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The mammalian or mechanistic target of rapamycin (mTOR) and associated phosphatidyl-inositiol 3-kinase (PI3K)/protein kinase B (Akt) pathways regulate cell growth, differentiation, migration, and survival, as well as angiogenesis and metabolism.,Dysregulation of these pathways is frequently associated with genetic/epigenetic alterations and predicts poor treatment outcomes in a variety of human cancers including cutaneous malignancies like melanoma and non-melanoma skin cancers.,Recently, the enhanced understanding of the molecular and genetic basis of skin dysfunction in patients with skin cancers has provided a strong basis for the development of novel therapeutic strategies for these obdurate groups of skin cancers.,This review summarizes recent advances in the roles of PI3K/Akt/mTOR and their targets in the development and progression of a broad spectrum of cutaneous cancers and discusses the current progress in preclinical and clinical studies for the development of PI3K/Akt/mTOR targeted therapies with nutraceuticals and synthetic small molecule inhibitors.

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.

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

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

The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC).,High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality.,In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (β-PV) play an important role in accelerating the early stages of skin tumorigenesis.,We investigated the effects of UV induced DNA damage in murine models of β-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain.,Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting.,Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry.,Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase.,Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding.,Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment.,The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci.,DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1.,We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation.,These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.,The online version of this article (doi:10.1186/s12943-015-0453-7) contains supplementary material, which is available to authorized users.

Certain cutaneous human papillomaviruses (HPVs), which are ubiquitous and acquired early during childhood, can cause a variety of skin tumors and are likely involved in the development of non-melanoma skin cancer, especially in immunosuppressed patients.,Hence, the burden of these clinical manifestations demands for a prophylactic approach.,To evaluate whether protective efficacy of a vaccine is potentially translatable to patients, we used the rodent Mastomys coucha that is naturally infected with Mastomys natalensis papillomavirus (MnPV).,This skin type papillomavirus induces not only benign skin tumours, such as papillomas and keratoacanthomas, but also squamous cell carcinomas, thereby allowing a straightforward read-out for successful vaccination in a small immunocompetent laboratory animal.,Here, we examined the efficacy of a virus-like particle (VLP)-based vaccine on either previously or newly established infections.,VLPs raise a strong and long-lasting neutralizing antibody response that confers protection even under systemic long-term cyclosporine A treatment.,Remarkably, the vaccine completely prevents the appearance of benign as well as malignant skin tumors.,Protection involves the maintenance of a low viral load in the skin by an antibody-dependent prevention of virus spread.,Our results provide first evidence that VLPs elicit an effective immune response in the skin under immunocompetent and immunosuppressed conditions in an outbred animal model, irrespective of the infection status at the time of vaccination.,These findings provide the basis for the clinical development of potent vaccination strategies against cutaneous HPV infections and HPV-induced tumors, especially in patients awaiting organ transplantation.

Conventional clinico-pathological features in melanoma patients should be integrated with new molecular diagnostic, predictive, and prognostic factors coming from the expanding genomic profiles.,Cutaneous melanoma (CM), even differing in biological behavior according to sun-exposure levels on the skin areas where it arises, is molecularly heterogeneous.,The next-generation sequencing (NGS) approaches are providing data on mutation landscapes in driver genes that may account for distinct pathogenetic mechanisms and pathways.,The purpose was to group and classify all somatic driver mutations observed in the main NGS-based studies.,Whole exome and whole genome sequencing approaches have provided data on spectrum and distribution of genetic and genomic alterations as well as allowed to discover new cancer genes underlying CM pathogenesis.,After evaluating the mutational status in a cohort of 686 CM cases from the most representative NGS studies, three molecular CM subtypes were proposed: BRAFmut, RASmut, and non-BRAFmut/non-RASmut.

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.

Multiple primary melanomas (MPM) occur up to 8% of patients with cutaneous malignant melanoma (CMM).,They are often sporadic harbouring several somatic mutations, but also familial cases harbouring a CDKN2A germline mutation have been describe in Caucasian populations.,The aim of this study was to investigate the incidence, the distribution patterns and the impact of known and unknown germline and somatic mutations in patients with MPM from Italy.,One-hundred and two MPM patients were enrolled for germline mutation analysis, and five patients with at least four MPMs were identified for somatic mutation analysis.,The demographic, pathologic and clinical features were retrieved from medical records.,Molecular analysis for both germline and somatic mutations was performed in genomic DNA from peripheral blood and tissue samples, respectively, through a next generation sequencing approach, using a specific multiple-gene panel constructed by the Italian Melanoma Intergroup for somatic analysis and a commercial cancer hotspot panel for somatic analysis.,CDKN2A mutations were detected in 6/16 (37.5%) and 3/86 (3.5%) MPM cases with and without family history for melanoma, respectively.,Furthermore, multiple MC1R and, to a lesser extent, ATM variants have been identified.,BAP1 variants were found only in MPM patients from southern Italy.,The most frequent somatic variants were the pathogenic BRAFV600E and TP53, followed by KIT, PIK3CA, KDR, and NRAS.,Single APC, ERBB4, MET, JAK3 and other variants with unknown function were also detected.,CDNK2A mutation is the most relevant susceptibility mutation in Italian patients with MPM, especially those with a family history for CMM.,The prevalence of this mutation and other sequence variants identified in this study varies among specific sub-populations.,Furthermore, some heterogeneity in driver somatic mutations between sporadic MPMs has been observed, as well as in a number of associated sequence variants the clinical impact of which needs to be further elucidated.,The online version of this article (10.1186/s12885-019-5984-7) contains supplementary material, which is available to authorized users.

Conjunctival malignant melanoma (CMM) is a rare malignancy and in the advanced setting there is no effective treatment.,In contrast, half of cutaneous melanomas have BRAF mutations and treatment with BRAF inhibitors is established for patients with disseminated disease.,The most common form of ocular melanoma, uveal melanoma, lacks these mutations, however, their presence has been reported for CMM.,We used the BRAF inhibitor vemurafenib to treat a 53 year-old female suffering from a BRAFV600E mutated metastatic CMM.,The patient benefited from the treatment, a response was evident within a week and she experienced a progression free survival of four months.,To our knowledge, this is the first described case of response to vemurafenib treatment in a patient with ocular melanoma.

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

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.

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.

Sentinel lymph node biopsy (SLNB) is a diagnostic procedure developed in the 1990s.,It is currently used to stage patients with primary cutaneous melanoma, provide prognostic information and guide management.,The Australian Clinical Practice Guidelines state that SLNB should be considered for patients with cutaneous melanoma >1 mm in thickness (or >0.8 mm with high-risk pathology features).,Until recently, sentinel lymph node (SLN) status was used to identify patients who might benefit from a completion lymph node dissection, a procedure that is no longer routinely recommended.,SLN status is now also being used to identify patients who might benefit from systemic adjuvant therapies such as anti-programmed cell death 1 (PD1) checkpoint inhibitor immunotherapy or BRAF-directed molecular targeted therapy, treatments that have significantly improved relapse-free survival for patients with resected stage III melanoma and improved overall survival of patients with unresectable stage III and stage IV melanoma.,Australian and international data indicate that approximately half of eligible patients receive an SLNB.,This mixed-methods study seeks to understand the structural, contextual and cultural factors affecting implementation of the SLNB guidelines.,Data collection will include: (1) cross-sectional questionnaires and semistructured interviews with general practitioners and dermatologists; (2) semistructured interviews with other healthcare professionals involved in the diagnosis and early definitive care of melanoma patients and key stakeholders including researchers, representatives of professional colleges, training organisations and consumer melanoma groups; and (3) documentary analysis of documents from government, health services and non-government organisations.,Descriptive analyses and multivariable regression models will be used to examine factors related to SLNB practices and attitudes.,Qualitative data will be analysed using thematic analysis.,Ethics approval has been granted by the University of Sydney.,Results will be disseminated through publications and presentations to clinicians, patients, policymakers and researchers and will inform the development of strategies for implementing SLNB guidelines in Australia.

Melanoma is the most aggressive form of skin cancer and one of the most treatment-refractory malignancies.,In metastatic melanoma cell lines, we analysed the anti-proliferative and pro-apoptotic potentials of a phenolic component of olive oil, the hydroxytyrosol.,In particular, through MTS assay, DeadEnd™ Colorimetric TUNEL assay, Annexin V binding and PI uptake, western blot experiment, intracellular reactive oxygen species (ROS) analysis, and the cell colony assay, we showed that the hydroxytyrosol treatment remarkably reduces the cell viability inducing the death for apoptosis of melanoma cells.,Moreover, we showed that the hydroxytyrosol treatment of melanoma cells leads to a significant increase of p53 and γH2AX expression, a significant decrease of AKT expression and the inhibition of cell colony formation ability.,Finally, we propose that the increased amount of intracellular reactive oxygen species (ROS) that may be related to the regulation of the pathways involved in the activation of apoptosis and in the inhibition of melanoma growth could be the strategy used by hydroxytyrosol to exert its functions in melanoma.,Therefore, for its role in melanoma growth inhibition, the hydroxytyrosol treatment could deeply interfere with melanoma progression as a promising therapeutic option for the treatment of this highly invasive tumour.

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.

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

Iron has been shown to trigger oxidative stress by elevating reactive oxygen species (ROS) and to participate in different modes of cell death, such as ferroptosis, apoptosis and necroptosis.,However, whether iron-elevated ROS is also linked to pyroptosis has not been reported.,Here, we demonstrate that iron-activated ROS can induce pyroptosis via a Tom20-Bax-caspase-GSDME pathway.,In melanoma cells, iron enhanced ROS signaling initiated by CCCP, causing the oxidation and oligomerization of the mitochondrial outer membrane protein Tom20.,Bax is recruited to mitochondria by oxidized Tom20, which facilitates cytochrome c release to cytosol to activate caspase-3, eventually triggering pyroptotic death by inducing GSDME cleavage.,Therefore, ROS acts as a causative factor and Tom20 senses ROS signaling for iron-driven pyroptotic death of melanoma cells.,Since iron activates ROS for GSDME-dependent pyroptosis induction and melanoma cells specifically express a high level of GSDME, iron may be a potential candidate for melanoma therapy.,Based on the functional mechanism of iron shown above, we further demonstrate that iron supplementation at a dosage used in iron-deficient patients is sufficient to maximize the anti-tumor effect of clinical ROS-inducing drugs to inhibit xenograft tumor growth and metastasis of melanoma cells through GSDME-dependent pyroptosis.,Moreover, no obvious side effects are observed in the normal tissues and organs of mice during the combined treatment of clinical drugs and iron.,This study not only identifies iron as a sensitizer amplifying ROS signaling to drive pyroptosis, but also implicates a novel iron-based intervention strategy for melanoma therapy.

Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy.,Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs.,Hence, alternative treatments are still required.,Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting.,To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures.,None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK.,Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma.,Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines.,Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.

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

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).

Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib.,However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type.,Better markers for targeted therapy are therefore urgently needed.,In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates.,In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.,Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue.,Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity.,While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors.,Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors.,The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.,Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity.,Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.

NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours.,Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma.,Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes.,The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

The immune checkpoint programmed death 1 receptor (PD-1) expressed on some tumor-infiltrating lymphocytes, and its ligand (PD-L1) expressed on tumor cells, enable cancers to evade the immune system.,Blocking PD-1 with the monoclonal antibody pembrolizumab is a promising immunotherapy strategy.,Thus, noninvasively quantifying the presence of PD-1 expression in the tumor microenvironment prior to initiation of immune checkpoint blockade may identify the patients likely to respond to therapy.,We have developed a 64Cu-pembrolizumab radiotracer and evaluated human dosimetry.,The tracer was utilized to image hPD-1 levels in two subcutaneous mouse models: (a) 293 T/hPD-1 cells xenografted into NOD-scid IL-2Rγnull mice (NSG/293 T/hPD-1) and (b) human peripheral blood mononuclear cells engrafted into NSG bearing A375 human melanoma tumors (hNSG/A375).,In each mouse model two cohorts were evaluated (hPD-1 blockade with pembrolizumab [blk] and non-blocked [nblk]), for a total of four groups (n = 3-5/group).,The xenograft-to-muscle ratio in the NSG/293 T/hPD-1 model at 24 h was significantly increased in the nblk group (7.0 ± 0.5) compared to the blk group (3.4 ± 0.9), p = 0.01.,The radiotracer dosimetry evaluation (PET/CT ROI-based and ex vivo) in the hNSG/A375 model revealed the highest radiation burden to the liver.,In summary, we validated the 64Cu-pembrolizumab tracer’s specific hPD-1 receptor targeting and predicted human dosimetry.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies.,This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets.,Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available.,Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas.,Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05).,KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%).,Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients.,Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas.,Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples.,This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1.,This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date.,The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes.,These findings expand our knowledge of this rare disease.

Down-regulation of the immune system facilitates tumor progression at different stages of cutaneous melanoma.,Sentinel nodes, the first lymph nodes on lymphatics draining directly from a primary melanoma are immune down-regulated by tumor-generated immune-suppressive cytokines including interleukin-10.,To better understand the kinetics of sentinel node suppression, we investigated interleukin-10 expression by melanoma cells and tumor-associated macrophages and lymphocytes at different stages of primary melanoma evolution.,We used reverse transcriptase in situ polymerase chain reaction to identify cellular sources of interleukin-10 mRNA in 39 melanomas.,Interleukin-10 mRNA was identified in tumor cells of 2/6 melanomas in situ (33%), 17/21 invasive melanomas (81%) and 11/12 metastatic melanomas (92%).,Higher interleukin-10 expression correlates with tumor progression, with differences between melanoma in situ, invasive melanoma and metastatic melanoma.,In primary melanomas, the interleukin-10 mRNA content of tumor cells correlates with Clark level.,There was significantly more interleukin-10 mRNA in vertical growth phase melanoma cells than in radial growth phase cells.,In a logistic regression model, moderate to high interleukin-10 mRNA expression by tumor cells was significantly associated with vertical growth phase melanoma.,Interleukin-10 mRNA was detected in melanoma-associated macrophages and lymphocytes.,In invasive melanomas, the interleukin-10 mRNA reactivity of macrophages declined as Clark level increased.,Alterations of immunity by interleukin-10 derived from melanoma cells and melanoma-associated macrophages and lymphocytes potentially facilitate evolution of the primary melanoma and render regional lymph nodes susceptible to metastases.

Screening for theranostic biomarkers is mandatory for the therapeutic management of cutaneous melanoma.,BRAF and NRAS genes must be tested in routine clinical practice.,The methods used to identify these alterations must be sensitive to detect mutant alleles in a background of wild type alleles, and specific to identify the correct mutation.,They should not require too much material, since in some cases the available samples are small biopsies.,Finally, they should also be quick enough to allow a rapid therapeutic management of patients.,Sixty five consecutive formalin-fixed paraffin-embedded (FFPE) melanoma samples were prospectively tested for BRAF mutations with the VE1 (anti-BRAF V600E) antibody and for both BRAF and NRAS mutations with the Idylla NRAS-BRAF-EGFR S492R Mutation Assay cartridges.,Results were compared to our routine laboratory practice, allele specific amplification and/or Sanger sequencing and discordant cases confirmed by digital PCR.,Excluding discordant by-design-mutations, system failures and DNA quantity or quality failures, BRAF IHC demonstrated an overall concordance of 89% for BRAF V600E mutation detection, the Idylla system gave a concordance of 100% for BRAF mutation detection and of 92.1% for NRAS mutation detection when compared to our reference.,When discrepancies were observed, all routine results were confirmed by digital PCR.,Finally, BRAF IHC positive predictive value (PPV) was of 82% and negative predictive value (NPV) of 92%.,The Idylla cartridges showed a PPV and NPV of both 100% for BRAF mutation detection and a PPV and NPV of 100% and 87% respectively, for NRAS mutation detection.,In conclusion, BRAF V600E immunohistochemistry is efficient for detecting the V600E mutation, but negative cases should be further evaluated by molecular approaches for other BRAF mutations.,Since 3 NRAS mutations have not been detected by the Idylla NRAS-BRAF-EGFR S492R Mutation Assay, these cartridges should not be used as a substitute for traditional molecular methods in the conventional patient therapeutic care process without the expertise needed to have a critical view of the produced results.

