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The full repertoire of human microRNAs (miRNAs) that could distinguish common (benign) nevi from cutaneous (malignant) melanomas remains to be established.,In an effort to gain further insight into the role of miRNAs in melanoma, we applied Illumina next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of nevi, thick primary (>4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines (both primary and metastatic) (n = 28).,From this data representing 698 known miRNAs, we defined a set of top-40 list, which properly classified normal from cancer; also confirming 23 (58%) previously discovered miRNAs while introducing an additional 17 (42%) known and top-15 putative novel candidate miRNAs deregulated during melanoma progression.,Surprisingly, the miRNA signature distinguishing specimens of melanoma from nevus was significantly different than that of melanoma cell lines from melanocytes.,Among the top list, miR-203, miR-204-5p, miR-205-5p, miR-211-5p, miR-23b-3p, miR-26a-5p and miR-26b-5p were decreased in melanomas vs. nevi.,In a validation cohort (n = 101), we verified the NGS results by qRT-PCR and showed that receiver-operating characteristic curves for miR-211-5p expression accurately discriminated invasive melanoma (AUC = 0.933), melanoma in situ (AUC = 0.933) and dysplastic (atypical) nevi (AUC = 0.951) from common nevi.,Target prediction analysis of co-transcribed miRNAs showed a cooperative regulation of key elements in the MAPK signaling pathway.,Furthermore, we found extensive sequence variations (isomiRs) and other non-coding small RNAs revealing a complex melanoma transcriptome.,Deep-sequencing small RNAs directly from clinically defined specimens provides a robust strategy to improve melanoma diagnostics.

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer.,A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells.,Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells.,To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities.,This screening revealed two major cohorts of differentially expressed miRNAs.,We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive.,This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma.,Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice.,Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings.,Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide.,It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required.,In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model.,DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis.,Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound.,Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells.,DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation.,Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs.,These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

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

The prognosis of melanoma patients is highly variable due to multiple factors conditioning immune response and driving metastatic progression.,In this study, we have correlated the expression of immune-related lncRNAs with patient survival, developed a prognostic model, and investigated the characteristics of immune response in the diverse groups.,The gene expression profiles and prognostic information of 470 melanoma patients were downloaded from TCGA database.,Significantly predictive lncRNAs were identified by multivariate Cox regression analyses, and a prognostic model based on these variables was constructed to predict survival.,Kaplan-Meier curves were plotted to estimate overall survival.,The predictive accuracy of the model was evaluated by the area under the ROC curve (AUC).,Principal component analysis was used to observe the distribution of immune-related genes.,CIBERSORT and ESTIMATE were used to evaluate the composition of immune cells and the immune microenvironment.,Eight immune-related lncRNAs were determined to be prognostic by multivariate COX regression analysis.,The patient scores were calculated and divided into high- and low-risk groups.,The model could effectively predict the prognosis in patients of different stages.,The AUC of the model is 0.784, which was significantly higher than that of the other variables.,There were significant differences in the distribution of immune-related genes between two groups; the immune score and immune function enrichment score were higher in the low risk group.

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

Cutaneous squamous cell carcinoma (cuSCC) comprises 15-20% of all skin cancers, accounting for over 700,000 cases in USA annually.,Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK).,To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model.,We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition.,Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible.,Cutaneous squamous cell of the skin is a common neoplasm that frequently arises from precancerous actinic keratoses.,Here, the authors carry out genomic analysis on matched sets of human lesions and compare with those in ultraviolet treated mice and identify conserved drivers of tumour development.

The precise mechanisms governing invasion at the leading edge of SCC and its subsequent metastasis are not fully understood.,We aimed to define the cancer related molecular changes that distinguish non-invasive tumor from invasive SCC.,To this end, we combined laser capture microdissection with cDNA microarray analysis.,We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared to normal epidermis.,There were 383 up- and 354 down-regulated genes in the “invasion set.”,SCC invasion was characterized by aberrant expression of various proteolytic molecules.,We noted increased expression of MMP7 and IL-24 in invasive SCC.,IL-24 induced the expression of MMP7 in SCC cells in culture.,In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture.,These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, though IL-24 has been suggested to have anti-tumor growth effects in other cancer types.,Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer.

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.

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

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer.,However, the function of Chi3l1 in T cell and its clinical implications are largely unknown.,Here we show that Chi3l1 expression was increased in activated T cells, especially in Th2 cells.,In addition, Chi3l1-deficient T cells are hyper-responsive to TcR stimulation and are prone to differentiating into Th1 cells.,Chi3l1-deficient Th1 cells show increased expression of anti-tumor immunity genes and decreased Th1 negative regulators.,Deletion of Chi3l1 in T cells in mice show reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung.,Furthermore, silencing of Chi3l1 expression in the lung using peptide-siRNA complex (dNP2-siChi3l1) efficiently inhibit lung metastasis with enhanced Th1 and CTL responses.,Collectively, this study demonstrates Chi3l1 is a regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.,Chitinase-3-like-1 (Chi3l1) has been involved in inflammation and pulmonary metastasis.,Here the authors show that Chi3l1 inhibits the T cell response by negatively regulating their activation and that, in a mouse model of melanoma, T cell-targeted silencing of Chi3l1 results in reduced lung metastasis.

Members of the 18 glycosyl hydrolase (GH 18) gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders.,This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1), which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling.,However, receptors that mediate the effects of GH 18 moieties have not been defined.,Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2) and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex.,We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms.,Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses.

Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with metastatic potential.,Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are novel regulators of gene expression.,To identify mRNAs, lncRNAs and circRNAs, which can be involved in cSCC, RNA-seq was performed on nine cSCCs and seven healthy skin samples.,Representative transcripts were validated by NanoString nCounter assays using an extended cohort, which also included samples from pre-cancerous skin lesions (actinic keratosis). 5,352 protein-coding genes, 908 lncRNAs and 55 circular RNAs were identified to be differentially expressed in cSCC.,Targets of 519 transcription factors were enriched among differentially expressed genes, 105 of which displayed altered level in cSCCs, including fundamental regulators of skin development (MYC, RELA, ETS1, TP63).,Pathways related to cell cycle, apoptosis, inflammation and epidermal differentiation were enriched.,In addition to known oncogenic lncRNAs (PVT1, LUCAT1, CASC9), a set of skin-specific lncRNAs were were identified to be dysregulated.,A global downregulation of circRNAs was observed in cSCC, and novel skin-enriched circRNAs, circ_IFFO2 and circ_POF1B, were identified and validated.,In conclusion, a reference set of coding and non-coding transcripts were identified in cSCC, which may become potential therapeutic targets or biomarkers.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and usually progresses from a UV-induced precancerous lesion termed actinic keratosis (AK).,Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain partially understood.,Here, we use Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status in healthy, AK and cSCC epidermis samples.,Importantly, we show that AK methylation patterns already display classical features of cancer methylomes and are highly similar to cSCC profiles.,Further analysis identifies typical features of stem cell methylomes, such as reduced DNA methylation age, non-CpG methylation, and stem cell-related keratin and enhancer methylation patterns.,Interestingly, this signature is detected only in half of the samples, while the other half shows patterns more closely related to healthy epidermis.,These findings suggest the existence of two subclasses of AK and cSCC emerging from distinct keratinocyte differentiation stages.,Cutaneous squamous cell carcinoma (cSCC) is a skin cancer that normally progresses from UV-induced actinic keratosis (AK).,Here, the authors investigate the epigenomics of cSCC and highlight two distinct subclasses of AK and cSCC originating from distinct keratinocyte differentiation stages.

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

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.

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

Supplemental Digital Content is available in the text.,The role of tumor-associated macrophages (TAMs) in cutaneous melanoma is controversial.,TAMs include immunogenic and immunosuppressive subtypes, and have distinct functions according to their microanatomical localization.,Our aim was to investigate TAMs in benign, premalignant, and malignant melanocytic lesions to determine possible associations with tumor progression and clinicopathological characteristics.,In total, 184 tissue samples, including benign and dysplastic nevi, in-situ melanomas, superficial (Breslow’s depth <1 mm), and deep (Breslow’s depth >4 mm) invasive melanomas and lymph node metastases, were analyzed for macrophage content.,Samples were stained immunohistochemically for CD68 and CD163, representing all TAMs and M2-macrophages, respectively.,Macrophages were counted by hotspot analysis, and assessed semiquantitatively from the tumor cell nests and stromal component of malignant cases.,CD68+ and CD163+ TAMs were more abundant in invasive melanomas compared with benign nevi.,The proportion of TAMs in the tumor nests was higher in deep melanomas and lymph node metastases compared with superficially invasive melanomas.,High amounts of CD68+ macrophages in tumor cell nests were associated with recurrence, whereas low CD163+ macrophage proportion in tumor stroma was associated with recurrence and in primary melanomas also with poor overall survival.,TAMs seem to promote tumor progression in cutaneous melanoma.,In particular, CD68+ TAMs and their abundance in tumor nests were associated with poor prognostic factors.,However, the correlation of low stromal CD163+ TAM proportion with a poor prognosis indicates that the role of TAMs depends on their subtype and microanatomical localization.

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

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

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type.,To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors.,Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2.,Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8.,SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas.,TERT aberrations and ATRX mutations are associated with alterations in telomere length.,Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.,Mucosal melanomas are challenging to treat partly because so little is known about the genetic drivers underpinning them.,Here, the authors perform a genomic landscape analysis of samples collected from three continents, revealing a potential role for CDK4/6 or MEK inhibition in the treatment of the disease.

While a number of autosomal dominant and autosomal recessive cancer syndromes have an associated spectrum of cancers, the prevalence and variety of cancer predisposition mutations in patients with multiple primary cancers have not been extensively investigated.,An understanding of the variants predisposing to more than one cancer type could improve patient care, including screening and genetic counselling, as well as advancing the understanding of tumour development.,A cohort of 57 patients ascertained due to their cutaneous melanoma (CM) diagnosis and with a history of two or more additional non-cutaneous independent primary cancer types were recruited for this study.,Patient blood samples were assessed by whole exome or whole genome sequencing.,We focussed on variants in 525 pre-selected genes, including 65 autosomal dominant and 31 autosomal recessive cancer predisposition genes, 116 genes involved in the DNA repair pathway, and 313 commonly somatically mutated in cancer.,The same genes were analysed in exome sequence data from 1358 control individuals collected as part of non-cancer studies (UK10K).,The identified variants were classified for pathogenicity using online databases, literature and in silico prediction tools.,No known pathogenic autosomal dominant or previously described compound heterozygous mutations in autosomal recessive genes were observed in the multiple cancer cohort.,Variants typically found somatically in haematological malignancies (in JAK1, JAK2, SF3B1, SRSF2, TET2 and TYK2) were present in lymphocyte DNA of patients with multiple primary cancers, all of whom had a history of haematological malignancy and cutaneous melanoma, as well as colorectal cancer and/or prostate cancer.,Other potentially pathogenic variants were discovered in BUB1B, POLE2, ROS1 and DNMT3A.,Compared to controls, multiple cancer cases had significantly more likely damaging mutations (nonsense, frameshift ins/del) in tumour suppressor and tyrosine kinase genes and higher overall burden of mutations in all cancer genes.,We identified several pathogenic variants that likely predispose to at least one of the tumours in patients with multiple cancers.,We additionally present evidence that there may be a higher burden of variants of unknown significance in ‘cancer genes’ in patients with multiple cancer types.,Further screens of this nature need to be carried out to build evidence to show if the cancers observed in these patients form part of a cancer spectrum associated with single germline variants in these genes, whether multiple layers of susceptibility exist (oligogenic or polygenic), or if the occurrence of multiple different cancers is due to random chance.

Although pilomatrixomas are frequently encountered by dermatologists and pathologists in the differential diagnosis of head and neck lesions, this is not usually the case among head and neck surgeons.,A pilomatrixoma (calcifying epithelioma of Malherbe) is a benign tumour of the hair matrix cells.,Histologically it is characterised by the presence of ghost cells, basophilic cells and foreign body cells.,It may sometimes be difficult to histologically distinguish it from its malignant counterpart, the pilomatrix carcinoma.,We report an interesting case of an ulcerated pilomatrixoma of the pinna in a middle-aged Caucasian female.,A 46-year-old Caucasian female presented with a one-month history of tender brownish lump on the pinna.,Initially it was thought to represent a pyogenic granuloma.,The lesion was treated by wide circular excision.,Histopathological evaluation reported a benign calcifying epithelioma of Malherbe.,A search of the world’s literature has led us to believe that this is a rare case of a calcifying epithelioma of Malherbe of the pinna.,The rapid growth and ulcerative nature of this tumour makes this case even more unique.

Calcifying epithelioma of Malherbe, or Pilomatricoma, is considered an uncommon cutaneous neoplasia, normally occurring in children as a solitary, firm, asymptomatic, hard, subcutaneous, slowly growing nodule on the face, neck, or proximal upper extremity.,In literature, two Pilomatricoma ultrasound patterns are described: the totally calcified nodule and the hypoechoic nodule with internal calcific foci.,High frequency ultrasound has not yet been applied for routine diagnosis of Pilomatricoma.,The aim of the study was to retrospectively identify specific ultrasound features.,We retrieved 124 histologically Pilomatricoma cases: 28 patients with 32 lesions were preoperatively evaluated with ultrasound.,22/32 have shown a solid formation, hypoechoic, with a sharp outline.,Of these 22, 10 lesions were completely calcifying and 12 partially calcified.,In 3/32 lesions with uncertain diagnosis, ultrasounds showed a complex/mixed pattern with pseudo-fluid areas and microspots. 7/32 lesions with US different diagnosis included 3 complex lesions, 2 cystic lesions and 2 solid nodular lesions.,In addition to well-known ultrasound patterns (completely calcified and partially calcified) we identified three new, not yet described, patterns that constitute the 31% of the cases: complex, pseudocistyc and pseudotumoral.

Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response.,Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks.,Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane.,This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells.,These features resemble the initial stages of invasive melanoma.,Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts.,This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment.,Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade.,Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163+PD-L1+ M2-like phenotype upon IL-10 blockade.,Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.,The online version of this article (10.1007/s00262-020-02626-4) contains supplementary material, which is available to authorized users.

Immune checkpoint inhibitors and adoptive cell transfer (ACT) of autologous tumor-infiltrating T cells have shown durable responses in patients with melanoma.,To study ACT and immunotherapies in a humanized model, we have developed PDXv2.0 - a melanoma PDX model where tumor cells and tumor-infiltrating T cells from the same patient are transplanted sequentially in non-obese diabetic/severe combined immune-deficient/common gamma chain (NOG/NSG) knockout mouse.,Key to T-cell survival/effect in this model is the continuous presence of interleukin-2 (IL-2).,Tumors that grow in PDXv2.0 are eradicated if the autologous tumor cells and T cells come from a patient that exhibited an objective response to ACT in the clinic.,However, T cells from patients that are non-responders to ACT cannot kill tumor cells in PDXv2.0.,Taken together, PDXv2.0 provides the potential framework to further model genetically diverse human cancers for assessing the efficacy of immunotherapies as well as combination therapies.,Combining different types of immune therapies might benefit certain patients.,Here, the authors develop an autologous immune-humanized melanoma mouse model that allows the preclinical assessment of cancer cell-T cell interactions from each individual patient and the benefits of immunotherapies combinations.

