had an increased risk of NMSC overall, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC): SOR (95%CI) were 1.48 (1.24-1.76), 1.39 (1.15-1.69) and 1.61 (1.35-1.91), respectively.,All of the investigated variants showed positive associations with NMSC, with consistent significant results obtained for V60L, D84E, V92M, R151C, R160W, R163Q and D294H: SOR (95%CI) ranged from 1.42 (1.19-1.70) for V60L to 2.66 (1.06-6.65) for D84E variant.,In stratified analysis, there was no consistent pattern of association between MC1R and NMSC by skin type, but we consistently observed higher SORs for subjects without red hair.,Our pooled-analysis highlighted a role of MC1R variants in NMSC development and suggested an effect modification by red hair colour phenotype.

Cancer cells, including melanoma, often metastasize regionally through lymphatics before metastasizing systemically through the blood1-4; however, the reason for this is unclear.,Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood.,Immunocompromised mice with patient-derived melanomas and immunocompetent mice with mouse melanomas had more melanoma cells per microliter of tumor-draining lymph than tumor-draining blood.,Cells metastasizing through blood, but not lymph, became dependent on the ferroptosis inhibitor GPX4.,Cells pre-treated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection.,We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid, and less free iron, in lymph.,Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumors.,Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than melanoma cells from subcutaneous tumors.,Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.

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.

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

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.

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.

Current therapeutic regimens attempt to eliminate all malignant cells of a melanoma lesion.,Pre-clinical data, however, indicate that melanoma is maintained by a minor subset of cancer cells, which are characterized by CD20 expression.,We attempted to eliminate those cells in a progressing, chemotherapy-refractory metastatic melanoma patient by lesional injections of the anti-CD20 therapeutic antibody rituximab and concomitant dacarbazine treatment, which was ineffective as monotherapy.,Although the frequency of CD20+ melanoma cells within the tumor lesions was initially about 2% and the bulk of tumor cells did not express CD20, rituximab treatment produced lasting remission accompanied by a decline of the melanoma serum marker S-100 to physiological levels.,Detailed in-depth-analyses revealed a switch of serum cytokines from a T helper-2 to a pro-inflammatory T helper-1 cell profile.,Apart from B cell elimination and decline in gammaglobulin levels, no grade 3/4 toxicity related to treatment was observed.,Data provide the first clinical evidence that targeting the minor subset of CD20+ “melanoma sustaining cells” produces regression of chemotherapy-refractory melanoma and highlight the potency of selective cancer cell targeting in the treatment of melanoma.

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

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

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

Germline mutations in the major melanoma susceptibility gene CDKN2A explain genetic predisposition in only 10-40% of melanoma‐prone families.,In our study we comprehensively characterized 488 melanoma cases from 451 non‐CDKN2A/CDK4 families for mutations in 30 established and candidate melanoma susceptibility genes using a custom‐designed targeted gene panel approach.,We identified (likely) pathogenic variants in established melanoma susceptibility genes in 18 families (n = 3 BAP1, n = 15 MITF p.E318K; diagnostic yield 4.0%).,Among the three identified BAP1‐families, there were no reported diagnoses of uveal melanoma or malignant mesothelioma.,We additionally identified two potentially deleterious missense variants in the telomere maintenance genes ACD and TERF2IP, but none in the POT1 gene.,MC1R risk variants were strongly enriched in our familial melanoma cohort compared to healthy controls (R variants: OR 3.67, 95% CI 2.88-4.68, p <0.001).,Several variants of interest were also identified in candidate melanoma susceptibility genes, in particular rare (pathogenic) variants in the albinism gene OCA2 were repeatedly found.,We conclude that multigene panel testing for familial melanoma is appropriate considering the additional 4% diagnostic yield in non‐CDKN2A/CDK4 families.,Our study shows that BAP1 and MITF are important genes to be included in such a diagnostic test.,What's new?,Germline mutations in CDKN2A are major contributors to familial melanoma.,These mutations, however, are responsible for only 10 to 40 percent of genetic susceptibility in melanoma‐prone families.,In this study, 30 established and candidate melanoma susceptibility genes were investigated for associations with the disease in patients from 451 non‐CDKN2A/CDK4 melanoma families.,From the candidate gene panel, (likely) pathogenic variants in BAP1 and MITF were identified in several families, and potentially deleterious variants were identified in the shelterin complex genes ACD and TERF2IP.,These genes appear to play a significant role in familial melanoma predisposition and are therefore promising candidates for incorporation into comprehensive genetic tests.

Both the combination of nivolumab + ipilimumab and single‐agent anti‐PD‐1 immunotherapy have demonstrated survival benefit for patients with advanced melanoma.,As the combination has a high rate of serious side effects, further analyses in randomized trials of combination versus anti‐PD‐1 immunotherapy are needed to understand who benefits most from the combination.,Clinical laboratory values that were routinely collected in randomized studies may provide information on the relative benefit of combination immunotherapy.,To prioritize which clinical laboratory factors to ultimately explore in these randomized studies, we performed a single‐center, retrospective analysis of patients with advanced melanoma who received nivolumab + ipilimumab either as part of a clinical trial (n = 122) or commercial use (n = 87).,Baseline routine laboratory values were correlated with overall survival (OS) and overall response rate (ORR).,Kaplan-Meier estimation and Cox regression were performed.,Median OS was 44.4 months, 95% CI (32.9, Not Reached).,A total of 110 patients (53%) responded (CR/PR).,Significant independent variables for favorable OS included the following: high relative eosinophils, high relative basophils, low absolute monocytes, low LDH, and a low neutrophil‐to‐lymphocyte ratio.,These newly identified factors, along with those previously reported to be associated with anti‐PD‐1 monotherapy outcomes, should be studied in the randomized trials of nivolumab + ipilimumab versus anti‐PD‐1 monotherapies to determine whether they help define the patients who benefit most from the combination versus anti‐PD‐1 alone.