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

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a multifunctional RNA-binding protein with an oncofetal pattern of expression shown to be implicated in the development of a variety of malignancies.,In this study, we explored the role and mechanisms of IGF2BP1 in melanoma development and progression.,In two different in vivo models, we showed that while genetic deletion or shRNA-mediated suppression of IGF2BP1 did not affect primary tumor formation, it drastically suppressed lung metastasis.,Here we demonstrated that extracellular vesicles (EVs) secreted by melanoma cells mediate the effects of IGF2BP1 on metastasis: EVs from the IGF2BP1 knockdown melanoma cells failed to promote metastasis whereas EVs isolated from IGF2BP1-overexpressed melanoma cells further accelerated EV-induced metastasis.,Moreover, the EVs from IGF2BP1 knockdown melanoma cells inhibited fibronectin deposition and accumulation of CD45+ cells in the lungs compared to control EVs, thus blocking the pre-metastatic niche formation potential of EVs.,IGF2BP1 knockdown did not affect size, number, or protein/RNA concentration of secreted EVs or their uptake by recipient cells in vitro or in vivo.,However, RNA-sequencing and proteomics analysis of the EVs revealed differential expression in a number of mRNA, proteins and miRNAs.,This suggested that IGF2BP1 is intimately involved in the regulation of the cargo of EVs, thereby affecting the pro-metastatic function of melanoma-derived EVs.,To the best of our knowledge, this is the first study that demonstrates the role of RNA-binding protein IGF2BP1 in EV-mediated promotion of melanoma metastasis and may provide novel avenues for the development of metastatic inhibitors.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, known to play important roles in melanoma formation and progression.,Recent advances in genome-wide methylation methods have provided the means to identify differentially methylated genes, methylation signatures, and potential biomarkers.,However, despite considerable effort and advances in cataloging methylation changes in melanoma, many questions remain unanswered.,The aim of this review is to summarize recent developments, emerging trends, and important unresolved questions in the field of aberrant DNA methylation in melanoma.,In addition to reviewing recent developments, we carefully synthesize the findings in an effort to provide a framework for understanding the current state and direction of the field.,To facilitate clarity, we divided the review into DNA methylation changes in melanoma, biomarker opportunities, and therapeutic developments.,We hope this review contributes to accelerating the utilization of the diagnostic, prognostic, and therapeutic potential of DNA methylation for the benefit of melanoma patients.

Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear.,Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden.,Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity.,However, single-cell-derived tumors with reduced ITH are swiftly rejected.,Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment.,Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity.,Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy.,These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.,•Novel mouse system to uncouple tumor mutational load and tumor heterogeneity•Lower tumor heterogeneity leads to decreased tumor growth because of immune rejection•Both clone numbers and their genetic diversity mediate tumor growth and rejection•Tumor heterogeneity is linked to patient survival and checkpoint blockade response,Novel mouse system to uncouple tumor mutational load and tumor heterogeneity,Lower tumor heterogeneity leads to decreased tumor growth because of immune rejection,Both clone numbers and their genetic diversity mediate tumor growth and rejection,Tumor heterogeneity is linked to patient survival and checkpoint blockade response,Syngeneic mouse models of melanoma and human patient data are used to explore the effect of intra-tumor heterogeneity on immune response and demonstrate that heterogeneity predicts immunotherapy outcomes better than mutational burden.

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

The extracellular matrix (ECM) plays an important role in the regulation of the tissue microenvironment and in the maintenance of cellular homeostasis.,Several proteins with a proteolytic activity toward several ECM components are involved in the regulation and remodeling of the ECM.,Among these, Matrix Metalloproteinases (MMPs) are a class of peptidase able to remodel the ECM by favoring the tumor invasive processes.,Of these peptidases, MMP-9 is the most involved in the development of cancer, including that of melanoma.,Dysregulations of the MAPKs and PI3K/Akt signaling pathways can lead to an aberrant overexpression of MMP-9.,Even ncRNAs are implicated in the aberrant production of MMP-9 protein, as well as other proteins responsible for the activation or inhibition of MMP-9, such as Osteopontin and Tissue Inhibitors of Metalloproteinases.,Currently, there are different therapeutic approaches for melanoma, including targeted therapies and immunotherapies.,However, no biomarkers are available for the prediction of the therapeutic response.,In this context, several studies have tried to understand the diagnostic, prognostic and therapeutic potential of MMP-9 in melanoma patients by performing clinical trials with synthetic MMPs inhibitors.,Therefore, MMP-9 may be considered a promising molecule for the management of melanoma patients due to its role as a biomarker and therapeutic target.

Tumor cell proliferation is a predictor of survival in cutaneous melanoma.,The aim of the present study was to evaluate the prognostic impact of mitotic count, Ki-67 expression and novel proliferation markers phosphohistone H3 (PHH3), minichromosome maintenance protein 4 (MCM4) and mitosin, and to compare the results with histopathological variables.,202 consecutive cases of nodular cutaneous melanoma were initially included.,Mitotic count (mitosis per mm2) was assessed on H&E sections, and Ki-67 expression was estimated by immunohistochemistry on standard sections.,PHH3, MCM4 and mitosin were examined by staining of tissue microarrays (TMA) sections.,Increased mitotic count and elevated Ki-67 expression were strongly associated with increased tumor thickness, presence of ulceration and tumor necrosis.,Furthermore, high mitotic count and elevated Ki-67 expression were also associated with Clark's level of invasion and presence of vascular invasion.,High expression of PHH3 and MCM4 was correlated with high mitotic count, elevated Ki-67 expression and tumor ulceration, and increased PHH3 frequencies were associated with tumor thickness and presence of tumor necrosis.,Univariate analyses showed a worse outcome in cases with elevated Ki-67 expression and high mitotic count, whereas PHH3, MCM4 and mitosin were not significant.,Tumor cell proliferation by Ki-67 had significant prognostic impact by multivariate analysis.,Ki-67 was a stronger and more robust prognostic indicator than mitotic count in this series of nodular melanoma.,PHH3, MCM4 and mitosin did not predict patient survival.

Tumor microenvironments are often characterized by an increase in oxidative stress levels.,We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis.,In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA).,A large fraction of KROS was inhibited by paxilline-a specific inhibitor of large-conductance Ca2+-activated BK channels.,The TEA-insensitive component was inhibited by senicapoc-a specific inhibitor of the Ca2+-activated KCa3.1 channel.,Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T.,Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole-two different inhibitors of the calcium-permeable TRPM2 channel.,Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T.,Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37.,The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma.,Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Cold Atmospheric Plasma Jet (CAPJ), with ion temperature close to room temperature, has tremendous potential in biomedical engineering, and can potentially offer a therapeutic option that allows cancer cell elimination without damaging healthy tissue.,We developed a hand-held flexible device for the delivery of CAPJ to the treatment site, with a modified high-frequency pulse generator operating at a RMS voltage of <1.2 kV and gas flow in the range 0.3-3 l/min.,The aims of our study were to characterize the CAPJ emitted from the device, and to evaluate its efficacy in elimination of cancer cells in-vitro and in-vivo.,The power delivered by CAPJ was measured on a floating or grounded copper target.,The power did not drastically change over distances of 0-14 mm, and was not dependent on the targets resistance.,Temperature of CAPJ-treated target was 23°-36° C, and was dependent on the voltage applied.,Spectroscopy indicated that excited OH- radicals were abundant both on dry and wet targets, placed at different distances from the plasma gun.,An in-vitro cell proliferation assay demonstrated that CAPJ treatment of 60 seconds resulted in significant reduction in proliferation of all cancer cell lines tested, and that CAPJ activated medium was toxic to cancer cells.,In-vivo, we treated cutaneous melanoma tumors in nude mice.,Tumor volume was significantly decreased in CAPJ-treated tumors relatively to controls, and high dose per fraction was more effective than low dose per fraction treatment.,Importantly, pathologic examination revealed that normal skin was not harmed by CAPJ treatment.,This preliminary study demonstrates the efficacy of flexible CAPJ delivery system against melanoma progression both in-vitro and in-vivo.,It is envisioned that adaptation of CAPJ technology for different kinds of neoplasms use may provide a new modality for the treatment of solid tumors.

Dynamic cellular heterogeneity underlies melanoma progression and therapy resistance.,Advances in single-cell technologies have revealed an increasing number of tumor and microenvironment cell states in melanoma, but little is understood about their function in vivo.,Zebrafish models are a powerful system for discovery, live imaging, and functional investigation of cell states throughout melanoma progression and treatment.,By capturing dynamic melanoma states in living animals, zebrafish have the potential to resolve the complexity of melanoma heterogeneity from a single cell through disease processes within the context of the whole body, revealing novel cancer biology and therapeutic targets.

Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid.,Non-tumorigenic cells did not undergo coalescence.,Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model.,Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model.,Normal melanocytes did not.,However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic.,A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence.,They also blocked coalescence of tumorigenic cells derived from a breast tumor.,These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs.

The clinical and pathological features of primary melanoma are not sufficiently sensitive to accurately predict which patients are at a greater risk of relapse.,Recently, a 31‐gene expression profile (DecisionDx‐Melanoma) test has shown promising results.,To evaluate the early prognostic performance of a genetic signature in a multicentre prospectively evaluated cohort.,Inclusion of patients with AJCC stages IB and II conducted between April 2015 and December 2016.,All patients were followed up prospectively to assess their risk of relapse.,Prognostic performance of this test was evaluated individually and later combined with the AJCC staging system.,Prognostic accuracy of disease‐free survival was determined using Kaplan-Meier curves and Cox regression analysis.,Results of the gene expression profile test were designated as Class 1 (low risk) and Class 2 (high risk).,Median follow‐up time was 26 months (IQR 22-30).,The gene expression profile test was performed with 86 patients; seven had developed metastasis (8.1%) and all of them were in the Class 2 group, representing 21.2% of this group.,Gene expression profile was an independent prognostic factor for relapse as indicated by multivariate Cox regression analysis, adjusted for AJCC stages and age.,This prospective multicentre cohort study, performed in a Spanish Caucasian cohort, shows that this 31‐gene expression profile test could correctly identify patients at early AJCC stages who are at greater risk of relapse.,We believe that gene expression profile in combination with the AJCC staging system could well improve the detection of patients who need intensive surveillance and optimize follow‐up strategies.

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.

Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers.,We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence.,Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation.,The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI).,Tumour and blood were analysed for prognostic and predictive markers.,Patients (n=1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18-88 years).,With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation].,OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82-1.16, P = 0.78).,At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74-0.99, P = 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78-1.07, P = 0.25).,Forty four percent of 682 melanomas assessed had a BRAFV600 mutation.,In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P = 0.06).,BRAF mutation positivity trended towards better OS with bevacizumab (P = 0.21).,Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS.,BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab.,ISRCTN 81261306; EudraCT Number: 2006-005505-64

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.

Melanin, the pigment produced by specialized cells, melanocytes, is responsible for skin and hair color.,Skin pigmentation is an important protective mechanism against the DNA damaging and mutagenic effects of solar ultraviolet radiation (UV).,It is acknowledged that exposure to UV is the main etiological environmental factor for all forms of skin cancer, including melanoma.,DNA repair capacity is another major factor that determines the risk for skin cancer.,Human melanocytes synthesize eumelanin, the dark brown form of melanin, as well as pheomelanin, which is reddish-yellow in color.,The relative rates of eumelanin and pheomelanin synthesis by melanocytes determine skin color and the sensitivity of skin to the drastic effects of solar UV.,Understanding the complex regulation of melanocyte function and how it responds to solar UV has a huge impact on developing novel photoprotective strategies to prevent skin cancer, particularly melanoma, the most fatal form, which originates from melanocytes.,This review provides an overview of the known differences in the photoprotective effects of eumelanin versus pheomelanin, how these two forms of melanin are regulated genetically and biochemically, and their impact on the DNA damaging effects of UV exposure.,Additionally, this review briefly discusses the role of paracrine factors, focusing on α-melanocortin (α-melanocyte stimulating hormone; α-MSH), in regulating melanogenesis and the response of melanocytes to UV, and describes a chemoprevention strategy based on targeting the melanocortin 1 receptor (MC1R) by analogs of its physiological agonist α-MSH.

We have recently reported a potential alternative tumor suppressor function for p16 relating to its capacity to regulate oxidative stress and observed that oxidative dysregulation in p16-depleted cells was most profound in melanocytes, compared to keratinocytes or fibroblasts.,Moreover, in the absence of p16 depletion or exogenous oxidative insult, melanocytes exhibited significantly higher basal levels of reactive oxygen species (ROS) than these other epidermal cell types.,Given the role of oxidative stress in melanoma development, we speculated that this increased susceptibility of melanocytes to oxidative stress (and greater reliance on p16 for suppression of ROS) may explain why genetic compromise of p16 is more commonly associated with predisposition to melanoma rather than other cancers.,Here we show that the presence of melanin accounts for this differential oxidative stress in normal and p16-depleted melanocytes.,Thus the presence of melanin in the skin appears to be a double-edged sword: it protects melanocytes as well as neighboring keratinocytes in the skin through its capacity to absorb UV radiation, but its synthesis in melanocytes results in higher levels of intracellular ROS that may increase melanoma susceptibility.

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking.,Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice.,Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression.,Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions.,Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment.,Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors.,Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.,Immune checkpoint therapies (ICT) are promising for treating various cancers, but response rates vary.,Here the authors show, in mouse models, that tumor-infiltrating mast cells colocalize with regulatory T cells, coincide with local reduction of MHC-I and CD8 T cells, and is associated with resistance to ICT, which can be reversed by c-kit inhibitor treatment.

Melanoma is the most aggressive form of skin cancer and one of the most treatment-refractory malignancies.,In metastatic melanoma cell lines, we analysed the anti-proliferative and pro-apoptotic potentials of a phenolic component of olive oil, the hydroxytyrosol.,In particular, through MTS assay, DeadEnd™ Colorimetric TUNEL assay, Annexin V binding and PI uptake, western blot experiment, intracellular reactive oxygen species (ROS) analysis, and the cell colony assay, we showed that the hydroxytyrosol treatment remarkably reduces the cell viability inducing the death for apoptosis of melanoma cells.,Moreover, we showed that the hydroxytyrosol treatment of melanoma cells leads to a significant increase of p53 and γH2AX expression, a significant decrease of AKT expression and the inhibition of cell colony formation ability.,Finally, we propose that the increased amount of intracellular reactive oxygen species (ROS) that may be related to the regulation of the pathways involved in the activation of apoptosis and in the inhibition of melanoma growth could be the strategy used by hydroxytyrosol to exert its functions in melanoma.,Therefore, for its role in melanoma growth inhibition, the hydroxytyrosol treatment could deeply interfere with melanoma progression as a promising therapeutic option for the treatment of this highly invasive tumour.

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

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

Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma.,The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult.,AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100.,AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100.,Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070.,Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver.,Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.

Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions.,In this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients.,Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome.,Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes.,Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied.,In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients.,The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease.,Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells.,The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance.,These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients.,On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients.

In melanoma, the lymphocytic infiltrate is a prognostic parameter classified morphologically into ‘brisk’, ‘non-brisk’ and ‘absent’ entailing a functional association that has never been proved.,Recently, it has been shown that lymphocytic populations can be very heterogeneous, and that anti-PD-1 immunotherapy supports activated T cells.,Here, we characterize the immune landscape in primary melanoma by high-dimensional single-cell multiplex analysis in tissue sections (MILAN technique) followed by image analysis, RT-PCR and shotgun proteomics.,We observed that the brisk and non-brisk patterns are heterogeneous functional categories that can be further sub-classified into active, transitional or exhausted.,The classification of primary melanomas based on the functional paradigm also shows correlation with spontaneous regression, and an improved prognostic value when compared to that of the brisk classification.,Finally, the main inflammatory cell subpopulations that are present in the microenvironment associated with activation and exhaustion and their spatial relationships are described using neighbourhood analysis.