Accumulating evidences indicated that plasmacytoma variant translocation 1 (PVT1) plays vital roles in several cancers.,However, the expression, functions, and clinical values of PVT1 in melanoma are still unknown.,In this study we measured the expression of PVT1 in clinical tissues and serum samples and explored the diagnostic value of PVT1 for melanoma and the effects of PVT1 on melanoma cell proliferation, cell cycle, and migration.,Our results, combined with publicly available PVT1 expression data, revealed that PVT1 is upregulated in melanoma tissues compared with nonneoplastic nevi tissues.,Serum PVT1 level is significantly increased in melanoma patients compared with age and gender-matched nonmelanoma controls with melanocytic nevus.,Receiver operating characteristic curve analyses revealed that serum PVT1 level could sensitively discriminate melanoma patients from controls.,Furthermore, serum PVT1 level indicted melanoma dynamics.,Functional experiments showed that overexpression of PVT1 promotes melanoma cells proliferation, cell cycle progression, and migration, while depletion of PVT1 significantly inhibits melanoma cells proliferation, cell cycle progression, and migration.,Collectively, our results indicate that PVT1 functions as an oncogene in melanoma and could be a potential diagnostic biomarker and therapeutic target for melanoma.

Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis.,To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner.,SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG).,When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG).,Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.

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

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

In this study, we incorporate analyses of genome-wide sequence and structural alterations with pre- and on-therapy transcriptomic and T cell repertoire features in immunotherapy-naive melanoma patients treated with immune checkpoint blockade.,Although tumor mutation burden is associated with improved treatment response, the mutation frequency in expressed genes is superior in predicting outcome.,Increased T cell density in baseline tumors and dynamic changes in regression or expansion of the T cell repertoire during therapy distinguish responders from non-responders.,Transcriptome analyses reveal an increased abundance of B cell subsets in tumors from responders and patterns of molecular response related to expressed mutation elimination or retention that reflect clinical outcome.,High-dimensional genomic, transcriptomic, and immune repertoire data were integrated into a multi-modal predictor of response.,These findings identify genomic and transcriptomic characteristics of tumors and immune cells that predict response to immune checkpoint blockade and highlight the importance of pre-existing T and B cell immunity in therapeutic outcomes.,Unmet need for integrated molecular models that interpret immunotherapy responseGenomic, transcriptomic, and T and B cell sequence data integration by machine learningT cell dynamism is a hallmark of response to immune checkpoint blockadeThe combined contributions of B, T, and tumor cell features predict clinical outcome,Unmet need for integrated molecular models that interpret immunotherapy response,Genomic, transcriptomic, and T and B cell sequence data integration by machine learning,T cell dynamism is a hallmark of response to immune checkpoint blockade,The combined contributions of B, T, and tumor cell features predict clinical outcome,Anagnostou et al. integrate genomic, expression, and immune cell repertoire analyses to gain insights into the crosstalk between cancer and immune cells during immunotherapy for melanoma.,These findings suggest that the complex phenotype of tumor immune infiltrates combined with genomic features of tumor cells are relevant for determining clinical outcomes.

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical.,Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated.,Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data.,Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism.,We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process.,We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants.,Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets.,Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.

Melanoma aggressiveness determines its growth and metastatic potential.,This study aimed at identifying new molecular pathways controlling melanoma cell malignancy.,Ten metastatic melanoma cell lines were characterized by their proliferation, migration and invasion capabilities.,The most representative cells were also characterized by spheroid formation assay, gene- and protein- expression profiling as well as cytokines secretion and the most relevant pathways identified through bioinformatic analysis were tested by in silico transcriptomic validation on datasets generated from biopsies specimens of melanoma patients.,Further, matrix metalloproteases (MMPs) activity was tested by zymography assays and TNF-alpha role was validated by anti-TNF cell-treatment.,An aggressiveness score (here named Melanoma AGgressiveness Score: MAGS) was calculated by measuring proliferation, migration, invasion and cell-doubling time in10human melanoma cell lines which were clustered in two distinct groups, according to the corresponding MAGS.,SK-MEL-28 and A375 cell lines were selected as representative models for the less and the most aggressive phenotype, respectively.,Gene-expression and protein expression data were collected for SK-MEL-28 and A375 cells by Illumina-, multiplex x-MAP-and mass-spectrometry technology.,The collected data were subjected to an integrated Ingenuity Pathway Analysis, which highlighted that cytokine/chemokine secretion, as well as Cell-To-Cell Signaling and Interaction functions as well as matrix metalloproteases activity were significantly different in these two cell types.,The key role of these pathways was then confirmed by functional validation.,TNF role was confirmed by exposing cells to the anti-TNF Infliximab antibody.,Upon such treatment melanoma cells aggressiveness was strongly reduced.,Metalloproteases activity was assayed, and their role was confirmed by comparing transcriptomic data from cutaneous melanoma patients (n = 45) and benign nevi (n = 18).,Inflammatory signals such as TNF and MMP-2 activity are key intrinsic players to determine melanoma cells aggressiveness suggesting new venue sin the identification of novel molecular targets with potential therapeutic relevance.,The online version of this article (10.1186/s13046-018-0982-1) contains supplementary material, which is available to authorized users.

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

In the search for novel, natural melanogenesis inhibitors, a new sesquiterpene, inularin, was isolated from the flowers of Inula britannica, and the structure was determined using spectroscopic and chemical methods.,The antimelanogenic effects of inularin on B16F10 melanoma cells and zebrafish embryos were evaluated.,Inularin dose-dependently reduced melanocyte-stimulating hormone-induced melanin production and L-DOPA oxidation in B16F10 cells.,Zebrafish embryos were used to confirm the antimelanogenic activity.,Inularin significantly decreased the pigmentation of embryos compared with untreated controls.

Sphere formation in conditioned serum‐free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor (tumorospheres) is considered useful for the enrichment of cancer stem‐like cells, also known as tumor‐initiating cells.,We used a gene expression microarray to investigate the gene expression profile of melanoma cancer stem‐like cells (MCSLCs).,The results showed that MCSLCs highly expressed the following Notch signaling pathway molecules: Notch3 (NM_008716), Notch4 (NM_010929), Dtx4 (NM_172442), and JAG2 (NM_010588).,Immunofluorescence staining showed tumorosphere cells highly expressed Notch4.,Notch4high B16F10 cells were isolated by FACS, and Western blotting showed that high Notch4 expression is related to the expression of epithelial-mesenchymal transition (EMT)‐associated proteins.,Reduced invasive and migratory properties concomitant with the downregulation of the EMT markers Twist1, vimentin, and VE‐cadherin and the overexpression of E‐cadherin was observed in human melanoma A375 and MUM‐2B cells.,In these cells, Notch4 was also downregulated, both by Notch4 gene knockdown and by application of the γ‐secretase inhibitor, DAPT.,Mechanistically, the re‐overexpression of Twist1 by the transfection of cells with a Twist1 expression plasmid led to an increase in VE‐cadherin expression and a decrease in E‐cadherin expression.,Immunohistochemical analysis of 120 human melanoma tissues revealed a significant correlation between the high expression of Notch4 and the metastasis of melanoma.,Taken together, our findings indicate that Notch4+ MCSLCs trigger EMT and promote the metastasis of melanoma cells.

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

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.

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

The initial use of BRAF targeted therapeutics in clinical trials has demonstrated encouraging responses in melanoma patients, although a rise in drug-resistant cells capable of advancing malignant disease has been described.,The current study uses BRAFV600E expressing WM793 melanoma cells to derive data aimed at investigating the molecular determinant of cell invasion following treatment with clinical BRAF inhibitors.,Small-molecule inhibitors targeting BRAF reduced MEK1/2-ERK1/2 pathway activation and cell survival; yet, viable cell subpopulations persisted.,The residual cells exhibited an elongated cell shape, prominent actin stress fibers and retained the ability to invade 3-D dermal-like microenvironments.,BRAF inhibitor treatments were associated with reduced expression of RND3, an antagonist of RHOA activation, and elevated RHOA-dependent signaling.,Restoration of RND3 expression or RHOA knockdown attenuated the migratory ability of residual cells without affecting overall cell survival.,The invasive ability of BRAF inhibitor treated cells embedded in collagen gels was diminished following RND3 re-expression or RHOA depletion.,Conversely, melanoma cell movement in the absence of BRAF inhibition was unaffected by RND3 expression or RHOA depletion.,These data reveal a novel switch in the requirement for RND3 and RHOA in coordinating the movement of residual WM793 cells that are initially refractive to BRAF inhibitor therapy.,These results have important clinical implications because they suggest that combining BRAF inhibitors with therapies that target the invasion of drug-resistant cells could aid in controlling disease relapse.

The incidence of cutaneous melanoma continues to increase in pale skinned peoples in Europe and elsewhere.,Epidemiological studies identified genetically determined phenotypes such as pale skin, freckles and red hair, and sunburn as risk factors for this cancer.,The development of many melanocytic naevi is also genetically determined and a strong melanoma risk phenotype.,Not surprisingly then, genome wide association studies have identified pigmentation genes as common risk genes, and to a lesser extent, genes associated with melanocytic naevi.,More unexpectedly, genes associated with telomere length have also been identified as risk genes.,Higher risk susceptibility genes have been identified, particularly >CDKN2A >as the most common cause, and very rarely genes such as >CDK4>, >POT1>, >TERT >and other genes in coding for proteins in the shelterin complex are found to be mutated.,Familial melanoma genes are associated with an increased number of melanocytic naevi but not invariably and the atypical naevus phenotype is therefore an imperfect marker of gene carrier status.,At a somatic level, the most common driver mutation is >BRAF>, second most common >NRAS>, third >NF1 >and increasing numbers of additional rarer mutations are being identified such as in >TP53>.,It is of note that the >BRAF >and >NRAS >mutations are not C>T accepted as characteristic of ultraviolet light induced mutations.

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

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.

This study investigated the capacity of genetic analysis of uveal melanoma samples to identify high-risk patients and discusses its clinical implications.,Patients with posterior uveal melanoma were prospectively enrolled.,Tumour samples were derived from enucleated globe, fine-needle aspirates or endoresection.,Chromosome 3 and 8 status was determined by array comparative genomic hybridisation (array-CGH).,Patients were followed after treatment to detect metastasis.,Four groups were classified by array-CGH.,Patients were divided into disomy 3 and normal chromosome 8 (D3/8nl), disomy 3 and 8q gain (D3/8g), monosomy 3 and normal chromosome 8 (M3/8nl) and monosomy 3 and 8 or 8q gain (M3/8g).,Median follow-up was 28 months (range: 1-147 months).,At the end of the study, 128 patients (33.7%) had developed metastasis and 96 patients had died.,Univariate Cox proportional hazard analysis showed that factors associated with metastasis included basal tumour diameter p=0.0007, tumour thickness p=0.01, mixed/epithelioid cell type p=0.0009 and genomic data p<0.0001.,High-risk profile was more strongly associated with metastasis than the other prognostic factors p<0.001.,Multivariate Cox modelling analysis showed that the status of chromosomes 3 and 8 were the only two variables that independently contributed to prognosis: monosomy 3 alone p=0.001 and monosomy 3 and 8q gain p<0.0001.,Array-CGH allowed identification of three prognostic groups with low, intermediate and high risk of developing metastasis.,Array-CGH is a reliable and inexpensive method for uveal melanoma prognosis.,This method is now currently used in France.

The incidence of malignant melanoma has continued to rise during the past decades.,However, in the last few years, treatment protocols have significantly been improved thanks to a better understanding of the key oncogenes and signaling pathways involved in its pathogenesis and progression.,Anticancer therapy would either kill tumor cells by triggering apoptosis or permanently arrest them in the G1 phase of the cell cycle.,Unfortunately, melanoma is often refractory to commonly used anticancer drugs.,More recently, however, some new anticancer strategies have been developed that are “external” to cancer cells, for example stimulating the immune system’s response or inhibiting angiogenesis.,In fact, the increasing knowledge of melanoma pathogenetic mechanisms, in particular the discovery of genetic mutations activating specific oncogenes, stimulated the development of molecularly targeted therapies, a form of treatment in which a drug (chemical or biological) is developed with the goal of exclusively destroying cancer cells by interfering with specific molecules that drive growth and spreading of the tumor.,Again, after the initial exciting results associated with targeted therapy, tumor resistance and/or relapse of the melanoma lesion have been observed.,Hence, very recently, new therapeutic strategies based on the modulation of the immune system function have been developed.,Since cancer cells are known to be capable of evading immune-mediated surveillance, i.e., to block the immune system cell activity, a series of molecular strategies, including monoclonal antibodies, have been developed in order to “release the brakes” on the immune system igniting immune reactivation and hindering metastatic melanoma cell growth.,In this review we analyze the various biological strategies underlying conventional chemotherapy as well as the most recently developed targeted therapies and immunotherapies, pointing at the molecular mechanisms of cell injury and death engaged by the different classes of therapeutic agents.

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

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.

In advanced cancers, transforming growth factor-beta (TGFβ) promotes tumor growth and metastases and suppresses host antitumor immunity.,GC1008 is a human anti-TGFβ monoclonal antibody that neutralizes all isoforms of TGFβ.,Here, the safety and activity of GC1008 was evaluated in patients with advanced malignant melanoma and renal cell carcinoma.,In this multi-center phase I trial, cohorts of patients with previously treated malignant melanoma or renal cell carcinoma received intravenous GC1008 at 0.1, 0.3, 1, 3, 10, or 15 mg/kg on days 0, 28, 42, and 56.,Patients achieving at least stable disease were eligible to receive Extended Treatment consisting of 4 doses of GC1008 every 2 weeks for up to 2 additional courses.,Pharmacokinetic and exploratory biomarker assessments were performed.,Twenty-nine patients, 28 with malignant melanoma and 1 with renal cell carcinoma, were enrolled and treated, 22 in the dose-escalation part and 7 in a safety cohort expansion.,No dose-limiting toxicity was observed, and the maximum dose, 15 mg/kg, was determined to be safe.,The development of reversible cutaneous keratoacanthomas/squamous-cell carcinomas (4 patients) and hyperkeratosis was the major adverse event observed.,One malignant melanoma patient achieved a partial response, and six had stable disease with a median progression-free survival of 24 weeks for these 7 patients (range, 16.4-44.4 weeks).,GC1008 had no dose-limiting toxicity up to 15 mg/kg.,In patients with advanced malignant melanoma and renal cell carcinoma, multiple doses of GC1008 demonstrated acceptable safety and preliminary evidence of antitumor activity, warranting further studies of single agent and combination treatments.,Clinicaltrials.gov NCT00356460

Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma.,Subsequent studies found TERT promoter mutations in a wide array of other major human cancers.,TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis.,TERT promoter mutations in cutaneous melanoma often show UV-signatures.,Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent.,We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing.,TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas.,The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies.,Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors.