Uveal melanoma (UM) development and progression is correlated with specific molecular changes.,Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q.,Hence, molecular analysis of UM is useful for diagnosis and prognosis.,The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.,A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes.,The status of chromosomes 3 and 8 were analysed with genomic probes.,The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.,Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM.,With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM.,Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM.,Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM.,Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.,Molecular analysis with dPCR is fast and sensitive.,Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples.,Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected.,Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.

Elevated levels of cell-free DNA (cfDNA) are frequently observed in tumor patients.,Activating mutations in exon 4 (R183) and exon 5 (Q209) of GNAQ and GNA11 are almost exclusively found in uveal melanoma, thus providing a highly specific marker for the presence of circulating tumor DNA (ctDNA).,To establish a reliable, noninvasive assay that might allow early detection and monitoring of metastatic disease, we determined the proportion of GNAQ or GNA11 mutant reads in cfDNA of uveal melanoma patients by ultradeep sequencing.,Cell-free DNA from 28 uveal melanoma patients with metastases or extraocular growth was isolated and quantified by real-time polymerase chain reaction (PCR) (7-1550 ng DNA/mL plasma).,GNAQ and GNA11 regions of interest were amplified in 22 of 28 patients and ultradeep sequencing of amplicons was performed to detect even low proportions of mutant reads.,We detected Q209 mutations (2-38% mutant reads) in either GNAQ or GNA11 in the plasma of 9 of 22 metastasized patients.,No correlation between the proportion of mutant reads and the concentration of cfDNA could be detected.,Among the nine ctDNA-positive patients, four had metastases in bone, whereas no metastases were detected in the 13 ctDNA-negative patients at this location (P = 0.025).,Furthermore, ctDNA-positive patients tended to be younger at initial diagnosis and show larger metastases.,The results show that ultradeep amplicon sequencing can be used to detect tumor DNA in plasma of metastasized uveal melanoma patients.,It remains to be shown if this approach can be used for early detection of disseminated tumor disease.

Avelumab is an anti-programmed cell death ligand 1 (PD-L1) antibody approved for treatment of Merkel cell carcinoma (MCC) and locally advanced or metastatic urothelial carcinoma.,It shares a similar side effect profile to other immune checkpoint inhibitors, including immune-related adverse reactions in the skin.,These adverse skin reactions can present as a morbilliform exanthem, lichenoid dermatitis, vitiligo, autoimmune bullous disorder, among others.,We describe a patient with advanced MCC successfully treated with avelumab who developed acute onset diffuse lichen planus-like keratoses (LPLK) at sites of existing seborrheic keratoses (SK) and lentigines.,Histopathology of an affected SK revealed papillomatous epidermal hyperplasia with lichenoid interface changes, numerous dyskeratotic keratinocytes and intermittent hypergranulosis.,The findings resembled lichen planus (LP) arising in an SK.,Onset of the skin symptoms corresponded with an inflammatory cancer response (clinical pseudo-progression), and the eruption improved as overall tumor burden decreased.,The patient’s pruritus was treated with topical steroids and cyrotherapy for individual symptomatic lesions.,Diffuse LPLK is a distinct immune-related reaction pattern associated with PD-L1/PD-1 checkpoint blockade.,This is an important side effect to be aware of as LPLK frequently mimic keratinocytic neoplasms.,Further observation is needed to assess the prevalence and significance of this immune therapy-associated adverse reaction.

Currently, there is no established standard of care for patients with metastatic CSCC.,Based on the mechanisms of CSCC carcinogenesis has been postulated that these tumors may be amenable to PD-1/PD-L1 blockade.,This case illustrates a patient with CSCC with nodal involvement and pulmonary metastases, refractory to two lines of platinum-based regimens and salvage surgery, for whom treatment with nivolumab was recommended.,His clinical course was marked by an atypical pattern of response, with initial reduction of soft tissue/visceral lesions, yet development of new bone findings, followed by overall improvement in subsequent scans and sustained disease control upon treatment continuation.,The case of patient with metastatic CSCC, refractory, received immunotherapy and evolved with atypical pattern of response.

Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse.,Here we show that a subpopulation of BRAFV600 mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity.,Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation.,Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells.,Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5′-untranslated region of some highly translated mRNAs.,Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance.,Melanoma persister cells are tolerant to anti-BRAF and anti-MEK inhibition and can trigger cancer relapse.,Here the authors show that a subset of N6-methyladenosine modified mRNAs is translationally activated in persister cells.,This preferential translation can be abrogated via eIF4A inhibition.