Metastasized or unresectable melanoma has been the first malignant tumor to be successfully treated with checkpoint inhibitors.,Nevertheless, about 40-50% of the patients do not respond to these treatments and severe side effects are observed in up to 60%.,Therefore, there is a high need to identify reliable biomarkers predicting response.,Tumor Mutation Burden (TMB) is a debated predictor for response to checkpoint inhibitors and early measurement of ctDNA can help to detect treatment failure to immunotherapy in selected melanoma patients.,However, it has not yet been clarified how TMB and ctDNA can be used to estimate response to combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma.,In this prospective biomarker study, we included 35 melanoma patients with ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) therapy.,In all patients, a tumor panel of 710 tumor-associated genes was applied (tumor vs. reference tissue comparison), followed by repetitive liquid biopsies.,Cell-free DNA was extracted and at least one driver mutation was monitored.,Treatment response was evaluated after about three months of therapy.,TMB was significantly higher in responders than in nonresponders and TMB > 23.1 Mut/Mb (TMB-high) was associated with a survival benefit compared to TMB ≤ 23.1 Mut/Mb (TMB-low or TMB-intermediate).,Furthermore, a > 50% decrease of cell-free DNA concentration or undetectable circulating tumor DNA (ctDNA), measured by tumor-specific variant copies/ml of plasma at first follow-up three weeks after treatment initiation were significantly associated with response to combined immunotherapy and improved overall survival, respectively.,It is noticeable that no patient with TMB ≤ 23.1 Mut/Mb and detectable or increasing ctDNA at first follow-up responded to immunotherapy.,High TMB, > 50% decrease of cell-free DNA concentration, and undetectable ctDNA at first follow-up seem to be associated with response and overall survival under combined immunotherapy.,The evaluation of ctDNA and cell-free DNA three weeks after treatment initiation may be suitable for early assessment of efficacy of immunotherapy.,The online version of this article (10.1186/s40425-019-0659-0) contains supplementary material, which is available to authorized users.

Identifying factors conferring responses to therapy in cancer is critical to select the best treatment for patients.,For immune checkpoint inhibition (ICI) therapy, mounting evidence suggests that the gut microbiome can determine patient treatment outcomes.,However, the extent to which gut microbial features are applicable across different patient cohorts has not been extensively explored.,We performed a meta-analysis of 4 published shotgun metagenomic studies (Ntot = 130 patients) investigating differential microbiome composition and imputed metabolic function between responders and nonresponders to ICI.,Our analysis identified both known microbial features enriched in responders, such as Faecalibacterium as the prevailing taxa, as well as additional features, including overrepresentation of Barnesiella intestinihominis and the components of vitamin B metabolism.,A classifier designed to predict responders based on these features identified responders in an independent cohort of 27 patients with the area under the receiver operating characteristic curve of 0.625 (95% CI: 0.348-0.899) and was predictive of prognosis (HR = 0.35, P = 0.081).,These results suggest the existence of a fecal microbiome signature inherent across responders that may be exploited for diagnostic or therapeutic purposes.,This work was funded by the Knut and Alice Wallenberg Foundation, BioGaia AB, and Cancerfonden.,A meta-analysis of studies investigating the impact of the gut microbiome on response to cancer immunotherapy in patients with metastatic melanoma

Growing evidence supports the importance of gut microbiota in the control of tumor growth and response to therapy.,Here, we select prebiotics that can enrich bacterial taxa that promote anti-tumor immunity.,Addition of the prebiotics inulin or mucin to the diet of C57BL/6 mice induces anti-tumor immune responses and inhibition of BRAF mutant melanoma growth in a subcutaneously implanted syngeneic mouse model.,Mucin fails to inhibit tumor growth in germ-free mice, indicating that the gut microbiota is required for the activation of the anti-tumor immune response.,Inulin and mucin drive distinct changes in the microbiota, as inulin, but not mucin, limits tumor growth in syngeneic mouse models of colon cancer and NRAS mutant melanoma and enhances the efficacy of a MEK inhibitor against melanoma while delaying the emergence of drug resistance.,We highlight the importance of gut microbiota in anti-tumor immunity and the potential therapeutic role for prebiotics in this process.

Melanoma can be classified based on the detection of relevant oncogenic driver mutations.,These mutations partially determine a patient's treatment options.,MEK inhibitors have demonstrated little efficacy in patients with NRAS‐mutated melanoma owing to primary and secondary resistance.,We report two patients with NRAS‐mutant metastatic melanoma with long‐term response to intermittent MEK‐inhibitor binimetinib therapy.,Intermittent dosing schedules could play a key role in preventing resistance to targeted therapy.,This article highlights the efficacy of an intermittent dosing schedule, toxicities associated with binimetinib, and possible mechanisms preventing resistance in targeted therapy.,Intermittent MEK‐inhibitor therapy may be considered in patients with NRAS‐mutated melanoma that have failed all standard therapies.,Melanomas harbor NRAS mutations in 10%-30% of the cases.,These mutations promote hyperactivation of the MAPK pathway, leading to proliferation and prolonged survival of tumor cells.Currently, drugs directly targeting NRAS are not available.,Downstream inhibition of the MAPK pathway can be considered as a therapeutic option after immunotherapeutic failure.Intermittent administration of kinase inhibitors might be the way to partially overcome the development of drug resistance by (a) inducing a fitness deficit for drug‐resistant cells on treatment break, (b) increasing the immunogenicity, and (c) inducing apoptosis and cell cycle arrest.,It also enhances expression of numerous immunomodulating molecules, and reduction of immunosuppressive factors, which suggests better access of the immune system to the tumor.,Melanomas harbor NRAS mutations in 10%-30% of the cases.,These mutations promote hyperactivation of the MAPK pathway, leading to proliferation and prolonged survival of tumor cells.,Currently, drugs directly targeting NRAS are not available.,Downstream inhibition of the MAPK pathway can be considered as a therapeutic option after immunotherapeutic failure.,Intermittent administration of kinase inhibitors might be the way to partially overcome the development of drug resistance by (a) inducing a fitness deficit for drug‐resistant cells on treatment break, (b) increasing the immunogenicity, and (c) inducing apoptosis and cell cycle arrest.,It also enhances expression of numerous immunomodulating molecules, and reduction of immunosuppressive factors, which suggests better access of the immune system to the tumor.,Case reports for two patients with NRAS‐mutant metastatic melanoma are presented, highlighting toxicities associated with binimetinib, as well as the efficacy of an intermittent dosing schedule and possible mechanisms preventing resistance in targeted therapy.

Finding additional functional targets for combination therapy could improve the outcome for melanoma patients.,In a spontaneous metastasis xenograft model of human melanoma a shRNA mediated knockdown of L1CAM more than sevenfold reduced the number of lung metastases after the induction of subcutaneous tumors for two human melanoma cell lines (MeWo, MV3).,Whole genome expression arrays of the initially L1CAM high MeWo subcutaneous tumors revealed unchanged or downregulated genes involved in epithelial to mesenchymal transition (EMT) except an upregulation of Jagged 1, indicating a compensatory change in Notch signaling especially as Jagged 1 expression showed an increase in MeWo L1CAM metastases and Jagged 1 was expressed in metastases of the initially L1CAM low MV3 cells as well.,Expression of 17 genes showed concordant regulation for L1CAM knockdown tumors of both cell lines.,The changes in gene expression indicated changes in the EMT network of the melanoma cells and an increase in p53/p21 and p38 activity contributing to the reduced metastatic potential of the L1CAM knockdowns.,Taken together, these data make L1CAM a highly interesting therapeutic target to prevent further metastatic spread in melanoma patients.

To estimate the proportion and numbers of cancers occurring in Australia attributable to solar ultraviolet radiation (UVR) and the proportion and numbers prevented by regular sun protection factor (SPF) 15+ sunscreen use.,We estimated the population attributable fraction (PAF) and numbers of melanomas and keratinocyte cancers (i.e. basal cell carcinomas and squamous cell carcinomas) due to exposure to ambient UVR resulting from residing in Australia versus residing in the UK (for melanoma) or Scandinavia (for keratinocyte cancers).,We also estimated the prevented fraction (PF): the proportion of cancers that would have occurred but were likely prevented by regular sunscreen use.,An estimated 7,220 melanomas (PAF 63%) and essentially all keratinocyte cancers occurring in Australia were attributable to high ambient UVR levels in Australia.,We estimated that regular sunscreen use prevented around 14,190 (PF 9.3%) and 1,730 (PF 14%) people from developing SCC and melanoma, respectively.,Although our approach was conservative, a high proportion of skin cancers in Australia are attributable to high ambient levels of UVR.,Prevailing levels of sunscreen use probably reduced skin cancer incidence by 10-15%.,Most skin cancers are preventable.,Sunscreen should be a component of a comprehensive sun protection strategy.

Non-melanoma skin cancer (NMSC), comprised of basal (BCC) and squamous (SCC) cell carcinomas, is the most common cancer in Caucasians.,Ultraviolet radiation (UVR) exposure is the most important environmental risk factor for NMSC.,However, the precise relationship between UVR and the risk of NMSC is complex, and the relationship may differ by skin cancer type.,A case-control study was conducted among Florida residents to investigate measures of patterns (intermittent vs. continuous) and timing (childhood vs. adulthood) of sunlight exposure in BCC and SCC.,Participants included 218 BCC and 169 SCC cases recruited from a university dermatology clinic and 316 controls with no history of skin or other cancers.,A history of blistering sunburn (a measure of intermittent sunlight exposure) was associated with both BCC (OR = 1.96, 95% CI = 1.27-3.03) and SCC (OR = 2.02, 95% CI = 1.22-3.33).,Additionally, having a job in the sun for ≥3 months for 10 years or longer (a measure of continuous sunlight exposure) was also associated with both BCC and SCC in our study population.,With the exception of younger age at first blistering sunburn, measures of younger age at sunlight exposure tended to be associated with SCC, but not BCC risk.,Results from the current study suggest that sunlight exposure is associated with both BCC and SCC risk regardless of the pattern in which the exposure was received (i.e. intermittent vs. continuous).,The data also suggest that sunlight exposure at a younger age may be more important for SCC but not BCC, however additional studies are needed to further characterize sunlight exposure-response relationships in different types of NMSC.

We report the case of a primitive nasal melanoma in an 82-year-old patient, showing how this rare malignancy, with non-specific signs and symptoms, can represent a challenging diagnosis for the physician.,A 82-year-old Caucasian patient presented for unilateral nasal obstruction and occasional epistaxis.,Computerized tomography (CT) and magnetic resonance imaging (MRI) of the facial massif revealed turbinate hypertrophy and a polypoid phlogistic tissue isointense in T1 with an intermediate signal in T2 and Short-TI Inversion Recovery (STIR)-T2, occupying the middle meatus and the anterior upper and lower left meatus with partial obliteration of the ostium and the infundibulum of the maxillary sinus.,The Positron emission tomography (PET) exam was negative for metastases.,Conservatory surgery in the left anterior video rhinoscopy was performed, allowing a radical 4-cm tumor excision.,Histology reported epithelioid cell melanoma, PanK−, CD45−, and PanMelanoma+.,Adjuvant radiotherapy was suggested, even considering a complete resection as the result of surgery.,No local or systemic relapse was noticed at the 2-month follow-up visit.,Although mucosal melanoma is a rare and aggressive malignancy characterized by a poor prognosis, early diagnosis allows a more conservative approach, with little surgical difficulty and no aesthetic effect.,Our case raises awareness of the importance of early intervention even in those cases where the clinic symptoms and diagnostic images show uncertain severity.

Post-translational histone modifications such as acetylation and methylation can affect gene expression.,Histone acetylation is commonly associated with activation of gene expression whereas histone methylation is linked to either activation or repression of gene expression.,Depending on the site of histone modification, several histone marks can be present throughout the genome.,A combination of these histone marks can shape global chromatin architecture, and changes in patterns of marks can affect the transcriptomic landscape.,Alterations in several histone marks are associated with different types of cancers, and these alterations are distinct from marks found in original normal tissues.,Therefore, it is hypothesized that patterns of histone marks can change during the process of tumorigenesis.,This review focuses on histone methylation changes (both removal and addition of methyl groups) in malignant melanoma, a deadly skin cancer, and the implications of specific inhibitors of these modifications as a combinatorial therapeutic approach.

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

Metastatic melanoma patients have a poor prognosis.,No chemotherapy regimen has improved overall survival.,More effective treatments are needed.,Docetaxel has clinical activity in melanoma and may be more active when combined with vinorelbine.,Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown activity as an adjuvant melanoma therapy.,We carried out a phase II study of these agents in patients with stage IV melanoma.,Patients had documented stage IV melanoma and may have had prior immuno or chemotherapy.,Previously treated brain metastases were allowed.,Docetaxel (40 mg/m2 IV) and vinorelbine (30 mg/m2 IV) were administered every 14 days, followed by GM-CSF (250 mg/m2 SC on days 2 to 12).,The primary endpoint of the study was 1-year overall survival (OS).,Secondary objectives were median overall survival, response rate (per RECIST criteria), and the toxicity profiles.,Fifty-two patients were enrolled; 80% had stage M1c disease.,Brain metastases were present in 21%.,Fifty-two percent of patients had received prior chemotherapy, including 35% who received prior biochemotherapy.,Toxicity was manageable.,Grade III/IV toxicities included neutropenia (31%), anemia (14%), febrile neutropenia (11.5%), and thrombocytopenia (9%).,DVS chemotherapy demonstrated clinical activity, with a partial response in 15%, and disease stabilization in 37%.,Six-month PFS was 37%.,Median OS was 11.4 months and 1-year OS rate was 48.1%.,The DVS regimen was active in patients with advanced, previously treated melanoma, with manageable toxicity.,The favorable 1-year overall survival and median survival rates suggest that further evaluation of the DVS regimen is warranted.

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

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

Body composition is an important predictor of drug toxicity and outcome.,Ipilimumab (Ipi), a monoclonal antibody used to treat metastatic melanoma, has specific toxicities.,No validated biomarkers that predict Ipi toxicity and efficacy exist.,Also, the impact of Ipi on body composition has not been established.,Patients with metastatic melanoma treated with Ipi between 2009 and 2015 were included.,Body composition was assessed by computed tomography at baseline and after four cycles of Ipi.,Sarcopenia and low muscle attenuation (MA) were defined using published cut-points.,All adverse events (AEs) and immune-related AEs (irAEs) were recorded (Common Terminology Criteria For Adverse Event V.4.0).,Eighty-four patients were included in this study (62% male, median age 54 years).,At baseline, 24% were sarcopenic and 33% had low MA.,On multivariate analysis, sarcopenia and low MA were significantly associated with high-grade AEs (OR=5.34, 95% CI: 1.15-24.88, P=0.033; OR=5.23, 95% CI: 1.41-19.30, P=0.013, respectively), and low MA was associated with high-grade irAEs (OR=3.57, 95% CI: 1.09-11.77, P=0.036).,Longitudinal analysis (n=59) revealed significant reductions in skeletal muscle area (SMA), total body fat-free mass, fat mass (all P<0.001) and MA (P=0.030).,Mean reduction in SMA was 3.3%/100 days (95% CI: −4.48 to −1.79%, P<0.001).,A loss of SMA ⩾7.5%/100 days (highest quartile) was a significant predictor of overall survival in multivariable Cox regression analysis (HR: 2.1, 95% CI: 1.02-4.56, P=0.046).,Patients with sarcopenia and low MA are more likely to experience severe treatment-related toxicity to Ipi.,Loss of muscle during treatment was predictive of worse survival.,Treatments to increase muscle mass and influence outcome warrant further investigation.

Ipilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma.,To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4+ and CD8+ T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry.,Microarray analysis of flow-cytometry purified CD4+ and CD8+ T cells was employed to assess gene profiling changes induced by ipilimumab.,Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens.,Ipilimumab up-regulated Ki67 and ICOS on CD4+ and CD8+ cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8+ at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4+ and CD8+ cells at 6-month post ipilimumab (p ≤ 0.001).,Increased EOMES+CD8+, GranzymeB+EOMES+CD8+ and decreased Ki67+EOMES+CD4+ T cells at 6 months were significantly associated with relapse (all p ≤ 0.03).,Decreased Ki67+CD8+ T cells were significantly associated with the development of irAE (p = 0.02).,At baseline, low Ki67+EOMES+CD8+ T cells were associated with relapse (p ≤ 0.001), and low Ki67+EOMES+CD4+ T cells were associated with irAE (p ≤ 0.008).,Up-regulation of proliferation and activation signals in CD4+ and CD8+ T cells were pharmacodynamic markers for ipilimumab.,Ki67+EOMES+CD8+ and Ki67+EOMES+CD4+T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.

