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

Background.,There are several circulatory biomarkers that are involved in forecasting the clinical outcome of cutaneous melanoma.,Serum/plasma vitamin D status is one of the markers intensively studied in this type of cutaneous cancer.,The combination of validated serum biomarkers (like LDH) with new biomarkers such as IL-8, angiogenic factor, and vitamin D is still at the dawn of research.,Hence, we are aiming to establish the predictive power of inflammatory biomarkers, such as IL-8, and metabolic ones, such as vitamin D.,These candidate biomarkers are intended to aid classical biomarkers, such as LDH, in the prognosis of cutaneous melanoma.,Methods.,Serum vitamin D and IL-8 were quantified in melanoma patients and in matching healthy controls.,Results.,Median serum vitamin D concentrations were significantly lower (p = 0.003) in melanoma patients as compared to healthy control subjects, while around 65% of the investigated patients have proven a severe circulatory deficiency of this vitamin.,IL-8 was found increased (p = 0.001) in melanoma patients as compared to controls.,Conclusion.,Upregulation of proangiogenic factors associated with vitamin D deficiency can prove to be potent future biomarkers candidates, enhancing the predictive power of classical LDH.

Previous studies suggested a protective effect of vitamin D against skin cancer development.,However, epidemiologic studies on orally taken vitamin D and risk of skin cancer (basal cell carcinoma [BCC], squamous cell carcinoma [SCC], and melanoma) are few.,We prospectively evaluated whether total, dietary and supplemental vitamin D intake were associated with skin cancer risk based on 63,760 women in the Nurses' Health Study (1984-2010) and 41,530 men in the Health Professionals Follow-up Study (1986-2010).,Dietary information on vitamin D intake was assessed every 2 to 4 years during the follow-up and cumulative averaged intake was used.,We used Cox proportional hazard models to compute the hazard ratios (HR) and 95% confidence intervals (CI).,Pooled HR of cohort-specific results were calculated using a random-effects model.,During the follow-up, we documented 20,840 BCC, 2,329 SCC and 1,320 melanoma cases.,Vitamin D consumption was not associated with the risk of SCC or melanoma but was modestly positively associated with BCC; the pooled HRs of BCC for extreme quintiles of vitamin D intake were 1.10 (95%CI = 1.05-1.15; Ptrend = 0.05) for total vitamin D and 1.13 (95% CI = 1.07 to 1.20; Ptrend <0.01) for dietary vitamin D.,Stratified analysis according to sun exposure related factors showed similar results.,In conclusion, vitamin D intake was positively associated with risk of BCC, while null associations were found with SCC and melanoma.,Our data do not support a beneficial role of orally taken vitamin D on skin cancer carcinogenesis.

Epidemiological evidence shows that people with thicker, or higher stage, melanomas have lower vitamin D status compared to those with thinner tumours.,Evidence from experimental studies is inconsistent, but some suggest that administration of vitamin D metabolites can decrease tumour aggressiveness.,Determine the relationship between vitamin D status at diagnosis and melanoma thickness (as an indicator of prognosis), in a subtropical setting with high melanoma incidence.,We recruited 100 melanoma patients in Brisbane, Australia within days of their diagnosis.,Data on factors previously associated with melanoma risk or prognosis were collected by questionnaire and physical examination.,Serum for 25-hydroxyvitamin D3 [25(OH)D] levels was collected prior to wider excision biopsy; histological indicators of prognosis were obtained from pathology reports.,We used multivariable logistic regression models to analyse the association between Breslow thickness (≥0.75 mm compared to <0.75 mm), Clark level (2-5 compared to 1) and presence of mitoses, and vitamin D status.,Serum 25(OH)D <50 nmol/L (versus ≥50 nmol/L) was associated with a nearly four-fold increase in risk of having a thicker tumour (Adjusted OR = 3.82, 95% CI: 1.03, 14.14; p = 0.04, adjusted for age, sex, skin phototype, body mass index and season at diagnosis).,There was no significant association with Clark level or presence of mitosis.,Serum 25(OH)D levels in the highest quartile (≥69.8 nmol/L) were not associated with a more favourable prognosis.,Vitamin D deficiency at the time of melanoma diagnosis is associated with thicker tumours that are likely to have a poorer prognosis.,Ensuring vitamin D levels of 50 nmol/L or higher in this population could potentially result in 18% of melanomas having Breslow thickness of <0.75 mm rather than ≥0.75 mm.

Oncogene-driven metabolic rewiring is an adaptation to low nutrient and oxygen conditions in the tumor microenvironment that enables cancer cells of diverse origin to hyperproliferate.,Aerobic glycolysis and enhanced reliance on glutamine utilization are prime examples of such rewiring.,However, tissue of origin as well as specific genetic and epigenetic changes determines gene expression profiles underlying these metabolic alterations in specific cancers.,In melanoma, activation of the MAPK pathway driven by mutant BRAF or NRAS is a primary cause of malignant transformation.,Activity of the MAPK pathway, as well as other factors, such as HIF1α, Myc and MITF, are among those that control the balance between non-oxidative and oxidative branches of central carbon metabolism.,Here, we discuss the nature of metabolic alterations that underlie melanoma development and affect its response to therapy.

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

Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition.,Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis.,We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions.,This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling.,We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability.,Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent.,These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.,The online version of this article (10.1186/s12943-018-0868-z) contains supplementary material, which is available to authorized users.

Extensive research has demonstrated a tumor‐promoting role of increased WNT5A expression in malignant melanoma.,However, very little light has been shed upon how WNT5A expression is up‐regulated in melanoma.,A potential regulator of WNT5A expression is the pro‐inflammatory cytokine Interleukin (IL)‐6, which shares the ability of WNT5A to increase melanoma cell invasion.,Here, we investigate whether IL‐6 can promote melanoma cell motility through an increased expression of WNT5A.,We clearly demonstrate that the WNT5A‐antagonistic peptide Box5 could inhibit IL‐6‐induced melanoma cell migration and invasion.,Furthermore, IL‐6 stimulation of the human melanoma cell lines HTB63 and A375 increased the expression of WNT5A in a dose‐dependent manner.,To identify the signaling mechanism responsible for this up‐regulation, we explored the involvement of the three main signals induced by IL‐6; STAT3, Akt and ERK 1/2.,Of these, only STAT3 was activated by IL‐6 in the melanoma cell lines tested.,However, the STAT3 inhibitor S3I‐201 failed to inhibit IL‐6‐induced WNT5A up‐regulation in HTB63 and A375 cells.,Nor did STAT3 siRNA silencing affect the expression of WNT5A.,In search of an alternative signaling mechanism, we detected IL‐6‐induced activation of p38‐MAPK in HTB63 and A375 cells.,The p38‐MAPK inhibitor SB203580 abolished the IL‐6‐induced WNT5A up‐regulation and blocked IL‐6‐induced melanoma cell invasion.,The latter effect could be rescued by the addition of recombinant WNT5A.,Notably, immunoprecipitation analysis revealed that only the p38α‐MAPK isoform was activated by IL‐6, and subsequent siRNA silencing of p38α‐MAPK abolished the IL‐6‐induced up‐regulation of WNT5A.,Taken together, we demonstrate a novel link between the two melanoma pro‐metastatic agents IL‐6 and WNT5A explaining how IL‐6 can increase melanoma cell invasion and thus promote the metastatic process.,This finding provides a basis for future therapeutic intervention of melanoma progression.,We provide a novel link between the melanoma pro‐metastatic agents IL‐6 and WNT5A.WNT5A signaling plays an important role in IL‐6‐induced melanoma cell motility.IL‐6 can increase WNT5A expression by STAT3‐independent signaling in melanoma cells.IL‐6‐induced WNT5A expression is mediated through p38α‐MAPK activation.,We provide a novel link between the melanoma pro‐metastatic agents IL‐6 and WNT5A.,WNT5A signaling plays an important role in IL‐6‐induced melanoma cell motility.,IL‐6 can increase WNT5A expression by STAT3‐independent signaling in melanoma cells.,IL‐6‐induced WNT5A expression is mediated through p38α‐MAPK activation.

Mitogen activated-protein kinase pathway inhibitors (MAPKis) improve treatment outcome in patients with disseminated BRAFV600 mutant cutaneous malignant melanoma (CMM) but responses are of limited duration due to emerging resistance.,Although extensive research in mechanisms of resistance is being performed, predictive biomarkers for durable responses are still lacking.,We used miRNA qPCR to investigate if different levels of extracellular microvesicle microRNA (EV miRNA) in matched plasma samples collected from patients with metastatic IV BRAFV600 mutated CMM before, during and after therapy with MAPKis could serve as predictive biomarkers.,EV miRNAs were extracted from plasma samples from 28 patients collected before and during therapy, measured by quantitative PCR-array and correlated to therapy outcome.,Increased levels of EV let-7g-5p during treatment compared to before treatment (EV let-7g-5p_delta) were associated with better disease control with MAPKis (odds ratio 8568.4, 95% CI = 4.8-1.5e+07, P = 0.000036).,Elevated levels of EV miR-497-5p during therapy were associated with prolonged progression free survival (PFS) (hazard ratio = 0.27, 95% CI = 0.13-0.52, P <0.000061).,EV miRNAs let-7g-5p and miR-497-5p were identified as putative novel predictive biomarkers of MAPKi treatment benefit in metastatic CMM patients highlighting the potential relevance of assessing EV miRNA during and after treatment to unravel novel mechanisms of resistance.