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

Ipilimumab has revolutionized malignant melanoma therapy, but a better understanding of the mechanisms behind treatment response and adverse effects is needed.,In this work, the immune system of ipilimumab treated patients was monitored to investigate potential mechanisms of action that may correlate with treatment outcome.,Blood samples from 43 advanced melanoma patients were taken before, during and at the end of treatment.,Hematological parameters were measured and flow cytometry analysis was performed in fresh samples within two hours of sample collection.,Strong differences in markers CD45RA, CCR7, HLA-DR and CD15 between fresh and cryopreserved samples were observed.,Ipilimumab treatment increased absolute lymphocyte counts, eosinophils, effector T cells and their activation status, whilst diminishing the suppressive side of the immune response, acting on regulatory T cells and myeloid derived suppressor cells (MDSCs).,These effects were visible after one ipilimumab infusion and, regarding eosinophil counts, correlated with onset of adverse events.,Monocytic MDSCs were decreased in response to treatment only in patients with clinical benefit; additionally, patients with a lower frequency of these cells after the first ipilimumab infusion experienced increased overall survival.,CD8 effector memory T cell frequencies at the end of treatment were higher in patients with clinical benefit and positively correlated with survival.,These data show that a clinical response to ipilimumab not only requires reshaping T cell populations, but additionally involves a reduction in suppressive cells such as monocytic MDSCs.,Our work could provide insight on predicting treatment outcome, assisting clinicians in offering the best personalized therapeutic approach.

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment.,Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery.,Tumor and blood biospecimens were obtained at baseline and at surgery.,Flow cytometry and immunohistochemistry for select biomarkers were performed.,Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2).,Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%).,Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2-19.2).,There was a significant decrease in circulating myeloid derived suppressor cells (MDSC).,Greater decrease in circulating monocyte gate MDSC Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS (p = 0.03).,There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034).,Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab.,In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02).,Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3+/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+).,The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS at one year.,Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

Tumour spheroids are common in vitro experimental models of avascular tumour growth.,Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth.,We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids.,We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region.,Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density.,Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size.,Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

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.

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.

Tumor cells evade the immune surveillance by up-regulating surface expression of PD-L1, which interacts with PD-1 on T cells to elicit the immune checkpoint response1,2.,Anti-PD-1 antibodies have shown remarkable promise in treating tumors, including metastatic melanoma2-4.,However, patient response rate is low4,5.,A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy.,Here we report that metastatic melanoma releases a high level of extracellular vesicles (EVs), mostly in the form of exosomes, that carry PD-L1 on their surface.,Interferon-γ (IFN-γ) up-regulates PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumor growth.,In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and changes during the course of anti-PD-1 therapy.,The magnitudes of the early on-treatment increase in circulating exosomal PD-L1, as an indicator of the adaptive response of the tumor cells to T cell re-invigoration, stratifies clinical responders from non-responders.,Our study unveils a mechanism by which tumor cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

There is an increasing global interest to support research areas that can assist in understanding disease and improving patient care.,The National Cancer Institute (NIH) has identified precision medicine-based approaches as key research strategies to expedite advances in cancer research.,The Cancer Moonshot program (https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative) is the largest cancer program of all time, and has been launched to accelerate cancer research that aims to increase the availability of therapies to more patients and, ultimately, to eradicate cancer.,Mass spectrometry-based proteomics has been extensively used to study the molecular mechanisms of cancer, to define molecular subtypes of tumors, to map cancer-associated protein interaction networks and post-translational modifications, and to aid in the development of new therapeutics and new diagnostic and prognostic tests.,To establish the basis for our melanoma studies, we have established the Southern Sweden Malignant Melanoma Biobank.,Tissues collected over many years have been accurately characterized with respect to the tumor and patient information.,The extreme variability displayed in the protein profiles and the detection of missense mutations has confirmed the complexity and heterogeneity of the disease.,It is envisaged that the combined analysis of clinical, histological, and proteomic data will provide patients with a more personalized medical treatment.,With respect to disease presentation, targeted treatment and medical mass spectrometry analysis and imaging, this overview report will outline and summarize the current achievements and status within malignant melanoma.,We present data generated by our cancer research center in Lund, Sweden, where we have built extensive capabilities in biobanking, proteogenomics, and patient treatments over an extensive time period.

Patient: Female, 50,Final Diagnosis: Adrenal insufficiency,Symptoms: Appetite loss • severe fatigue,Medication: -,Clinical Procedure: -,Specialty: Endocrinology and Metabolic,Unusual clinical course,Ipilimumab is a therapeutic human monoclonal antibody that targets the T-cell inhibitory molecule, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and is classified as an immune checkpoint inhibitor that has been shown to improve prognosis in patients with advanced melanoma.,However, several immune-related adverse events have been reported to be associated with ipilimumab Treatment.,A case of acute exacerbation of chronic adrenal insufficiency is presented that highlights that glucocorticoid dosage for patients undergoing steroid treatment at the time of ipilimumab treatment has yet to be established.,A 50-year-old Japanese woman was diagnosed with malignant melanoma on the sole of her right foot.,During her second course of ipilimumab treatment, she developed acute adrenal insufficiency caused by isolated adrenocorticotropic hormone (ACTH) deficiency, which required treatment with oral hydrocortisone.,However, the symptoms of her adrenal insufficiency worsened, and she commenced treatment with 12 courses of nivolumab, a therapeutic human monoclonal antibody that blocks programmed cell death protein 1 (PD-1) on the surface of T-cells.,She did not require corticosteroid support during nivolumab treatment.,This case report highlights the risk of exacerbating adrenal insufficiency during treatment with ipilimumab.,The differences in clinical outcome in this patient between ipilimumab and nivolumab treatment might be explained by the different mechanisms between ipilimumab and nivolumab on immune function.