The introduction of programmed cell death protein 1 (PD-1) blockers (i.e. nivolumab and pembrolizumab) has significantly improved the prognosis of patients with advanced melanoma.,However, the long treatment duration (i.e. two years or longer) has a high impact on patients and healthcare systems in terms of (severe) toxicity, health-related quality of life (HRQoL), resource use, and healthcare costs.,While durable tumour responses have been observed and PD-1 blockade is discontinued on an individual basis, no consensus has been reached on the optimal treatment duration.,The objective of the Safe Stop trial is to evaluate whether early discontinuation of first-line PD-1 blockade is safe in patients with advanced and metastatic melanoma who achieve a radiological response.,The Safe Stop trial is a nationwide, multicentre, prospective, single-arm, interventional study in the Netherlands.,A total of 200 patients with advanced and metastatic cutaneous melanoma and a confirmed complete response (CR) or partial response (PR) according to response evaluation criteria in solid tumours (RECIST) v1.1 will be included to early discontinue first-line monotherapy with nivolumab or pembrolizumab.,The primary objective is the rate of ongoing responses at 24 months after discontinuation of PD-1 blockade.,Secondary objectives include best overall and duration of response, need and outcome of rechallenge with PD-1 blockade, and changes in (serious) adverse events and HRQoL.,The impact of treatment discontinuation on healthcare resource use, productivity losses, and hours of informal care will also be assessed.,Results will be compared to those from patients with CR or PR who completed 24 months of treatment with PD-1 blockade and had an ongoing response at treatment discontinuation.,It is hypothesised that it is safe to early stop first-line nivolumab or pembrolizumab at confirmed tumour response while improving HRQoL and reducing costs.,From a patient, healthcare, and economic perspective, shorter treatment duration is preferred and overtreatment should be prevented.,If early discontinuation of first-line PD-1 blockade appears to be safe, early discontinuation of PD-1 blockade may be implemented as the standard of care in a selected group of patients.,The Safe Stop trial has been registered in the Netherlands Trial Register (NTR), Trial NL7293 (old NTR ID: 7502), https://www.trialregister.nl/trial/7293.,Date of registration September 30, 2018.

Supplemental Digital Content is available in the text.,CheckMate 218, a North American expanded access program (EAP), investigated nivolumab plus ipilimumab in patients with advanced melanoma.,Safety and efficacy, including 2-year survival in clinically relevant patient subgroups, are reported.,Eligible patients were aged ≥18 years with unresectable stage III/IV melanoma, an Eastern Cooperative Oncology Group performance status of 0/1, and no prior checkpoint inhibitors.,Patients received nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks for 4 cycles (induction) followed by nivolumab 3 mg/kg every 2 weeks (maintenance) until progression or unacceptable toxicity or a maximum of 48 weeks.,Safety and overall survival (OS) data were collected.,This EAP included 754 treated patients from the USA (n = 580) and Canada (n = 174).,Median follow-up time was 17.8 months.,All-grade and grade 3-4 treatment-related adverse events were reported in 96% and 53% of patients and led to treatment discontinuation in 36% and 26% of patients, respectively.,OS rates at 12 and 24 months were 82% [95% confidence interval (CI) 79-84] and 70% (95% CI 66-74), respectively.,Twenty-four-month OS rates were 63% in patients aged ≥75 years, 56% in patients with elevated lactate dehydrogenase levels, 73% in patients with BRAF wild-type tumors, 70% in patients with BRAF mutant tumors, and 56% in patients with mucosal melanoma.,In this EAP, nivolumab plus ipilimumab demonstrated high survival rates and safety outcomes consistent with those from randomized clinical trials, further supporting the use of this combination for advanced melanoma across multiple subgroups.

Skin cutaneous melanoma (SCM) is a common malignant tumor of the skin and its pathogenesis still needs to be studied.,In this work, we constructed a co-expression network and screened for hub genes by weighted gene co-expression network analysis (WGCNA) using the GSE98394 dataset.,The relationship between the mRNA expression of hub genes and the prognosis of patients with melanoma was validated by Gene Expression Profiling Interactive Analysis (GEPIA) database.,Furthermore, immunohistochemistry in the Human Protein Atlas was used to validate hub genes and grayscale analysis was performed using ImageJ software.,It was found that the yellow module was most significantly associated with the difference between common nevus and SCM, and 13 genes whose expression correlation >0.9 were candidate hub genes.,The expression of three genes (STK26, KCNT2, CASP12) was correlated with the prognosis of SCM.,STK26 (P = 0.0024) and KCNT2 (P < 0.0001) were significantly different between normal skin and SCM.,These three hub genes have potential value as predictors for accurate diagnosis and prognosis of SCM in the future.

Several studies have reported that cigarette smoking is inversely associated with the risk of melanoma.,This study further tested whether incorporating genetic factors will provide another level of evaluation of mechanisms underlying the association between smoking and risk of melanoma.,We investigated the association between SNPs selected from genome-wide association studies (GWAS) on smoking behaviors and risk of melanoma using 2,298 melanoma cases and 6,654 controls.,Among 16 SNPs, three (rs16969968 [A], rs1051730 [A] and rs2036534 [C] in the 15q25.1 region) reached significance for association with melanoma risk in men (0.01 < = P values < = 0.02; 0.85 < = Odds Ratios (ORs) <= 1.20).,There was association between the genetic scores based on the number of smoking behavior-risk alleles and melanoma risk with P-trend = 0.005 among HPFS.,Further association with smoking behaviors indicating those three SNPs (rs16969968 [A], rs1051730 [A] and rs2036534 [C]) significantly associated with number of cigarettes smoked per day, CPD, with P = 0.009, 0.011 and 0.001 respectively.,The SNPs rs215605 in the PDE1C gene and rs6265 in the BDNF gene significantly interacted with smoking status on melanoma risk (interaction P = 0.005 and P = 0.003 respectively).,Our study suggests that smoking behavior-related SNPs are likely to play a role in melanoma development and the potential public health importance of polymorphisms in the CHRNA5-A3-B4 gene cluster.,Further larger studies are warranted to validate the findings.

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

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 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.

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.

Mucosal melanomas (MM) are rare aggressive cancers in humans, and one of the most common forms of oral cancers in dogs.,Similar biological and histological features are shared between MM in both species, making dogs a powerful model for comparative oncology studies of melanomas.,Although exome sequencing recently identified recurrent coding mutations in canine MM, little is known about changes in non-coding gene expression, and more particularly, in canine long non-coding RNAs (lncRNAs), which are commonly dysregulated in human cancers.,Here, we sampled a large cohort (n = 52) of canine normal/tumor oral MM from three predisposed breeds (poodles, Labrador retrievers, and golden retrievers), and used deep transcriptome sequencing to identify more than 400 differentially expressed (DE) lncRNAs.,We further prioritized candidate lncRNAs by comparative genomic analysis to pinpoint 26 dog-human conserved DE lncRNAs, including SOX21-AS, ZEB2-AS, and CASC15 lncRNAs.,Using unsupervised co-expression network analysis with coding genes, we inferred the potential functions of the DE lncRNAs, suggesting associations with cancer-related genes, cell cycle, and carbohydrate metabolism Gene Ontology (GO) terms.,Finally, we exploited our multi-breed design to identify DE lncRNAs within breeds.,This study provides a unique transcriptomic resource for studying oral melanoma in dogs, and highlights lncRNAs that may potentially be diagnostic or therapeutic targets for human and veterinary medicine.

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

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.

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.

Cutaneous squamous cell carcinoma (cSCC) has a high tumour mutational burden (50 mutations per megabase DNA pair).,Here, we combine whole-exome analyses from 40 primary cSCC tumours, comprising 20 well-differentiated and 20 moderately/poorly differentiated tumours, with accompanying clinical data from a longitudinal study of immunosuppressed and immunocompetent patients and integrate this analysis with independent gene expression studies.,We identify commonly mutated genes, copy number changes and altered pathways and processes.,Comparisons with tumour differentiation status suggest events which may drive disease progression.,Mutational signature analysis reveals the presence of a novel signature (signature 32), whose incidence correlates with chronic exposure to the immunosuppressive drug azathioprine.,Characterisation of a panel of 15 cSCC tumour-derived cell lines reveals that they accurately reflect the mutational signatures and genomic alterations of primary tumours and provide a valuable resource for the validation of tumour drivers and therapeutic targets.,It is known cutaneous squamous cell carcinoma (cSCC) involves a high tumour mutation burden.,Here the authors identify common cSCC mutated genes, copy number changes, altered pathways and report the presence of a novel mutation signature associated with chronic exposure to the immunosuppressive drug azathioprine.

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

The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients.,Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use.,Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells.,Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level.,Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution.,At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance.,However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution.,Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process.,However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis.,Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.

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.

Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti‐cancer drug trials for several decades.,However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans.,Therefore, anti‐tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials.,Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre‐clinical animal model for assessing anti‐cancer drugs.,However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours.,Interleukin (IL)‐2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL‐2, IL‐4, IL‐7, IL‐9, IL‐15 and IL‐21, is required for proper lymphoid development.,IL2RG−/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis.,Compared to age‐matched wild‐type pigs, IL2RG −/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T‐ and NK‐cell deficiency.,Human melanoma Mel888 cells generated tumours in IL2RG −/Y pigs but not in wild‐type littermates.,The human tumours grew faster in IL2RG −/Y pigs than in nude mice.,Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti‐cancer drugs.

RUNX2, a master osteogenic transcript ion factor, is overexpressed in several cancer cells; in melanoma it promotes cells migration and invasion as well as neoangiogenesis.,The annual mortality rates related to metastatic melanoma are high and novel agents are needed to improve melanoma patients’ survival.,It has been shown that lectins specifically target malignant cells since they present the Thomsen-Friedenreich antigen.,This disaccharide is hidden in normal cells, while it allows selective lectins binding in transformed cells.,Recently, an edible lectin named BEL β-trefoil has been obtained from the wild mushroom Boletus edulis.,Our previous study showed BEL β-trefoil effects on transcription factor RUNX2 downregulation as well as on the migration ability in melanoma cells treated in vitro.,Therefore, to better understand the role of this lectin, we investigated the BEL β-trefoil effects in a zebrafish in vivo model, transplanted with human melanoma cells expressing RUNX2.,Our data showed that BEL β-trefoil is able to spread in the tissues and to reduce the formation of metastases in melanoma xenotransplanted zebrafish.,In conclusion, BEL β-trefoil can be considered an effective biomolecule to counteract melanoma disease.

The incidence of melanoma is increasing over the years with a still poor prognosis and the lack of a cure able to guarantee an adequate survival of patients.,Although the new immuno-based coupled to target therapeutic strategy is encouraging, the appearance of targeted/cross-resistance and/or side effects such as autoimmune disorders could limit its clinical use.,Alternative therapeutic strategies are therefore urgently needed to efficiently kill melanoma cells.,Ferroptosis induction and execution were evaluated in metastasis-derived wild-type and oncogenic BRAF melanoma cells, and the process responsible for the resistance has been dissected at molecular level.,Although efficiently induced in all cells, in an oncogenic BRAF- and ER stress-independent way, most cells were resistant to ferroptosis execution.,At molecular level we found that: resistant cells efficiently activate NRF2 which in turn upregulates the early ferroptotic marker CHAC1, in an ER stress-independent manner, and the aldo-keto reductases AKR1C1 ÷ 3 which degrades the 12/15-LOX-generated lipid peroxides thus resulting in ferroptotic cell death resistance.,However, inhibiting AKRs activity/expression completely resensitizes resistant melanoma cells to ferroptosis execution.,Finally, we found that the ferroptotic susceptibility associated with the differentiation of melanoma cells cannot be applied to metastatic-derived cells, due to the EMT-associated gene expression reprogramming process.,However, we identified SCL7A11 as a valuable marker to predict the susceptibility of metastatic melanoma cells to ferroptosis.,Our results identify the use of pro-ferroptotic drugs coupled to AKRs inhibitors as a new valuable strategy to efficiently kill human skin melanoma cells.

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

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

Genetics-based basket trials have emerged to test targeted therapeutics across multiple cancer types.,However, while vemurafenib is FDA-approved for BRAF-V600E melanomas, the non-melanoma basket trial was unsuccessful, suggesting mutation status is insufficient to predict response.,We hypothesized that proteomic data would complement mutation status to identify vemurafenib-sensitive tumors and effective co-treatments for BRAF-V600E tumors with inherent resistance.,Reverse Phase Proteomic Array (RPPA, MD Anderson Cell Lines Project), RNAseq (Cancer Cell Line Encyclopedia) and vemurafenib sensitivity (Cancer Therapeutic Response Portal) data for BRAF-V600E cancer cell lines were curated.,Linear and nonlinear regression models using RPPA protein or RNAseq were evaluated and compared based on their ability to predict BRAF-V600E cell line sensitivity (area under the dose response curve).,Accuracies of all models were evaluated using hold-out testing.,CausalPath software was used to identify protein-protein interaction networks that could explain differential protein expression in resistant cells.,Human examination of features employed by the model, the identified protein interaction networks, and model simulation suggested anti-ErbB co-therapy would counter intrinsic resistance to vemurafenib.,To validate this potential co-therapy, cell lines were treated with vemurafenib and dacomitinib (a pan-ErbB inhibitor) and the number of viable cells was measured.,Orthogonal partial least squares (O-PLS) predicted vemurafenib sensitivity with greater accuracy in both melanoma and non-melanoma BRAF-V600E cell lines than other leading machine learning methods, specifically Random Forests, Support Vector Regression (linear and quadratic kernels) and LASSO-penalized regression.,Additionally, use of transcriptomic in place of proteomic data weakened model performance.,Model analysis revealed that resistant lines had elevated expression and activation of ErbB receptors, suggesting ErbB inhibition could improve vemurafenib response.,As predicted, experimental evaluation of vemurafenib plus dacomitinb demonstrated improved efficacy relative to monotherapies.,Conclusions: Combined, our results support that inclusion of proteomics can predict drug response and identify co-therapies in a basket setting.

Although some fluoroquinolones have been found to exert anti-tumor activity, studies on the effect of these drugs on melanoma cells are relatively rare.,The aim of this study was to examine the effect of lomefloxacin on cell viability, reactive oxygen species production, redox balance, cell cycle distribution, DNA fragmentation, and apoptosis in COLO829 melanoma cells.,Lomefloxacin decreases the cell viability in a dose- and time-dependent manner.,For COLO829 cells treated with the drug for 24, 48, and 72 h, the values of IC50 were found to be 0.51, 0.33, and 0.25 mmol/L, respectively.,The analyzed drug also altered the redox signaling pathways, as shown by intracellular reactive oxygen species overproduction and endogeneous glutathione depletion.,After lomefloxacin treatment, the cells were arrested in S- and G2/M-phase, suggesting a mechanism related to topoisomerase II inhibition.,DNA fragmentation was observed when the cells were exposed to increasing lomefloxacin concentrations and a prolongation of incubation time.,Moreover, it was demonstrated that the drug induced mitochondrial membrane breakdown as an early hallmark of apoptosis.,The obtained results provide a strong molecular basis for the pharmacologic effect underlying the potential use of lomefloxacin as a valuable agent for the treatment of melanoma in vivo.

The effects of UVR on the skin include tanning, carcinogenesis, immunomodulation, and synthesis of vitamin D, among others.,Melanocortin 1 receptor polymorphisms correlate with skin pigmentation, UV sensitivity, and skin cancer risk.,This article reviews pathways through which UVR induces cutaneous stress and the pigmentation response.,Modulators of the UV tanning pathway include sunscreen agents, MC1R activators, adenylate cyclase activators, phosphodiesterase 4D3 inhibitors, T oligos, and MITF regulators such as histone deacetylase (HDAC)-inhibitors.,UVR, as one of the most ubiquitous carcinogens, represents both a challenge and enormous opportunity in skin cancer prevention.