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

Effective treatment options are limited for patients with advanced (metastatic or unresectable) melanoma who progress after immune checkpoint inhibitors and targeted therapies.,Adoptive cell therapy using tumor-infiltrating lymphocytes has demonstrated efficacy in advanced melanoma.,Lifileucel is an autologous, centrally manufactured tumor-infiltrating lymphocyte product.,We conducted a phase II open-label, single-arm, multicenter study in patients with advanced melanoma who had been previously treated with checkpoint inhibitor(s) and BRAF ± MEK targeted agents.,Lifileucel was produced from harvested tumor specimens in central Good Manufacturing Practice facilities using a streamlined 22-day process.,Patients received a nonmyeloablative lymphodepletion regimen, a single infusion of lifileucel, and up to six doses of high-dose interleukin-2.,The primary end point was investigator-assessed objective response rate (ORR) per RECIST, version 1.1.,Sixty-six patients received a mean of 3.3 prior therapies (anti-programmed death 1 [PD-1] or programmed death ligand 1 [PD-L1]: 100%; anticytotoxic T-lymphocyte-associated protein-4: 80%; BRAF ± MEK inhibitor: 23%).,The ORR was 36% (95% CI, 25 to 49), with two complete responses and 22 partial responses.,Disease control rate was 80% (95% CI, 69 to 89).,Median duration of response was not reached after 18.7-month median study follow-up (range, 0.2-34.1 months).,In the primary refractory to anti-PD-1 or PD-L1 therapy subset, the ORR and disease control rate were 41% (95% CI, 26 to 57) and 81% (95% CI, 66 to 91), respectively.,Safety profile was consistent with known adverse events associated with nonmyeloablative lymphodepletion and interleukin-2.,Lifileucel demonstrated durable responses and addresses a major unmet need in patients with metastatic melanoma with limited treatment options after approved therapy, including the primary refractory to anti-PD-1 or PD-L1 therapy subset.

Supplemental Digital Content is available in the text.,The overall survival (OS) of patients with metastatic uveal melanoma is short, the evidence for effectiveness of treatments is limited, and no consensus on the choice of treatment exists.,We aimed to advance interpretation of OS as an outcome by pooling peer-reviewed data.,The design is a systematic review and meta-analysis.,We searched PubMed from 1 January 1980, to 29 March 2017, for articles reporting patient-level survival in Kaplan-Meier or numerical form.,We digitized survival graphs, pooled individual survival times, calculated median OS by treatment modality, and compared each modality by the log-rank test and Cox regression using conventional chemotherapy (CHT) as a reference.,Individual-level data were obtained from 78 articles with 2494 patients.,The median OS across all treatment modalities was 1.07 years (range: 0.59-2.50 years).,Pooled OS reported after isolated hepatic perfusion [median OS: 1.34 years; hazard ratio (HR): 0.92, 95% confidence interval (CI): 0.87-0.97, P = 0.0040], immunoembolization (median OS: 1.63; HR: 0.97, 95% CI: 0.95-1.00, P = 0.0080), and surgery (median OS: 1.43; HR: 0.94, 95% CI: 0.92-0.96, P < 0.0001) was longer, and after checkpoint inhibitor shorter (median OS: 0.59; HR: 1.13, 95% CI: 1.06-1.20, P < 0.0001) than after CHT (median OS: 0.91 years), but subject to identifiable confounding factors.,OS following other modalities did not differ from CHT.,Reported OS was unassociated with the decade of publication, but depended on the percentage of first-line treated patients.,Our results suggest no clinically significant difference in OS by treatment modality or decade.,Most of the difference in reported OS likely is attributable to surveillance, selection, and publication bias rather than treatment-related prolongation.,Our pooled data provide benchmarks for future trials.

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging.,Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood.,The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers.,The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis.,The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches.,We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy.,We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

MicroRNA (miRNA) signatures are not only found in cancer tissue but also in blood of cancer patients.,Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool.,Using a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients.,We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set.,A hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls.,The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81).,Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis.,Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis.,MiRNA microarray data were validated by qRT-PCR.,Our study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma.

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.

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

NLRP3 inflammasome was introduced as a double-edged sword in tumorigenesis and influenced immunotherapy response by modulating host immunity.,However, a systematic assessment of the NLRP3-inflammasome-related genes across human cancers is lacking, and the predictive role of NLRP3 inflammasome in cancer immunotherapy (CIT) response remains unexplored.,Thus, in this study, we performed a pan-cancer analysis of NLRP3-inflammasome-related genes across 24 human cancers.,Out of these 24 cancers, 15 cancers had significantly different expression of NLRP3-inflammasome-related genes between normal and tumor samples.,Meanwhile, Cox regression analysis showed that the NLRP3 inflammasome score could be served as an independent prognostic factor in skin cutaneous melanoma.,Further analysis indicated that NLRP3 inflammasome may influence tumor immunity mainly by mediating tumor-infiltrating lymphocytes and macrophages, and the effect of NLRP3 inflammasome on immunity is diverse across tumor types in tumor microenvironment.,We also found that the NLRP3 inflammasome score could be a stronger predictor for immune signatures compared with tumor mutation burden (TMB) and glycolytic activity, which have been reported as immune predictors.,Furthermore, analysis of the association between NLRP3 inflammasome and CIT response using six CIT response datasets revealed the predictive value of NLRP3 inflammasome for immunotherapy response of patients in diverse cancers.,Our study illustrates the characterization of NLRP3 inflammasome in multiple cancer types and highlights its potential value as a predictive biomarker of CIT response, which can pave the way for further investigation of the prognostic and therapeutic potentials of NLRP3 inflammasome.

Cutaneous melanoma represents one of the deadliest types of skin cancer.,The prognosis strongly depends on the disease stage, thus early detection is crucial.,New therapies, including BRAF and MEK inhibitors and immunotherapies, have significantly improved the survival of patients in the last decade.,However, intrinsic and acquired resistance is still a challenge.,In this review, we discuss two major aspects that contribute to the aggressiveness of melanoma, namely, the embryonic origin of melanocytes and melanoma cells and cellular plasticity.,First, we summarize the physiological function of epidermal melanocytes and their development from precursor cells that originate from the neural crest (NC).,Next, we discuss the concepts of intratumoral heterogeneity, cellular plasticity, and phenotype switching that enable melanoma to adapt to changes in the tumor microenvironment and promote disease progression and drug resistance.,Finally, we further dissect the connection of these two aspects by focusing on the transcriptional regulators MSX1, MITF, SOX10, PAX3, and FOXD3.,These factors play a key role in NC initiation, NC cell migration, and melanocyte formation, and we discuss how they contribute to cellular plasticity and drug resistance in melanoma.

Gamma-delta (γδ) T lymphocytes are primed to potently respond to pathogens and transformed cells by recognizing a broad range of antigens.,However, adoptive immunotherapy with γδT cells has exhibited mixed treatment responses.,Better understanding of γδT cell biology and stratifying healthy donors for allogeneic adoptive therapy is clinically needed to fully realize the therapeutic potential of γδT cells.,We examine 98 blood samples from healthy donors and measure their expansion capacity after zoledronate stimulation, and test the migration and cytotoxic effector function of expanded γδT cells in 2D culture, 3D tumor spheroid and patient-derived melanoma organoid assays.,We find that γδT cell expansion capacity is independent of expansion methods, gender, age and HLA type.,Basal γδT cell levels in Peripheral blood mononuclear cell (PBMC) correlate well with their expansion, migration and cytotoxic effector capacity in vitro.,Circulating γδT cells with lower expression of PD-1, CTLA-4, Eomes, T-bet and CD69, or higher IFN-γ production expand better. γδT cells with central memory and effector memory phenotypes are significantly more abundant in good expanders.,A cut-off level of 0.82% γδT cells in PBMC stratifies good versus poor γδT cell expansion with a sensitivity of 97.78%, specificity of 90.48% and area under the curve of 0.968 in a healthy individual.,Donors with higher Vδ2 Index Score in PBMC have greater anti-tumor functions including migratory function and cytotoxicity.,Our results demonstrate that the interindividual γδT cell functions correlate with their circulating levels in healthy donors.,Examination of circulating γδT cell level may be used to select healthy donors to participate in γδT-based immunotherapies.

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.

To evaluate the results of restaging completely resected stage IIIB/C melanoma prior to start of adjuvant therapy.,One hundred twenty patients with stage IIIB or IIIC (AJCC 2009) melanoma who underwent complete surgical resection were screened for inclusion in our trial investigating adjuvant dendritic cell therapy (NCT02993315).,All patients underwent imaging to exclude local relapse or metastasis before entering the trial.,The frequency of recurrent disease within 12 weeks after resection and the method of detection were investigated.,Sixty-nine (58%) stage IIIB and 51 (43%) stage IIIC melanoma patients were screened.,Median age was 54 (range 27-79) years.,Twenty-two (18%) of 120 patients with completely resected stage IIIB/C melanoma had evidence of early recurrent disease, despite exclusion thereof by prior imaging.,Median interval between resection and detection of relapse was 7.4 (range 4.3-10.7) weeks.,Recurrence was asymptomatic in 17 (77%) patients, but metastasis was noticed by the patient or physician in 5 (23%).,Eight patients with local relapse received local treatment with curative intent, and one was treated with systemic therapy.,The remaining patients had distant metastasis, 1 of whom underwent resection of a solitary liver metastasis while 12 patients received systemic treatment.,Patients with completely resected stage IIIB/C melanoma have high risk of early recurrence before start of adjuvant therapy.,Restaging should be considered for high-risk melanoma patients before start of adjuvant therapy.

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

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

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.

Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%.,The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAFV600E.,However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments.,It is therefore critical to elucidate alternative mechanisms regulating melanoma progression.,Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition.,We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAFWT) and WM35 (BRAFV600E), with in vitro environmental stress and nutrient deprivation.,Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells.,Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo.,Our results demonstrate that, in BRAFWT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAFWT melanomas.,Implications: Expression of CXCR3 on BRAFWT melanoma cells may be a mediator of melanoma progression.

While immunotherapies are rapidly becoming mainstays of cancer treatment, significant gaps remain in our understanding of how to optimally target them, alone or in combination.,Here we describe a novel method to monitor levels of immune cells and pathways in expression data from solid tumors using pre-defined groups or modules of co-regulated immune genes.,We show that expression of an interconnected sub-network of type I interferon-stimulated genes (ISGs) in melanomas at the time of diagnosis significantly predicted patient survival, as did, to a lesser extent, sub-networks of T helper/T regulatory and NK/T Cytotoxic cell genes.,As a group, poor prognosis tumors with reduced ISG and immune gene levels exhibited significant copy number loss of the interferon gene cluster located at chromosome 9p21.3.,Our studies demonstrate a link between type I interferon action and immune cell levels in melanomas, and suggest that therapeutic approaches augmenting both activities may be most beneficial.

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury.,YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis.,However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved.,We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity.,We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling.,Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal β-catenin activation.,Lastly, we found prominent nuclear activity of GLI2, YAP and β-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC.,Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development.