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.

Interaction between malignant cells and immune cells that reside within the tumor microenvironment (TME) modulate different aspects of tumor development and progression.,Recent works showed the importance of miRNA-containing extracellular vesicles in this crosstalk.,Interested in understanding the interplay between melanoma and immune-related TME cells, we characterized the TCGA’s metastatic melanoma samples according to their tumor microenvironment profiles, HLA-I neoepitopes, transcriptome profile and classified them into three groups.,Moreover, we combined our results with melanoma single-cell gene expression and public miRNA data to better characterize the regulatory network of circulating miRNAs and their targets related to immune evasion and microenvironment response.,The group associated with a worse prognosis showed phenotypic characteristics that favor immune evasion, including a strong signature of suppressor cells and less stable neoantigen:HLA-I complexes.,Conversely, the group with better prognosis was marked by enrichment in lymphocyte and MHC signatures.,By analyzing publicly available melanoma single-cell RNA and microvesicle microRNAs sequencing data we identified circulating microRNAs potentially involved in the crosstalk between tumor and TME cells.,Candidate miRNA/target gene pairs with previously reported roles in tumor progression and immune escape mechanisms were further investigated and demonstrated to impact patient’s overall survival not only in melanoma but across different tumor types.,Our results underscore the impact of tumor-microenvironment interactions on disease outcomes and reveal potential non-invasive biomarkers of prognosis and treatment response.

The contributions of the humoral immune response to melanoma are now widely recognized, with reports of positive prognostic value ascribed to tumor-infiltrating B cells (TIL-B) and increasing evidence of B cells as key predictors of patient response to treatment.,There are disparate views as to the pro- and anti-tumor roles of B cells.,B cells appear to play an integral role in forming tumor-associated tertiary lymphoid structures (TLSs) which can further modulate T cell activation.,Expressed antibodies may distinctly influence tumor regulation in the tumor microenvironment, with some isotypes associated with strong anti-tumor immune response and others with progressive disease.,Recently, B cells have been evaluated in the context of cancer immunotherapy.,Checkpoint inhibitors (CPIs), targeting T cell effector functions, have revolutionized the management of melanoma for many patients; however, there remains a need to accurately predict treatment responders.,Increasing evidence suggests that B cells may not be simple bystanders to CPI immunotherapy.,Mature and differentiated B cell phenotypes are key positive correlates of CPI response.,Recent evidence also points to an enrichment in activatory B cell phenotypes, and the contribution of B cells to TLS formation may facilitate induction of T cell phenotypes required for response to CPI.,Contrastingly, specific B cell subsets often correlate with immune-related adverse events (irAEs) in CPI.,With increased appreciation of the multifaceted role of B cell immunity, novel therapeutic strategies and biomarkers can be explored and translated into the clinic to optimize CPI immunotherapy in melanoma.

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses.,Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads.,To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells.,Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα.,Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions.,Variable regions were then cloned and expressed as human IgG1/k antibodies.,Like the original clone, engineered antibodies from single cells recognized native FRα.,To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells.,Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher.,In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively.,From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads.,This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T‐cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms.,After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multidisciplinary setting.,It has confirmed recognition in well‐known documented conditions such as graft‐vs.‐host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease.,In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology.,This was done using the recognized and approved guidelines of EDF for this task.,All authors had the opportunity to review each contribution as it was added.,These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion.,The guidelines were divided into two parts: PART I covers Cutaneous T‐cell lymphoma, chronic graft‐vs.‐host disease and acute graft‐vs.‐host disease, while PART II will cover scleroderma, solid organ transplantation, Crohn’s disease, use of ECP in paediatric patients, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (L-CTCL) that arises from malignant clonally derived skin-homing CD4+ T cells.,Based on advancements in our understanding of the mechanisms underlying L-CTCL, boosting the suppressed immune response emerges as a promising strategy in SS management.,Immune checkpoint inhibitory molecules have already demonstrated efficacy in a wide spectrum of malignancies.,Currently, agents targeting the programmed death-1 (PD-1) axis are under evaluation in L-CTCL.,Here we investigated the expression of PD-1 and its ligands, PD-L1 and PD-L2 in blood and skin from patients with L-CTCL.,We demonstrate that PD-1 expression is markedly increased on tumor T cells compared to non-tumor CD4+ T cells from SS patients and to CD4+ cells from healthy individuals.,In contrast, PD-L1 shows decreased expression on tumor T cells, while PD-L2 expression is low without significant differences between these groups.,Functional PD-1 blockade in vitro resulted in reduced Th2 phenotype of non-tumor T lymphocytes, but enhanced the proliferation of tumor T cells from SS patients.,Our study sheds some light on the PD-1 axis in both peripheral blood and skin compartments in SS patients, which may be relevant for the treatment of L-CTCL with immune checkpoint inhibitor.