Carvedilol is reported to prevent cancers in humans and animal models.,However, a molecular mechanism has yet to be established, and the extent to which other β-blockers are chemopreventive remains relatively unknown.,A comparative pharmacological approach was utilized with the expectation that a mechanism of action could be devised.,JB6 Cl 41-5a (JB6 P+) murine epidermal cells were used to elucidate the chemopreventative properties of β-blockers, as JB6 P+ cells recapitulate in vivo tumor promotion and chemoprevention.,The initial hypothesis was that β-blockers that are GRK/β-arrestin biased agonists, like carvedilol, are chemopreventive.,Sixteen β-blockers of different classes, isoproterenol, and HEAT HCl were individually co-administered with epidermal growth factor (EGF) to JB6 P+ cells to examine the chemopreventative properties of each ligand.,Cytotoxicity was examined to ensure that the anti-transformation effects of each ligand were not due to cellular growth inhibition.,Many of the examined β-blockers suppressed EGF-induced JB6 P+ cell transformation in a non-cytotoxic and concentration-dependent manner.,However, the IC50 values are high for the most potent inhibitors (243, 326, and 431 nM for carvedilol, labetalol, and alprenolol, respectively) and there is no correlation between pharmacological properties and inhibition of transformation.,Therefore, the role of α1- and β2-adrenergic receptors (AR) was examined by standard competition assays and shRNA targeting β2-ARs, the only β-AR expressed in JB6 P+ cells.,The results reveal that pharmacological inhibition of α1- and β2-ARs and genetic knockdown of β2-ARs did not abrogate carvedilol-mediated inhibition of EGF-induced JB6 P+ cell transformation.,Furthermore, topical administration of carvedilol protected mice from UV-induced skin damage, while genetic ablation of β2-ARs increased carvedilol-mediated effects.,Therefore, the prevailing hypothesis that the chemopreventive property of carvedilol is mediated through β-ARs is not supported by this data.

Melanoma incidence rates have continued to increase in the United States, and risk behaviors remain high.,Melanoma is responsible for the most skin cancer deaths, with about 9,000 persons dying from it each year.,CDC analyzed current (2011) melanoma incidence and mortality data, and projected melanoma incidence, mortality, and the cost of treating newly diagnosed melanomas through 2030.,Finally, CDC estimated the potential melanoma cases and costs averted through 2030 if a comprehensive skin cancer prevention program was implemented in the United States.,In 2011, the melanoma incidence rate was 19.7 per 100,000, and the death rate was 2.7 per 100,000.,Incidence rates are projected to increase for white males and females through 2019.,Death rates are projected to remain stable.,The annual cost of treating newly diagnosed melanomas was estimated to increase from $457 million in 2011 to $1.6 billion in 2030.,Implementation of a comprehensive skin cancer prevention program was estimated to avert 230,000 melanoma cases and $2.7 billion in initial year treatment costs from 2020 through 2030.,If additional prevention efforts are not undertaken, the number of melanoma cases is projected to increase over the next 15 years, with accompanying increases in health care costs.,Much of this morbidity, mortality, and health care cost can be prevented.,Substantial reductions in melanoma incidence, mortality, and cost can be achieved if evidence-based comprehensive interventions that reduce ultraviolet (UV) radiation exposure and increase sun protection are fully implemented and sustained.

Studies evaluating a relationship of vitamin D in patients with primary melanoma have consistently identified an inverse correlation with Breslow thickness, but an inconsistent impact on survival.,Vitamin D in later stages of melanoma has been less studied.,Vitamin D was measured in serum from 341 patients with resected stage IIB-IIIC melanoma recruited to the AVAST-M adjuvant melanoma randomised trial, collected prior to randomisation, then at 3 and 12 months.,Vitamin D levels were compared with patient demographics, known melanoma prognostic factors, disease-free interval (DFI) and overall survival (OS).,A total of 73% patients had stage III melanoma, 32% were enroled (and therefore tested) >1 year after primary melanoma diagnosis.,Median pre-randomisation vitamin D level was 56.5 (range 12.6-189.0 nmol/L).,Vitamin D levels did not significantly vary over 12 months (p = 0.24).,Individual pre-randomisation vitamin D levels did not differ significantly for Breslow thickness, tumour ulceration, or disease stage.,Neither did pre-randomisation vitamin D predict for DFI (HR = 0.98 per 10 nmol/L increase; 95% confidence interval (CI) 0.93-1.04, p = 0.59) or OS (HR = 0.96 per 10 nmol/L increase, 95% CI 0.90-1.03, p = 0.31).,For stage II patients, DFI improved with higher pre-randomisation vitamin D levels for those on bevacizumab (HR = 0.74 per 10 nmol nmol/L increase; 95% CI 0.56-0.97), but not for the observation arm (HR = 1.07 per 10 nmol/L increase; 95% CI 0.85-1.34).,In this stage II/III melanoma cohort, vitamin D did not correlate with known prognostic markers, nor predict for DFI or OS, but there was some evidence of benefit for patients with stage II disease treated with bevacizumab.

The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell.,We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi.,Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both.,They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro.,Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.,Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR).,Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice.,However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo.,Aldh1a1 knockdown reduced the high levels of CD133+/CD29+/CD44+ cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells.,Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells.,In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells.,Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression.,Since AhR may antagonize the protumoral effects of Aldh1a1, the AhRlow-Aldh1a1high phenotype could be indicative of bad outcome in melanoma.,The online version of this article (doi:10.1186/s12943-015-0419-9) contains supplementary material, which is available to authorized users.

TP53 is considered the most commonly-altered gene in cutaneous squamous cell carcinoma (cSCC).,Conversely, RAS mutations have been reported in a low percentage of cSCC.,The objective of our study was to evaluate the frequency of p53 expression and RAS mutations in cSCC and correlate them with clinicopathological features and patient outcome.,We performed immunohistochemistry for p53 and genetic profiling for RAS mutations in a retrospective series of cSCC.,The predictive value of p53 expression, RAS mutations, and clinicopathological parameters was assessed using logistic regression models.,The overall frequency of RAS mutations was 9.3% (15/162), and 82.1% of the cases (133/162) had p53 overexpression.,RAS mutations rate was 3.2% (1/31) of in situ cSCCs and 10.7% (14/131) of invasive cSCCs.,RAS mutations were more frequently associated with an infiltrative than an expansive pattern of invasion (p = 0.046). p53 overexpression was a predictor of recurrence in the univariate analysis.,Our results indicate that RAS mutations associate with features of local aggressiveness.,Larger studies with more recurrent and metastatic cSCCs are necessary to further address the prognostic significance of p53 overexpression in patients’ risk stratification.

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.

Malignant tumors shed DNA into the circulation.,The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw.,However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors.,In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined.,Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively).,Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls.,Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively).,Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer.,These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

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

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

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

The activity of the M isoform of microphthalmia-associated transcription factor (MITF-M) has been attributed to regulation of differentiation, proliferation, survival and senescence of melanoma cells.,MITF expression was shown to be antagonized by the activation of transcription factor NF-κB.,Parthenolide, an inhibitor of NF-κB, has not been yet reported to affect MITF-M expression.,Our results obtained in patient-derived melanoma cell populations indicate that parthenolide efficiently decreases the MITF-M level.,This is neither dependent on p65/NF-κB signaling nor RAF/MEK/ERK pathway activity as inhibition of MEK by GSK1120212 (trametinib) and induction of ERK1/2 activity by parthenolide itself do not interfere with parthenolide-triggered depletion of MITF-M in both wild-type BRAF and BRAFV600E melanoma populations.,Parthenolide activity is not prevented by inhibitors of caspases, proteasomal and lysosomal pathways.,As parthenolide reduces MITF-M transcript level and HDAC1 protein level, parthenolide-activated depletion of MITF-M protein may be considered as a result of transcriptional regulation, however, the influence of parthenolide on other elements of a dynamic control over MITF-M cannot be ruled out.,Parthenolide induces diverse effects in melanoma cells, from death to senescence.,The mode of the response to parthenolide is bound to the molecular characteristics of melanoma cells, particularly to the basal MITF-M expression level but other cell-autonomous differences such as NF-κB activity and MCL-1 level might also contribute.,Our data suggest that parthenolide can be developed as a drug used in combination therapy against melanoma when simultaneous inhibition of MITF-M, NF-κB and HDAC1 is needed.

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

Melanoma is the most lethal form of skin cancer, and develops from mutation of pigment-producing cells.,As it becomes malignant, it usually grows in size, changes proportions, and develops an irregular border.,We introduce a system for early detection of such changes, which enables whole-body screening, especially useful in patients with atypical mole syndrome.,The paper proposes a procedure to build a 3D model of the patient, relate the high-resolution skin images with the model, and orthorectify these images to enable detection of size and shape changes in nevi.,The novelty is in the application of image encoding indices and barycentric coordinates of the mesh triangles.,The proposed procedure was validated with a set of markers of a specified geometry.,The markers were attached to the body of a volunteer and analyzed by the system.,The results of quantitative comparison of original and corrected images confirm that the orthorectification allows for more accurate estimation of size and proportions of skin nevi.

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.

Autophagy is a resistance mechanism to BRAF/MEK inhibition in BRAFV600-mutant melanoma.,Here we used hydroxychloroquine (HCQ) to inhibit autophagy in combination with dabrafenib 150 mg twice daily and trametinib 2 mg every day (D+T).,We conducted a phase I/II clinical trial in four centers of HCQ + D+T in patients with advanced BRAFV600-mutant melanoma.,The primary objectives were the recommended phase II dose (RP2D) and the one-year progression-free survival (PFS) rate of >53%.,Thirty-four patients were evaluable for one-year PFS rate.,Patient demographics were as follows: elevated lactate dehydrogenase: 47%; stage IV M1c/M1d: 52%; prior immunotherapy: 50%.,In phase I, there was no dose-limiting toxicity.,HCQ 600 mg orally twice daily with D+T was the RP2D.,The one-year PFS rate was 48.2% [95% confidence interval (CI), 31.0%-65.5%], median PFS was 11.2 months (95% CI, 5.4-16.9 months), and response rate (RR) was 85% (95% CI, 64%-95%).,The complete RR was 41% and median overall survival (OS) was 26.5 months.,In a patient with elevated LDH (n = 16), the RR was 88% and median PFS and OS were 7.3 and 22 months, respectively.,HCQ + D+T was well tolerated and produced a high RR but did not meet criteria for success for the one-year PFS rate.,There was a high proportion of patients with pretreated and elevated LDH, an increasingly common demographic in patients receiving targeted therapy.,In this difficult-to-treat population, the RR and PFS were encouraging.,A randomized trial of D+T + HCQ or placebo in patients with BRAFV600-mutant melanoma with elevated LDH and previous immunotherapy is being conducted.

The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma and transformed outcomes for patients with metastatic disease.,The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition, prompting evaluation of a new combination incorporating a CDK4/6 inhibitor.,Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition has recently entered clinical trials for the treatment of BRAFV600 melanoma.,Interestingly, while BRAF- and MEK-targeted therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity.,Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and can be both immune-potentiating and immune-inhibitory.,BRAFV600 melanoma patients are also eligible to receive immunotherapy, specifically checkpoint inhibitors against PD-1 and CTLA-4.,The immunomodulatory activity of BRAF/MEK-targeted therapies has prompted interest in combination therapies incorporating these with immune checkpoint inhibitors, however recent clinical trials investigating this approach have produced variable results.,Here, we summarize the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibitors, shedding light on the prospective utility of this combination alone and in conjunction with immune checkpoint blockade.,Understanding the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimizing novel combination and scheduling approaches to combat melanoma and improve patient outcomes.

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.

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

To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases.,Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types.,Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2).,Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases.,Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature.,Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor.,Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors.,Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.

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.

Primary malignant melanoma of the lung is an extremely rare pulmonary carcinoma.,Only 45 cases have been reported in the literature.,Herein, we report a case of a 46‐year‐old male patient with an O Rh negative blood type who presented with pulmonary bronchial symptoms and underwent lobectomy.,Genetic testing was also performed but no targetable mutations were found, and the patient's PD‐L1 RNA level was low.,He developed brain metastasis four months after surgery and received radiotherapy but died 21 months after diagnosis.,We review the published cases of this rare pulmonary lesion.

In the lung, melanoma is mostly arranged as patterns of multiple nodules, solitary nodules, or miliary invasions.,Very rarely, it also displays a “crazy paving” pattern (also described as a “paving stone,” “flagstone,” or “slabstone” pattern), which is rarer still in discrete bilateral nodules.,This pattern is considered to be caused by pulmonary alveolar proteinosis, but its association with various diseases is unclear.,A 60-year-old man was diagnosed with pulmonary melanoma.,Computed tomography revealed discrete bilateral nodules surrounded by a “paving” pattern.,A literature review found more than 40 types of diseases that have presented with “paving” patterns in the lung-predominantly pulmonary alveolar proteinosis, viral pneumonia, exogenous lipoid pneumonia, bacterial pneumonia, pulmonary alveolar microlithiasis, interstitial pneumonia, ARDS, squalene aspiration pneumonia, radiation pneumonitis, drug-induced pneumonitis, pulmonary leptospirosis, pulmonary hemorrhage, and pulmonary nocardiosis.,We describe the first case of pulmonary melanoma in the form of discrete bilateral nodules accompanied with a computed tomography paving pattern.,Although pulmonary paving patterns are rare, more than 40 diseases reportedly display them; clinicians should consider melanoma of the lung in differential diagnoses for patients who show such a pattern.

Melanoma was again a focus of attention at the 2015 American Society of Clinical Oncology (ASCO) Annual Meeting, in particular the use of combination treatment strategies involving immunotherapies and/or targeted agents.,New data on targeted therapies confirmed previous findings, with combined BRAF inhibitor (vemurafenib) plus MEK inhibitor (cobimetinib) improving progression-free survival (PFS) compared to vemurafenib monotherapy in patients with BRAFV600 mutation-positive tumors (CoBRIM trial).,Positive results were also seen with combined dabrafenib and trametinib in patients with BRAF V600E/K metastatic melanoma and encorafenib plus binimetinib in BRAFV600-mutant cutaneous melanoma.,Even more interesting news centered on the use of combination immunotherapy, in particular the randomized, double-blind CheckMate 067 study in which median PFS with nivolumab plus ipilimumab was 11.5 months, compared to 2.9 months with ipilimumab alone (HR 0.42) and 6.9 months with nivolumab alone (HR 0.57).,Of interest, in patients with ≥5% PD-L1 expression, median PFS was 14 months with the combination or with nivolumab alone compared with 3.9 months in the ipilimumab group, while in the PD-L1 negative cohort, the combination remained superior to both monotherapies.,Given that combination therapy was accompanied by a high occurrence of side-effects, this raises the suggestion that combination therapy might be reserved for PD-L1 negative patients only, with PD-L1 positive patients achieving the same benefit from nivolumab monotherapy.,However, overall survival data are awaited and the equivalence of single agent to the combination remains unconvincing.,Interesting data were also reported on the combination of T-VEC (talimogene laherparepvec) with ipilimumab, and the anti-PD-1 agent MEDI4736 (durvolumab) combined with dabrafenib plus trametinib.,Emerging data also suggested that predictive markers based on immunoprofiling and mismatch repair deficiency may be of clinical use.,In conclusion, the use of combination approaches to treat patients with melanoma, as well as other cancers, is no longer a just a wish for the future but is today a clinical reality with a rapidly growing evidence-base.,Moreover, the most exciting consideration is that this is far from the end of the story, but rather a fantastic introduction.

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis.,We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype.,This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process.,We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process.,Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation.,Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells.,Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion.,Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube.,In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies.,These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure.,An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors.,However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC.,In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes.,To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure.,Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA.,Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival.,Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival.,These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC.,Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer.,The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells.,To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST.,MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1).,Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells.,Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells.,Both NF-κB and MYC have been shown to regulate MCT1 expression.,While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels.,Several MCC lines had high levels of MYCL and MYCN but not MYC.,Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells.,Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.