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.

PARP inhibitors niraparib and talazoparib are FDA approved for special cases of breast cancer.,PARP is an interesting repair protein which is frequently affected in cancer cells.,We studied the combined action of talazoparib or niraparib with ionizing radiation in melanoma cells and healthy fibroblasts.,Homologous recombination (HR) status in six different melanoma cell lines and healthy fibroblasts was assessed.,Cell cultures were treated with PARP inhibitors talazoparib or niraparib and ionizing radiation (IR).,Apoptosis, necrosis and cell cycle distribution was analyzed via flow cytometry.,Cell migration was studied by scratch assays.,Studied melanoma cell cultures are HR deficient.,Studied healthy fibroblasts are HR proficient.,Talazoparib and niraparib have congruent effects within the same cell cultures.,In all cell cultures, combined treatment increases cell death and G2/M arrest compared to IR.,Combined treatment in melanoma cells distinctly increases G2/M arrest.,Healthy fibroblasts are less affected by G2/M arrest.,Treatment predominantly decelerates or does not modify migration.,In two cell cultures migration is enhanced under the inhibitors.,Although the two PARP inhibitors talazoparib and niraparib appear to be suitable for a combination treatment with ionizing radiation in our in vitro studies, a combination treatment cannot generally be recommended.,There are clear interindividual differences in the effect of the inhibitors on different melanoma cells.,Therefore, the effect on the cancer cells should be studied prior to a combination therapy.,Since melanoma cells increase more strongly than fibroblasts in G2/M arrest, the fractional application of combined treatment should be further investigated.

Drugs such as gemcitabine that increase replication stress are effective chemotherapeutics in a range of cancer settings.,These drugs effectively block replication and promote DNA damage, triggering a cell cycle checkpoint response through the ATR-CHK1 pathway.,Inhibiting this signalling pathway sensitises cells to killing by replication stress‐inducing drugs.,Here, we investigated the effect of low‐level replication stress induced by low concentrations (> 0.2 mm) of the reversible ribonucleotide reductase inhibitor hydroxyurea (HU), which slows S‐phase progression but has little effect on cell viability or proliferation.,We demonstrate that HU effectively synergises with CHK1, but not ATR inhibition, in > 70% of melanoma and non‐small‐cell lung cancer cells assessed, resulting in apoptosis and complete loss of proliferative potential in vitro and in vivo.,Normal fibroblasts and haemopoietic cells retain viability and proliferative potential following exposure to CHK1 inhibitor plus low doses of HU, but normal cells exposed to CHK1 inhibitor combined with submicromolar concentrations of gemcitabine exhibited complete loss of proliferative potential.,The effects of gemcitabine on normal tissue correlate with irreversible ATR-CHK1 pathway activation, whereas low doses of HU reversibly activate CHK1 independently of ATR.,The combined use of CHK1 inhibitor and subclinical HU also triggered an inflammatory response involving the recruitment of macrophages in vivo.,These data indicate that combining CHK1 inhibitor with subclinical HU is superior to combination with gemcitabine, as it provides equal anticancer efficacy but with reduced normal tissue toxicity.,These data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination.

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

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.

Erasing histone H3 trimethylation marks suppresses the metastasis of human melanomas.,Metabolic reprogramming is a major factor in transformation, and particular metabolic phenotypes correlate with oncogenotype, tumor progression, and metastasis.,By profiling metabolites in 17 patient-derived xenograft melanoma models, we identified durable metabolomic signatures that correlate with biological features of the tumors.,BRAF mutant tumors had metabolomic and metabolic flux features of enhanced glycolysis compared to BRAF wild-type tumors.,Tumors that metastasized efficiently from their primary sites had elevated levels of metabolites related to protein methylation, including trimethyllysine (TML).,TML levels correlated with histone H3 trimethylation at Lys9 and Lys27, and methylation at these sites was also enhanced in efficiently metastasizing tumors.,Erasing either of these marks by genetically or pharmacologically silencing the histone methyltransferase SETDB1 or EZH2 had no effect on primary tumor growth but reduced cellular invasiveness and metastatic spread.,Thus, metabolite profiling can uncover targetable epigenetic requirements for the metastasis of human melanoma cells.

MAPK and AKT pathways are frequently co-activated in melanoma through overexpression of receptor tyrosine kinases, mutations in their signaling surrogates, such as RAS and BRAF, or loss of negative regulators such as PTEN.,Since RAS can be a positive upstream regulator of PI3-K, it has been proposed that the loss of PTEN and the activation of RAS are redundant events in melanoma pathogenesis (Tsao et al., 2000).,Here, in genetically engineered mouse models of cutaneous melanomas, we sought to better understand the genetic interactions between HRAS activation and PTEN inactivation in melanoma genesis and progression in vivo.,We showed that HRAS activation cooperates with Pten+/- and Ink4a/Arf-/- to increase melanoma penetrance and promote metastasis.,Correspondingly, gain- and loss-of-function studies established that Pten loss increases invasion and migration of melanoma cells and non-transformed melanocytes, and that such biological activity correlates with a shift to phosphorylation of AKT2 isoform and E-cadherin down-regulation.,Thus, Pten inactivation can drive the genesis and promote the metastatic progression of RAS activated Ink4a/Arf deficient melanomas.

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.

To investigate the feasibility of using bevacizumab to improve the survival of American Joint Committee on Cancer (AJCC) stage III melanoma patients, we investigated how a single bevacizumab treatment affected nodal disease and a panel of biomarkers in clinically fluorodeoxyglucose positron emission tomography (FDG-PET)/computed tomography (CT)-staged, stage III melanoma patients, prior to therapeutic lymph node dissection (TLND).,Four weeks before TLND, nine patients (median age 50, range 28.8-62.1 years; two male, seven female) with palpable lymph node metastases received 7.5 mg/kg bevacizumab.,Before and after this treatment, all patients were assessed by measurements of the maximum standardized uptake value (SUVmax) by FDG-PET scan, and serum S-100B and lactate dehydrogenase (LDH).,After TLND, the dissection specimen was analyzed for number of removed lymph nodes, number of metastatic lymph nodes, and tumor necrosis.,Median follow-up was 15.5 (2.2-32.9) months.,Histopathological analysis revealed tumor necrosis in six patients, of whom five had an S-100B decline and one had an unchanged S-100B level after bevacizumab.,The other three patients showed an S-100B increase and no necrosis.,Tumor necrosis was correlated with S-100B decrease (P = 0.048).,No association was found between necrosis and the markers SUVmax and LDH.,No wound healing disturbances were encountered.,Tumor necrosis in dissection specimens was associated with declining S-100B levels, while elevated S-100B was only found in cases with no necrosis.,Bevacizumab might be useful in treating AJCC stage III melanoma patients prior to TLND, and S100-B appears to be a useful marker for assessment of treatment effects.

Mucosal melanoma is a rare and poorly characterized subtype of human melanoma.,Here we perform a cross-species analysis by sequencing tumor-germline pairs from 46 primary human muscosal, 65 primary canine oral and 28 primary equine melanoma cases from mucosal sites.,Analysis of these data reveals recurrently mutated driver genes shared between species such as NRAS, FAT4, PTPRJ, TP53 and PTEN, and pathogenic germline alleles of BRCA1, BRCA2 and TP53.,We identify a UV mutation signature in a small number of samples, including human cases from the lip and nasal mucosa.,A cross-species comparative analysis of recurrent copy number alterations identifies several candidate drivers including MDM2, B2M, KNSTRN and BUB1B.,Comparison of somatic mutations in recurrences and metastases to those in the primary tumor suggests pervasive intra-tumor heterogeneity.,Collectively, these studies suggest a convergence of some genetic changes in mucosal melanomas between species but also distinctly different paths to tumorigenesis.,Mucosal melanoma is a rare melanoma subtype that is poorly characterised.,Here, the authors sequenced human, canine, and equine melanoma samples and performed a cross-species analysis, which revealed candidate driver genes, recurrent copy number alterations in regions syntenic between species, extensive intra-tumour heterogeneity and potential germline predisposing alleles

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

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion.,This process is important for immunotherapy based on PD-1 blockade.,We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells.,These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter.,PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter.,Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors.,Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.,Garcia-Diaz et al. performed a small hairpin RNA screen and genetic and functional studies to map the signaling pathways that result in reactive PD-L1 and PD-L2 on melanoma cells upon interferon gamma exposure.,The authors highlight the importance of the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis for clinical responses to PD-1 blockade therapy.

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options.,The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15).,Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis.,To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes.,We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025).,Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008).,Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas.,Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively.,Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells.,Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents.

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

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

Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells.,Those cells are considered as responsible for tumor resistance to therapies.,Moreover, melanoma cells are characterized by their high phenotypic plasticity.,Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure.,By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells.,Natural compounds were the focus of the present study.,We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity.,Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed.,Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM.,Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells.,On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter.,Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected.,Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC.,Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.

Growing evidence suggests that the cancer stem cell phenotype in melanoma is dynamically regulated.,Therefore, effective therapies have to target simultaneously bulk tumor cells and melanoma stem-like cells.,The aim of the present study was to investigate the effects of parthenolide on heterogeneous cancer cell populations from anchorage-independent melanospheres.,Cells derived from nodular melanoma specimens were grown under serum-free sphere-forming conditions.,The effects of parthenolide on cellular viability, immunophenotype and self-renewing capacity were assessed with cells from dissociated melanospheres.,Its penetration capacity was evaluated with intact melanospheres.,In melanoma cells that survived treatment with parthenolide, a different immunophenotype than that in untreated control was found.,The frequency of cells expressing the ABCB5 transporter was markedly reduced.,Most importantly, melanoma cells that survived parthenolide treatment lost their self-renewing capacity.,Significantly lower influence of drug on cellular viability and frequency of ABCB5-positive cells was observed in intact melanospheres.,The potential clinical significance of our findings is based on the ability of parthenolide to affect both bulk and melanoma stem-like cells with clonogenic capacity and high expression of the ABCB5 transporter.,Its low penetration capacity, however, may limit its action to easily accessible melanoma cells, either circulating in the blood or those in the vicinity to blood vessels within the tumor.,Because of limited penetration capacity of parthenolide, this drug should be further explored as a part of multimodal therapies rather than as a stand-alone therapeutic agent.

Uveal melanoma is the second most common form of melanoma and a predominant intraocular malignant tumor in adults.,The development of uveal melanoma is a multistep process involving genetic and epigenetic alteration of proto-oncogenes and tumor-suppressor genes.,Recent discoveries have shed a new light on the involvement of a class of noncoding RNA known as microRNAs (miRNAs) in uveal melanoma.,A lot of miRNAs show differential expressions in uveal melanoma tissues and cell lines.,Genes coding for these miRNAs have been characterized as novel oncogene and tumor-suppressor genes based on findings that these miRNAs control malignant phenotypes of uveal melanoma cells.,Several studies have confirmed that dysregulation of miRNAs promotes cell-cycle progression, confers resistance to apoptosis, and enhances invasiveness and metastasis.,Moreover, several miRNAs have also been shown to correlate with uveal melanoma initiation and progression, and thus may be used as biomarkers for early diagnosis and prognosis.,Elucidating the biological aspects of miRNA dysregulation may help us better understand the pathogenesis of uveal melanoma and promote the development of miRNA directed-therapeutics against this disease.

Up to 50% of patients with uveal melanoma (UM) develop metastatic disease with limited treatment options.,The immunomodulating agent ipilimumab has shown an overall survival (OS) benefit in patients with cutaneous metastatic melanoma in two phase III trials.,As patients with UM were excluded in these studies, the Dermatologic Cooperative Oncology Group (DeCOG) conducted a phase II to assess the efficacy and safety of ipilimumab in patients with metastatic UM.,We undertook a multicenter phase II study in patients with different subtypes of metastatic melanoma.,Here we present data on patients with metastatic UM (pretreated and treatment-naïve) who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals.,Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria.,Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0.,Primary endpoint was the OS rate at 12 months.,Forty five pretreated (85%) and eight treatment-naïve (15%) patients received at least one dose of ipilimumab. 1-year and 2-year OS rates were 22% and 7%, respectively.,Median OS was 6.8 months (95% CI 3.7-8.1), median progression-free survival 2.8 months (95% CI 2.5-2.9).,The disease control rate at weeks 12 and 24 was 47% and 21%, respectively.,Sixteen patients had stable disease (47%), none experienced partial or complete response.,Treatment-related AEs were observed in 35 patients (66%), including 19 grade 3-4 events (36%).,One drug-related death due to pancytopenia was observed.,Ipilimumab has very limited clinical activity in patients with metastatic UM.,Toxicity was manageable when treated as per protocol-specific guidelines.,ClinicalTrials.gov NCT01355120

Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells.,Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels.,We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRASQ61K in an Ink4a deficient background, two frequent driver mutations in human melanoma.,Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain and liver metastases.,Whole genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis.,Notably, loss or knockdown of STAT3 in mouse or human cells resulted in up-regulation of MITF and induction of cell proliferation.,Mechanistically we show that STAT3-induced CEBPa/b expression was sufficient to suppress MITF transcription.,Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus.,Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression.,We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.