Despite that basal cell carcinoma (BCC) is curative in the vast majority of cases, some patients are at high risk of recurrence and, in a few patients, lesions can progress to a point unsuitable for local therapy and prognosis is quite poor.,The aim of the present work is to review clinical and pathologic characteristics as well as classical and new treatment options for high-risk, metastatic and locally advanced BCC.,Surgery and radiotherapy remain the selected treatments for the majority of high-risk lesions.,However, some patients are located on a blurry clinical boundary between high-risk and locally advanced BCC.,Treatment of these patients is challenging and need an individualized and highly specialized approach.,The treatment of locally advanced BCC, in which surgery or radiotherapy is unfeasible, inappropriate or contraindicated, and metastatic BCC has changed with new Hedgehog pathway inhibitors of which vismodegib is the first drug approved by FDA and EMA.

Aberrant expression of miRNAs and their biogenesis factors has been frequently observed in different types of cancer.,We recently reported that expression of DICER1 is reduced in metastatic melanoma.,Nevertheless, so far very little is known about the expression pattern of other miRNA biogenesis factors in this type of malignancy.,Here, we investigated the expression pattern of DROSHA in a large set of melanocytic lesions by tissue microarray and immunohistochemistry (n = 409).,We found that nuclear expression of DROSHA is markedly reduced in the early stages of melanoma progression (P = 0.0001) and is inversely correlated with melanoma thickness (P = 0.0001), AJCC stages (P = 0.0001), and ulceration status (P = 0.002).,We also confirmed the reduced expression of nuclear DROSHA by a second specific antibody raised against a different region of the DROSHA protein.,In addition, we observed that the reduced nuclear expression of DROSHA during melanoma progression is accompanied by an increased cytoplasmic expression of this protein (P = 0.0001).,Finally, we found that expression pattern of DROSHA varies from that of DICER1 and concomitant loss of expression of both DICER1 and DROSHA confers the worse outcome for melanoma patients.,Our results demonstrate a reduced nuclear expression of DROSHA which further highlights a perturbed miRNA biogenesis pathway in melanoma.,In addition, the aberrant subcellular localization of DROSHA indicates possible deregulation in the mechanisms responsible for its proper localization in the nucleus.

MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas.,We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region.,Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222.,Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount.,To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions.,In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells.,Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns.,They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes.,Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context.,Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined.,MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF).,By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211.,Expression of this miRNA is often reduced in melanoma samples.,Here, we investigated functional roles of miR-211 by identifying and studying new target genes.,We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively.,MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion.,These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

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

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.

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

The coronavirus disease 2019 (COVID-19) pandemic has led to lockdowns for much of the world.,In Italy, all health procedures not directly related to COVID-19 were reduced or suspended, thus limiting patient access to hospitals.,Any delay in cancer treatment presents the additional risk of tumors progressing from being curable to incurable.,Specifically, melanoma survival rate strictly depends on tumor thickness, which, in turn, is a function of time.,To estimate the impact on melanoma progression caused by the reduction in dermatologic services during the COVID-19 lockdown, a retrospective observational cohort study was conducted.,This study was designed to compare the clinical and histologic characteristics of the primary melanomas removed in the first 2 months after the end of the lockdown (May-July 2020) in 12 Italian centers characterized by different COVID-19 case frequencies.,The control group was represented by the melanomas removed during the same period in the previous 3 years.,Overall, 1,124 melanomas were considered: 237 as part of the study group and 887 from the control group (average, 295), with a 20% reduction.,Breslow thickness, as well as high-risk histotypes and melanomas with vertical growth, increased for all melanomas.,Ulcerated and high mitotic index melanomas increased, particularly in northern Italy.,In Italy, the lockdown led to a significant worsening of melanoma severity, causing a staging jump, with a consequent worsening of outcomes.

The UK healthcare system, including skin cancer departments, has been profoundly affected by the COVID‐19 pandemic.,Despite service capacity and a worldwide increase in incidence, anecdotal reports suggest a decline in skin cancer diagnoses following COVID‐19.,To determine if there has been a decrease in skin cancer diagnosis in the UK in the COVID‐19 era, we analysed data from the Northern Cancer Network from 23 March 2020 to 23 June 2020 and compared it with the same period in 2019 (pre‐COVID).,In the COVID period, there was a decrease of 68.61% in skin cancer diagnoses, from 3619 to 1136 (P < 0.01).,Surprisingly, skin cancer waiting times were also reduced in the COVID period compared to the pre‐COVID period (median of 8 and 12 days, respectively; P < 0.001).,Collectively, these data highlight a statistically significant reduction in both skin cancer diagnoses and waiting times during the COVID period.

Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis.,Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states.,Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state.,Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation.,We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated.,CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival.,These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.,Phenotype switching is a form of plasticity that allows melanoma cancer cells that leave the primary tumour to invade secondary sites, to switch from an invasive to a proliferative state.,Here the authors identify EDN3, and its synthetic enzyme ECE2, as a regulator of melanoma plasticity in the microenvironment.

Live imaging and genetic studies of the initial interactions between leukocytes and transformed cells in zebrafish larvae indicate an attractant role for H2O2 and suggest that blocking these early interactions reduces expansion of transformed cell clones.,It has not previously been possible to live image the earliest interactions between the host environment and oncogene-transformed cells as they initiate formation of cancers within an organism.,Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin.,Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden.,Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages.,We show that a major component of the signal drawing inflammatory cells to oncogenic HRASG12V-transformed cells is H2O2, which is also a key damage cue responsible for recruiting neutrophils to a wound.,Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair.,These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble non-healing wounds.

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

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients.,We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified.,PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP).,The functional activity of these T lymphocytes was characterized and compared with that of TILs.,In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed.,An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC’s samples obtained in 7 cutaneous and ocular metastatic melanoma patients.,Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs.,Most MLTCs were enriched in non-terminally differentiated TEM cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137).,A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients.,In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases).,Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients.,Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol.,This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.,The online version of this article (doi:10.1007/s00262-011-1179-z) contains supplementary material, which is available to authorized users.

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

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

During embryonic development Wnt family members and bone morphogenetic proteins (BMPs) cooperatively induce epithelial-mesenchymal transition (EMT) in the neural crest.,Wnt and BMPs are reactivated during malignant transformation in melanoma.,We previously demonstrated that the BMP-antagonist noggin blocked the EMT phenotype of melanoma cells in the neural crest and malignant invasion of melanoma cells in the chick embryo; vice-versa, malignant invasion was induced in human melanocytes in vivo by pre-treatment with BMP-2.,Although there are conflicting results in the literature about the role of β-catenin for invasion of melanoma cells, we found Wnt/β-catenin signaling to be analogously important for the EMT-like phenotype of human metastatic melanoma cells in the neural crest and during invasion: β-catenin was frequently expressed at the invasive front of human primary melanomas and Wnt3a expression was inversely correlated with survival of melanoma patients.,Accordingly, cytoplasmic β-catenin levels were increased during invasion of melanoma cells in the rhombencephalon of the chick embryo.,Fibroblast derived Wnt3a reduced melanoma cell adhesion and enhanced migration, while the β-catenin inhibitor PKF115-584 increased adhesion and reduced migration in vitro and in the chick embryonic neural crest environment in vivo.,Similarly, knockdown of β-catenin impaired intradermal melanoma cell invasion and PKF115-584 efficiently reduced liver metastasis in a chick chorioallantoic membrane model.,Our observations were accompanied by specific alterations in gene expression which are linked to overall survival of melanoma patients.,We present a novel role for Wnt-signaling in neural crest like melanoma cell invasion and metastasis, stressing the crucial role of embryonic EMT-inducing neural crest signaling for the spreading of malignant melanoma.,The online version of this article (10.1186/s12943-018-0773-5) contains supplementary material, which is available to authorized users.

Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia.,Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis.,It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.,By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia.,We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis.,In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants.,The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein.,We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions.,Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.,We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.

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

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.

The identification of novel antimetastatic therapeutic targets is necessary for improved treatment of patients with acquired BRAF inhibitor‐resistant (BRAFi‐R) melanoma, in whom metastasis is a major concern.,Our present study focused on the identification of such targets to explore novel antimetastatic therapeutic options for BRAFi‐R melanoma patients.,We confirmed the development of BRAFi resistance in our BRAFi‐treated melanoma cell lines by demonstrating reduced sensitivity to BRAF inhibitors, increased ERK1/2 activity and increased WNT5A expression.,Here, we demonstrated for the first time that high secretion of interleukin‐6 (IL‐6) was associated with increased invasive migration of BRAFi‐R melanoma cells.,This finding could be readily explained by the increased expression of WNT5A in BRAFi‐R melanoma cells and the presence of an IL‐6/WNT5A positive feedback loop in parental melanoma cells.,Surprisingly, however, we found that the IL‐6/WNT5A positive feedback loop present in parental melanoma cells was lost during the development of acquired BRAFi resistance, meaning that IL‐6 and WNT5A signalling were independent events in BRAFi‐R melanoma cells.,Despite the absence of an IL‐6/WNT5A loop, we found that both an IL‐6 blocking antibody and the WNT5A antagonist Box5 alone impaired the elevated invasive migration of BRAFi‐R melanoma cells, but combined use of the two was more effective.,This impaired invasive migration of BRAFi‐R melanoma cells correlated well with the reduction in Cdc42‐GTPase activity and alterations of the actin cytoskeleton in these cells.,In summary, our novel identification of IL‐6 as a key independent promoter of the invasive migration of BRAFi‐R melanoma cells stresses that a combination of a blocking IL‐6 antibody and administration of the WNT5A antagonist Box5 might be an attractive antimetastatic approach for future treatment of BRAFi‐R melanoma patients.

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

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.