Aberrant DNA methylation pattern is a well-known epigenetic marker of cancer cells.,Recently, aberrant methylation was also reported in the peripheral blood of cancer patients and it could potentially serve as a biomarker for cancer risk.,We investigated the methylation pattern of LINE-1 and other repetitive DNA elements in peripheral blood of cutaneous melanoma patients in order to search for an association with clinical characteristics.,The patient cohort was composed by 69 unrelated melanoma patients, 28 of whom were hereditary cases (with or without CDKN2A mutations) and 41 were isolated (sporadic) melanoma cases.,Methylation of LINE-1 was evaluated by pyrosequencing, whereas additional repetitive DNA sequences were assessed using Illumina 450K methylation microarray.,Melanoma patients exhibited a higher, albeit heterogeneous, LINE-1 methylation level compared with controls.,Hereditary melanoma patients carrying CDKN2A mutations showed a hypermethylated pattern of both LINE-1 and repetitive DNA elements compared with other patients.,In particular, the methylation level at one specific CpG of LINE-1 was found to be correlated with the occurrence of metastasis.,Our data suggest that LINE-1 hypermethylation in peripheral blood of melanoma patients is a potential epigenetic biomarker for metastasis occurrence.

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

Drug addiction denotes the dependency of tumors on the same therapeutic drugs to which they have acquired resistance.,Observations from cultured cells1-3, animal models4 and patients5-7 raise the possibility that cancer drug addiction can instigate a potential cancer vulnerability, which may be used therapeutically.,However, for this trait to become of clinical interest, it is imperative to first define the underlying mechanism.,Therefore, we performed an unbiased CRISPR-Cas9 knockout screen to functionally mine the genome of melanoma cells that are both resistant and addicted to BRAF inhibition for “addiction genes”.,Here, we describe a signaling pathway comprising ERK2, JUNB and FRA1, disruption of which allows tumor cells to reverse addiction and survive upon treatment discontinuation.,This occurred both in culture and mice, and was irrespective of the acquired drug resistance mechanism.,In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of EMT.,In melanoma, this caused shutdown of the lineage survival oncoprotein MITF, restoration of which reversed both phenotype switching and drug addiction-associated lethality.,In melanoma patients who had progressed on BRAF inhibition, treatment cessation was followed by increased expression of the phenotype switch-associated receptor tyrosine kinase AXL.,Drug discontinuation synergized with the melanoma chemotherapeutic dacarbazine by further suppressing MITF and its prosurvival target BCL2 while inducing DNA damage.,Our results uncover a pathway driving cancer drug addiction, which may guide alternating therapeutic strategies for enhanced clinical responses of drug-resistant cancers.

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

The aim of the study was to analyse efficacy, safety, and health-related quality of life (HRQoL) for sorafenib treatment in patients with metastatic uveal melanoma.,A multicentre, single-arm phase II trial was conducted.,The primary objective was to determine the non-progression rate (RECIST) at 24 weeks for patients receiving sorafenib at a dose of 800 mg per day.,Secondary endpoints included progression-free survival (PFS), overall survival (OS), toxicity, and HRQoL.,Thirty-two patients were included.,Ten patients showed non-progression at 24 weeks (31.2%) without objective tumour responses.,The estimated 24-week PFS was 31.2% (95% CI: 14.8%-47.6%) and the estimated 24-week OS was 62.5% (95% CI: 45.4%-79.6%).,Ten patients (34.3%) had at least one grade 3 or 4 adverse reaction and 12 patients (41.4%) required dose modifications due to toxicity.,At 24 weeks, no patient had an improvement in global HRQoL and 87.5% experienced a permanent increase in physical fatigue.,Sorafenib demonstrated non-progression at 24 weeks in 31.2% of patients.,However, 41.4% of patients required dose modifications due to toxicity and no improvement in HRQoL was demonstrated.

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

Development of T cell-directed immune checkpoint inhibitors (ICI) has revolutionized metastatic melanoma (MM) therapy, but <50% of treated patients experience durable responses.,This phase I trial (NCT01946373) investigates the safety/feasibility of tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) combined with dendritic cell (DC) vaccination in MM patients progressing on ICI.,An initial cohort (5 patients) received TIL therapy alone to evaluate safety and allow for optimization of TIL expansion protocols.,A second cohort (first-in-man, 5 patients) received TIL combined with autologous tumor lysate-loaded DC vaccination.,All patients received cyclophosphamide/fludarabine preconditioning prior to, and intravenous (i.v.),IL-2 after, TIL transfer.,The DC vaccine was given as five intradermal injections after TIL and IL-2 administration.,[18F]-FDG PET/CT radiology was performed to evaluate clinical response, according to RECIST 1.1 (on the CT part).,Immunological monitoring was performed by flow cytometry and T-cell receptor (TCR) sequencing.,In the safety/optimization cohort, all patients had a mixed response or stable disease, but none durable.,In the combination cohort, two patients experienced complete responses (CR) that are still ongoing (>36 and >18 months, respectively).,In addition, two patients had partial responses (PR), one still ongoing (>42 months) with only a small bone-lesion remaining, and one of short duration (<4 months).,One patient died early during treatment and did not receive DC.,Long-lasting persistency of the injected TILs was demonstrated in blood.,In summary, we report clinical responses by TIL therapy combined with DC vaccination in 4 out of 4 treated MM patients who previously failed ICI.

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers.,Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses.,Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial.,Ten patients were treated with TIL therapy.,Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed.,Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products.,Five out of 10 patients, who were all anti-PD-1 naïve at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years.,Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed.,For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion.,The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity.,In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial.

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

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi+MEKi) therapies have significantly improved clinical outcomes in patients with metastatic melanoma.,Unfortunately, the efficacy is beset by the acquisition of drug resistance1-6.,Here we investigated molecular mechanisms underlying acquired resistance to BRAFi (BRAFi resistance, “BR”) and BRAFi+MEKi (combination therapy resistance, “CR”).,Consistent with previous studies, BR is mediated by ERK pathway re-activation.,CR is, however, mediated by mechanisms independent of re-activation of ERK in many therapy-resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in acquired drug resistant cells and play a pivotal role in mediating both BR and CR.,Our screening using reverse phase protein array (RPPA) revealed distinct mechanisms by which PAKs mediate BR and CR.,In BR, PAKs phosphorylate CRAF and MEK to reactivate ERK.,In CR, PAKs regulate JNK and β-catenin phosphorylation, mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK.,Together, our results provide new insights into molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Cutaneous melanoma shows a high metastatic potential based on its ability to overcome the immune system’s control.,The mechanisms activated for these functions vary extremely and are also represented by the production of a number of extracellular vesicles including exosomes.,Other vesicles showing a potential role in the melanoma progression include oncosomes and melanosomes and the majority of them mediate tumor processes including angiogenesis, immune regulation, and modifications of the micro-environment.,Moreover, a number of epigenetic modifications have been described in melanoma and abundant production of altered microRNAs (mi-RNAs), non-coding RNAs, histones, and abnormal DNA methylation have been associated with different phases of melanoma progression.,In addition, exosomes, miRNAs, and other molecular factors have been used as potential biomarkers reflecting disease evolution while others have been suggested to be potential druggable molecules for therapeutic application.

Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids.,Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells.,Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells.,However, the characteristics and functions of exosomes derived from NK cells remain unknown.,In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo.,Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads.,The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays.,Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation.,NK-92 Exo were characterized by transmission electron microscopy and western blotting.,We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells.,The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays.,To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line.,Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells.,In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo.,We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging.,Tumor mass was monitored after in vivo experiments.,Results: RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells.,B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay.,For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes.,The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX.,We demonstrated by western blot analysis that NK-92 Exo presented two functional NK proteins, namely perforin and FasL.,Moreover, we confirmed the membrane expression of FasL.,The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway.,The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p < 0.001) and CCK-8 assays (p < 0.001).,Furthermore, in normal healthy cells, even after 24 h of co-culture, NK-92 Exo did not exhibit significant side effects.,In the in vivo experiments, tumors in the vehicle control group were significantly increased, compared with those in the NK-92 Exo-treated group (p < 0.05).,Conclusion: The results of the current study suggest that exosomes derived from NK cells exert cytotoxic effects on melanoma cells and thus warrant further development as a potential immunotherapeutic strategy for cancer.

As widely acknowledged, 40-50% of all melanoma patients harbour an activating BRAF mutation (mostly BRAF V600E).,The identification of the RAS-RAF-MEK-ERK (MAP kinase) signalling pathway and its targeting has represented a valuable milestone for the advanced and, more recently, for the completely resected stage III and IV melanoma therapy management.,However, despite progress in BRAF-mutant melanoma treatment, the two different approaches approved so far for metastatic disease, immunotherapy and BRAF+MEK inhibitors, allow a 5-year survival of no more than 60%, and most patients relapse during treatment due to acquired mechanisms of resistance.,Deep insight into BRAF gene biology is fundamental to describe the acquired resistance mechanisms (primary and secondary) and to understand the molecular pathways that are now being investigated in preclinical and clinical studies with the aim of improving outcomes in BRAF-mutant patients.

To explore the safety and tolerability of combining two epigenetic drugs: decitabine (a DNA methyltransferase inhibitor) and panobinostat (a histone deacetylase inhibitor), with chemotherapy with temozolomide (an alkylating agent).,The purpose of such combination is to evaluate the use of epigenetic priming to overcome resistance of melanoma to chemotherapy.,A Phase I clinical trial enrolling patients aged 18 years or older, with recurrent or unresectable stage III or IV melanoma of any site.,This trial was conducted with full Institutional Review Board approval and was registered with the National Institutes of Health under the clinicaltrials.gov identifier NCT00925132.,Patients were treated with subcutaneous decitabine 0.1 or 0.2 mg/kg three times weekly for 2 weeks (starting on day 1), in combination with oral panobinostat 10, 20, or 30 mg every 96 h (starting on day 8), and oral temozolomide 150 mg/m2/day on days 9 through 13.,In cycle 2, temozolomide was increased to 200 mg/m2/day if neutropenia or thrombocytopenia had not occurred.,Each cycle lasted 6 weeks, and patients could receive up to six cycles.,Patients who did not demonstrate disease progression were eligible to enter a maintenance protocol with combination of weekly panobinostat and thrice-weekly decitabine until tumor progression, unacceptable toxicity, or withdrawal of consent.,Twenty patients were initially enrolled, with 17 receiving treatment.,The median age was 56 years.,Eleven (65 %) were male, and 6 (35 %) were female.,Eleven (64.7 %) had cutaneous melanoma, 4 (23.5 %) had ocular melanoma, and 2 (11.8 %) had mucosal melanoma.,All patients received at least one treatment cycle and were evaluable for toxicity.,Patients received a median of two 6-week treatment cycles (range 1-6).,None of the patients experienced DLT.,MTD was not reached.,Adverse events attributed to treatment included grade 3 lymphopenia (24 %), anemia (12 %), neutropenia (12 %), and fatigue (12 %), as well as grade 2 leukopenia (30 %), neutropenia (23 %), nausea (23 %), and lymphopenia (18 %).,The most common reason for study discontinuation was disease progression.,This triple agent of dual epigenetic therapy in combination with traditional chemotherapy was generally well tolerated by the cohort and appeared safe to be continued in a Phase II trial.,No DLTs were observed, and MTD was not reached.

In human mutant BRAF melanoma cells, the stemness transcription factor FOXD3 is rapidly induced by inhibition of ERK1/2 signaling and mediates adaptive resistance to RAF inhibitors.,However, the mechanism underlying ERK signaling control of FOXD3 expression remains unknown.,Here we show that SOX10 is both necessary and sufficient for RAF inhibitor-induced expression of FOXD3 in mutant BRAF melanoma cells.,SOX10 activates the transcription of FOXD3 by binding to a regulatory element in FOXD3 promoter.,Phosphorylation of SOX10 by ERK inhibits its transcription activity toward multiple target genes by interfering with the sumoylation of SOX10 at K55, which is essential for its transcription activity.,Finally, depletion of SOX10 sensitizes mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo.,Thus, our work discovers a novel phosphorylation-dependent regulatory mechanism of SOX10 transcription activity and completes an ERK1/2/SOX10/FOXD3/ERBB3 axis that mediates adaptive resistance to RAF inhibitors in mutant BRAF melanoma cells.,In BRAF mutant melanoma, inhibition of ERK1/2 induces FOXD3 and mediates RAF inhibitor resistance.,Here, the authors show that ERK1/2 mediated phosphorylation regulates sumoylation of SOX10 which activates FOXD3, and depletion of SOX10 sensitises BRAF mutant melanoma cells to RAF inhibitors.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF).,Accordingly, downregulation of MITF induces invasion in melanoma cells, however little is known about the underlying mechanisms.,Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C.,Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion.,We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells.,Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity, and lung colonization.,Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells.,Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide.,Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory.,While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored.,Here we investigated the function of ERK5 signaling in melanoma.,We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells.,Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E).,We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5.,Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF.,Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation.,Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts.,Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.

Melanoma is one of the most aggressive types of human cancer, characterized by enhanced heterogeneity and resistance to conventional therapy at advanced stages.,We and others have previously shown that HEDGEHOG-GLI (HH-GLI) signaling is required for melanoma growth and for survival and expansion of melanoma-initiating cells (MICs).,Recent reports indicate that HH-GLI signaling regulates a set of genes typically expressed in embryonic stem cells, including SOX2 (sex-determining region Y (SRY)-Box2).,Here we address the function of SOX2 in human melanomas and MICs and its interaction with HH-GLI signaling.,We find that SOX2 is highly expressed in melanoma stem cells.,Knockdown of SOX2 sharply decreases self-renewal in melanoma spheres and in putative melanoma stem cells with high aldehyde dehydrogenase activity (ALDHhigh).,Conversely, ectopic expression of SOX2 in melanoma cells enhances their self-renewal in vitro.,SOX2 silencing also inhibits cell growth and induces apoptosis in melanoma cells.,In addition, depletion of SOX2 progressively abrogates tumor growth and leads to a significant decrease in tumor-initiating capability of ALDHhigh MICs upon xenotransplantation, suggesting that SOX2 is required for tumor initiation and for continuous tumor growth.,We show that SOX2 is regulated by HH signaling and that the transcription factors GLI1 and GLI2, the downstream effectors of HH-GLI signaling, bind to the proximal promoter region of SOX2 in primary melanoma cells.,In functional studies, we find that SOX2 function is required for HH-induced melanoma cell growth and MIC self-renewal in vitro.,Thus SOX2 is a critical factor for self-renewal and tumorigenicity of MICs and an important mediator of HH-GLI signaling in melanoma.,These findings could provide the basis for novel therapeutic strategies based on the inhibition of SOX2 for the treatment of a subset of human melanomas.

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.

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.

Cutaneous melanoma is a lethal disease, even when diagnosed in advanced stages.,Although recent progress in biology and treatment has dramatically improved survival rates, new therapeutic approaches are still needed.,Deregulation of epigenetics, which mainly controls DNA methylation status and chromatin remodeling, is implied not only in cancer initiation and progression, but also in resistance to antitumor drugs.,Epigenetics in melanoma has been studied recently in both melanoma preclinical models and patient samples, highlighting its potential role in different phases of melanomagenesis, as well as in resistance to approved drugs such as immune checkpoint inhibitors and MAPK inhibitors.,This review summarizes what is currently known about epigenetics in melanoma and dwells on the recognized and potential new targets for testing epigenetic drugs, alone or together with other agents, in advanced melanoma patients.

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression.,However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive.,Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability.,In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces.,Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner.,Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo.,Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.,Soft 3D gels can promote the growth of tumour-repopulating cells, a self-renewing subpopulation of cancer cells critical in cancer progression.,Here, the authors investigate the mechanism behind this phenomenon and show that the histone 3 lysine residue 9 methylation and Sox2 are controlling this process.

Genomic alterations occurring during melanoma progression and the resulting genomic heterogeneity between metastatic deposits remain incompletely understood.,Analyzing 86 metastatic melanoma deposits from 53 patients with whole-exome sequencing (WES), we show a low branch to trunk mutation ratio and little intermetastatic heterogeneity, with driver mutations almost completely shared between lesions.,Branch mutations consistent with UV damage indicate that metastases may arise from different subclones in the primary tumor.,Selective gain of mutated BRAF alleles occurs as an early event, contrasting whole-genome duplication (WGD) occurring as a late truncal event in about 40% of cases.,One patient revealed elevated mutational diversity, probably related to previous chemotherapy and DNA repair defects.,In another patient having received radiotherapy toward a lymph node metastasis, we detected a radiotherapy-related mutational signature in two subsequent distant relapses, consistent with secondary metastatic seeding.,Our findings add to the understanding of genomic evolution in metastatic melanomas.,As melanoma progresses, it evolves.,Here, in advanced melanoma the authors study genomic evolution, highlighting trunk mutations dominated by the ultraviolet damage signature, common late truncal whole-genome duplication events, as well as selective copy number gain of mutant BRAF.