The randomized phase III coBRIM study (NCT01689519) demonstrated improved progression-free survival (PFS) and overall survival (OS) with addition of cobimetinib to vemurafenib compared with vemurafenib in patients with previously untreated BRAFV600 mutation-positive advanced melanoma.,We report long-term follow-up of coBRIM, with at least 5 years since the last patient was randomized.,Eligible patients were randomized 1:1 to receive either oral cobimetinib (60 mg once daily on days 1-21 in each 28-day cycle) or placebo in combination with oral vemurafenib (960 mg twice daily).,495 patients were randomized to cobimetinib plus vemurafenib (n = 247) or placebo plus vemurafenib (n = 248).,Median follow-up was 21.2 months for cobimetinib plus vemurafenib and 16.6 months for placebo plus vemurafenib.,Median OS was 22.5 months (95% CI, 20.3-28.8) with cobimetinib plus vemurafenib and 17.4 months (95% CI, 15.0-19.8) with placebo plus vemurafenib; 5-year OS rates were 31% and 26%, respectively.,Median PFS was 12.6 months (95% CI, 9.5-14.8) with cobimetinib plus vemurafenib and 7.2 months (95% CI, 5.6-7.5) with placebo plus vemurafenib; 5-year PFS rates were 14% and 10%, respectively.,OS and PFS were longest in patients with normal baseline lactate dehydrogenase levels and low tumor burden, and in those achieving complete response.,The safety profile remained consistent with previously published reports.,Extended follow-up of coBRIM confirms the long-term clinical benefit and safety profile of cobimetinib plus vemurafenib compared with vemurafenib monotherapy in patients with BRAFV600 mutation-positive advanced melanoma.

The BRIM-3 trial showed improved progression-free survival (PFS) and overall survival (OS) for vemurafenib compared with dacarbazine in treatment-naive patients with BRAFV600 mutation-positive metastatic melanoma.,We present final OS data from BRIM-3.,Patients were randomly assigned in a 1 : 1 ratio to receive vemurafenib (960 mg twice daily) or dacarbazine (1000 mg/m2 every 3 weeks).,OS and PFS were co-primary end points.,OS was assessed in the intention-to-treat population, with and without censoring of data for dacarbazine patients who crossed over to vemurafenib.,Between 4 January 2010 and 16 December 2010, a total of 675 patients were randomized to vemurafenib (n = 337) or dacarbazine (n = 338, of whom 84 crossed over to vemurafenib).,At the time of database lock (14 August 2015), median OS, censored at crossover, was significantly longer for vemurafenib than for dacarbazine {13.6 months [95% confidence interval (CI) 12.0-15.4] versus 9.7 months [95% CI 7.9-12.8; hazard ratio (HR) 0.81 [95% CI 0.67-0.98]; P = 0.03}, as was median OS without censoring at crossover [13.6 months (95% CI 12.0-15.4) versus 10.3 months (95% CI 9.1-12.8); HR 0.81 (95% CI 0.68-0.96); P = 0.01].,Kaplan-Meier estimates of OS rates for vemurafenib versus dacarbazine were 56% versus 46%, 30% versus 24%, 21% versus 19% and 17% versus 16% at 1, 2, 3 and 4 years, respectively.,Overall, 173 of the 338 patients (51%) in the dacarbazine arm and 175 of the 337 (52%) of those in the vemurafenib arm received subsequent anticancer therapies, most commonly ipilimumab.,Safety data were consistent with the primary analysis.,Vemurafenib continues to be associated with improved median OS in the BRIM-3 trial after extended follow-up.,OS curves converged after ≈3 years, likely as a result of crossover from dacarbazine to vemurafenib and receipt of subsequent anticancer therapies.,NCT01006980.

Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment.,Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib.,Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines.,Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy.,We found that five out of six post-treatment cells had higher migration activity than pretreatment cells.,However, no unequivocal correlation between increased migration and classic epithelial-mesenchymal transition (EMT) markers could be identified.,In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively.,Interestingly, high EGFR expression was associated with elevated migration but not with proliferation.,Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression.,Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition.,Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

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 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.

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

Despite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by residual disease that ultimately results in relapse.,This residual disease is often characterized by non-genetic adaptive resistance, that in melanoma is characterised by altered metabolism.,Here, we examine how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNA interference (RNAi) screen and global gene expression profiling.,Using this systematic approach we demonstrate post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation.,As proof of concept we demonstrate the RNA processing kinase U2AF homology motif kinase 1 (UHMK1) associates with mRNAs encoding metabolism proteins and selectively controls their transport and translation during adaptation to BRAF-targeted therapy.,UHMK1 inactivation induces cell death by disrupting therapy induced metabolic reprogramming, and importantly, delays resistance to BRAF and MEK combination therapy in multiple in vivo models.,We propose selective mRNA processing and translation by UHMK1 constitutes a mechanism of non-genetic resistance to targeted therapy in melanoma by controlling metabolic plasticity induced by therapy.,Different adaptive mechanisms have been reported to reduce the efficacy of mutant BRAF inhibition in melanoma.,Here, the authors show BRAF inhibition induces the translational regulation of metabolic genes leading to acquired therapy resistance.

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.

Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma.,Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi.,miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR.,The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA.,ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting.,Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines.,Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development.,VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA.,miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity.,VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines.,ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance.,At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not.,In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2.,Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity.,Circulating VEGF-A is a promising biomarker for predicting patients’ response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance.,The online version of this article (10.1186/s13046-019-1238-4) contains supplementary material, which is available to authorized users.