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

Drastically elevated glycolytic activity is a prominent metabolic feature of cancer cells.,Until recently it was thought that tumor cells shift their entire energy production from oxidative phosphorylation (OXPHOS) to glycolysis.,However, new evidence indicates that many cancer cells still have functional OXPHOS, despite their increased reliance on glycolysis.,Growing pre-clinical and clinical evidence suggests that targeting mitochondrial metabolism has anti-cancer effects.,Here, we analyzed mitochondrial respiration and the amount and activity of OXPHOS complexes in four melanoma cell lines and normal human dermal fibroblasts (HDFs) by Seahorse real-time cell metabolic analysis, immunoblotting, and spectrophotometry.,We also tested three clinically approved antibiotics, one anti-parasitic drug (pyrvinium pamoate), and a novel anti-cancer agent (ONC212) for effects on mitochondrial respiration and proliferation of melanoma cells and HDFs.,We found that three of the four melanoma cell lines have elevated glycolysis as well as OXPHOS, but contain dysfunctional mitochondria.,The antibiotics produced different effects on the melanoma cells and HDFs.,The anti-parasitic drug strongly inhibited respiration and proliferation of both the melanoma cells and HDFs.,ONC212 reduced respiration in melanoma cells and HDFs, and inhibited the proliferation of melanoma cells.,Our findings highlight ONC212 as a promising drug for targeting mitochondrial respiration in cancer.

Inherited CDKN2A mutation is a strong risk factor for cutaneous melanoma.,Moreover, carriers have been found to have poor melanoma-specific survival.,In this study, responses to novel immunotherapy agents in CDKN2A mutation carriers with metastatic melanoma were evaluated.,CDKN2A mutation carriers that have developed metastatic melanoma and undergone immunotherapy treatments were identified among carriers enrolled in follow-up studies for familial melanoma.,The carriers’ responses were compared with responses reported in phase III clinical trials for CTLA-4 and PD-1 inhibitors.,From publicly available data sets, melanomas with somatic CDKN2A mutation were analysed for association with tumour mutational load.,Eleven of 19 carriers (58%) responded to the therapy, a significantly higher frequency than observed in clinical trials (p=0.03, binomial test against an expected rate of 37%).,Further, 6 of the 19 carriers (32%) had complete response, a significantly higher frequency than observed in clinical trials (p=0.01, binomial test against an expected rate of 7%).,In 118 melanomas with somatic CDKN2A mutations, significantly higher total numbers of mutations were observed compared with 761 melanomas without CDKN2A mutation (Wilcoxon test, p<0.001).,Patients with CDKN2A mutated melanoma may have improved immunotherapy responses due to increased tumour mutational load, resulting in more neoantigens and stronger antitumorous immune responses.

How targeted therapies and immunotherapies shape tumours and thereby influence subsequent therapeutic responses is poorly understood.,Here, we show in melanoma patients and mouse models that when tumours relapse following targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies.,We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumour microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response.,Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumours to immunotherapy.,Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which is not only reactivated, but also exhibits an increased transcriptional output that drives immune evasion.,Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.

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.

There is growing evidence that tripartite motif-containing protein 44 (TRIM44) plays crucial role in tumor development.,However, the underlying mechanism of this deubiquitinating enzyme remains unclear.,Large clinical samples were used to detect TRIM44 expression and its associations with clinicopathological features and prognosis.,Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to elucidate the function and underlying mechanisms of TRIM44 induced tumor progression.,Co-immunoprecipitation (Co-IP) assays and mass spectrometric analyses were applied to verify the interacting proteins of TRIM44.,We found that TRIM44 was commonly amplified in melanoma tissues compared with paratumoral tissues.,TRIM44 expression also positively correlated with more aggressive clinicopathological features, such as Breslow depth (p = 0.025), distant metastasis (p = 0.012), and TNM stage (p = 0.002).,Importantly, we found that TRIM44 was an independent indicator of prognosis for melanoma patients.,Functionally, overexpression of TRIM44 facilitated cell invasion, migration, apoptosis resistance and proliferation in vitro, and promoted lung metastasis and tumorigenic ability in vivo.,Importantly, high level of TRIM44 induced melanoma cell epithelial-mesenchymal transition (EMT), which is one of the most important mechanisms for the promotion of tumor metastasis.,Mechanistically, high levels of TRIM44 increased the levels of p-AKT (T308) and p-mTOR (S2448), and a specific AKT inhibitor inhibited TRIM44-induced tumor progression.,Co-IP assays and mass spectrometric analyses indicated that TRIM44 overexpression induces cell EMT through activating AKT/mTOR pathway via directly binding and stabilizing TOLL-like receptor 4 (TLR4), and TLR4 interference impeded TRIM44 induced tumor progression.,Moreover, we demonstrated that TRIM44 is the target of miR-26b-5p, which is significantly downregulated in melanoma tissues and may be responsible for the overexpression of TRIM44.,TRIM44, regulated by miR-26b-5p, promotes melanoma progression by stabilizing TLR4, which then activates the AKT/mTOR pathway.,TRIM44 shows promise as a prognostic predictor and a therapeutic target for melanoma patients.,The online version of this article (10.1186/s13046-019-1138-7) contains supplementary material, which is available to authorized users.

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

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

Cutaneous melanoma 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.

Organ transplant recipients (OTRs) have a 100‐fold increased risk of cutaneous squamous cell carcinoma (cSCC).,We prospectively evaluated the association between β genus human papillomaviruses (βPV) and keratinocyte carcinoma in OTRs.,Two OTR cohorts without cSCC were assembled: cohort 1 was transplanted in 2003‐2006 (n = 274) and cohort 2 was transplanted in 1986‐2002 (n = 352).,Participants were followed until death or cessation of follow‐up in 2016. βPV infection was assessed in eyebrow hair by using polymerase chain reaction-based methods. βPV IgG seroresponses were determined with multiplex serology.,A competing risk model with delayed entry was used to estimate cumulative incidence of histologically proven cSCC and the effect of βPV by using a multivariable Cox regression model.,Results are reported as adjusted hazard ratios (HRs).,OTRs with 5 or more different βPV types in eyebrow hair had 1.7 times the risk of cSCC vs OTRs with 0 to 4 different types (HR 1.7, 95% confidence interval 1.1‐2.6).,A similar risk was seen with high βPV loads (HR 1.8, 95% confidence interval 1.2‐2.8).,No significant associations were seen between serum antibodies and cSCC or between βPV and basal cell carcinoma.,The diversity and load of βPV types in eyebrow hair are associated with cSCC risk in OTRs, providing evidence that βPV is associated with cSCC carcinogenesis and may present a target for future preventive strategies.,In two cohorts of organ transplant recipients, those with five and more different beta‐genus human papillomavirus types and high virus loads in eyebrow hairs subsequently develop significantly more cutaneous squamous cell carcinomas than others, suggesting that these virus types may increase the risk of cutaneous squamous cell carcinoma in these patients.

The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established.,However, far less is known about the E6 protein from the β human papillomavirus genus (β-HPVs).,Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance.,Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage.,Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair.,Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB).,Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation.,We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation.,We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected.,Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA.,Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest.,In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.

Cancer cell phenotype largely depends on oxygen availability.,The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue.,Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2).,A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines.,Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner.,Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells.,Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner.,In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

Melanoma arises from neural crest‐derived melanocytes which reside mostly in the skin in an adult organism.,Epithelial-mesenchymal transition (EMT) is a tumorigenic programme through which cells acquire mesenchymal, more pro‐oncogenic phenotype.,The reversible phenotype switching is an event still not completely understood in melanoma.,The EMT features and increased invasiveness are associated with lower levels of the pivotal lineage identity maintaining and melanoma‐specific transcription factor MITF (microphthalmia‐associated transcription factor), whereas increased proliferation is linked to higher MITF levels.,However, the precise role of MITF in phenotype switching is still loosely characterized.,To exclude the changes occurring upstream of MITF during MITF regulation in vivo, we employed a model whereby MITF expression was inducibly regulated by shRNA in melanoma cell lines.,We found that the decrease in MITF caused only moderate attenuation of proliferation of the whole cell line population.,Proliferation was decreased in five of 15 isolated clones, in three of them profoundly.,Reduction in MITF levels alone did not generally produce EMT‐like characteristics.,The stem cell marker levels also did not change appreciably, only a sharp increase in SOX2 accompanied MITF down‐regulation.,Oppositely, the downstream differentiation markers and the MITF transcriptional targets melastatin and tyrosinase were profoundly decreased, as well as the downstream target livin.,Surprisingly, after the MITF decline, invasiveness was not appreciably affected, independently of proliferation.,The results suggest that low levels of MITF may still maintain relatively high proliferation and might reflect, rather than cause, the EMT‐like changes occurring in melanoma.

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

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

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

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

The progressive infiltration of immune cells is associated with the progression of melanoma.,Specifically, Th17 cells in melanoma microenvironment have both antitumor and protumor effects.,It is now necessary to understand the contradictory data associated with how Th17 cells play a role in melanoma.,This review will summarize the current knowledge regarding the potential mechanisms that may be involved in the effects of Th17 cells in melanoma progression.,Currently, since adoptive transferring Th17 cells has been successful in eradicating melanoma in mice, it offers promise for next-generation adoptive cell transfer, as ex vivo expanded stemness-like memory Th17 cells which are induced by distinct cytokines or pharmacologic reagents may be infused into melanoma patients to potentiate treatment outcome.

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

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

Ipilimumab, a fully human monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4, has demonstrated an improvement in overall survival in two phase III trials of patients with advanced melanoma.,The primary objective of the current trial was to prospectively explore candidate biomarkers from the tumor microenvironment for associations with clinical response to ipilimumab.,In this randomized, double-blind, phase II biomarker study (ClinicalTrials.gov NCT00261365), 82 pretreated or treatment-naïve patients with unresectable stage III/IV melanoma were induced with 3 or 10 mg/kg ipilimumab every 3 weeks for 4 doses; at Week 24, patients could receive maintenance doses every 12 weeks.,Efficacy was evaluated per modified World Health Organization response criteria and safety was assessed continuously.,Candidate biomarkers were evaluated in tumor biopsies collected pretreatment and 24 to 72 hours after the second ipilimumab dose.,Polymorphisms in immune-related genes were also evaluated.,Objective response rate, response patterns, and safety were consistent with previous trials of ipilimumab in melanoma.,No associations between genetic polymorphisms and clinical activity were observed.,Immunohistochemistry and histology on tumor biopsies revealed significant associations between clinical activity and high baseline expression of FoxP3 (p = 0.014) and indoleamine 2,3-dioxygenase (p = 0.012), and between clinical activity and increase in tumor-infiltrating lymphocytes (TILs) between baseline and 3 weeks after start of treatment (p = 0.005).,Microarray analysis of mRNA from tumor samples taken pretreatment and post-treatment demonstrated significant increases in expression of several immune-related genes, and decreases in expression of genes implicated in cancer and melanoma.,Baseline expression of immune-related tumor biomarkers and a post-treatment increase in TILs may be positively associated with ipilimumab clinical activity.,The observed pharmacodynamic changes in gene expression warrant further analysis to determine whether treatment-emergent changes in gene expression may be associated with clinical efficacy.,Further studies are required to determine the predictive value of these and other potential biomarkers associated with clinical response to ipilimumab.