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.

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.

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.

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.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and frequently progresses from an actinic keratosis (AK), a sun-induced keratinocyte intraepithelial neoplasia (KIN).,Epigenetic mechanisms involved in the phenomenon of progression from AK to cSCC remain to be elicited.,Expression of microRNAs in sun-exposed skin, AK and cSCC was analysed by Agilent microarrays.,DNA methylation of miR-204 promoter was determined by bisulphite treatment and pyrosequencing.,Identification of miR-204 targets and pathways was accomplished in HaCat cells.,Immunofluorescence and immunohistochemistry were used to analyze STAT3 activation and PTPN11 expression in human biopsies.,cSCCs display a marked downregulation of miR-204 expression when compared to AK.,DNA methylation of miR-204 promoter was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC.,In HaCaT cells miR-204 inhibits STAT3 and favours the MAPK signaling pathway, likely acting through PTPN11, a nuclear tyrosine phosphatase that is a direct miR-204 target.,In non-peritumoral AK lesions, activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartments, whereas AK lesions adjacent to cSCCs display activated STAT3 in the nuclei.,Our data suggest that miR-204 may act as a “rheostat” that controls the signalling towards the MAPK pathway or the STAT3 pathway in the progression from AK to cSCC.,The online version of this article (doi:10.1186/s12943-016-0537-z) contains supplementary material, which is available to authorized users.

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

We assessed the utility of droplet digital PCR (ddPCR) to evaluate the potential of using circulating tumour DNA (ctDNA) as a post therapy monitoring tool in melanoma by comparing it to serum LDH levels and RECIST scores. ddPCR was shown to be reliable in distinguishing mutant from wild type alleles with no false positives.,Subsequently, we quantified ctDNA (V600EBRAF,V600KBRAF or Q61HNRAS) in 6 stage IV melanoma patients across several time points during their treatment course.,All tested patients had detectable ctDNA, which exhibited dynamic changes corresponding to the changes in their disease status.,The ctDNA levels fell upon treatment response and rose with detectable disease progression.,In our group of patients, ctDNA was more consistent and informative than LDH as a blood-based biomarker.,In addition, BRAF mutant ctDNA as detected by ddPCR could be used diagnostically where the tumour block was unavailable.,In conclusion, this study demonstrates the applicability of using ddPCR to detect and quantify ctDNA in the plasma of melanoma patients.

The major challenge in antigen‐specific immunotherapy of cancer is to select the most relevant tumor antigens to target.,To this aim, understanding their mode of expression by tumor cells is critical.,We previously identified a melanoma‐specific antigen, melanoma‐overexpressed antigen 1 (MELOE‐1)-coded for by a long noncoding RNA-whose internal ribosomal entry sequence (IRES)‐dependent translation is restricted to tumor cells.,This restricted expression is associated with the presence of a broad‐specific T‐cell repertoire that is involved in tumor immunosurveillance in melanoma patients.,In the present work, we explored the translation control of MELOE‐1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP‐A1) binds to the MELOE‐1 IRES and acts as an IRES trans‐activating factor (ITAF) to promote the translation of MELOE‐1 in melanoma cells.,In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP‐A1 cytoplasmic translocation, enhances MELOE‐1 translation and recognition of melanoma cells by a MELOE‐1‐specific T‐cell clone.,These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress‐induced tumor antigens such as MELOE‐1.,Our study demonstrates that reticular stress in melanoma cells promoted the translocation of the IRES transacting factor hnRPN‐A1 to the cytoplasm.,By binding to the IRES site of meloe mRNA, hnRPN‐A1 promoted the translation of the melanoma‐specific antigen MELOE‐1 and led to the expression of a new immunogenic HLA/peptide complex at the cell surface.,Altogether, our data suggest that pharmacological stimulation of stress pathways may enhance the efficacy of T‐cell based immunotherapies in targeting stress‐induced tumor antigens, such as MELOE‐1 in patients with melanoma.

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.

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.

Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide.,Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release.,We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway.,Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells.,Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET.,In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex.,Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI.,In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype.,Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.

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

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients.,Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44.,However, its role in regulating BMP signalling in melanoma is not clear.,In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells.,Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression.,Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP.,Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B.,Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients.,Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

Melanoma was again a focus of attention at the 2015 American Society of Clinical Oncology (ASCO) Annual Meeting, in particular the use of combination treatment strategies involving immunotherapies and/or targeted agents.,New data on targeted therapies confirmed previous findings, with combined BRAF inhibitor (vemurafenib) plus MEK inhibitor (cobimetinib) improving progression-free survival (PFS) compared to vemurafenib monotherapy in patients with BRAFV600 mutation-positive tumors (CoBRIM trial).,Positive results were also seen with combined dabrafenib and trametinib in patients with BRAF V600E/K metastatic melanoma and encorafenib plus binimetinib in BRAFV600-mutant cutaneous melanoma.,Even more interesting news centered on the use of combination immunotherapy, in particular the randomized, double-blind CheckMate 067 study in which median PFS with nivolumab plus ipilimumab was 11.5 months, compared to 2.9 months with ipilimumab alone (HR 0.42) and 6.9 months with nivolumab alone (HR 0.57).,Of interest, in patients with ≥5% PD-L1 expression, median PFS was 14 months with the combination or with nivolumab alone compared with 3.9 months in the ipilimumab group, while in the PD-L1 negative cohort, the combination remained superior to both monotherapies.,Given that combination therapy was accompanied by a high occurrence of side-effects, this raises the suggestion that combination therapy might be reserved for PD-L1 negative patients only, with PD-L1 positive patients achieving the same benefit from nivolumab monotherapy.,However, overall survival data are awaited and the equivalence of single agent to the combination remains unconvincing.,Interesting data were also reported on the combination of T-VEC (talimogene laherparepvec) with ipilimumab, and the anti-PD-1 agent MEDI4736 (durvolumab) combined with dabrafenib plus trametinib.,Emerging data also suggested that predictive markers based on immunoprofiling and mismatch repair deficiency may be of clinical use.,In conclusion, the use of combination approaches to treat patients with melanoma, as well as other cancers, is no longer a just a wish for the future but is today a clinical reality with a rapidly growing evidence-base.,Moreover, the most exciting consideration is that this is far from the end of the story, but rather a fantastic introduction.

The management of melanoma has evolved due to improved understanding of its molecular drivers.,To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed.,Mutations were identified in 43 of the 46 genes on the panel.,The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%).,Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype.,Concurrent TP53 mutations were the most frequent event in tumors with BRAFV600 and NRAS mutations.,Melanomas with BRAFNon-V600 mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%).,The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location.,Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600 and NRAS mutations.,These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma.

Accurate prognostic biomarkers in early-stage melanoma are urgently needed to stratify patients for clinical trials of adjuvant therapy.,We applied a previously developed open source deep learning algorithm to detect tumor-infiltrating lymphocytes (TILs) in hematoxylin and eosin (H&E) images of early-stage melanomas.,We tested whether automated digital (TIL) analysis (ADTA) improved accuracy of prediction of disease specific survival (DSS) based on current pathology standards.,ADTA was applied to a training cohort (n = 80) and a cutoff value was defined based on a Receiver Operating Curve.,ADTA was then applied to a validation cohort (n = 145) and the previously determined cutoff value was used to stratify high and low risk patients, as demonstrated by Kaplan-Meier analysis (p ≤ 0.001).,Multivariable Cox proportional hazards analysis was performed using ADTA, depth, and ulceration as co-variables and showed that ADTA contributed to DSS prediction (HR: 4.18, CI 1.51-11.58, p = 0.006).,ADTA provides an effective and attainable assessment of TILs and should be further evaluated in larger studies for inclusion in staging algorithms.

Acquired melanocytic nevi are commonly found in sun exposed and unexposed human skin, but the potential for their transformation into invasive melanoma is not clear.,Therefore, a mouse model of nevus initiation and progression was developed in C3H/HeN mice using a modified chemical carcinogenesis protocol.,Nevi develop due to DNA damage initiated by dimethylbenz(a) anthracene (DMBA) followed by chronic promotion with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA).,Dysplastic pigmented skin lesions appeared in 7-9 wk with 100% penetrance.,Nests of melanocytic cells appeared in a subset of skin draining lymph nodes (dLN) by 25 wk, but not in age matched controls.,Immunohistochemistry, real‐time PCR, and flow cytometric analyses confirmed their melanocytic origin.,Transformed cells were present in a subset of nevi and dLNs, which exhibited anchorage‐independent growth, tumor development, and metastasis in nude mice.,Approximately 50% of the cell lines contained H‐Ras mutations and lost tumor suppressor p16Ink4a expression.,While most studies of melanoma focus on tumor progression in transgenic mouse models where the mutations are present from birth, our model permits investigation of acquired mutations at the earliest stages of nevus initiation and promotion of nevus cell transformation.,This robust nevus/melanoma model may prove useful for identifying genetic loci associated with nevus formation, novel oncogenic pathways, tumor targets for immune‐prevention, screening therapeutics, and elucidating mechanisms of immune surveillance and immune evasion.,© 2015 The Authors.,Molecular Carcinogenesis, published by Wiley Periodicals, Inc.

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.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, known to play important roles in melanoma formation and progression.,Recent advances in genome-wide methylation methods have provided the means to identify differentially methylated genes, methylation signatures, and potential biomarkers.,However, despite considerable effort and advances in cataloging methylation changes in melanoma, many questions remain unanswered.,The aim of this review is to summarize recent developments, emerging trends, and important unresolved questions in the field of aberrant DNA methylation in melanoma.,In addition to reviewing recent developments, we carefully synthesize the findings in an effort to provide a framework for understanding the current state and direction of the field.,To facilitate clarity, we divided the review into DNA methylation changes in melanoma, biomarker opportunities, and therapeutic developments.,We hope this review contributes to accelerating the utilization of the diagnostic, prognostic, and therapeutic potential of DNA methylation for the benefit of melanoma patients.

The lncRNA biomarkers in melanoma remain to be further explored.,The lncRNAs with different expression levels in melanoma tissue were identified by microarray analysis.,To investigate the biological functions of target lncRNA, several in-vivo and in-vitro studies were performed.,Potential mechanisms of competitive endogenous RNAs (ceRNAs) were predicted by using bioinformatics analysis and explored by western blot assay, fluorescence in situ hybridization assay, real-time quantitative PCR (RT-qPCR) array, RNA pull-down analysis, AGO2-RIP assay, and dual-luciferase reporter assay.,The results demonstrated decreased LINC00459 in melanoma cell lines and tissues.,According to the in-vitro and in-vivo experiments, up-regulated LINC00459 had inhibitory effect on cell proliferation and invasion.,Bioinformatics analyses suggested that miR-218 could be a direct target of LINC00459.,In addition, miR-218 was proved to be able to directly target the dickkopf-related protein 3 (DKK3) gene.,In conclusion, our analysis suggested that the LINC00459 could sponge miR-218 and increase the expression of DKK3 gene, thus inhibiting the invasion and proliferation of melanoma cells, which indicated that the LINC00459 could be an effective biomarker for melanoma and its potential as the therapeutic target.

Dysregulated microRNA (miR)-625 expression has been observed in several kinds of cancer.,MicroRNAs are important factors in the development and progression of malignant melanoma, though the clinical significance and function of miR-625 in human malignant melanoma remain unclear.,Levels of miR-625 expression were therefore determined in 36 pairs of malignant melanoma and adjacent non-tumor tissue using qPCR.,The effects of miR-625 dysregulation on malignant melanoma cell proliferation, wound healing, migration and invasion in vitro and tumorigenicity in vivo were investigated using CCK-8, transwell assays, and a nude mouse subcutaneous tumor model.,Bioinformatics analysis and luciferase reporter system were used to predict and confirm the target gene of miR-625. miR-625 levels were frequently decreased in malignant melanoma.,Ectopic expression of miR-625 suppressed proliferation, wound healing, migration, and tumorgenicity in malignant melanoma.,Moreover, miR-625 acted, at least in part, by suppressing potential target SOX2.,These results show that miR-625 is a tumor suppressor that inhibits the development and progression of malignant melanoma, which suggests miR-625 is potentially a new diagnostic marker and therapeutic target of malignant melanoma.

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

Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide.,The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway.,However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease.,The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance.,We have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi).,The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors.,Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application.,In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks.,Eight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi.,Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells.,First in vivo studies confirmed these observations.,Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance.,Here, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells.,These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.,The online version of this article (10.1186/s13046-019-1038-x) contains supplementary material, which is available to authorized users.

An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas.,Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas.,However, acquired drug resistance invariably develops after the initial treatment.,Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of melanomas to BRAF inhibitors.,Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma.,AEBP1 is shown to be highly upregulated in PLX4032-resistant melanoma cells because of the hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway.,This upregulates AEBP1 expression and thus leads to the activation of NF-κB via accelerating IκBa degradation.,In addition, inhibition of the PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant phenotype of melanoma cells.,Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor.,Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway, particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma.

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.

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

Melanoma remains as the deadliest form of skin cancer with limited and inefficient treatment options available for patients with metastatic disease.,Within the last decade, the thrombin receptor, Protease Activated Receptor-1, has been described as an essential gene involved in the progression of human melanoma.,PAR-1 is known to activate adhesive, invasive and angiogenic factors to promote melanoma metastasis.,It is overexpressed not only in metastatic melanoma cell lines but is also highly expressed in metastatic lesions as compared to primary nevi and normal skin.,Recently, PAR-1 has been described to regulate the gap junction protein Connexin 43 and the tumor suppressor gene Maspin to promote the metastatic melanoma phenotype.,Herein, we review the role of PAR-1 in the progression of melanoma as well as utilizing PAR-1-regulated genes as potential therapeutic targets for melanoma treatment.

To examine the association between level and patterns of baseline intra-tumoural BRAFV600E protein expression and clinical outcome of BRAFV600E melanoma patients treated with selective BRAF inhibitors.,Fifty-eight BRAFV600E metastatic melanoma patients treated with dabrafenib or vemurafenib on clinical trials had pre-treatment tumour BRAFV600E protein expression immunohistochemically (IHC) assessed using the BRAF V600E mutant-specific antibody VE1.,Sections were examined for staining intensity (score 1-3) and percentage of immunoreactive tumour cells, and from this an immunoreactive score (IRS) was derived (intensity × per cent positive/10).,The presence of intra-tumoural heterogeneity for BRAFV600E protein expression was also assessed.,BRAFV600E expression was correlated with RECIST response, time to best response (TTBR), progression-free survival (PFS) and overall survival (OS).,Expression was generally high (median IRS 28 (range 5-30)) and homogeneous (78%).,Expression of mutated protein BRAFV600E as measured by intensity, per cent immunoreactive cells, or IRS did not correlate with RECIST response, TTBR, PFS or OS, including on multivariate analysis.,Heterogeneity of staining was seen in 22% of cases and did not correlate with outcome.,In the current study population, IHC-measured pre-treatment BRAFV600E protein expression does not predict response or outcome to BRAF inhibitor therapy in BRAFV600E metastatic melanoma patients.

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

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

Melanoma is one of the most deadly and therapy-resistant cancers.,Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy.,FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice.,FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway.,Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2.,Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity.,Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.,FTO is an m6A demethylase.,Here, the authors show that FTO promotes melanoma tumorigenicity and contributes to resistance to anti-PD1 blockade, while FTO inhibition sensitizes melanoma to anti-PD1 blockade.