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

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

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

Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogen‐activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance.,To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy.,Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours.,Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations.,Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib‐induced cell death and CD271 expression.,MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib‐resistant melanoma cell survival and invasion, respectively.,CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi.,In vitro studies demonstrate MEKi of BRAF‐mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271‐expressing subpopulations.,Trametinib‐induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance.,Treatment of CD271‐expressing melanoma subpopulations with RNA interference and small‐molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9‐tetrahydrocannabinol and Vps34 - reduced survival of MEKi‐resistant melanoma cells.,Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi‐resistant cells in an in vivo zebrafish xenograft.,These results highlight a novel mechanism of MEKi‐induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug‐resistant melanoma cells to the cytotoxic effects of MEKi.,What's already known about this topic?,The targeted mitogen‐activated protein kinase kinase (MEK) inhibitor (MEKi) trametinib is used for the treatment of patients with BRAF‐mutant metastatic melanoma in combination with BRAF inhibitors, but many tumours rapidly develop resistance to these drugs.Resistance of melanoma to trametinib has been associated with the induction of prosurvival autophagy, but it is unclear how stem‐cell markers, such as CD271 contribute to the transitionary phase of drug resistance.,The targeted mitogen‐activated protein kinase kinase (MEK) inhibitor (MEKi) trametinib is used for the treatment of patients with BRAF‐mutant metastatic melanoma in combination with BRAF inhibitors, but many tumours rapidly develop resistance to these drugs.,Resistance of melanoma to trametinib has been associated with the induction of prosurvival autophagy, but it is unclear how stem‐cell markers, such as CD271 contribute to the transitionary phase of drug resistance.,What does this study add?,Transient induction of the low‐affinity neurotrophin receptor CD271 is a critical regulator of the adaptive drug‐‐response phase of melanoma cells to trametinib.Genetic or chemical inhibition of CD271 prevents emergence of trametinib‐induced drug‐resistant melanoma subpopulations.Progression to trametinib resistance by melanoma subpopulations results in loss of CD271 expression and subsequent resistance to CD271 inhibitors, but not to the cytotoxic effects of autophagy modulation.,Transient induction of the low‐affinity neurotrophin receptor CD271 is a critical regulator of the adaptive drug‐‐response phase of melanoma cells to trametinib.,Genetic or chemical inhibition of CD271 prevents emergence of trametinib‐induced drug‐resistant melanoma subpopulations.,Progression to trametinib resistance by melanoma subpopulations results in loss of CD271 expression and subsequent resistance to CD271 inhibitors, but not to the cytotoxic effects of autophagy modulation.,What is the translational message?,Autophagy modulation either through inhibition of vacuolar protein sorting 34, or activation of cytotoxic autophagy with tetrahydrocannabinol effectively resensitizes drug‐resistant melanoma cells to trametinib in vitro and in vivo.Combined autophagy modulation and MEK inhibition offers a novel personalized therapeutic strategy to overcome MEKi‐induced drug resistance for patients with BRAF‐mutant metastatic melanoma.,Autophagy modulation either through inhibition of vacuolar protein sorting 34, or activation of cytotoxic autophagy with tetrahydrocannabinol effectively resensitizes drug‐resistant melanoma cells to trametinib in vitro and in vivo.,Combined autophagy modulation and MEK inhibition offers a novel personalized therapeutic strategy to overcome MEKi‐induced drug resistance for patients with BRAF‐mutant metastatic melanoma.,Plain language summary available online,Respond to this article

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

Malignant melanoma is the most aggressive and deadly form of skin cancer.,Dacarbazine (DTIC) has been the approved first-line treatment for metastatic melanoma in routine clinical practice.,However, response rates with single-agent DTIC are low.,The objective of this study was to compare the efficacy and safety of DTIC with or without placebo and DTIC-based combination therapies in patients with advanced metastatic melanoma.,We searched from electronic databases such as The Cochrane Library, MEDLINE, EBSCO, EMBASE, Ovid, CNKI, and CBMDisc from 2003 to 2013.,The primary outcome measures were overall response and 1-year survival, and the secondary outcome measurements were adverse events.,Nine randomized controlled trials (RCTs) involving 2,481 patients were included in the meta-analysis.,DTIC-based combination therapies was superior to DTIC alone in overall response (combined risk ratio [RR] = 1.60, 95% confidence interval [CI]: 1.27-2.01) and 1-year survival (combined RR = 1.26, 95% CI: 1.14-1.39).,Patients with DTIC-based combination therapies had higher incidence of adverse events including nausea (combined RR = 1.23, 95% CI: 1.10-1.36), vomiting (combined RR = 1.73, 95% CI: 1.41-2.12) and neutropenia (combined RR = 1.75, 95% CI: 1.42-2.16) compared to the group for DTIC alone.,These data suggested that DTIC-based combination therapies could moderately improve the overall response and the 1-year survival but increased the incidence of adverse events.,Further large-scale, high-quality, placebo-controlled, double-blind trials are needed to confirm this conclusion.

Tremelimumab, a fully human cytotoxic T-lymphocyte antigen 4 monoclonal antibody, and PF-3512676, a Toll-like receptor-9 agonist, are targeted immune modulators that elicit durable single-agent antitumour activity in advanced cancer.,To determine the maximum tolerated dose (MTD) of these agents combined during this phase I study, patients received intravenous tremelimumab (6.0, 10.0, or 15.0 mg kg−1) every 12 weeks plus subcutaneous PF-3512676 (0.05, 0.10, or 0.15 mg kg−1) weekly.,Primary end points were safety and tolerability; secondary end points included pharmacokinetics and antitumour activity.,Twenty-one patients with stage IV melanoma (n=17) or advanced solid tumours (n=4) were enrolled.,Injection-site reactions (n=21; 100%), influenza-like illness (n=18; 86%), and diarrhoea (n=13; 62%) were the most common treatment-related adverse events (TAEs).,Grade ⩾3 TAEs were reported (n=7; 33%).,Dose-limiting toxicities (prespecified 6-week observation) occurred in one of the six patients in the 10 mg kg−1 tremelimumab plus 0.05 mg kg−1 PF-3512676 cohort (grade 3 hypothalamopituitary disorder) and two of the six patients in the 15 mg kg−1 tremelimumab plus 0.05 mg kg−1 PF-3512676 cohort (grade 3 diarrhoea).,Consequently, 15 mg kg−1 tremelimumab plus 0.05 mg kg−1 PF-3512676 exceeded the MTD.,Two melanoma patients achieved durable (⩾170 days) partial response.,No human antihuman antibody responses to tremelimumab were observed.,Weekly PF-3512676 (⩽0.15 mg kg−1) plus tremelimumab (⩽10 mg kg−1 every 12 weeks) was tolerable.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy.,Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples.,Tumor cells reveal novel subclonal genomic complexity and transcriptional states.,Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4.,V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response.,An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity.,This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.,Uveal melanoma is highly metastatic and unresponsive to checkpoint immunotherapy.,Here, the authors present single-cell transcriptomics of 59,915 cells in 8 primary and 3 metastatic samples, highlighting the diversity of the tumour microenvironment.

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.

This descriptive study was aimed to examine trends in the incidence of melanoma and nonmelanoma in South Korea.,The nationwide incidence data for melanoma and non-melanoma skin cancer was obtained from the Korea Central Cancer Registry.,Age-standardized rates were calculated and analyzed, using a Joinpoint regression model.,The incidence of basal cell carcinoma has increased dramatically both in men (average annual percentage change [AAPC], 8.0 [95% confidence interval (CI), 6.0 to 10.1]) and women (AAPC, 9.0 [95% CI, 7.5 to 10.4]).,Squamous cell carcinoma has also steadily increased both in men (AAPC, 3.3 [95% CI, 2.6 to 4.0]) and women (AAPC, 6.8 [95% CI, 5.3 to 8.4]).,Cutaneous melanoma increased continuously from 1999 to 2014 inwomen (AAPC, 3.5 [95% CI, 2.4 to 4.6]), whilst rapidly increasing in men until 2005 (APC, 7.9 [95% CI, 2.4 to 13.7]) after which no increase has been observed (APC, -0.2 [95% CI, -2.3 to 2.0]).,The incidence rates of melanoma and non-melanoma skin cancer have increased over the past years, with the exception of melanoma in men.,Further studies are required to investigate the reasons for the increased incidence of these skin cancers in South Korea.

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

Computer-aided diagnosis (CADx) software that provides a second opinion has been widely used to assist physicians with various tasks.,In dermatology, however, CADx has been mostly limited to melanoma or melanocytic skin cancer diagnosis.,The frequency of non-melanocytic skin cancers and the accessibility of regular digital macrographs have raised interest in developing CADx for broader applications.,To investigate the feasibility of using CADx to diagnose both melanocytic and non-melanocytic skin lesions based on conventional digital photographic images.,This study was approved by an institutional review board, and the requirement to obtain informed consent was waived.,In total, 769 conventional photographs of melanocytic and non-melanocytic skin lesions were retrospectively reviewed and used to develop a CADx system.,Conventional and new color-related image features were developed to classify the lesions as benign or malignant using support vector machines (SVMs).,The performance of CADx was compared with that of dermatologists.,The clinicians' overall sensitivity, specificity, and accuracy were 83.33%, 85.88%, and 85.31%, respectively.,New color correlation and principal component analysis (PCA) features improved the classification ability of the baseline CADx (p = 0.001).,The estimated area under the receiver operating characteristic (ROC) curve (Az) of the proposed CADx system was 0.949, with a sensitivity and specificity of 85.63% and 87.65%, respectively, and a maximum accuracy of 90.64%.,We have developed an effective CADx system to classify both melanocytic and non-melanocytic skin lesions using conventional digital macrographs.,The system's performance was similar to that of dermatologists at our institute.,Through improved feature extraction and SVM analysis, we found that conventional digital macrographs were feasible for providing useful information for CADx applications.,The new color-related features significantly improved CADx applications for skin cancer.

Dermoscopy is one of the major imaging modalities used in the diagnosis of melanoma and other pigmented skin lesions.,In current practice, dermatologists determine lesion area by manually drawing lesion borders.,Therefore, automated assessment tools for dermoscopy images have become an important research field mainly because of inter- and intra-observer variations in human interpretation.,One of the most important steps in dermoscopy image analysis is automated detection of lesion borders.,To our knowledge, in our 2010 study we achieved one of the highest accuracy rates in the automated lesion border detection field by using modified density based clustering algorithm.,In the previous study, we proposed a novel method which removes redundant computations in well-known spatial density based clustering algorithm, DBSCAN; thus, in turn it speeds up clustering process considerably.,Our previous study was heavily dependent on the pre-processing step which creates a binary image from original image.,In this study, we embed a new distance measure to the existing algorithm.,This provides twofold benefits.,First, since new approach removes pre-processing step, it directly works on color images instead of binary ones.,Thus, very important color information is not lost.,Second, accuracy of delineated lesion borders is improved on 75% of 100 dermoscopy image dataset.,Previous and improved methods are tested within the same dermoscopy dataset along with the same set of dermatologist drawn ground truth images.,Results revealed that the improved method directly works on color images without any pre-processing and generates more accurate results than existing method.

Metastatic melanoma is the most aggressive and obstinate skin cancer with poor prognosis.,Variant novel applicable regimens have emerged during the past decades intensively, while the most profound approaches are oncogene-targeted therapy and T-lymphocyte mediated immunotherapy.,Although targeted therapies generated remarkable and rapid clinical responses in the majority of patients, acquired resistance was developed promptly within months leading to tumor relapse.,By contrast, immunotherapies elicited long-term tumor regression.,However, the overall response rate was limited.,In view of the above, either targeted therapy or immunotherapy cannot elicit durable clinical responses in large range of patients.,Interestingly, the advantages and limitations of these regimens happened to be complementary.,An increasing number of preclinical studies and clinical trials proved a synergistic antitumor effect with the combination of targeted therapy and immunotherapy, implying a promising prospect for the treatment of metastatic melanoma.,In order to achieve a better therapeutic effectiveness and reduce toxicity in patients, great efforts need to be made to illuminate multifaceted interplay between targeted therapy and immunotherapy.

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

Ceralasertib is a potent and selective oral inhibitor of the serine/threonine protein kinase ataxia telangiectasia and Rad3-related (ATR) protein.,Eligible patients with solid tumors, enriched for melanoma, received ceralasertib in combination with a fixed dose of paclitaxel (80 mg/m2 on D1, D8, D15) in 28-day cycles.,The dose of ceralasertib was escalated to reach an MTD in a rolling 6 design.,The starting dose of ceralasertib was 40 mg QD.,Fifty-seven patients (33 patients with melanoma who failed prior PD1/L1 treatment) were enrolled in 7 dose cohorts ranging from 40 mg QD to 240 mg BD plus weekly paclitaxel.,The RP2D was established as ceralasertib 240 mg BD days 1-14 plus paclitaxel 80 mg/m2 on D1, D8, D15 every 28 days.,The most common toxicities were neutropenia (n = 39, 68%), anemia (n = 25, 44%), and thrombocytopenia (n = 21, 37%).,In the full analysis set of 57 patients, the overall response rate (ORR) was 22.6% (95% CI, 12.5-35.3).,In 33 patients with melanoma, resistant to prior anti-PD1 therapy, the ORR was 33.3% (95% CI, 18.0-51.8).,In the melanoma subset, the mPFS was 3.6 months (95% CI, 2.0-5.8), the median duration of response was 9.9 months (95% CI, 3.7-23.2), and the mOS was 7.4 months (95% CI, 5.7-11.9).,Ceralasertib in combination with paclitaxel was well tolerated in patients with advanced malignancies and showed evidence of antitumor activity.,Durable responses were observed in patients with advanced cutaneous, acral, and mucosal melanoma resistant to anti-PD1/L1 treatment.,See related commentary by Ashworth, p.,4667

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.

Unlike other tumours, TP53 is rarely mutated in melanoma; however, it fails to function as a tumour suppressor.,We assume that its functions might be altered through interactions with several families of proteins, including p53/p73, NME and GLI.,To elucidate the potential interplay among these families we analysed the expression profiles of aforementioned genes and proteins in a panel of melanoma cell lines, metastatic melanoma specimens and healthy corresponding tissue.,Using qPCR a higher level of NME1 gene expression and lower levels of Δ40p53β, ΔNp73, GLI1, GLI2 and PTCH1 were observed in tumour samples compared to healthy tissue.,Protein expression of Δ133p53α, Δ160p53α and ΔNp73α isoforms, NME1 and NME2, and N′ΔGLI1, GLI1FL, GLI2ΔN isoforms was elevated in tumour tissue, whereas ∆Np73β was downregulated.,The results in melanoma cell lines, in general, support these findings.,In addition, we correlated expression profiles with clinical features and outcome.,Higher Δ133p53β and p53α mRNA and both GLI1 mRNA and GLI3R protein expression had a negative impact on the overall survival.,Shorter overall survival was also connected with lower p53β and NME1 gene expression levels.,In conclusion, all examined genes may have implications in melanoma development and functional inactivity of TP53.

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood.,Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin.,Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14.,The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation.,NME1‐induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1‐deficient parental lines.,NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA‐mediated knock‐down.,Increased synthesis and ECM deposition of fibronectin was necessary for NME1‐induced cell spreading, as knock‐down of fibronectin opposed the effects of NME1 on cell morphology.,Fibronectin knock‐down also reversed the ability of NME1 to promote aggregation when cells were plated on a non‐adherent substratum.,Similarly, inhibiting activation of the fibronectin receptor integrin α4β1 with an anti‐α4 antibody reversed the motility‐suppressing effect of NME1.,A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression.,In summary, these findings indicate fibronectin is an important effector of the motility‐suppressing function of NME1 in melanoma cells.

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.