Melanoma accounts for 1.7% of global cancer diagnoses and is the fifth most common cancer in the US.,Melanoma incidence is rising in developed, predominantly fair-skinned countries, growing over 320% in the US since 1975.,However, US mortality has fallen almost 30% over the past decade with the approval of 10 new targeted or immunotherapy agents since 2011.,Mutations in the signaling-protein BRAF, present in half of cases, are targeted with oral BRAF/MEK inhibitor combinations, while checkpoint inhibitors are used to restore immunosurveillance likely inactivated by UV radiation.,Although the overall 5-year survival has risen to 93.3% in the US, survival for stage IV disease remains only 29.8%.,Melanoma is most common in white, older men, with an average age of diagnosis of 65.,Outdoor UV exposure without protection is the main risk factor, although indoor tanning beds, immunosuppression, family history and rare congenital diseases, moles, and obesity contribute to the disease.,Primary prevention initiatives in Australia implemented since 1988, such as education on sun-protection, have increased sun-screen usage and curbed melanoma incidence, which peaked in Australia in 2005.,In the US, melanoma incidence is not projected to peak until 2022-2026.,Fewer than 40% of Americans report practicing adequate protection (sun avoidance from 10 a.m.-4 p.m. and regular application of broad-spectrum sunscreen with an SPF > 30).,A 2-4-fold return on investment is predicted for a US sun-protection education initiative.,Lesion-directed skin screening programs, especially for those at risk, have also cost-efficiently reduced melanoma mortality.

Cutaneous melanoma is the solid neoplasia with the highest growing incidence among all tumors.,It spreads predictably to the lymphatic vessels and sentinel lymph node, and when the latter is affected the prognosis worsens dramatically.,Sentinel lymph node biopsy is considered when thickness of the primary tumor exceeds 1mm and/or when there are adverse features in thinner melanomas.,When there is nodal metastasis, current evidence in the literature recommends complete lymphadenectomy, although this procedure has its intrinsic risks (i.e., lymphedema and cellulitis), and there are no published clinical trials proving additional overall survival benefits.,The current in-depth literature review thus aims to identify patients that will benefit most from the procedure, including those with the highest likelihood of presenting additional affected lymph nodes in the same nodal basin.,The authors also discuss techniques for identification of the sentinel lymph node, false-negative rates, and predictive models for lymph node involvement.,In conclusion, complete elective lymphadenectomy should always be discussed on a case-by-case basis when metastases are detected in the sentinel lymph node.

Uveal melanoma (UM), the most common ocular malignancy, is characterized by GNAQ/11 mutations.,Hippo/YAP and Ras/mitogen-activated protein kinase (MAPK) emerge as two important signaling pathways downstream of G protein alpha subunits of the Q class (GαQ/11)-mediated transformation, although whether and how they contribute to UM genesis in vivo remain unclear.,Here, we adapt an adeno-associated virus (AAV)-based ocular injection method to directly deliver Cre recombinase into the mouse uveal tract and demonstrate that Lats1/2 kinases suppress UM formation specifically in uveal melanocytes.,We find that genetic activation of YAP, but not Kras, is sufficient to initiate UM.,We show that YAP/TAZ activation induced by Lats1/2 deletion cooperates with Kras to promote UM progression via downstream transcriptional reinforcement.,Furthermore, dual inhibition of YAP/TAZ and Ras/MAPK synergizes to suppress oncogenic growth of human UM cells.,Our data highlight the functional significance of Lats-YAP/TAZ in UM initiation and progression in vivo and suggest combination inhibition of YAP/TAZ and Ras/MAPK as a new therapeutic strategy for UM.

Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics.,In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/β-catenin-mediated transcriptional program.,We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression.,In vivo efficacy of ICG-001 was evaluated in a UM xenograft model.,ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration.,Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition.,Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling.,Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC.,Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome.,Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice.,Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell.,Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.

Clinical translation of therapeutic nuclear acid, particularly those targeting tumor progression, has been hampered by the intrinsic weaknesses of nuclear acid therapeutic including poor systemic stability, rapid clearance, low membrane permeability and lack of targeting ability.,Small nuclear acid engineered into carrier-free nanodrugs with structural stability and disease targeting may be viable to overcome pharmaceutical obstacles of nuclear acid.,A general method through a mild and simple chemistry was established to convert therapeutic miRNA into an infinite Auric-sulfhydryl coordination supramolecular miRNA termed IacsRNA with near-spherical nanostructure, high colloid as well as anti-hydrolysis stability and low macrophage uptakes.,IacsRNA presented the increased half-life period in circulation and accumulation at tumor sites in comparison to normal miRNA.,Moreover, Iacs-miR-30c showed no toxicity of viscera and sanguis system in the 5-time injection dosage of the treatment.,More importantly, Iacs-miR-30c potently suppressed the Wnt signaling pathway in vitro and in vivo, and effectively sensitized both potency of 5-Fu in PDX model of colon cancer and Anti-PD1 in B16F10 homograft model of melanoma.,Collectively, this work amply confirmed the design of IacsRNA as a general and viable strategy of nano-pharmaceutic to concert flimsy therapeutic miRNA into potential drugs.,Considering from a broader perspective, the miRNA-initiated infinite coordination self-assembly strategy has distinct advantages in resurrecting nuclear acid therapeutics, probably bringing new inspiration to RNA-derived therapeutics of a great variety of human diseases including cancer.,The online version contains supplementary material available at 10.1186/s12951-021-01212-9.

Melanoma treatment has been revolutionized by antibody-based immunotherapies.,IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells.,Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models.,Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity.,JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ.,Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma.,Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies.,Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions.,Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.,IFNγ secretion by CD8+ T cells is critical for immunotherapy efficacy.,In this study, the authors show that melanoma patients can become resistant to immunotherapy by acquiring chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2.

No standard treatment has been defined for metastatic uveal melanoma (mUM).,Although clinical trials testing Nivolumab/Pembrolizumab for cutaneous melanoma did not include mUM, anti PD-1 agents are commonly used for this disease.,In this prospective observational cohort single arm study, we investigated efficacy and safety of Pembrolizumab as first-line therapy for mUM.,The efficacy was evaluated in terms of progression-free survival (PFS), response rate and overall survival (OS).,Toxicity was also assessed.,Seventeen patients were enrolled.,A median of 8 cycles were administered (range 2-28).,Two patients achieved partial response (11.7%), 6 a disease stabilization (35.3%), whereas 9 (53%) had a progression.,No complete response was observed.,PFS of the overall population was 3.8 months.,PFS was 9.7 months for patients with an interval higher than 5 years from diagnosis of primary tumor to metastatic disease and 2.6 months for patients with an interval lower than 5 years [p = 0.039, HR 0.2865 (95% CI 0.0869-0.9443)].,Median OS was not reached.,The two responding patients were still on treatment with Pembrolizumab at the time of data analysis.,Survival was 12.8 months for patients with clinical benefit, while OS for progressive patients was 3.1 months.,PD-L1 expression and genomic abnormalities predictive of relapse after diagnosis of primary tumor were not associated with PFS.,Toxicity was mild, without grade 3-4 side effects.,The efficacy of Pembrolizumab does not seem particularly different when compared to other agents for mUM, but responding patients had a remarkable disease control.

Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease.,Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas.,For the majority of mucosal melanomas oncogenic driver mutations remain unknown.,In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma.,NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively.,Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples.,Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma.,RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type.,BRAF and KIT mutations were rare with frequencies below 10% each.,Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

The prognosis of patients with metastatic melanoma has substantially improved over the last years with the advent of novel treatment strategies, mainly immune checkpoint inhibitors and BRAF and MEK inhibitors.,Given the survival benefit provided in the metastatic setting and the evidence from prospective clinical trials in the early stages, these drugs have been introduced as adjuvant therapies for high-risk resected stage III disease.,Several studies have also investigated immune checkpoint inhibitors, as well as BRAF and MEK inhibitors, for neoadjuvant treatment of high-risk stage III melanoma, with preliminary evidence suggesting this could be a very promising approach in this setting.,However, even with new strategies, the risk of disease recurrence varies widely among stage III patients, and no available biomarkers for predicting disease recurrence have been established to date.,Improved risk stratification is particularly relevant in this setting to avoid unnecessary treatment for patients who have minimum risk of disease recurrence and to reduce toxicities and costs.,Research for predictive and prognostic biomarkers in this setting is ongoing to potentially shed light on the complex interplay between the tumor and the host immune system, and to further personalize treatment.,This review provides an insight into available data on circulating and tissue biomarkers, including the tumor microenvironment and associated gene signatures, and their predictive and prognostic role during neoadjuvant and adjuvant treatment for cutaneous high-risk melanoma patients.

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

The heterogeneous behavior of patients with melanoma makes prognostication challenging.,To address this, a gene expression profile (GEP) test to predict metastatic risk was previously developed.,This study evaluates the GEP’s prognostic accuracy in an independent cohort of cutaneous melanoma patients.,This multi-center study analyzed primary melanoma tumors from 523 patients, using the GEP to classify patients as Class 1 (low risk) and Class 2 (high risk).,Molecular classification was correlated to clinical outcome and assessed along with AJCC v7 staging criteria.,Primary endpoints were recurrence-free (RFS) and distant metastasis-free (DMFS) survival.,The 5-year RFS rates for Class 1 and Class 2 were 88% and 52%, respectively, and DMFS rates were 93% versus 60%, respectively (P < 0.001).,The GEP was a significant predictor of RFS and DMFS in univariate analysis (hazard ratio [HR] = 5.4 and 6.6, respectively, P < 0.001 for each), along with Breslow thickness, ulceration, mitotic rate, and sentinel lymph node (SLN) status (P < 0.001 for each).,GEP, tumor thickness and SLN status were significant predictors of RFS and DMFS in a multivariate model that also included ulceration and mitotic rate (RFS HR = 2.1, 1.2, and 2.5, respectively, P < 0.001 for each; and DMFS HR = 2.7, 1.3 and 3.0, respectively, P < 0.01 for each).,The GEP test is an objective predictor of metastatic risk and provides additional independent prognostic information to traditional staging to help estimate an individual’s risk for recurrence.,The assay identified 70% of stage I and II patients who ultimately developed distant metastasis.,Its role in consideration of patients for adjuvant therapy should be examined prospectively.,The online version of this article (10.1186/s12885-018-4016-3) contains supplementary material, which is available to authorized users.