It has been shown that the response of V600EBRAF melanoma cells to targeted therapeutics is affected by growth factors.,We have investigated the influence of three different growth factors, bFGF, EGF and HGF used either alone or in combination, on the response of V600EBRAF melanoma cell populations established from surgical specimens to vemurafenib and trametinib, targeting V600EBRAF and MEK1/2, respectively.,We report that proliferation and phenotype of V600EBRAF melanoma cell populations were not detectably influenced by exogenous growth factors.,Neither cell distribution in cell cycle and CCND1 expression nor activity of signaling pathways crucial for melanoma development and maintenance, including the RAF/MEK/ERK pathway, WNT/β-catenin pathway and NF-κB signaling, were affected by the presence of different growth factors.,We furthermore show that vemurafenib and trametinib abrogated the activity of ERK1/2, arrested cells in G0/G1 cell cycle phase, triggered apoptosis, induced changes in the expression of CXCL8, CCND1 and CTGF and the frequency of Ki-67high and CD271high cells.,These effects were, however, similar in the presence of different growth factors.,Interestingly, comparable results were also obtained for melanoma cells grown without exogenous growth factors bFGF, EGF and HGF for a period as long as 4 months prior the drug treatment.,We conclude that the composition or lack of exogenous growth factors bFGF, EGF and HGF do not markedly influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro.,Our results question the necessity of these growth factors in the medium that is used for culturing V600EBRAF melanoma cells.

The incidence of Merkel cell carcinoma (MCC), a rare form of skin cancer with a poor prognosis, has increased in Italy in recent decades.,Avelumab, an anti-programmed death ligand 1 monoclonal antibody, is approved for the treatment of metastatic MCC (mMCC) based on the results of the phase 2 JAVELIN Merkel 200 trial.,The global avelumab expanded access program (EAP) was designed to provide compassionate use of avelumab prior to approval for patients with mMCC who had limited treatment options.,We report findings from a subgroup of Italian patients enrolled in the avelumab EAP.,Eligible patients had mMCC and progressive disease following ≥ 1 prior line of chemotherapy or were ineligible for chemotherapy or clinical trial participation.,Patients received avelumab 10 mg/kg intravenously every 2 weeks.,Treating physicians were provided with an initial 3-month supply of avelumab; resupply was permitted if the patient achieved a complete response, partial response, stable disease, or other clinical benefit per physician assessment.,Safety and efficacy data for the EAP were reported at the treating physician’s discretion.,Between April 1, 2016, and September 14, 2018, 109 requests for avelumab were received from Italy, and 102 were approved.,All but 1 of the approved patients had received ≥ 1 prior line of therapy.,At data cutoff (March 22, 2019), 95 patients had been supplied with avelumab and response data were available for 55 patients.,The objective response rate in response-evaluable patients was 29.1%, including 6 patients (10.9%) who achieved a complete response and 10 patients (18.2%) who achieved a partial response; in the total population supplied with avelumab (n = 95), the proportion who had an objective response was 16.8%.,The median duration of treatment in responding patients was 9.7 months (range, 3.5-41.7 months).,The most frequently reported treatment-related adverse events were infusion-related reaction (single preferred term; n = 3 [3.2%]) and pyrexia (n = 2 [2.1%]).,Results from Italian patients enrolled in the avelumab EAP are consistent with the findings of the JAVELIN Merkel 200 trial and confirm the efficacy and safety of avelumab treatment in this population.

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

Since deterioration of the immune apparatus is closely associated with cancer, we examined the effect of aging on the growth and metastasis of intraocular melanomas in mice.,Murine B16LS9 melanoma cells were transplanted into the posterior compartment of the eye (vitreous chamber) and intraocular tumor growth and development of liver metastases were evaluated in young (8-10 weeks of age) and old (>18 months of age) mice.,Liver metastases were also induced by intrasplenic injection of melanoma cells.,Natural killer (NK) cells from the livers of mice harboring liver metastases were evaluated in vitro for their cytolytic activity.,Tumors grew more rapidly in the eyes of young mice than old mice, yet old mice developed significantly more liver metastases.,Increased liver metastasis in old mice was evident even when melanoma cells were injected intrasplenically as a means of bypassing the influence of the ocular immunosuppressive environment.,Increased liver metastases in old mice correlated with reduced cytolytic activity of liver NK cells.,Lethally irradiated young mice reconstituted with bone marrow from old donors developed significantly more liver metastases than young mice reconstituted with bone marrow from young donors, indicating that bone marrow-derived cells were the root cause of the heightened development of metastases in old mice.,Aging affects the growth and metastasis of intraocular melanomas.,Even though intraocular melanomas grow slower in old mice, the development of liver metastases is exacerbated and correlates with a reduction in liver NK cell activity in the old mouse.