Combination treatment with BRAF (BRAFi) plus MEK inhibitors (MEKi) has demonstrated survival benefit in patients with advanced melanoma harboring activating BRAF mutations.,Previous preclinical studies suggested that an intermittent dosing of these drugs could delay the emergence of resistance.,Contrary to expectations, the first published phase 2 randomized study comparing continuous versus intermittent schedule of dabrafenib (BRAFi) plus trametinib (MEKi) demonstrated a detrimental effect of the “on−off” schedule.,Here we report confirmatory data from the Phase II randomized open-label clinical trial comparing the antitumoral activity of the standard schedule versus an intermittent combination of vemurafenib (BRAFi) plus cobimetinib (MEKi) in advanced BRAF mutant melanoma patients (NCT02583516).,The trial did not meet its primary endpoint of progression free survival (PFS) improvement.,Our results show that the antitumor activity of the experimental intermittent schedule of vemurafenib plus cobimetinib is not superior to the standard continuous schedule.,Detection of BRAF mutation in cell free tumor DNA has prognostic value for survival and its dynamics has an excellent correlation with clinical response, but not with progression.,NGS analysis demonstrated de novo mutations in resistant cases.,Whether intermittent strategies of delivering drugs can improve cancer patients survival is still unclear.,Here, the authors reports the results of a randomized phase II clinical trial aimed to compare the efficacy and safety of two dosing regimens (continuous and intermittent) of vemurafenib and cobimetinib combination as first-line treatment of patients with unresectable or metastatic advanced melanoma with BRAFV600 mutation

A point mutation in the BRAF gene, leading to a constitutively active form of the protein, is present in 45%-60% of patients and acts as a key driver in melanoma.,Shortly after therapy induction, resistance to MAPK pathway-specific inhibitors develops, indicating that pathway inhibition is circumvented by epigenetic mechanisms.,Here, we mimicked epigenetic modifications in melanoma cells by reprogramming them into metastable induced pluripotent cancer cells (iPCCs) with the ability to terminally differentiate into non-tumorigenic lineages. iPCCs and their differentiated progeny were characterized by an increased resistance against targeted therapies, although the cells harbor the same oncogenic mutations and signaling activity as the parental melanoma cells.,Furthermore, induction of a pluripotent state allowed the melanoma-derived cells to acquire a non-tumorigenic cell fate, further suggesting that tumorigenicity is influenced by the cell state.,•Human melanoma cells reprogrammed toward an iPSC-like state (iPCCs)•iPCCs differentiated into neurons and fibroblasts•iPCC-derived fibroblasts show no tumorigenic potential•iPCCs and iPCC-derived fibroblasts lose oncogene addiction,Human melanoma cells reprogrammed toward an iPSC-like state (iPCCs),iPCCs differentiated into neurons and fibroblasts,iPCC-derived fibroblasts show no tumorigenic potential,iPCCs and iPCC-derived fibroblasts lose oncogene addiction,Aberrant activation of the MAPK pathway is a major cause of melanoma.,Resistance to MAPK pathway-specific inhibitors hampers successful treatment of melanoma.,By reprogramming human melanoma cells toward pluripotency and subsequent differentiation, Utikal and colleagues demonstrate that the tumorigenic phenotype and oncogene addiction are linked to a certain differentiation lineage.

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.

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.

In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome.,For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients.,On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion.,Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin.,Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination.,Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2.,Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD).,The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months.,Temozolomide reduced circulating Foxp3+Treg cells in all patients.,A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%.,Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%.,Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells.,Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2.,However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

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.

miRNAs are key regulatory small non-coding RNAs involved in critical steps of melanoma tumorigenesis; however, the relationship between sequence specific variations at the 5′ or 3′ termini (isomiR) of a miRNA and cancer phenotype remains unclear.,Deep-sequencing and qRT-PCR showed reduced expression of miR-144/451a cluster and most abundant isomiR (miR451a.1) in dysplastic nevi, in-situ and invasive melanomas compared to common nevi and normal skin (n = 101). miRNA in situ hybridization reproducibly confirmed lost miR-451a.1 in melanoma compared to nevus cells or adjacent keratinocytes.,Significantly higher expression of miR-451a.1 was associated with amelanotic phenotype in melanomas (n = 47).,In contrast, miR-451a was associated with melanotic phenotype, absent pagetoid scatter of intraepidermal melanocytes, superficial spreading histological subtype and tumor inflammation.,Sequencing miRNAs from cultured melanocytes with cytoplasmic melanin gradient (light, medium to dark) showed absent miR-451a while revealing other melanin-associated miRNAs, e.g. miR-30b, miR-100 and miR-590 in darkly and let-7a, let-7i and let-7f in lightly to moderately pigmented cultured melanocytes.,Ectopic expression of miR-144/451a in melanoma cell lines resulted in markedly higher levels of mature miR-451a.1 than miR451a or miR-144; and significantly retarded cell migration and inhibited invasion in a glucose-sensitive manner.,Surprisingly, these effects were not mediated by calcium binding protein 39 (CAB39), a proven miR451a gene target. miR-144/miR-451a cluster is a novel miRNA locus with tumor suppressive activity in melanoma.

Cutaneous melanoma (CM) is a very aggressive disease, often characterized by unresponsiveness to conventional therapies and high mortality rates worldwide.,The identification of the activating BRAFV600 mutations in approximately 50% of CM patients has recently fueled the development of novel small‐molecule inhibitors that specifically target BRAFV600 ‐mutant CM.,In addition, a major progress in CM treatment has been made by monoclonal antibodies that regulate the immune checkpoint inhibitors.,However, although target‐based therapies and immunotherapeutic strategies have yielded promising results, CM treatment remains a major challenge.,In the last decade, accumulating evidence points to the aberrant expression of different types of noncoding RNAs (ncRNAs) in CM.,While studies on microRNAs have grown exponentially leading to significant insights on CM biology, the role of circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) in this tumor is less understood, and much remains to be discovered.,Here, we summarize and critically review the available evidence on the molecular functions of circRNAs and lncRNAs in BRAFV600 ‐mutant CM and CM immunogenicity, providing recent updates on their functional role in targeted therapy and immunotherapy resistance.,In addition, we also include an evaluation of several algorithms and databases for prediction and validation of circRNA and lncRNA functional interactions.,In the last decade, accumulating evidence points to the aberrant expression of different types of noncoding RNAs in cutaneous melanoma (CM).,Here, we summarize and critically review the available evidence on the molecular functions of circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) in BRAFV600 ‐mutant CM and CM immunogenicity, providing recent updates on their functional role in targeted therapy and immunotherapy resistance.,In addition, we also include an evaluation of several algorithms and databases for prediction and validation of circRNA and lncRNA functional interactions.

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

The incidence of malignant melanoma has continued to rise during the past decades.,However, in the last few years, treatment protocols have significantly been improved thanks to a better understanding of the key oncogenes and signaling pathways involved in its pathogenesis and progression.,Anticancer therapy would either kill tumor cells by triggering apoptosis or permanently arrest them in the G1 phase of the cell cycle.,Unfortunately, melanoma is often refractory to commonly used anticancer drugs.,More recently, however, some new anticancer strategies have been developed that are “external” to cancer cells, for example stimulating the immune system’s response or inhibiting angiogenesis.,In fact, the increasing knowledge of melanoma pathogenetic mechanisms, in particular the discovery of genetic mutations activating specific oncogenes, stimulated the development of molecularly targeted therapies, a form of treatment in which a drug (chemical or biological) is developed with the goal of exclusively destroying cancer cells by interfering with specific molecules that drive growth and spreading of the tumor.,Again, after the initial exciting results associated with targeted therapy, tumor resistance and/or relapse of the melanoma lesion have been observed.,Hence, very recently, new therapeutic strategies based on the modulation of the immune system function have been developed.,Since cancer cells are known to be capable of evading immune-mediated surveillance, i.e., to block the immune system cell activity, a series of molecular strategies, including monoclonal antibodies, have been developed in order to “release the brakes” on the immune system igniting immune reactivation and hindering metastatic melanoma cell growth.,In this review we analyze the various biological strategies underlying conventional chemotherapy as well as the most recently developed targeted therapies and immunotherapies, pointing at the molecular mechanisms of cell injury and death engaged by the different classes of therapeutic agents.

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

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.

PD-L1 (programmed cell death 1 ligand 1) expression in melanoma has been associated with a better response to anti-PD-1 (programmed cell death 1) therapy.,However, patients with PD-L1-negative melanomas can respond to anti-PD-1 blockade, suggesting that the other PD-1 ligand, PD-L2 (programmed cell death 1 ligand 2), might also be relevant for efficacy of PD-1 inhibition.,We investigated PD-L2 expression and methylation as a prognostic and predictive biomarker in melanoma.,DNA methylation at five CpG loci and gene expression of PD-L2 were evaluated with regard to survival in 470 melanomas from The Cancer Genome Atlas.,PD-L2 promoter methylation in correlation with PD-L2 mRNA and protein expression was analyzed in human melanoma cell lines.,Prognostic and predictive value of PD-L2 methylation was validated using quantitative methylation-specific PCR in a multicenter cohort of 129 melanoma patients receiving anti-PD-1 therapy. mRNA sequencing data of 121 melanoma patients receiving anti-PD-1 therapy provided by Liu et al. were analyzed for PD-L2 mRNA expression.,We found significant correlations between PD-L2 methylation and mRNA expression levels in melanoma tissues and cell lines.,Interferon-γ inducible PD-L2 protein expression correlated with PD-L2 promoter methylation in melanoma cells.,PD-L2 DNA promoter hypomethylation and high mRNA expression were found to be strong predictors of prolonged overall survival.,In pre-treatment melanoma samples from patients receiving anti-PD-1 therapy, low PD-L2 DNA methylation and high PD-L2 mRNA expression predicted longer progression-free survival.,PD-L2 expression seems to be regulated via DNA promoter methylation.,PD-L2 DNA methylation and mRNA expression may predict progression-free survival in melanoma patients receiving anti-PD-1 immunotherapy.,Assessment of PD-L2 should be included in further clinical trials with anti-PD-1 antibodies.

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.

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

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

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

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade.,A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations.,In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes.,We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1.,This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients.,In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy.,We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies.,Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes.,Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire.,Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells.,In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches.

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved.,We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting.,In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1.,All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification.,The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108 pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients.,This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment.,Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment.,Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity.,Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1.,Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer.,Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration.,Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies.,Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.,There are many patients who do not respond to immune checkpoint inhibitor (ICI) immunotherapy.,Here, the authors show a significant negative correlation between sphingosine kinase-1 (SK1) expression and survival for ICI-treated melanoma patients, and further show that targeting SK1 improves response to ICI in mouse cancer models.

Chimeric antigen receptor (CAR)-engineered T cells have demonstrated promising clinical efficacy in patients with B cell lymphoma.,However, the application of CAR-T cell therapy in the treatment of other solid tumors has been limited.,We incorporated 4-1BB into the anti-GD2 CAR-T cells to test their cytotoxicity in melanoma in vitro and in vivo.,Moreover, we reported the expression of ganglioside GD2 in non-Caucasian melanoma populations for the first time, thus providing a basis for future clinical research.,This study included tumor samples from 288 melanoma patients at the Peking University Cancer Hospital & Institute.,Clinical data were collected.,Immunohistochemical assays using antibodies against ganglioside GD2 were performed on formalin-fixed, paraffin-embedded specimens.,The ability of ganglioside GD2 CAR-T cells to kill ganglioside GD2+ melanoma cells was evaluated in vitro and in a patient-derived xenograft (PDX) model.,Among the 288 samples, 49.3% of cases (142/288) demonstrated positive staining with ganglioside GD2.,The median survival time in patients exhibiting ganglioside GD2 expression was significantly shorter than that in patients without ganglioside GD2 expression (31 vs.,47.1 months, P < 0.001).,In the present study, CAR was constructed using a GD2-specific scFv (14.,G2a), T cell receptor CD3ζ chain, and the CD137 (4-1BB) costimulatory motif.,In addition, the GD2.,BBζ CAR-T cells demonstrated specific lysis of ganglioside GD2-expressing melanoma cells in vitro.,In two PDX models, mice that received intravenous or local intratumor injections of GD2.,BBζ CAR-T cells experienced rapid tumor regression.,These data demonstrate that the rate of GD2 expression in Chinese patients is 49.3%.,GD2.,BBζ CAR-T cells can both efficiently lyse melanoma in a GD2-specific manner and release Th1 cytokines in an antigen-dependent manner in vitro and in vivo.,Anti-GD2/4-1BB CAR-T cells represent a clinically appealing treatment strategy for Chinese melanoma patients exhibiting GD2 expression and provide a basis for future studies of the clinical application of immunotherapy for melanoma.,The online version of this article (10.1186/s13045-017-0548-2) contains supplementary material, which is available to authorized users.

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.

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.

Histone deacetylase (HDAC) inhibitors are widely used in clinical investigation as novel drug targets.,For example, panobinostat and vorinostat have been used to treat patients with melanoma.,However, HDAC inhibitors are small-molecule compounds without a specific target, and their mechanism of action is unclear.,Therefore, it is necessary to investigate which HDACs are required for the proliferation and metastasis of melanoma cells.,We used overexpression and knocking down lentivirus to clarify the influence of HDAC5 and HDAC6 in melanoma development.,Also, we introduced stable HDAC5 or HDAC6 knockdown cells into null mice and found that the knockdown cells were unable to form solid tumors.,Finally, we tested HDAC5 and HDAC6 expression and sub-location in clinical melanoma tissues and tumor adjacent tissues.,In this study, and found that HDAC5 and HDAC6 were highly expressed in melanoma cells but exhibited low expression levels in normal skin cells.,Furthermore, we knocked down HDAC5 or HDAC6 in A375 cells and demonstrated that both HDAC5 and HDAC6 contributed to the proliferation and metastasis of melanoma cells.,This study demonstrated both HDAC5 and HDAC6 were required for melanoma cell proliferation and metastasis through different signaling pathways.,The online version of this article (doi:10.1186/s12967-015-0753-0) contains supplementary material, which is available to authorized users.

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

Genetically modified T cells to recognize tumor-associated antigens by transgenic TCRs or chimeric antigen receptors (CAR) have been successfully applied in clinical trials.,However, the disadvantages of either TCR mismatching or the requirement of a surface tumor antigen limit their wider applications in adoptive T cell therapy.,A TCR-like chimeric receptor, specific for the melanoma-related gp100/HLA-A2 complex was created by joining a TCR-like antibody GPA7 with the endodomains of CD28 and CD3-ζ chain.,This TCR-like CAR, GPA7-28z, was subsequently introduced into human T cells.,Retargeted T cells expressing GPA7-28z could exhibit efficient cytotoxic activities against human melanoma cells in vitro in the context with HLA-A2.,Furthermore, infusion of GPA7-28z-transduced T cells suppressed melanoma progression in a xenograft mouse model.,Redirecting human T cells with TCR-like CARs would be a promising alternative approach to TCR-mediated therapy for melanoma patients, which is also feasible for targeting a variety of other tumor antigens.

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.

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection.,However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution.,Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations.,Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence.,Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations.,Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection.,This implies that the metastatic proclivity of UM is “set in stone” early in tumor evolution and may explain why advances in primary treatment have not improved survival.,Uveal melanoma (UM), the most common primary eye cancer, is strongly linked to mutations in the tumor suppressor BAP1.,Here, the authors analyze 151 primary UM samples to find that BAP1 and other canonical genomic aberrations arise in an early punctuated burst followed by neutral tumor evolution.