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

We characterized the mutational landscape of human skin cutaneous melanoma (SKCM) using data obtained from The Cancer Genome Atlas (TCGA) project.,We analyzed next-generation sequencing data of somatic copy number alterations and somatic mutations in 303 metastatic melanomas.,We were able to confirm preeminent drivers of melanoma as well as identify new melanoma genes.,The TCGA SKCM study confirmed a dominance of somatic BRAF mutations in 50% of patients.,The mutational burden of melanoma patients is an order of magnitude higher than of other TCGA cohorts.,A multi-step filter enriched somatic mutations while accounting for recurrence, conservation, and basal rate.,Thus, this filter can serve as a paradigm for analysis of genome-wide next-generation sequencing data of large cohorts with a high mutational burden.,Analysis of TCGA melanoma data using such a multi-step filter discovered novel and statistically significant potential melanoma driver genes.,In the context of the Pan-Cancer study we report a detailed analysis of the mutational landscape of BRAF and other drivers across cancer tissues.,Integrated analysis of somatic mutations, somatic copy number alterations, low pass copy numbers, and gene expression of the melanogenesis pathway shows coordination of proliferative events by Gs-protein and cyclin signaling at a systems level.

Recently, melanoma has become the most malignant and commonly occurring skin cancer.,Melanoma is not only the major source (75%) of deaths related to skin cancer, but also it is hard to be treated by the conventional drugs.,Recent research indicated that angiogenesis is an important factor for tumor initiation, expansion, and response to therapy.,Thus, we proposed a novel multi-scale agent-based computational model that integrates the angiogenesis into tumor growth to study the response of melanoma cancer under combined drug treatment.,Our multi-scale agent-based model can simulate the melanoma tumor growth with angiogenesis under combined drug treatment.,The significant synergistic effects between drug Dox and drug Sunitinib demonstrated the clinical potential to interrupt the communication between melanoma cells and its related vasculatures.,Also, the sensitivity analysis of the model revealed that diffusivity related to the micro-vasculatures around tumor tissues closely correlated with the spread, oscillation and destruction of the tumor.,Simulation results showed that the 3D model can represent key features of melanoma growth, angiogenesis, and its related micro-environment.,The model can help cancer researchers understand the melanoma developmental mechanism.,Drug synergism analysis suggested that interrupting the communications between melanoma cells and the related vasculatures can significantly increase the drug efficacy against tumor cells.

Annual solar ultraviolet radiation (UVR) is mostly determined by latitude and altitude.,Over the last decades, increasing UVR ground levels have been observed.,Exposure to UVR is associated with a life-time risk to develop melanoma, a malign skin cancer.,Thus, we hypothesized that melanoma incidence in Austria is associated with altitude of place of living and time of diagnosis.,We investigated this hypothesis in an ecological study by district and year for Austrian melanoma incidence (1990-2010) and mortality (1970-2011) data.,As expected, incidence rates increased with altitude (about 2% per 10 m) and year (about 2%).,Additionally, melanoma incidence rates were about 50% higher in urban than in rural districts.,In contrast, mortality rates decreased with altitude (for males: 0.4% per 10 m, for women: 0.7% per 10 m, respectively).,The observed discrepancy between incidence and mortality data could partly be explained by melanoma diagnosis at earlier tumor stage in districts with higher altitude.,Possible reasons for this finding include higher awareness of patients, better diagnostic performance of medical professionals working at higher altitudes, or slower tumor growth due to protective effects of sun light-associated vitamin D synthesis.

Unprotected leisure time exposure to ultraviolet radiation from the sun or artificial tanning beds is the most important environmental risk factor for melanoma, a malignant skin cancer with increasing incidences over the past decades.,The aim of the present study was to assess the impact of skin health information provided by several sources and different publishing issues on knowledge, risk perception, and sun protective behavior of sunbathers.,A cross-sectional questionnaire survey was conducted among Austrian residents (n=563) spending leisure time outdoors in August 2010.,Print media, television, and family were perceived as the most relevant sources of information on skin health, whereas the source physician was only ranked as fourth important source.,Compared to other sources, information provided by doctors positively influenced participants' knowledge on skin risk and sun protective behavior resulting in higher scores in the knowledge test (p=0.009), higher risk perception (p<0.001), and more sun protection (p<0.001).,Regarding gender differences, internet was more often used by males as health information source, whereas females were more familiar with printed information material in general.,The results of this survey put emphasis on the demand for information provided by medical professionals in order to attain effective, long-lasting promotion of photoprotective habits.

To identify CD8+ T cell-related factors and the co-expression network in melanoma and illustrate the interactions among CD8+ T cell-related genes in the melanoma tumor microenvironment.,We obtained melanoma and paracancerous tissue mRNA matrices from TCGA-SKCM and GSE65904.,The CIBERSORT algorithm was used to assess CD8+ T cell proportions, and the “estimate” package was used to assess melanoma tumor microenvironment purity.,Weighted gene co-expression network analysis was used to identify the most related co-expression modules in TCGA-SKCM and GSE65904.,Subsequently, a co-expression network was built based on the joint results in the two cohorts.,Subsequently, we identified the core genes of the two most relevant modules of CD8+T lymphocytes according to the module correlation, and constructed the signature using ssGSEA.,Later, we compared the signature with the existing classical pathways and gene sets, and confirmed the important prognostic significance of the signature in this paper.,Nine co-expressed genes were identified as CD8+ T cell-related genes enriched in the cellular response to interferon−gamma process and antigen processing and presentation of peptide antigen.,In the low expression level group, inflammation and immune responses were weaker.,Single-cell sequencing and immunohistochemistry indicated that these nine genes were highly expressed in CD8+ T cells group.,We identified nine-gene signature, and the signature is considered as the biomarker for T lymphocyte response and clinical response to immune checkpoint inhibitors for melanoma

The ETS family modulates immune response and drug efficiency to targeted therapies, but their role in melanoma is largely unclear.,In this study, the ETS family was systematically analyzed in multiple public data sets.,Bioinformatics tools were used to characterize the function of ETV7 in melanoma.,A prognostic model was constructed using the LASSO Cox regression method.,We found that ETV7 was the only differentially expressed gene with significant prognostic relevance in melanoma.,Enrichment analysis of seven independent data sets indicated ETV7 participation in various immune-related pathways.,ETV7 particularly showed a strong positive correlation with CD8+ T cell infiltration.,The prognostic model based on ETV7 and its hub genes showed a relatively good predictive value in training and testing data sets.,Thus, ETV7 can potentially regulate the immune microenvironment in melanoma.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear.,We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification.,HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC).,Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation.,Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification.,Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX.,These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells.,Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF.,The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

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

Lysyl oxidase-like 3 (LOXL3) is a member of the lysyl oxidase family comprising multifunctional enzymes with depicted roles in extracellular matrix maturation, tumorigenesis, and metastasis.,In silico expression analyses followed by experimental validation in a comprehensive cohort of human cell lines revealed a significant upregulation of LOXL3 in human melanoma.,We show that LOXL3 silencing impairs cell proliferation and triggers apoptosis in various melanoma cell lines.,Further supporting a pro-oncogenic role in melanoma, LOXL3 favors tumor growth in vivo and cooperates with oncogenic BRAF in melanocyte transformation.,Upon LOXL3 depletion, melanoma cells display a faulty DNA damage response (DDR), characterized by ATM checkpoint activation and inefficient ATR activation leading to the accumulation of double-strand breaks (DSBs) and aberrant mitosis.,Consistent with these findings, LOXL3 binds to proteins involved in the maintenance of genome integrity, in particular BRCA2 and MSH2, whose levels dramatically decrease upon LOXL3 depletion.,Moreover, LOXL3 is required for efficient DSB repair in melanoma cells.,Our results reveal an unexpected role for LOXL3 in the control of genome stability and melanoma progression, exposing its potential as a novel therapeutic target in malignant melanoma, a very aggressive condition yet in need for more effective treatment options.

Melanomas are highly radioresistant tumors, mainly due to efficient DNA double-strand break (DSB) repair.,Dbait (which stands for DNA strand break bait) molecules mimic DSBs and trap DNA repair proteins, thereby inhibiting repair of DNA damage induced by radiation therapy (RT).,First, the cytotoxic efficacy of Dbait in combination with RT was evaluated in vitro in SK28 and 501mel human melanoma cell lines.,Though the extent of RT-induced damage was not increased by Dbait, it persisted for longer revealing a repair defect.,Dbait enhanced RT efficacy independently of RT doses.,We further assayed the capacity of DT01 (clinical form of Dbait) to enhance efficacy of “palliative” RT (10 × 3 Gy) or “radical” RT (20 × 3 Gy), in an SK28 xenografted model.,Inhibition of repair of RT-induced DSB by DT01 was revealed by the significant increase of micronuclei in tumors treated with combined treatment.,Mice treated with DT01 and RT combination had significantly better tumor growth control and longer survival compared to RT alone with the “palliative” protocol [tumor growth delay (TGD) by 5.7-fold; median survival: 119 vs 67 days] or the “radical” protocol (TGD by 3.2-fold; median survival: 221 vs 109 days).,Only animals that received the combined treatment showed complete responses.,No additional toxicity was observed in any DT01-treated groups.,This preclinical study provides encouraging results for a combination of a new DNA repair inhibitor, DT01, with RT, in the absence of toxicity.,A first-in-human phase I study is currently under way in the palliative management of melanoma in-transit metastases (DRIIM trial).