The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma.,MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma.,How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model.,Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity.,We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease.,Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis.,These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

People with pale skin, red hair, freckles, and an inability to tan-the “redhair/fairskin” phenotype- are at highest risk of developing melanoma, compared to all other pigmentation types1.,Genetically, this phenotype is frequently the product of inactivating polymorphisms in the Melanocortin 1 receptor (MC1R) gene.,MC1R encodes a cAMP stimulating G-protein coupled receptor that controls pigment production.,Minimal receptor activity, as in redhair/fairskin polymorphisms, produces red/yellow pheomelanin pigment, while increasing MC1R activity stimulates production of black/brown eumelanin2.,Pheomelanin has weak UV shielding capacity relative to eumelanin and has been shown to amplify UVA-induced reactive oxygen species (ROS) 3-5.,Several observations, however, complicate the assumption that melanoma risk is completely UV dependent.,For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and UV signature mutations are infrequently oncogenic drivers6.,While linkage of melanoma risk to UV exposure is beyond doubt, UV-independent events are also likely to play a significant role1,7.,Here, we introduced into mice carrying an inactivating mutation in the Mc1r gene (who exhibit a phenotype analogous to redhair/fairskin humans), a conditional, melanocyte-targeted allele of the most commonly mutated melanoma oncogene, BRafV600E.,We observed a high incidence of invasive melanomas without providing additional gene aberrations or UV exposure.,To investigate the mechanism of UV-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r e/e background.,Selective absence of pheomelanin synthesis was protective against melanoma development.,In addition, normal Mc1re/e mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1re/e mouse skin.,These data suggest that the pheomelanin pigment pathway produces UV-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage.,While UV protection remains important, additional strategies may be required for optimal melanoma prevention.

Uveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available.,This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening.,Six UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions.,3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas.,PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation.,Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid.,In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay.,All six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages.,PUM spheroids were genetically identical to the original sampled tumor.,In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells.,Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells.,UM cell lines and PUM samples can successfully generate uniform 3D spheroids.,PUM spheroids retain histological and genetic characteristics of the primary tumor.,3D spheroids are an important system for use in future high-throughput drug testing.,The use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients.

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.

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

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

Dysplastic nevi (DN) is a strong risk factor for cutaneous malignant melanoma (CMM), and it frequently occurs in melanoma-prone families.,To identify genetic variants for DN, we genotyped 677 tagSNPs in 38 melanoma candidate genes that are involved in pigmentation, DNA repair, cell cycle control, and melanocyte proliferation pathways in a total of 504 individuals (310 with DN, 194 without DN) from 53 melanoma-prone families (23 CDKN2A mutation positive and 30 negative).,Conditional logistic regression, conditioning on families, was used to estimate the association between DN and each SNP separately, adjusted for age, sex, CMM and CDKN2A status.,P-values for SNPs in the same gene were combined to yield gene-specific p-values.,Two genes, CDK6 and XRCC1, were significantly associated with DN after Bonferroni correction for multiple testing (P=0.0001 and 0.00025, respectively), whereas neither gene was significantly associated with CMM.,Associations for CDK6 SNPs were stronger in CDKN2A mutation positive families (rs2079147, Pinteraction=0.0033), whereas XRCC1 SNPs had similar effects in mutation-positive and negative families.,The association for one of the associated SNPs in XRCC1 (rs25487) was replicated in two independent datasets (random effect meta-analysis: P<0.0001).,Our findings suggest that some genetic variants may contribute to DN risk independently of their association with CMM in melanoma-prone families.

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

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.

The long non-coding RNA ANRIL, antisense to the CDKN2B locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms.,Despite the identification of multiple isoforms of ANRIL, expression of certain transcripts has been found to be tissue-specific and the characterisation of ANRIL transcripts remains incomplete.,Several functions have been associated with ANRIL.,In our judgement, studies on ANRIL functionality are premature pending a more complete appreciation of the profusion of isoforms.,We found differential expression of ANRIL exons, which indicates that multiple isoforms exist in melanoma cells.,In addition to linear isoforms, we identified circular forms of ANRIL (circANRIL).,Further characterisation of circANRIL in two patient-derived metastatic melanoma cell lines (NZM7 and NZM37) revealed the existence of a rich assortment of circular isoforms.,Moreover, in the two melanoma cell lines investigated, the complements of circANRIL isoforms were almost completely different.,Novel exons were also discovered.,We also found the family of linear ANRIL was enriched in the nucleus, whilst the circular isoforms were enriched in the cytoplasm and they differed markedly in stability.,With respect to the variable processing of circANRIL species, bioinformatic analysis indicated that intronic Arthrobacter luteus (Alu) restriction endonuclease inverted repeats and exon skipping were not involved in selection of back-spliced exon junctions.,Based on our findings, we hypothesise that “ANRIL” has wholly distinct dual sets of functions in melanoma.,This reveals the dynamic nature of the locus and constitutes a basis for investigating the functions of ANRIL in melanoma.

Cancer research continues to highlight the extensive genetic diversity that exists both between and within tumors.,This intrinsic heterogeneity poses one of the central challenges to predicting patient clinical outcome and the personalization of treatments.,Despite progress in some individual tumor types, it is not yet possible to prospectively, accurately classify patients by expected survival.,One hypothesis proposed to explain this is that the prognostic classifiers developed to date are insufficiently sensitive and specific; however it is also possible that patients are not equally easy to classify by any given biomarker.,We demonstrate in a cohort of 45 AJCC stage III melanoma patients that clinico-pathologic biomarkers can identify those patients that are most likely to be misclassified by a molecular biomarker.,The process of modelling the classifiability of patients was then replicated in a cohort of 49 stage II breast cancer patients and 53 stage III colon cancer patients.,A multi-step procedure incorporating this information not only improved classification accuracy but also indicated the specific clinical attributes that had made classification problematic in each cohort.,These findings show that, even when cohorts are of moderate size, including features that explain the patient-specific performance of a prognostic biomarker in a classification framework can improve the modelling and estimation of survival.

Intravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720.,Initially, melanoma cells respond to PLX4720, but rapid reactivation of ERK/MAPK is observed in areas of high stromal density.,This is linked to “paradoxical” activation of melanoma-associated fibroblasts by PLX4720 and the promotion of matrix production and remodeling leading to elevated integrin β1/FAK/Src signaling in melanoma cells.,Fibronectin-rich matrices with 3-12 kPa elastic modulus are sufficient to provide PLX4720 tolerance.,Co-inhibition of BRAF and FAK abolished ERK reactivation and led to more effective control of BRAF-mutant melanoma.,We propose that paradoxically activated MAFs provide a “safe haven” for melanoma cells to tolerate BRAF inhibition.,•BRAF mutant melanoma cells respond to PLX4720 heterogeneously in vivo•BRAF inhibition activates MAFs, leading to FAK-dependent melanoma survival signaling•ECM-derived signals can support residual disease•BRAF and FAK inhibition synergize in pre-clinical models,BRAF mutant melanoma cells respond to PLX4720 heterogeneously in vivo,BRAF inhibition activates MAFs, leading to FAK-dependent melanoma survival signaling,ECM-derived signals can support residual disease,BRAF and FAK inhibition synergize in pre-clinical models,Hirata et al. show that the BRAF inhibitor PLX4720 promotes melanoma-associated fibroblasts in BRAF-mutant melanomas to produce and remodel matrix, leading to integrin β1-FAK-Src signaling and reactivation of ERK and MAPK in melanoma cells.,Co-inhibition of BRAF and FAK blocks ERK reactivation.

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

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

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

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

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear1,2.,The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event3.,To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a V600EBRAF mouse model.,In mice expressing V600EBRAF in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden.,We show that sunscreen (UVA superior: UVB SPF50) delayed the onset of UVR-driven melanoma, but only provided partial protection.,The UVR-exposed tumours presented increased numbers of single nucleotide variants (SNVs) and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in ~40% of cases.,TP53 is an accepted UVR target in non-melanoma skin cancer, but is not thought to play a major role in melanoma4.,However, we show that mutant Trp53 accelerated V600EBRAF-driven melanomagenesis and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma.,Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans5.,We identify TP53/Trp53 as a UVR-target gene that cooperates with V600EBRAF to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis.,Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

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

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

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

Sebaceous carcinoma is an aggressive adnexal skin tumor with a predilection for the eyelids and sebaceous glands of the head and neck.,A 73 year-old man presented with confusion and was found to have widely disseminated sebaceous carcinoma with metastases to brain, lungs, liver, bowel, lymph nodes, and bone.,Following initial treatment of the brain metastases with surgery he received post-operative radiosurgery.,He then began systemic immunotherapy with pembrolizumab.,After 6 months, he developed a near complete response to therapy by irRECIST and RECIST v.1.1.,The response was associated with circulating CD8+ T cells with central memory (CM) and effector memory (EM) phenotype and mature CD16 + CD57+ NK cells.,During treatment the patient developed adrenal insufficiency requiring high-dose systemic corticosteroids and later adrenal replacement therapy.,After 12-months of follow-up he showed imaging evidence of progression in liver, mediastinum, and abdominal lymph nodes.,Given persistent, strong PD-L1 expression he resumed pembrolizumab therapy and showed radiographic evidence of an ongoing response to therapy.,This is the first report describing objective clinical and radiographic responses following immunotherapy for widely metastatic sebaceous carcinoma.,The dramatic therapeutic response to pembrolizumab was associated with peripheral blood circulating memory T cells and mature Natural Killer cells after 6 months (24 weeks) of therapy.,This report supports prospective clinical trials of anti-PD1 checkpoint blockade for metastatic sebaceous carcinoma.,The online version of this article (10.1186/s40425-018-0357-3) contains supplementary material, which is available to authorized users.

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

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

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

Promising antitumor activities of nivolumab, a fully humanized IgG4 inhibitor antibody against the programmed death‐1 protein, were suggested in previous phase 1 studies.,The present phase 2, single‐arm study (JAPIC‐CTI #111681) evaluated the antitumor activities of nivolumab and explored its predictive correlates in advanced melanoma patients at 11 sites in Japan.,Intravenous nivolumab 2 mg/kg was given repeatedly at 3‐week intervals to 35 of 37 patients enrolled from December 2011 to May 2012 until they experienced unacceptable toxicity, disease progression, or complete response.,Primary endpoint was objective response rate.,Serum levels of immune modulators were assessed at multiple time points.,As of 21 October 2014, median response duration, median progression‐free survival, and median overall survival were 463 days, 169 days, and 18.0 months, respectively.,The overall response rate and 1‐ and 2‐year survival rates were 28.6%, 54.3%, and 42.9%, respectively.,Thirteen patients remained alive at the end of the observation period and no deaths were drug related.,Grade 3-4 drug‐related adverse events were observed in 31.4% of patients.,Pretreatment serum interferon‐γ, and interleukin‐6 and ‐10 levels were significantly higher in the patients with objective tumor responses than in those with tumor progression.,In conclusion, giving repeated i.v. nivolumab had potent and durable antitumor effects and a manageable safety profile in advanced melanoma patients, strongly suggesting the usefulness of nivolumab for advanced melanoma and the usefulness of pretreatment serum cytokine profiles as correlates for predicting treatment efficacy.

Limited adjuvant treatment options exist for patients with high-risk surgically resected melanoma.,This first-in-human study investigated the safety, tolerability and immunologic correlates of Melanoma GVAX, a lethally irradiated granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting allogeneic whole-cell melanoma vaccine, administered in the adjuvant setting.,Patients with stage IIB-IV melanoma were enrolled following complete surgical resection.,Melanoma GVAX was administered intradermally once every 28 days for four cycles, at 5E7 cells/cycle (n = 3), 2E8 cells/cycle (n = 9), or 2E8 cells/cycle preceded by cyclophosphamide 200 mg/m2 to deplete T regulatory cells (Tregs; n = 8).,Blood was collected before each vaccination and at 4 and 6 months after treatment initiation for immunologic studies.,Vaccine injection site biopsies and additional blood samples were obtained 2 days after the 1st and 4th vaccines.,Among 20 treated patients, 18 completed 4 vaccinations.,Minimal treatment-related toxicity was observed.,One patient developed vitiligo and patches of white hair during the treatment and follow-up period.,Vaccine site biopsies demonstrated complex inflammatory infiltrates, including significant increases in eosinophils and PD-1+ lymphocytes from cycle 1 to cycle 4 (P < 0.05).,Serum GM-CSF concentrations increased significantly in a dose-dependent manner 48 h after vaccination (P = 0.0086), accompanied by increased numbers of activated circulating monocytes (P < 0.0001) and decreased percentages of myeloid-derived suppressor cells among monocytes (CD14+ , CD11b+ , HLA-DR low or negative; P = 0.002).,Cyclophosphamide did not affect numbers of circulating Tregs.,No significant changes in anti-melanoma immunity were observed in peripheral T cells by interferon-gamma ELIPSOT, or immunoglobulins by serum Western blotting.,Melanoma GVAX was safe and tolerable in the adjuvant setting.,Pharmacodynamic testing revealed complex vaccine site immune infiltrates and an immune-reactive profile in circulating monocytic cell subsets.,These findings support the optimization of Melanoma GVAX with additional monocyte and dendritic cell activators, and the potential development of combinatorial treatment regimens with synergistic agents.,Trial registration: NCT01435499,The online version of this article (doi:10.1186/s12967-015-0572-3) contains supplementary material, which is available to authorized users.

The skin’s tendency to sunburn rather than tan is a major risk factor for skin cancer.,Here we report a large genome-wide association study of ease of skin tanning in 176,678 subjects of European ancestry.,We identify significant association with tanning ability at 20 loci.,We confirm previously identified associations at six of these loci, and report 14 novel loci, of which ten have never been associated with pigmentation-related phenotypes.,Our results also suggest that variants at the AHR/AGR3 locus, previously associated with cutaneous malignant melanoma the underlying mechanism of which is poorly understood, might act on disease risk through modulation of tanning ability.,The skin’s tanning response to sun exposure shows great interindividual variability.,Here, Visconti et al. perform a genome-wide association study for ease of skin tanning and identify 20 genetic loci, ten of which had not previously been associated with pigmentation-related traits.

Cutaneous squamous cell carcinoma represents the second most common cutaneous malignancy, affecting 7-11% of Caucasians in the United States.,The genetic determinants of susceptibility to cutaneous squamous cell carcinoma remain largely unknown.,Here we report the results of a two-stage genome-wide association study of cutaneous squamous cell carcinoma, totalling 7,404 cases and 292,076 controls.,Eleven loci reached genome-wide significance (P<5 × 10−8) including seven previously confirmed pigmentation-related loci: MC1R, ASIP, TYR, SLC45A2, OCA2, IRF4 and BNC2.,We identify an additional four susceptibility loci: 11q23.3 CADM1, a metastasis suppressor gene involved in modifying tumour interaction with cell-mediated immunity; 2p22.3; 7p21.1 AHR, the dioxin receptor involved in anti-apoptotic pathways and melanoma progression; and 9q34.3 SEC16A, a putative oncogene with roles in secretion and cellular proliferation.,These susceptibility loci provide deeper insight into the pathogenesis of squamous cell carcinoma.,Cutaneous squamous cell carcinoma is the second most common type of skin cancer.,In this genome-wide association study, which includes over 7,000 cases, the authors identify 4 new susceptibility loci for this cancer and also provide independent replication of 9 previously reported susceptibility loci.

The current study defines a fibroblast-derived niche that facilitates the therapeutic escape of melanoma cells from BRAF inhibition.,Vemurafenib treatment led to the release of TGF-β from the melanoma cells that increased the differentiation state of the fibroblasts; an affect associated with fibronectin deposition, increase in α-smooth muscle actin (α-SMA) expression and the release of neuregulin (NRG).,At the same time, vemurafenib directly activated the fibroblasts through paradoxical stimulation of the MAPK pathway, causing them to secrete hepatocyte growth factor (HGF).,Treatment with the BRAF/MEK inhibitor combination reversed the release of HGF.,Adhesion of melanoma cells to fibronectin was critical in amplifying the fibroblast-derived NRG and HGF-mediated PI3K/AKT survival signaling in the melanoma cells following BRAF inhibition.,In co-culture studies, combination treatment with inhibitors of BRAF/MET/HER kinase was ineffective at reversing the fibroblast-mediated therapeutic escape from BRAF inhibition.,Instead, it was noted that combined BRAF/PI3K inhibition overcame fibroblast-mediated drug resistance in vitro and was associated with enhanced anti-tumor effects in an in vivo xenograft model.,Thus, we show melanoma cells and fibroblasts remodel their microenvironment in response to BRAF inhibition and that these adaptations allow tumor cells to evade therapy through increased PI3K/AKT survival signaling.

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