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage.,To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation.,We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7.,BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo.,MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers.,Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy.,BRG1 also regulates the dynamics of MITF genomic occupancy.,MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.,DOI:http://dx.doi.org/10.7554/eLife.06857.001,Melanocytes are pigment-producing cells found primarily in the skin.,Many of the genes that help these cells to develop are also thought to affect the development of melanomas: an aggressive form of skin cancer that originates in these cells.,One such gene encodes a protein called MITF.,This protein binds to DNA and regulates genes that control the development, survival, and spread of melanocytes; it is also linked to the invasive properties of melanomas.,The MITF protein works together with partner proteins to control numerous genes, activating some while inhibiting others, by binding to nearby stretches of DNA that act as regulatory elements.,Its interactions are therefore widespread and complex.,Now, Laurette, Strub et al. have used techniques called tandem affinity purification and mass spectrometry to identify the proteins that interact with MITF.,This investigation found many new protein partners for MITF, including proteins involved in DNA damage, repair, and replication.,MITF also associates with two proteins-one of which is called BRG1-that are involved in modifying how tightly DNA is packaged inside cells.,DNA wrapped around proteins is known as chromatin, and if chromatin is tightly packed, the genes in that stretch of DNA cannot be easily accessed or activated.,Removing BRG1 from melanocytes and melanoma cells caused the cells to die or stop growing.,When BRG1 was removed from developing mouse embryos, melanocytes failed to form.,Further investigation revealed that MITF, together with another protein, localize BRG1 to sites in the melanocyte's DNA to open up the chromatin and regulate nearby genes.,Furthermore, Laurette, Strub et al. report that BRG1 binds to many such elements in a characteristic manner, in which two BRG1 proteins flank the stretch of DNA bound by MITF and several other key DNA-binding proteins that together regulate many aspects of melanocyte and melanoma cell physiology.,Laurette, Strub et al. have therefore revealed many details about the molecules that activate genes in melanomas and melanocyte cells, as well as the interactions between these molecules.,The results could also help researchers to understand how the BRG1 protein organises chromatin packing in other cell types.,DOI:http://dx.doi.org/10.7554/eLife.06857.002

To relate conjunctival melanoma characteristics to local control.,Retrospective, registry-based interventional study with data gathered from 10 ophthalmic oncology centres from 9 countries on 4 continents.,Conjunctival melanoma patients diagnosed between January 2001 and December 2013 were enrolled in the study.,Primary treatments included local excision, excision with cryotherapy and exenteration.,Adjuvant treatments included topical chemotherapy, brachytherapy, proton and external beam radiotherapy (EBRT).,Cumulative 5-year and 10-year Kaplan-Meier local recurrence rates were related to clinical and pathological T-categories of the eighth edition of the American Joint Committee on Cancer (AJCC) staging system.,288 patients had a mean initial age of 59.7±16.8 years.,Clinical T-categories (cT) were cT1 (n=218,75.7%), cT2 (n=34, 11.8%), cT3 (n=15, 5.2%), cTx (n=21,7.3%) with no cT4.,Primary treatment included local excision (n=161/288, 55.9%) followed by excision biopsy with cryotherapy (n=108/288, 37.5%) and exenteration (n=5/288, 1.7%).,Adjuvant therapies included topical mitomycin (n=107/288, 37.1%), plaque-brachytherapy (n=55/288, 19.1%), proton-beam (n=36/288, 13.5%), topical interferon (n=20/288, 6.9%) and EBRT (n=15/288, 5.2%).,Secondary exenteration was performed (n=11/283, 3.9%).,Local recurrence was noted in 19.1% (median=3.6 years).,Cumulative local recurrence was 5.4% (3.2-8.9%), 19.3% (14.4-25.5%) and 36.9% (26.5-49.9%) at 1, 5 and 10 years, respectively. cT3 and cT2 tumors were twice as likely to recur than cT1 tumours, but only cT3 had statistically significantly greater risk of local recurrence than T1 (p=0.013).,Factors such as tumour ulceration, plica or caruncle involvement and tumour thickness were not significantly associated with an increased risk of local recurrence.,This multicentre international study showed that eighth edition of AJCC tumour staging was related to the risk of local recurrence of conjunctival melanoma after treatment.,The 10-year cumulative local recurrence remains high despite current management.

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

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

Melanoma 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.

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.

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to play critical roles in tumor development and progression.,The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence the transcriptional activation of targeted genes.,We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression.,Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence.,Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly and activation of DNA repair pathways through direct transcriptional regulatory mechanisms.,Additionally, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells following combination treatment with cisplatin.,Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

The DecisionDx-Melanoma test provides prognostic information for patients with cutaneous melanoma (CM).,Using formalin-fixed paraffin-embedded primary tumor tissue, the RT-PCR-based test classifies patients into a low- (Class 1) or high-risk (Class 2) category for recurrence based on expression of 31 genes.,The current study was designed to assess the analytical validity of this test.,Inter-assay, inter-instrument, and inter-operator studies were performed to evaluate reliability of the 31-gene expression test results, sample stability and reagent stability.,From March 2013 through June 2016, the gene expression test was performed on 8244 CM tumors.,De-identified data from Pathology Reports were used to assess technical success.,Robust sample and reagent stability was observed.,Inter-assay concordance on 168 specimens run on 2 consecutive days was 99% and matched probability scores were significantly correlated (R2 = 0.96).,Inter-instrument concordance was 95%, and probability scores had a correlation R2 of 0.99 (p < 0.001).,From 8244 CM specimens submitted since 2013, 85% (7023) fulfilled pre-specified tumor content parameters.,In these samples with sufficient tumor requirements, the technical success of the test was 98%.,DecisionDx-Melanoma is a robust gene expression profile test that demonstrates strong reproducibility between experiments and has high technical reliability on clinical samples.,The online version of this article (10.1186/s13000-018-0690-3) contains supplementary material, which is available to authorized users.

Recently, a 23‐gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions.,The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions.,A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory.,Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists.,A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses.,The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis.,The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%.,These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice.,Cancer 2017;123:617-628.,© 2016 American Cancer Society.,A 23‐gene signature has recently been developed to differentiate malignant and benign melanocytic lesions.,This study shows that the gene expression signature differentiates benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5% in comparison with a triple‐concordant histopathologic review.

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

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

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

Wnt5a has been implicated in melanoma progression and metastasis, although the exact downstream signaling events that contribute to melanoma metastasis are poorly understood.,Wnt5a signaling results in acyl protein thioesterase 1 (APT1) mediated depalmitoylation of pro-metastatic cell adhesion molecules CD44 and MCAM, resulting in increased melanoma invasion.,The mechanistic details that underlie Wnt5a-mediated regulation of APT1 activity and cellular function remain unknown.,Here, we show Wnt5a signaling regulates APT1 activity through induction of APT1 phosphorylation and we further investigate the functional role of APT1 phosphorylation on its depalmitoylating activity.,We found phosphorylation increased APT1 depalmitoylating activity and reduced APT1 dimerization.,We further determined APT1 phosphorylation increases melanoma invasion in vitro, and also correlated with increased tumor grade and metastasis.,Our results further establish APT1 as an important regulator of melanoma invasion and metastatic behavior.,Inhibition of APT1 may represent a novel way to treat Wnt5a driven cancers.

Germline mutations in BAP1 have been associated with BAP1-Tumor Predisposition Syndrome (BAP1-TPDS), a predisposition to multiple tumors within a family that includes uveal melanoma (UM), cutaneous melanoma, malignant mesothelioma and renal cell carcinoma.,Alternatively, somatic mutations in BAP1 in UM have been associated with high risk for metastasis.,In this study, we compare the risk of metastasis in UM that carry germline versus somatic BAP1 mutations and mutation-negative tumors.,DNA extracted from 142 UM and matched blood samples was sequenced using Sanger or next generation sequencing to identify BAP1 gene mutations.,Eleven of 142 UM (8%) carried germline BAP1 mutations, 43 (30%) had somatic mutations, and 88 (62%) were mutation-negative.,All BAP1 mutations identified in blood samples were also present in the matched UM.,There were 52 unique mutations in 54 tumors.,All were pathogenic or likely pathogenic.,A comparison of tumors carrying somatic vs. germline mutations, or no mutations, showed a higher frequency of metastasis in tumors carrying somatic mutations: 74% vs.,36%, P=0.03 and 74% vs.,26% P<0.001, respectively.,Tumors with a somatic mutation compared to mutation-negative had an older age of diagnosis of (61.8 vs.,52.2 years, P=0.002), and shorter time to metastasis (16 vs. 26 months, P=0.04).,Kaplan-Meier analysis further showed that tumors with somatic (vs. germline) mutations demonstrated a greater metastatic risk (P=0.03).,Cox multivariate analysis showed in addition to chromosome-3 monosomy and larger tumor diameter, the presence of BAP1 somatic, but not germline mutations, was significantly associated with risk of metastasis(P=0.02).,Personal or family history of BAP1-TPDS was available for 79 of the cases.,All eight cases with germline mutations reported a history of BAP1-TPDS, which was significantly greater than what was observed in cases with somatic mutations (10 of 23, P=0.009) or mutation-negative cases (11 of 48, P<0.001).,Defining germline vs. somatic nature of BAP1 mutations in UM can inform the individual about both the risk of metastasis, and the time to metastasis, which are critically important outcomes for the individual.,This information can also change the cascade screening and surveillance of family members.,The online version of this article (10.1186/s12885-018-5079-x) contains supplementary material, which is available to authorized users.

The global incidence of melanoma has been increasing faster than any other form of cancer.,New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy.,Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma.,They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination.,We investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines.,Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRT-PCR.,Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells.,Ectopic expression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate.,We have identified and characterised a novel DNA damage response mechanism in melanoma.,Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNA polymerase zeta to allow replication to proceed.,This response also resulted in synthetic lethality to the PARP inhibitor olaparib.,This unusual DNA damage response may be a more appropriate strategy for an aggressive and rapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results in increased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib.,The online version of this article (10.1186/s12885-017-3864-6) contains supplementary material, which is available to authorized users.

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

We compared gene expression signatures of aggressive amelanotic (Amela) melanomas with those of slowly growing pigmented melanomas (Mela), identifying pathways potentially responsible for the aggressive Amela phenotype.,Both tumors develop in mice upon conditional deletion in melanocytes of Ink4a/Arf tumor suppressor genes with concomitant expression of oncogene H-RasG12V and a known tumor antigen.,We previously showed that only the aggressive Amela tumors were highly infiltrated by leukocytes concomitant with local and systemic inflammation.,We report that Amela tumors present a pattern of de-differentiation with reduced expression of genes involved in pigmentation.,This correlates with reduced and enhanced expression, respectively, of microphthalmia-associated (Mitf) and Pou3f2/Brn-2 transcription factors.,The reduced expression of Mitf-controlled melanocyte differentiation antigens also observed in some human cutaneous melanoma has important implications for immunotherapy protocols that generally target such antigens.,Induced Amela tumors also express Epithelial-Mesenchymal-Transition (EMT)-like and TGFβ-pathway signatures.,These are correlated with constitutive Smad3 signaling in Amela tumors and melanoma cell lines.,Signatures of infiltrating leukocytes and some chemokines such as chemotactic cytokine ligand 2 (Ccl2) that contribute to leukocyte recruitment further characterize Amela tumors.,Inhibition of the mitogen-activated protein kinase (MAPK) activation pathway in Amela tumor lines leads to reduced expression of EMT hallmark genes and inhibits both proinflammatory cytokine Ccl2 gene expression and Ccl2 production by the melanoma cells.,These results indicate a link between EMT-like processes and alterations of immune functions, both being controlled by the MAPK pathway.,They further suggest that targeting the MAPK pathway within tumor cells will impact tumor-intrinsic oncogenic properties as well as the nature of the tumor microenvironment.

This secondary analysis of a randomized clinical trial examines the association between immune-related adverse events and recurrence-free survival among patients with high-risk stage III melanoma who were treated with pembrozimulab therapy or placebo in the EORTC 1325/KEYNOTE-054 study.,Are immune-related adverse events associated with recurrence-free survival in patients with high-risk stage III melanoma treated with pembrolizumab therapy compared with placebo?,In this secondary analysis of a randomized clinical trial of 1019 adults with stage III melanoma, the occurrence of an immune-related adverse event was associated with longer recurrence-free survival among both men and women in the pembrolizumab arm.,However, in the placebo arm, this association was not significant.,These findings suggest that the occurrence of an immune-related adverse event is an indicator of pembrolizumab activity in patients with high-risk stage III melanoma.,Whether immune-related adverse events (irAEs) indicate drug activity in patients treated with immune checkpoint inhibitors remains unknown.,To investigate the association between irAEs and recurrence-free survival (RFS) in the double-blind EORTC 1325/KEYNOTE-054 clinical trial comparing pembrolizumab therapy and placebo for the treatment of patients with high-risk stage III melanoma.,A total of 1019 adults with stage III melanoma were randomly assigned on a 1:1 ratio to receive treatment with pembrolizumab therapy or placebo.,Eligible patients were adults 18 years and older with complete resection of cutaneous melanoma metastatic to lymph nodes, classified with stage IIIA (at least 1 micrometastasis measuring >1 mm in greatest diameter), IIIB, or IIIC (without in-transit metastasis) cancer.,Patients were randomized from August 26, 2015, to November 14, 2016.,The clinical cutoff for the data set was October 2, 2017.,Analyses were then performed on the database, which was locked on November 28, 2017.,Participants were scheduled to receive 200 mg of pembrolizumab or placebo every 3 weeks for a total of 18 doses for approximately 1 year or until disease recurrence, unacceptable toxic effects, major protocol violation, or withdrawal of consent.,The association between irAEs and RFS was estimated using a Cox model adjusted for sex, age, and AJCC-7 stage, with a time-varying covariate that had a value of 0 before irAE onset and 1 after irAE onset.,Of 1011 patients who began treatment with pembrolizumab therapy or placebo, 622 (61.5%) were men and 389 (38.5%) were women; 386 patients (38.2%) were aged 50 to 64 years, 377 (37.3%) were younger than 50 years, and 248 (24.5%) were 65 years and older.,Consistent with the reported main analysis in the intent-to-treat population, RFS was longer in the pembrolizumab arm compared with the placebo arm (hazard ratio [HR], 0.56; 98.4% CI, 0.43-0.74) among patients who started treatment.,The incidence of irAEs was 190 (37.4%) in the pembrolizumab arm (n = 509) and 45 (9.0%) in the placebo arm (n = 502); in each treatment group, the incidence was similar for men and women.,The occurrence of an irAE was associated with a longer RFS in the pembrolizumab arm (HR, 0.61; 95% CI, 0.39-0.95; P = .03) in both men and women.,However, in the placebo arm, this association was not significant.,Compared with the placebo arm, the reduction in the hazard of recurrence or death in the pembrolizumab arm was greater after the onset of an irAE than without or before an irAE (HR, 0.37; 95% CI, 0.24-0.57 vs HR, 0.61; 95% CI, 0.49-0.77, respectively; P = .03).,In this study, the occurrence of an irAE was associated with a longer RFS in the pembrolizumab arm.,ClinicalTrials.gov identifier: NCT02362594; EudraCT identifier: 2014-004944-37

The incidence of melanoma among those of an Asian ethnicity is lower than in Caucasians; few large‐scale Asian studies that include follow‐up data have been reported.,To investigate the clinical characteristics of Japanese patients with melanoma and to evaluate the prognostic factors.,Detailed patient information was collected from the database of Japanese Melanoma Study Group of the Japanese Skin Cancer Society.,The American Joint Committee on Cancer seventh Edition system was used for TNM classification.,The Kaplan‐Meier method and Cox proportional hazards model were used to estimate the impact of clinical and histological parameters on disease‐specific survival in patients with invasive melanoma.,In total, 4594 patients were included in this analysis.,The most common clinical type was acral lentiginous melanoma (40.4%) followed by superficial spreading melanoma (20.5%), nodular melanoma (10.0%), mucosal melanoma (9.5%), and lentigo maligna melanoma (8.1%).,The 5‐year disease‐specific survival for each stage was as follows: IA = 98.0%, IB = 93.9%, IIA = 94.8%, IIB = 82.4%, IIC = 71.8%, IIIA = 75.0%, IIIB = 61.3%, IIIC = 41.7%, and IV = 17.7%.,Although multivariate analysis showed that clinical classifications were not associated with survival across all stages, acral type was an independent poor prognostic factor in stage IIIA.,Our study revealed the characteristics of melanoma in the Japanese population.,The 5‐year disease‐specific survival of each stage showed a similar trend to that of Caucasians.,While clinical classification was not associated with survival in any stages, acral type was associated with poor survival in stage IIIA.,Our result might indicate the aggressiveness of acral type in certain populations.