Melanomas are malignant tumors that originate from melanocytes.,They are most frequently localized in the skin, but 5% of all melanomas interest also extracutaneous sites as mucosal surfaces, parenchymatous organs, the retroperitoneum area, and the ocular ball.,The purpose of this study was to investigate the epidemiologic and morphologic data of mucosal melanomas diagnosed at Emergency City Hospital (Timisoara, Romania) during a period of 12 years.,The study included 17 cases of extracutaneous, extraocular melanomas, with 16 primary melanomas and one secondary melanoma.,All our patients were older than 53 years and were mostly men.,Most of the patients presented with localized disease; only one case had regional lymph node metastases, and another one had systemic metastases at the time of diagnosis.,Regarding localization, nine of 16 melanomas were in the head and neck region, six were diagnosed in the gastrointestinal and urogenital tracts (three cases each), and one case had a rare localization (retroperitoneum).,The most common histologic type was represented by epithelioid cells, and the majority of the tumors were achromic.,Mucosal melanoma is a tumor associated with aging, all our patients being older than 53 years.,Because of unspecific symptoms and low incidence, the diagnosis is often delayed and requires teamwork among the clinician, pathologist, radiologist, and oncologist.,Different genetic fingerprints impose a correct diagnosis to offer the patient the best novel, personalized therapy.

Our understanding of the molecular pathways that underlie melanoma remains incomplete.,Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies.,Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.,Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules.,Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients.,The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis.,An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study.,Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active.,Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.,This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis.,Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response).,The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC).,MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes.,Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown.,We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material.,We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA.,Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ.,In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR.,Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters.,Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.

TOC Summary: This virus may be part of normal human flora and harmless in most adults.,Merkel cell polyomavirus (MCV) is a recently discovered virus that causes 80% of Merkel cell carcinomas.,We examined data for 564 gay/bisexual male participants >18 years of age in the Multicenter AIDS Cohort Study in Pittsburgh, Pennsylvania, USA, and found that 447 (79.3%) were MCV-antibody positive at initial enrollment.,Of the 117 MCV-seronegative men, 31 subsequently seroconverted over a 4-year follow-up period, corresponding to a 6.6% annual conversion rate.,MCV immunoglobulin G levels remained detectable up to 25 years after exposure.,No signs, symptoms, or routine diagnostic test results were associated with MCV infection, and no correlation between HIV infection or AIDS progression and MCV infection was noted.,An initial correlation between chronic hepatitis B virus infection and MCV prevalence could not be confirmed among MCV seroconverters or in studies of a second hepatitis B virus-hyperendemic cohort from Qidong, China.,In adults, MCV is typically an asymptomatic, common, and commensal viral infection that initiates rare cancers after virus (rather than host cell) mutations.

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

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

Melanoma is a highly aggressive malignant skin tumor as well as the primary reason for skin cancer‐specific deaths.,We first identified immune‐related long noncoding RNA (lncRNA) prognostic signature and found potential immunotherapeutic targets for melanoma cancer.,RNA‐seq data and clinical features of melanoma samples were obtained from The Cancer Genome Atlas.,Samples of melanoma were randomly assigned to the training and testing cohort.,The immune‐related lncRNA signature was then obtained via using univariate, LASSO, and multivariate Cox analysis of patients in the training cohort.,Eight significant immune‐related lncRNA signature was then subsequently obtained through correlation analysis between immune‐related genes and lncRNAs.,The association between risk score and immune cell infiltration was finally assessed using TIMER and CIBERSORT.,Three hundred and fifty‐six immune‐related lncRNAs were obtained.,Among them, eight immune‐related lncRNAs were identified to build a prognostic risk signature model.,The model's performance was then confirmed using the Kaplan-Meier curves, risk plots, and time‐dependent receiver‐operating characteristic curves in the training cohort.,The risk score was identified and confirmed as an independent prognostic factor through univariate and multivariate Cox regression analyses.,These results were further verified in the testing and whole cohorts.,CIBERSORT algorithm showed that the infiltration levels of T cells CD8, M1 macrophages, plasma cells, T cells CD4 memory activated, T cells gamma delta, and mast cells activated were significantly lower in the high‐risk group while the infiltration level of macrophages M0 was significantly lower in the low‐risk group.,The immune‐related lncRNA signature offers prognostic markers and potential immunotherapeutic targets for melanoma.,We applied univariate Cox, LASSO, and multivariate Cox regression analysis to identify immune‐related lncRNAs with remarkable prognostic potential and constructed a multiple immune‐related lncRNAs signature to predict melanoma' prognosis. lncRNA signatures of immune‐related offers prognostic markers and potential immunotherapeutic targets for melanoma.

Growing evidence suggests that long non‐coding RNAs NORAD and miR‐205 play a significant role in regulating cancer progression and metastasis.,In this study, high expression of NORAD was firstly observed in melanoma tissues and human malignant melanoma cell lines, our aim was to study the interaction of them in the process of invasion and migration of malignant melanoma cells.,NORAD, miR‐205, and EGLN2 mRNA level in MM cells was detected by qRT‐PCR.,In situ hybridization (ISH) was performed to detect NORAD expression in MM tissues specimens.,Effects of NORAD and miR‐205 on Prolyl hydroxylase 2 (EGLN2) expression was explored by western blot in MM cells line.,Dual‐luciferase reporter assay was performed to verify the interaction relationship between NORAD and miR‐205, as well as, miR‐205 and EGLN2.,Transwell assay was conducted to explore the effects of NORAD and miR‐205 in vitro.,Xenografts in nude mice experiment were used to confirm the role of NORAD and miR‐205 in vivo.,In vitro, NORAD knockdown significantly inhibited migration and invasion of malignant melanoma cells and elevated the expression of miR‐205, there was an interaction between miR‐205 and NORAD in the RNA‐induced silencing complex.,Upregulation of miR‐205 induced significant inhibition of migratory and invasive ability compared with the scrambled control.,However, downregulating NORAD largely reversed this effect.,Furthermore, the regulatory effects of miR‐205 on EGLN2 levels and the induction of endoplasmic reticulum stress were reversed by NORAD.,In vivo, deletion of miR‐205 induced tumor growth in nude mice.,NORAD may play critical roles in tumorigenesis and progression of malignant melanoma by regulating of the miR‐205‐EGLN2 pathway, and may serve as a new therapeutic target.

Our research has focused on in vitro and in vivo evaluations of a new Carmustine (BCNU)-loaded clinoptilolite-based delivery system.,Two clinoptilolite ionic forms-hydrogen form (HCLI) and sodium form (NaCLI)-were prepared, allowing a loading degree of about 5-6 mg BCNU/g of zeolite matrix due to the dual porous feature of clinoptilolite.,Clinoptilolite-based delivery systems released 35.23% of the load in 12 h for the BCNU@HCLI system and only 10.82% for the BCNU@NaCLI system.,The BCNU@HCLI system was chosen to develop gel and cream semisolid dosage forms.,The cream (C_BCNU@HCLI) released 29.6% of the loaded BCNU after 12 h in the Nylon synthetic membrane test and 31.6% in the collagen membrane test, higher by comparison to the gel.,The new cream was evaluated in vivo in a chemically induced model of skin cancer in mice.,Quantitative immunohistochemistry analysis showed stronger inhibition of B-cell lymphoma-2 (bcl-2) and cyclooxygenase 2 (cox-2) protein expression, known markers for cancer survival and aggressiveness, after the treatment with C_BCNU@HCLI by comparison to all the control treatment types, including an off-label magistral formula commercially available Carmustine cream as reference, bringing evidence that a clinoptilolite-based delivery systems could be used as a cancer drug carriers and controlled release systems (skin-targeted topical delivery systems).

Anthracycline chemotherapeutics, e.g. doxorubicin and daunorubicin, are active against a broad spectrum of cancers.,Their cytotoxicity is mainly attributed to DNA intercalation, interference with topoisomerase activity, and induction of double-stranded DNA breaks.,Since modification of anthracyclines can profoundly affect their pharmacological properties we attempted to elucidate the mechanism of action, and identify possible molecular targets, of bis-anthracycline WP760 which previously demonstrated anti-melanoma activity at low nanomolar concentrations.,We studied the effect of WP760 on several human melanoma cell lines derived from tumors in various development stages and having different genetic backgrounds.,WP760 inhibited cell proliferation (IC50 = 1-99 nM), impaired clonogenic cell survival (100 nM), and inhibited spheroid growth (≥300 nM).,WP760 did not induce double-stranded DNA breaks but strongly inhibited global transcription.,Moreover, WP760 caused nucleolar stress and led to activation of the p53 pathway.,PCR array analysis showed that WP760 suppressed transcription of ten genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) associated with, inter alia, cytoprotective mechanisms initiated in cancer cells during chemotherapy.,Furthermore, WP760 downregulated IGF1R and upregulated PLK2 expression in most of the tested melanoma cell lines.,These results suggest that WP760 exerts anti-melanoma activity by targeting global transcription and activation of the p53 pathway and could become suitable as an effective therapeutic agent.,The online version of this article (doi:10.1007/s10637-017-0465-9) contains supplementary material, which is available to authorized users.

Upregulation of the histone methyltransferase enzyme EZH2 and its histone modification H3K27me3 has been linked to melanoma progression, metastasis, and resistance to immune checkpoint blockade (ICB).,In clinical trials, EZH2 inhibitors are currently tested to overcome resistance to ICB.,The aim of this study is to evaluate expression patterns and the predictive value of H3K27me3 and EZH2 in metastatic melanoma samples prior to ICB.,As H3K27me3 expression has been associated with a dedifferentiated, invasive melanoma phenotype, we also investigated the prognostic value of H3K27me3 expression in primary melanomas.,H3K27me3 and EZH2 expression were evaluated in a cohort of 44 metastatic melanoma samples before ICB using immunohistochemistry (IHC). 29/44 (66%) of melanomas showed H3K27me3 expression, and 6/44 (14%) showed EZH2 expression.,No predictive value for therapeutic response to anti-PD-1 therapy could be found for H3K27me3 or EZH2 expression on melanoma cells.,To investigate the prognostic significance of H3K27me3, we analyzed H3K27me3 expression in a representative cohort of 136 primary melanomas with known sentinel lymph node status.,H3K27me3 expression is associated with increased tumor thickness and nodal involvement.,Melanoma metastases showed a higher expression of H3K27me3 in comparison to primary melanomas.,In human melanoma cell lines, TNFα and INFγ could not induce H3K27me3 expression.,Our study shows that H3K27me3 expression is more frequent than EZH2 and is associated with a more invasive and metastatic melanoma cell phenotype.,We suggest that H3K27me3 expression by IHC might be a suitable method to evaluate the activity of EZH2 inhibitors in clinical trials.