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

N-acetyltransferase 10 (NAT10) has been considered a target for the treatment of human diseases such as cancer and laminopathies; however, its functional role in the biology of melanocytes is questionable.,Using a small molecule or small interfering RNA targeting NAT10, we examined the effect of NAT10 inhibition on melanogenesis and melanoma growth in human and mouse melanoma cells.,Genetic silencing or chemical inhibition of NAT10 resulted in diminished melanin synthesis through the suppression of melanogenesis-stimulating genes such as those encoding dopachrome tautomerase (DCT) and tyrosinase in B16F10 melanoma cells.,In addition, NAT10 inhibition significantly increased cell cycle arrest in S-phase, thereby suppressing the growth and proliferation of malignant melanoma cells in vitro and in vivo.,These results demonstrate the potential role of NAT10 in melanogenesis and melanoma growth through the regulation of microphthalmia-associated transcription factor (MITF) expression and provide a promising strategy for the treatment of various skin diseases (melanoma) and pigmentation disorders (chloasma and freckles).

Melanoma is a cancer with rising incidence and new therapeutics are needed.,For this, it is necessary to understand the molecular mechanisms of melanoma development and progression.,Melanoma differs from other cancers by its ability to produce the pigment melanin via melanogenesis; this biosynthesis is essentially regulated by microphthalmia-associated transcription factor (MITF).,MITF regulates various processes such as cell cycling and differentiation.,MITF shows an ambivalent role, since high levels inhibit cell proliferation and low levels promote invasion.,Hence, well-balanced MITF homeostasis is important for the progression and spread of melanoma.,Therefore, it is difficult to use MITF itself for targeted therapy, but elucidating its complex regulation may lead to a promising melanoma-cell specific therapy.,We systematically analyzed the regulation of MITF with a novel established transcription factor based gene regulatory network model.,Starting from comparative transcriptomics analysis using data from cells originating from nine different tumors and a melanoma cell dataset, we predicted the transcriptional regulators of MITF employing ChIP binding information from a comprehensive set of databases.,The most striking regulators were experimentally validated by functional assays and an MITF-promoter reporter assay.,Finally, we analyzed the impact of the expression of the identified regulators on clinically relevant parameters of melanoma, i.e. the thickness of primary tumors and patient overall survival.,Our model predictions identified SOX10 and SOX5 as regulators of MITF.,We experimentally confirmed the role of the already well-known regulator SOX10.,Additionally, we found that SOX5 knockdown led to MITF up-regulation in melanoma cells, while double knockdown with SOX10 showed a rescue effect; both effects were validated by reporter assays.,Regarding clinical samples, SOX5 expression was distinctively up-regulated in metastatic compared to primary melanoma.,In contrast, survival analysis of melanoma patients with predominantly metastatic disease revealed that low SOX5 levels were associated with a poor prognosis.,MITF regulation by SOX5 has been shown only in murine cells, but not yet in human melanoma cells.,SOX5 has a strong inhibitory effect on MITF expression and seems to have a decisive clinical impact on melanoma during tumor progression.,The online version of this article (doi:10.1186/s12920-016-0170-0) contains supplementary material, which is available to authorized users.

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

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

Increased or decreased expression of LIF receptor (LIFr) has been reported in several human cancers, including skin cancer, but its role in melanoma is unknown.,In this study, we investigated the expression pattern of LIFr in melanoma and assessed its prognostic value.,Using tissue microarrays consisting of 441 melanomas and 96 nevi, we found that no normal nevi showed high LIFr expression.,LIFr staining was significantly increased in primary melanoma compared to dysplastic nevi (P = 0.0003) and further increased in metastatic melanoma (P = 0.0000).,Kaplan-Meier survival curve and univariate Cox regression analyses showed that increased expression of LIFr was correlated with poorer 5-year patient survival (overall survival, P = 0.0000; disease-specific survival, P = 0.0000).,Multivariate Cox regression analyses indicated that increased LIFr expression was an independent prognostic marker for primary melanoma (P = 0.036).,LIFr knockdown inhibited melanoma cell migration in wound healing assays and reduced stress fiber formation.,LIFr knockdown correlated with STAT3 suppression, but not YAP, suggesting that LIFr activation might stimulate melanoma cell migration through the STAT3 pathway.,Our data indicate that strong LIFr expression identifies potentially highly malignant melanocytic lesions at an early stage and LIFr may be a potential target for the development of early intervention therapeutics.

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.

Melanoma is an aggressive malignancy of melanocytes and most commonly arises in the skin.,In 2002, BRAF gene mutations were identified in melanoma, and this finding resulted in the development of several small-molecule molecular inhibitors that specifically targeted the BRAF V600E mutation.,The development of targeted therapies for advanced-stage melanoma, including tyrosine kinase inhibitors (TKIs) of the BRAF (V600E) kinase, vemurafenib and dabrafenib, have been approved for the treatment of advanced melanoma leading to improved clinical outcomes.,However, the development of BRAF inhibitor (BRAFi) resistance has significantly reduced the therapeutic efficacy after prolonged treatment.,Recent studies have identified the molecular mechanisms for BRAFi resistance.,This review aims to describe the impact of BRAFi resistance on the pathogenesis of melanoma, the current status of molecular pathways involved in BRAFi resistance, including intrinsic resistance, adaptive resistance, and acquired resistance.,This review will discuss how an understanding of the mechanisms associated with BRAFi resistance may aid the identification of useful strategies for overcoming the resistance to BRAF-targeted therapy in patients with advanced-stage melanoma.