New therapeutic modalities for melanoma promise benefit in selected individuals.,Efficacy appears greater in patients with lower tumor burden, suggesting an important role for risk‐stratified surveillance.,Robust predictive markers might permit optimization of agent to patient, while low‐risk prognostic markers might guide more conservative management.,This review evaluates protein, gene, and multiplexed marker panels that may contribute to better risk assessment and improved management of patients with cutaneous melanoma.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency.,Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles.,This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients.,Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

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

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

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

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

Wnt5a has been implicated in melanoma progression and metastasis, although the exact downstream signaling events that contribute to melanoma metastasis are poorly understood.,Wnt5a signaling results in acyl protein thioesterase 1 (APT1) mediated depalmitoylation of pro-metastatic cell adhesion molecules CD44 and MCAM, resulting in increased melanoma invasion.,The mechanistic details that underlie Wnt5a-mediated regulation of APT1 activity and cellular function remain unknown.,Here, we show Wnt5a signaling regulates APT1 activity through induction of APT1 phosphorylation and we further investigate the functional role of APT1 phosphorylation on its depalmitoylating activity.,We found phosphorylation increased APT1 depalmitoylating activity and reduced APT1 dimerization.,We further determined APT1 phosphorylation increases melanoma invasion in vitro, and also correlated with increased tumor grade and metastasis.,Our results further establish APT1 as an important regulator of melanoma invasion and metastatic behavior.,Inhibition of APT1 may represent a novel way to treat Wnt5a driven cancers.

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

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

Melanoma treatment has been revolutionized over the past decade.,Long-term results with immuno-oncology (I-O) agents and targeted therapies are providing evidence of durable survival for a substantial number of patients.,These results have prompted consideration of how best to define long-term benefit and cure.,Now more than ever, oncologists should be aware of the long-term outcomes demonstrated with these newer agents and their relevance to treatment decision-making.,As the first tumor type for which I-O agents were approved, melanoma has served as a model for other diseases.,Accordingly, discussions regarding the value and impact of long-term survival data in patients with melanoma may be relevant in the future to other tumor types.,Current findings indicate that, depending on the treatment, over 50% of patients with melanoma may gain durable survival benefit.,The best survival outcomes are generally observed in patients with favorable prognostic factors, particularly normal baseline lactate dehydrogenase and/or a low volume of disease.,Survival curves from melanoma clinical studies show a plateau at 3 to 4 years, suggesting that patients who are alive at the 3-year landmark (especially in cases in which treatment had been stopped) will likely experience prolonged cancer remission.,Quality-of-life and mixture-cure modeling data, as well as metrics such as treatment-free survival, are helping to define the value of this long-term survival.,In this review, we describe the current treatment landscape for melanoma and discuss the long-term survival data with immunotherapies and targeted therapies, discussing how to best evaluate the value of long-term survival.,We propose that some patients might be considered functionally cured if they have responded to treatment and remained treatment-free for at least 2 years without disease progression.,Finally, we consider that, while there have been major advances in the treatment of melanoma in the past decade, there remains a need to improve outcomes for the patients with melanoma who do not experience durable survival.

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

Uveal melanoma 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.

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

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).

HSP90 (heat shock protein 90) is an ATP-dependent molecular chaperone involved in a proper folding and maturation of hundreds of proteins.,HSP90 is abundantly expressed in cancer, including melanoma.,HSP90 client proteins are the key oncoproteins of several signaling pathways controlling melanoma development, progression and response to therapy.,A number of natural and synthetic compounds of different chemical structures and binding sites within HSP90 have been identified as selective HSP90 inhibitors.,The majority of HSP90-targeting agents affect N-terminal ATPase activity of HSP90.,In contrast to N-terminal inhibitors, agents interacting with the middle and C-terminal domains of HSP90 do not induce HSP70-dependent cytoprotective response.,Several inhibitors of HSP90 were tested against melanoma in pre-clinical studies and clinical trials, providing evidence that these agents can be considered either as single or complementary therapeutic strategy.,This review summarizes current knowledge on the role of HSP90 protein in cancer with focus on melanoma, and provides an overview of structurally different HSP90 inhibitors that are considered as potential therapeutics for melanoma treatment.

Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context.,In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression.,Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds.,In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles.,This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy.,Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors.

We performed harmonized molecular and clinical analysis on 1,048 melanomas and discovered markedly different global genomic properties among subtypes (BRAF, (N)RAS, NF1, Triple Wild-Type), subtype-specific preferences for secondary driver genes, and active mutational processes previously unreported in melanoma.,Secondary driver genes significantly enriched in specific subtypes reflected preferential dysregulation of additional pathways, such as induction of TGF-β signaling in BRAF melanomas and inactivation of the SWI/SNF complex in (N)RAS melanomas, and select co-mutation patterns coordinated selective response to immune checkpoint blockade.,We also defined the mutational landscape of Triple Wild-Type melanomas and identified enrichment of DNA repair defect signatures in this subtype, which were associated with transcriptional downregulation of key DNA repair genes and may revive previously discarded or currently unconsidered therapeutic modalities for genomically stratified melanoma patient subsets.,Broadly, harmonized meta-analysis of melanoma whole-exomes identified distinct molecular drivers that may point to multiple opportunities for biological and therapeutic investigation.

Immune checkpoint inhibition (ICI) is an essential treatment option in melanoma.,Its outcome may be improved by a preceding radiation of metastases.,This study aimed to investigate the impact of a preceding radiotherapy on the clinical outcome of ICI treatment.,This multicenter retrospective cohort study included patients who received anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or anti-programmed cell death protein 1 (PD-1) ICI with or without preceding radiotherapy for unresectable metastatic melanoma.,ICI therapy outcome was measured as best overall response (BOR), progression-free (PFS) and overall survival (OS).,Response and survival analyses were adjusted for confounders identified by directed acyclic graphs.,Adjusted survival curves were calculated using inverse probability treatment weighting.,835 patients who received ICI (anti-CTLA-4, n=596; anti-PD-1, n=239) at 16 centers were analyzed, whereof 235 received a preceding radiotherapy of metastatic lesions in stage IV disease.,The most frequent organ sites irradiated prior to ICI therapy were brain (51.1%), lymph nodes (17.9%) and bone (17.9%).,After multivariable adjustment for confounders, no relevant differences in ICI therapy outcome were observed between cohorts with and without preceding radiotherapy.,BOR was 8.7% vs 13.0% for anti-CTLA-4 (adjusted relative risk (RR)=1.47; 95% CI=0.81 to 2.65; p=0.20), and 16.5% vs 25.3% for anti-PD-1 (RR=0.93; 95% CI=0.49 to 1.77; p=0.82).,Survival probabilities were similar for cohorts with and without preceding radiotherapy, for anti-CTLA-4 (PFS, adjusted HR=1.02, 95% CI=0.86 to 1.25, p=0.74; OS, HR=1.08, 95% CI=0.81 to 1.44, p=0.61) and for anti-PD-1 (PFS, HR=0.84, 95% CI=0.57 to 1.26, p=0.41; OS, HR=0.73, 95% CI=0.43 to 1.25, p=0.26).,Patients who received radiation last before ICI (n=137) revealed no better survival than those who had one or more treatment lines between radiation and start of ICI (n=86).,In 223 patients with brain metastases, we found no relevant survival differences on ICI with and without preceding radiotherapy.,This study detected no evidence for a relevant favorable impact of a preceding radiotherapy on anti-CTLA-4 or anti-PD-1 ICI treatment outcome in metastatic melanoma.

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

Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types.,Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this “difficult-to-treat” cancer type has not been investigated so far.,We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients.,Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes.,The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion.,Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells.,The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects.,In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth.,In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.

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

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.

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

The risk of developing cutaneous squamous cell carcinoma (SCC) is markedly increased in organ transplant recipients (OTRs) compared to the normal population.,Next to sun exposure, the immunosuppressive regimen is an important risk factor for the development of SCC in OTRs.,Various gene mutations (e.g.,TP53) and genetic alterations (e.g. loss of CDKN2A, amplification of RAS) have been found in SCCs.,The aim of this genome-wide study was to identify pathways and genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs).,To perform the analysis in an isogenic background, RNA and DNA were isolated from SCC, AK and normal (unexposed) epidermis (NS) from each of 13 OTRs.,Samples were subjected to genome-wide expression analysis and genome SNP analysis using Illumina’s HumanWG-6 BeadChips and Infinium II HumanHap550 Genotyping BeadChips, respectively. mRNA expression results were verified by quantitative PCR.,Hierarchical cluster analysis of mRNA expression profiles showed SCC, AK and NS samples to separate into three distinct groups.,Several thousand genes were differentially expressed between epidermis, AK and SCC; most upregulated in SCCs were hyperproliferation related genes and stress markers, such as keratin 6 (KRT6), KRT16 and KRT17.,Matching to oncogenic pathways revealed activation of downstream targets of RAS and cMYC in SCCs and of NFκB and TNF already in AKs.,In contrast to what has been reported previously, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles.,Vast differences in gene expression profiles exist between SCC, AK and NS from immunosuppressed OTRs.,Moreover, several pathways activated in SCCs were already activated in AKs, confirming the assumption that AKs are the precursor lesions of SCCs.,Since the drastic changes in gene expression appeared unlinked to specific genomic gains or losses, the causal events driving SCC development require further investigation.,Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs.,Further study is required to identify the mechanisms of early activation of NFκB and TNF, and to establish whether these pathways offer a feasible target for preventive intervention among OTRs.

Genetically engineered T cells expressing a T-cell receptor (TCR) are powerful tools for cancer treatment and have shown significant clinical effects in sarcoma patients.,However, mismatch of the introduced TCR α/β chains with endogenous TCR may impair the expression of transduced TCR, resulting in an insufficient antitumor capacity of modified T cells.,Here, we report the development of immunotherapy using human lymphocytes transduced with a codon-optimized melanoma-associated antigen (MAGE)-A4 and HLA-A*2402-restricted TCR, which specifically downregulate endogenous TCR by small interfering RNA (si-TCR).,We evaluated the efficacy of this immunotherapy in both NOD-SCID mice and uterine leiomyosarcoma patients.,Our results revealed that transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR, enhanced cytotoxic activity against antigen-expressing tumor cells, and increased interferon-γ production by specific MAGE-A4 peptide stimulation.,Retarded tumor growth was also observed in NOD-SCID mice inoculated with human tumor cell lines expressing both MAGE-A4 and HLA-A*2402.,Furthermore, we report the successful management of a case of uterine leiomyosarcoma treated with MAGE-A4 si-TCR/HLA-A*2402 gene-modified T cells.,Our results indicate that the TCR-modified T cell therapy is a promising novel strategy for cancer treatment.

To characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures.,Primary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures.,These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed.,In monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells.,In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids.,This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines.,Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures.,This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo.,This study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor.,Differences in the cell culture method does plays an important role in phospholipid composition of the cells.,The online version of this article (doi:10.1186/s40659-017-0117-8) contains supplementary material, which is available to authorized users.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT).,Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy.,To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCβ4, and CYSLTR2 mutations in plasma DNA.,Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression.,In total, 42 primary UM and 11 metastatic patients were enrolled.,All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls.,Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients.,Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors.,Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

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.

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.

Microenvironment cues involved in melanoma progression are largely unknown.,Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host.,A role of exosomes in driving melanoma progression under microenvironmental acidity was never described.,We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7.,To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis.,Functional roles of exosomes were tested in migration and invasion tests.,Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression.,We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive.,We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes.,In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis.,A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases.,A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes.,Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.,The online version of this article (10.1186/s13046-018-0915-z) contains supplementary material, which is available to authorized users.

Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized.,In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors.,We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation.,Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB.,Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data.,Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response.,Analysis of fully clinically annotated and sequenced melanoma tumor samples collected before anti-PD1 treatment suggests that determinants of response differ on the basis of previous anti-CTLA4 therapy, and that tumor mutational burden may not be a strong predictor of response across melanoma subtypes.

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

Solid tumors are complex systems characterized by dynamic interactions between neoplastic cells, non-tumoral cells, and extracellular components.,Among all the stromal cells that populate tumor microenvironment, fibroblasts are the most abundant elements and are critically involved in disease progression.,Cancer-associated fibroblasts (CAFs) have pleiotropic functions in tumor growth and extracellular matrix remodeling implicated in local invasion and distant metastasis.,CAFs additionally participate in the inflammatory response of the tumor site by releasing a variety of chemokines and cytokines.,It is becoming clear that understanding the dynamic, mutual melanoma-fibroblast relationship would enable treatment options to be amplified.,To better characterize melanoma-associated fibroblasts, here we analyzed low-passage primary CAFs derived from advanced-stage primary skin melanomas, focusing on the immuno-phenotype.,Furthermore, we assessed the expression of several CAF markers and the production of growth factors.,To deepen the study of CAF-melanoma cell crosstalk, we employed CAF-derived supernatants and trans-well co-culture systems to evaluate the influences of CAFs on (i) the motogenic ability of melanoma cells, (ii) the chemotherapy-induced cytotoxicity, and (iii) the release of mediators active in modulating tumor growth and spread.

The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma.,Mutation specific antibodies against NRASQ61R and BRAFV600E proteins could offer additional data on tumor heterogeneity.,The specificity and sensitivity of NRASQ61R immunohistochemistry have recently been reported excellent.,We aimed to determine the utility of immunohistochemistry using SP174 anti-NRASQ61R and VE1 anti-BRAFV600E antibodies in the theranostic mutation screening of melanomas.,142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry.,23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively.,The 23 NRASQ61R and 23 BRAFV600E-mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative.,Proportions and intensities of staining were varied.,Other NRASQ61L, NRASQ61K, BRAFV600K and BRAFV600R mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data.,Technical limitations are discussed to explain those discrepancies.,Anyway we could not rule out real tumor heterogeneity.,In our study, we showed that combining immunohistochemistry analysis targeting NRASQ61R and BRAFV600E proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.,The online version of this article (doi:10.1186/s13000-015-0359-0) contains supplementary material, which is available to authorized users.

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.

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.

Adjuvant targeted therapy (TT) improves relapse free survival in patients with resected BRAF mutant stage III melanoma.,The outcomes and optimal management of patients who relapse after adjuvant TT is unknown.,Patients from twenty-one centres with recurrent melanoma after adjuvant TT were included.,Disease characteristics, adjuvant therapy, recurrence, treatment at relapse and outcomes were examined.,Eighty-five patients developed recurrent melanoma; nineteen (22%) during adjuvant TT.,Median time to first recurrence was 18 months and median follow-up from first recurrence was 31 months.,Fifty-eight (68%) patients received immunotherapy (IT) or TT as 1st line systemic therapy at either first or subsequent recurrence and had disease that was assessable for response.,Response to anti-PD-1 (±trial agent), combination ipilimumab-nivolumab, TT rechallenge and ipilimumab monotherapy was 63%, 62% 25% and 10% respectively.,Twenty-eight (33%) patients had died at census, all from melanoma.,Two-year OS was 84% for anti-PD-1 therapy (±trial agent), 92% for combination ipilimumab and nivolumab, 49% for TT and 45% for ipilimumab monotherapy (p = 0.028).,Patients who relapse after adjuvant TT respond well to subsequent anti-PD-1 based therapy and have outcomes similar to those seen when first line anti-PD-1 therapy is used in stage IV melanoma.

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

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

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

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

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

Aquaporin 3 (AQP3) and phospholipase D2 (PLD2) are abnormally expressed and/or localized in squamous cell carcinoma (SCC).,AQP3 transports glycerol to PLD2 for the synthesis of lipid second messenger, which can mediate the effect of the AQP3/PLD2 signaling module in the regulation of keratinocyte proliferation and differentiation.,However, the role of the AQP3/PLD2 signaling module in the pathogenesis of SCC remains to be fully elucidated.,In the present study, the expression levels of AQP3 and PLD2 in tissue samples were examined using immunohistochemistry, it was found that the expression levels of AQP3 and PLD2 in tissue samples of actinic keratosis (AK), Bowen's disease (BD) and SCC were significantly increased.,AQP3 small interfering RNA (siRNA) and PLD2 siRNA were constructed and used for transfection into the human A431 SCC cell line, and their anticancer effect on SCC was examined.,The mRNA expression and protein expression levels of AQP3 and PLD2 were significantly downregulated following siRNA transfection.,AQP3 siRNA and PLD2 siRNA inhibited the proliferation and promoted the apoptosis of A431 cells.,Taken together, the findings of the present study suggested that increased levels of AQP3 and PLD2 were correlated with tumor progression and development in SCC.,AQP3 siRNA and PLD2 siRNA significantly downregulated the mRNA and protein levels of AQP3 and PLD2 in the A431 cells; inhibiting proliferation and promoting apoptosis in vitro.,The concomitant effects of AQP3/PLD2 signaling by inhibiting the expression of siRNA may be important for the treatment of SCC in the future.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer.,Inflammation is a typical feature in cSCC progression.,Analysis of the expression of inflammasome components in cSCC cell lines and normal human epidermal keratinocytes revealed upregulation of the expression of AIM2 mRNA and protein in cSCC cells.,Elevated levels of AIM2 mRNA were noted in cSCCs in vivo compared with normal skin.,Strong and moderate tumor cell specific expression of AIM2 was detected with immunohistochemistry (IHC) in sporadic human cSCCs in vivo, whereas expression of AIM2 was moderate in cSCC in situ (cSCCIS) and low or absent in actinic keratosis (AK) and normal skin.,IHC of cSCCs, cSCCIS and AKs from organ transplant recipients also revealed strong and moderate tumor cell specific expression of AIM2 in cSCCs.,Knockdown of AIM2 resulted in reduction in viability of cSCC cells and onset of apoptosis.,RNA-seq and pathway analysis after knockdown of AIM2 in cSCC cells revealed downregulation of the biofunction category Cell cycle and upregulation of the biofunction category Cell Death and Survival.,Knockdown of AIM2 also resulted in reduction in invasion of cSCC cells and downregulation in production of invasion proteinases MMP1 and MMP13.,Knockdown of AIM2 resulted in suppression of growth and vascularization of cSCC xenografts in vivo.,These results provide evidence for the role of AIM2 in the progression of cSCC and identify AIM2 inflammasome function as a potential therapeutic target in these invasive and metastatic tumors.

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

Patients with advanced melanoma have a compromised anti-tumor immune response leading to tumor immune tolerance and a tumor microenvironment conducive to disease progression.,Immunotherapy that successfully overcomes this tumor-mediated immune suppression has made the greatest impact in the management of this disease over the past few years.,This progress through immunotherapy builds upon earlier successes that interferon-α had in the treatment of melanoma in the adjuvant setting, as well as that of high-dose interleukin-2 in advanced melanoma.,The development of immune checkpoint inhibitors has led to dramatic clinical activity in advanced melanoma.,In particular, anti-CTLA4 and anti-PD1 monoclonal antibodies have taken us forward into the realm of longer survival and durable responses with the possibility of cure in a continuously increasing proportion of patients.,Combination immunotherapeutic strategies and novel immunotherapeutic agents are being tested at an accelerated pace where the outlook for long-term survival benefits for the majority of patients appears brighter than ever.

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

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

Inflammasomes are mediators of inflammation, and constitutively activated NLRP3 inflammasomes have been linked to IL-1β-mediated tumorigenesis in human melanoma.,Whereas NLRP3 regulation of caspase-1 activation requires the adaptor protein ASC, caspase-1 activation by another danger-signaling sensor NLRP1 does not require ASC because NLRP1 contains a C-terminal CARD domain that facilitates direct caspase-1 activation via CARD-CARD interaction.,We hypothesized that NLRP1 has additional biological activities besides IL-1β maturation and investigated its role in melanoma tumorigenesis.,NLRP1 expression in melanoma was confirmed by analysis of 216 melanoma tumors and 13 human melanoma cell lines.,Unlike monocytic THP-1 cells with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm.,Knocking down NLRP1 revealed a tumor promoting property of NLRP1 both in vitro and in vivo.,Mechanistic studies showed that caspase-1 activity, IL-1β production, IL-1β secretion, and NF-kB activity were reduced by knocking down of NLRP1 in human metastatic melanoma cell lines 1205Lu and HS294T, indicating that NLRP1 inflammasomes are active in metastatic melanoma.,However, unlike previous reports showing that NLRP1 enhances pyroptosis in macrophages, NLRP1 in melanoma behaved differently in the context of cell death.,Knocking down NLRP1 increased caspase-2, -9, and -3/7 activities and promoted apoptosis in human melanoma cells.,Immunoprecipitation revealed interaction of NLRP1 with CARD-containing caspase-2 and -9, whereas NLRP3 lacking a CARD motif did not interact with the caspases.,Consistent with these findings, NLRP1 activation but not NLRP3 activation reduced caspase-2, -9, and -3/7 activities and provided protection against apoptosis in human melanoma cells, suggesting a suppressive role of NLRP1 in caspase-3/7 activation and apoptosis via interaction with caspase-2 and -9.,In summary, we showed that NLRP1 promotes melanoma growth by enhancing inflammasome activation and suppressing apoptotic pathways.,Our study demonstrates a tumor-promoting role of NLRP1 in cancer cells.

A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells.,Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability.,In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported.,The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.,We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal.,Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated.,Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells.,In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells.,CXCR6+ cells produced more aggressive tumors.,CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.,The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology.,Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development.,Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Although recent therapeutic advances based on our understanding of molecular phenomena have prolonged the survival of melanoma patients, the prognosis of melanoma remains dismal and further understanding of the underlying mechanism of melanoma progression is needed.,In this study, differential expression analyses revealed that many genes, including AKT1 and CDK2, play important roles in melanoma.,Functional analyses of differentially expressed genes (DEGs), obtained from the GEO (Gene Expression Omnibus) database, indicated that high proliferative and metastatic abilities are the main characteristics of melanoma and that the PI3K and MAPK pathways play essential roles in melanoma progression.,Among these DEGs, major facilitator superfamily domain-containing 12 (MFSD12) was found to have significantly and specifically upregulated expression in melanoma, and elevated MFSD12 level promoted cell proliferation by promoting cell cycle progression.,Mechanistically, MFSD12 upregulation was found to activate PI3K signaling, and a PI3K inhibitor reversed the increase in cell proliferation endowed by MFSD12 upregulation.,Clinically, high MFSD12 expression was positively associated with shorter overall survival (OS) and disease-free survival (DFS) in melanoma patients, and MFSD12 was an independent prognostic factor for OS and DFS in melanoma patients.,Therapeutically, in vivo assays further confirmed that MFSD12 interference inhibited tumor growth and lung metastasis in melanoma.,In conclusion, elevated MFSD12 expression promotes melanoma cell proliferation, and MFSD12 is a valuable prognostic biomarker and promising therapeutic target in melanoma.

The full repertoire of human microRNAs (miRNAs) that could distinguish common (benign) nevi from cutaneous (malignant) melanomas remains to be established.,In an effort to gain further insight into the role of miRNAs in melanoma, we applied Illumina next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of nevi, thick primary (>4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines (both primary and metastatic) (n = 28).,From this data representing 698 known miRNAs, we defined a set of top-40 list, which properly classified normal from cancer; also confirming 23 (58%) previously discovered miRNAs while introducing an additional 17 (42%) known and top-15 putative novel candidate miRNAs deregulated during melanoma progression.,Surprisingly, the miRNA signature distinguishing specimens of melanoma from nevus was significantly different than that of melanoma cell lines from melanocytes.,Among the top list, miR-203, miR-204-5p, miR-205-5p, miR-211-5p, miR-23b-3p, miR-26a-5p and miR-26b-5p were decreased in melanomas vs. nevi.,In a validation cohort (n = 101), we verified the NGS results by qRT-PCR and showed that receiver-operating characteristic curves for miR-211-5p expression accurately discriminated invasive melanoma (AUC = 0.933), melanoma in situ (AUC = 0.933) and dysplastic (atypical) nevi (AUC = 0.951) from common nevi.,Target prediction analysis of co-transcribed miRNAs showed a cooperative regulation of key elements in the MAPK signaling pathway.,Furthermore, we found extensive sequence variations (isomiRs) and other non-coding small RNAs revealing a complex melanoma transcriptome.,Deep-sequencing small RNAs directly from clinically defined specimens provides a robust strategy to improve melanoma diagnostics.

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

Malignant melanoma of the skin (CMM) is associated with ultraviolet radiation exposure, but the mechanisms and even the wavelengths responsible are unclear.,Here we use a mammalian model to investigate melanoma formed in response to precise spectrally defined ultraviolet wavelengths and biologically relevant doses.,We show that melanoma induction by ultraviolet A (320-400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes.,In contrast, ultraviolet B radiation (280-320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage.,Thus, we identified two ultraviolet wavelength-dependent pathways for the induction of CMM and describe an unexpected and significant role for melanin within the melanocyte in melanomagenesis.,Exposure to ultraviolet light is responsible for a large proportion of melanomas but the molecular mechanisms are unknown.,In this study, melanoma is found to be induced in mice by UVA and UVB light in a pigment-dependent and -independent manner, respectively, resulting in different types of DNA damage.

Metformin is one of the biguanides commonly used in patients with type II Diabetes Mellitus.,Apart from its hypoglycemic properties, metformin also inhibits the cell cycle by restricting protein synthesis and cell proliferation via regulating the LKB1/AMPL pathway.,Furthermore, it also enhances the PD-1 blockade through a reduction of tumor hypoxia.,Metformin has shown a significant favorable impact on treatment-related outcomes in solid tumors, but these outcomes have not been replicated in the limited clinical studies done on malignant melanoma.,Moreover, none of these studies have reported on the efficacy of the combined use of metformin and immune checkpoint inhibitors (ICIs).,This is a retrospective cohort study that includes patients diagnosed with metastatic malignant melanoma and treated with ipilimumab, nivolumab, and/or pembrolizumab (Cohort A); or ipilimumab, nivolumab, and/or pembrolizumab plus metformin (Cohort B) between January 1st 2011 through December 15th 2017.,In this study, patients are stratified based on anti-PD-1 only and anti-CTLA4/anti-PD-1 combination therapies in each cohort.,Objective response rate (ORR) is the primary endpoint.,Disease control rate (DCR), overall survival (OS) and progression-free survival (PFS) are the secondary endpoints.,Cohort A had 33 patients (60%), while cohort B had 22 (40%).,Overall patient characteristics were similar between both cohorts.,ORR was higher in cohort B (68.2% vs.,54.5%, P = 0.31).,The DCR was higher in cohort B as well (77.3% vs.,60.6%, P = 0.19).,Median OS (46.7 months vs. 28 months), and median PFS (19.8 months vs. 5 months) were longer in cohort B.,However, on univariate and multivariate analyses, none of these differences were statistically significant.,The mean number of new metastatic sites which appeared during therapy were significantly higher in cohort A (A:1.51 vs.,B:0.59, P = 0.009).,We have observed favorable treatment-related outcomes (ORR, DCR, median PFS and median OS) in patients who have received metformin in combination with ICIs without reaching significance, probably, due to small sample size.,Hence, large prospective clinical trials are required to study the synergistic effect of metformin in combination with ICIs before it can be recommended as routine additive therapy.

Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling.,This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover.,Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules.,To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives.,Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile.,In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability.,Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles.,Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover.,To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B).,We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib.,Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.

Oleanolic and ursolic acids are natural triterpenic compounds with pentacyclic cholesterol-like structures which gives them very low water solubility, a significant disadvantage in terms of bioavailability.,We previously reported the synthesis of inclusion complexes between these acids and cyclodextrins, as well as their in vivo evaluation on chemically induced skin cancer experimental models.,In this study the synergistic activity of the acid mixture included inside hydroxypropyl-gamma-cyclodextrin (HPGCD) was monitored using in vitro tests and in vivo skin cancer models.,The coefficient of drug interaction (CDI) was used to characterize the interactions as synergism, additivity or antagonism.,Our results revealed an increased antitumor activity for the mixture of the two triterpenic acids, both single and in complex with cyclodextrin, thus proving their complementary biologic activities.

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

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

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

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

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

Epithelial-mesenchymal transition (EMT) is involved in physiologic processes such as embryogenesis and wound healing.,A similar mechanism occurs in some tumors where cells leave the epithelial layer and gain mesenchymal particularities in order to easily migrate to other tissues.,This process can explain the invasiveness and aggressiveness of these tumors which metastasize, by losing the epithelial phenotype (loss of E-cadherin, desmoplakin, and laminin-1) and acquiring mesenchymal markers (N-cadherin).,Complex changes and interactions happen between the tumor cells and the microenvironment involving different pathways, transcription factors, altered expression of adhesion molecules, reorganization of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in specific microRNAs.,The purpose of this review is to determine particularities of the EMT process in the most common malignant cutaneous tumors (squamous cell carcinoma, basal cell carcinoma, and melanoma) which still have an increasingly high incidence.,More studies are required on this topic in order to establish clear correlations.,High costs related to skin cancer therapies in general as well as high impact on patients' quality of life demand finding new, reliable prognostic and therapeutic markers with significant public health impact.

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