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

The emergence of human intratumoural immunotherapy (HIT-IT) is a major step forward in the management of unresectable melanoma.,The direct injection of treatments into melanoma lesions can cause cell lysis and induce a local immune response, and might be associated with a systemic immune response.,Directly injecting immunotherapies into tumours achieves a high local concentration of immunostimulatory agent while minimising systemic exposure and, as such, HIT-IT agents are associated with lower toxicity than systemic immune checkpoint inhibitors (CPIs), enabling their potential use in combination with other therapies.,Consequently, multiple HIT-IT agents, including oncolytic viruses, pattern-recognition receptor agonists, injected CPIs, cytokines and immune glycolipids, are under investigation.,This review considers the current clinical development status of HIT-IT agents as monotherapy and in combination with systemic CPIs, and the practical aspects of administering and assessing the response to these agents.,The future of HIT-IT probably lies in its use in combination with systemic CPIs; data from Phase 2 trials indicate a synergy between HIT-IT and CPIs.,Data also suggest that the addition of HIT-IT to a CPI might generate responses in CPI-refractory tumours, thereby overcoming resistance and addressing a current unmet need in unresectable and metastatic melanoma for treatment options following progression after CPI treatment.

One of the problems in antigen-specific cancer immunotherapy is the low density of the tumor antigen-derived peptide endogenously presented on tumor cell surface major histocompatibility complex class I molecules.,To overcome this, we are engaged in research on peptide intra-tumor injection to enhance tumor cell antigenicity.,In in vivo studies using immunodeficient mice, the peptide injected into a solid mass of subcutaneous tumor was revealed to be loaded onto human leukocyte antigen class I molecules of tumor cells.,In a peptide vaccine model and an adoptive cell transfer model using C57BL/6 mice, peptide intra-tumor injection was effective in terms of tumor growth inhibition and prolongation of survival time.,Moreover, an antigen-spreading effect was detected after peptide intra-tumor injection.,Peptide intra-tumor injection is an effective method of enhancing tumor cell antigenicity.,It can induce additional peptide loading onto tumor cells, making tumor cells more antigenic for specific cytotoxic T-lymphocyte activity.,Peptide intra-tumor injection may be a useful option for improvement of antigen-specific immunotherapy against solid tumors.

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

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

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine cutaneous malignancy with poor prognosis.,In Europe, approved systemic therapies are limited to the PD-L1 inhibitor avelumab.,For avelumab-refractory patients, efficient and safe treatment options are lacking.,At three different sites in Germany, clinical and molecular data of patients with metastatic MCC being refractory to the PD-L1 inhibitor avelumab and who were later on treated with combined IPI/NIVO were retrospectively collected and evaluated.,Five patients treated at three different academic sites in Germany were enrolled.,Three out of five patients investigated for this report responded to combined IPI/NIVO according to RECIST 1.1.,Combined immunotherapy was well tolerated without any grade II or III immune-related adverse events.,Two out of three responders to IPI/NIVO received platinum-based chemotherapy in between avelumab and combined immunotherapy.,In this small retrospective study, we observed a high response rate and durable responses to subsequent combined immunotherapy with IPI/NIVO in avelumab-refractory metastatic MCC patients.,In conclusion, our data suggest a promising activity of second- or third-line PD-1- plus CTLA-4-blockade in patients with anti-PD-L1-refractory MCC.

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 aim of this study was to compare the efficacy and safety of interferon (IFN) combined with dacarbazine (DTIC) (experimental group) versus DTIC alone (control group) in cutaneous malignant melanoma.,After searching all available databases, eligible articles were identified and subjected to quality assessment.,Meta-analysis was performed using RevMan 5.3; combined relative risk (RR) and 95% confidence intervals (95% CIs) were calculated for survival rates, response rates, and adverse events.,Eight randomized controlled trials published between 1990 and 2014 involving 795 patients were included in the meta-analysis.,Compared with DTIC alone, IFN combined with DTIC significantly increased the overall response rate (RR = 1.59, 95% CI 1.21-2.08, P = 0.0008),the complete response rate (RR = 3.30, 95% CI 1.89-5.76, P < 0.0001), 2-year survival (RR = 1.59, 95% CI 0.99-2.54, P = 0.050) grade ≥3 hematologic toxicity (RR = 2.30, 95% CI 1.32-4.02, P = 0.003), neurotoxicity (RR = 18.15, 95% CI 5.34-61.74, P < 0.00001), and flu-like symptoms (RR = 6.31, 95% CI 1.95-20.39, P = 0.002).,The partial response rate, grade ≥3 nausea and vomiting, treatment-related, and 1- and 3-year survival were not significantly different between IFN combined with DTIC and DTIC alone.,IFN combined with DTIC may moderately improve the complete response rate, but increases the incidence of adverse events and has no significant effect on 1- and 3-year survival in cutaneous malignant melanoma.

Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study.,In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed.,Thirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously.,Tumor BRAFV600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR).,Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples.,The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation.,One patient had a RECIST partial response (PR) lasting 175 days.,Three patients experienced stable disease (SD) with a mean duration of 37 weeks.,Routine BRAFV600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors.,No correlation was seen between BRAFV600E mutational status and clinical activity.,No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples.,Sorafenib monotherapy has limited activity in advanced melanoma patients.,BRAFV600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib.,Clinical Trials.gov NCT00119249