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

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.

Excessive extracellular matrix (ECM) remodeling and a reactive stroma can affect T-cell infiltration and T-cell activity in the tumor and hereby influence response to immune checkpoint inhibitors (ICI).,In the pursuit of finding biomarkers that predict treatment response, we evaluated the association between serum biomarkers of collagen and vimentin turnover and outcomes in metastatic melanoma patients treated with the anti-CTLA-4 antibody ipilimumab (IPI).,Type III collagen formation (PRO-C3), MMP-degraded type I, type III and type IV collagens (C1M, C3M and C4M), and citrullinated and MMP-degraded vimentin (VICM) were measured with ELISAs in serum from metastatic melanoma patients before (n = 66) and 3 weeks after (n = 52) initiation of IPI treatment.,Biomarker levels were associated with Disease Control Rate (DCR) and survival outcomes.,We found that baseline levels of PRO-C3 (p = 0.011), C1M (p = 0.003), C3M (p = 0.013) and C4M (p = 0.027) were significantly elevated in patients with progressive disease (PD).,Univariate Cox regression analysis identified high PRO-C3 (p = 0.021) and C4M (p = 0.008) as predictors of poor overall survival (OS) and the biomarkers remained significant when evaluated with other covariates (PRO-C3 (p = 0.049) and C4M (p = 0.046)).,Multivariate analysis identified VICM as a predictor of longer OS (p = 0.026).,Similarly, a high C3M/PRO-C3 ratio predicted for increased OS (p = 0.034).,Only C3M (p = 0.003) and VICM (p < 0.0001) increased 3 weeks after treatment.,ECM and tissue remodeling quantified in pre-treatment serum were associated with response and survival outcomes in metastatic melanoma patients treated with IPI.,This highlights the importance of addressing the ECM and stromal component non-invasively in future ICI studies.,The online version of this article (10.1186/s40425-018-0474-z) contains supplementary material, which is available to authorized users.

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

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

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

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

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.

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.

Tumour blood vessels are gateways for distant metastasis.,Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts.,We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells.,However, the contribution of TECs to metastasis has not been elucidated.,Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs.,Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours.,Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal-regulated kinase 1/2.,Biglycan expression was upregulated by DNA demethylation in TECs.,Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis.

The incidence of malignant melanoma is increasing.,The majority of patients are diagnosed in early stages when the disease is highly curable.,However, the more advanced or metastatic cases have always been a challenge for clinicians.,The poor prognosis for patients with melanoma is now changing as numerous of promising approaches have appeared recently.,The discovery of aberrations of pathways responsible for intracellular signal transduction allowed us to introduce agents specifically targeting the mutated cascades.,Numerous clinical studies have been conducted to improve effectiveness of melanoma treatment.,From 2011 until now, the U.S.,FDA has approved seven novel agents, such as BRAF-inhibitors (vemurafenib 2011, dabrafenib 2013), MEK-inhibitors (trametinib 2013), anti-PD1 antibodies (nivolumab 2014, pembrolizumab 2014), anti-CTLA-4 antibody (ipilimumab 2011), or peginterferon-alfa-2b (2011) intended to be used in most advanced cases of melanoma.,Nevertheless, clinicians continue working on new possible methods of treatment as resistance to the novel drugs is a commonly observed problem.,This paper is based on latest data published until the end of January 2015.

Melanoma is a highly aggressive disease that is difficult to treat due to rapid tumor growth, apoptotic resistance, and high metastatic potential.,The MET tyrosine kinase receptor promotes many of these cellular processes, and while MET is often overexpressed in melanoma, the mechanism driving this overexpression is unknown.,Since the MET gene is rarely mutated or amplified in melanoma, MET overexpression may be driven by to increased activation through promoter elements.,In this report, we find that transcription factors PAX3 and ETS1 directly interact to synergistically activate MET expression.,Inhibition of PAX3 and ETS1 expression in melanoma cells leads to a significant reduction of MET receptor levels.,The 300 bp 5′ proximal MET promoter contains a PAX3 response element and two ETS1 consensus motifs.,While ETS1 can moderately activate both of these sites without cofactors, robust MET promoter activation of the first site is PAX-dependent and requires the presence of PAX3, while the second site is PAX-independent.,The induction of MET by ETS1 via this second site is enhanced by HGF-dependent ETS1 activation, thereby MET indirectly promotes its own expression.,We further find that expression of a dominant negative ETS1 reduces the ability of melanoma cells to grow both in culture and in vivo.,Thus, we discover a pathway where ETS1 advances melanoma through the expression of MET via PAX-dependent and independent mechanisms.

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

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.

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.

Phenotypic changes during cancer progression are associated with alterations in gene expression, which can be exploited to build molecular signatures for tumor stage identification and prognosis.,However, it is not yet known whether the relative abundance of transcript isoforms may be informative for clinical stage and survival.,Using information theory and machine learning methods, we integrated RNA sequencing and clinical data from The Cancer Genome Atlas project to perform the first systematic analysis of the prognostic potential of transcript isoforms in 12 solid tumors to build new signatures for stage and prognosis.,This study was also performed in breast tumors according to estrogen receptor (ER) status and melanoma tumors with proliferative and invasive phenotypes.,Transcript isoform signatures accurately separate early from late-stage groups and metastatic from non-metastatic tumors, and are predictive of the survival of patients with undetermined lymph node invasion or metastatic status.,These signatures show similar, and sometimes better, accuracies compared with known gene expression signatures in retrospective data and are largely independent of gene expression changes.,Furthermore, we show frequent transcript isoform changes in breast tumors according to ER status, and in melanoma tumors according to the invasive or proliferative phenotype, and derive accurate predictive models of stage and survival within each patient subgroup.,Our analyses reveal new signatures based on transcript isoform abundances that characterize tumor phenotypes and their progression independently of gene expression.,Transcript isoform signatures appear especially relevant to determine lymph node invasion and metastasis and may potentially contribute towards current strategies of precision cancer medicine.,The online version of this article (doi:10.1186/s13073-016-0339-3) contains supplementary material, which is available to authorized users.

A key focus in cancer research is the discovery of biomarkers that accurately diagnose early lesions in non-invasive tissues.,Several studies have identified malignancy-associated DNA methylation changes in blood, yet no general cancer biomarker has been identified to date.,Here, we explore the potential of blood DNA methylation as a biomarker of pan-cancer (cancer of multiple different origins) in 41 female cancer discordant monozygotic (MZ) twin-pairs sampled before or after diagnosis using the Illumina HumanMethylation450 BeadChip.,We analysed epigenome-wide DNA methylation profiles in 41 cancer discordant MZ twin-pairs with affected individuals diagnosed with tumours at different single primary sites: the breast, cervix, colon, endometrium, thyroid gland, skin (melanoma), ovary, and pancreas.,No significant global differences in whole blood DNA methylation profiles were observed.,Epigenome-wide analyses identified one novel pan-cancer differentially methylated position at false discovery rate (FDR) threshold of 10 % (cg02444695, P = 1.8 × 10−7) in an intergenic region 70 kb upstream of the SASH1 tumour suppressor gene, and three suggestive signals in COL11A2, AXL, and LINC00340.,Replication of the four top-ranked signals in an independent sample of nine cancer-discordant MZ twin-pairs showed a similar direction of association at COL11A2, AXL, and LINC00340, and significantly greater methylation discordance at AXL compared to 480 healthy concordant MZ twin-pairs.,The effects at cg02444695 (near SASH1), COL11A2, and LINC00340 were the most promising in biomarker potential because the DNA methylation differences were found to pre-exist in samples obtained prior to diagnosis and were limited to a 5-year period before diagnosis.,Gene expression follow-up at the top-ranked signals in 283 healthy individuals showed correlation between blood methylation and gene expression in lymphoblastoid cell lines at PRL, and in the skin tissue at AXL.,A significant enrichment of differential DNA methylation was observed in enhancer regions (P = 0.03).,We identified DNA methylation signatures in blood associated with pan-cancer, at or near SASH1, COL11A2, AXL, and LINC00340.,Three of these signals were present up to 5 years prior to cancer diagnosis, highlighting the potential clinical utility of whole blood DNA methylation analysis in cancer surveillance.,The online version of this article (doi:10.1186/s13148-016-0172-y) contains supplementary material, which is available to authorized users.

SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial.,In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies.,Using murine allograft melanoma models, our results suggest increased systemic expression of SIRT2 promotes tumor progression.,In this study, SIRT2-overexpressing mice exhibited enhanced tumor growth and larger tumor volumes compared to their wild-type littermates.,Mechanistically, systemic overexpression of SIRT2 reduces the number of tumor-infiltrating natural killer (NK) cells and suppresses NK cell function and proliferation within the tumor microenvironment (TME).,Furthermore, despite the enhancing effect of NK cell depletion on tumor volume and growth rate in wild-type littermate mice, this effect was diminished in SIRT2-overexpressing mice.,Lastly, pharmacological inhibition of SIRT2 increases NK cell tumor infiltration and suppresses allograft melanoma tumor growth.,The findings of this study identify a dynamic functional interaction between systemic SIRT2 and NK cell activity, which controls melanoma tumor progression.,Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could present a new opportunity to mediate immunotherapy response and resistance.

Hepatic metastasis develops in ~ 50% of uveal melanoma (UM) patients with no effective treatments.,Although GNAQ/GNA11 mutations are believed to confer pathogenesis of UM, the underlying mechanism of liver metastasis remains poorly understood.,Given that profound epigenetic evolution may occur in the long journey of circulating tumor cells (CTCs) to distant organs, we hypothesized that EZH2 endowed tumor cells with enhanced malignant features (e.g., stemness and motility) during hepatic metastasis in UM.,We aimed to test this hypothesis and explore whether EZH2 was a therapeutic target for hepatic metastatic UM patients.,Expression of EZH2 in UM was detected by qRT-PCR, Western blotting and immunohistochemistry staining.,Proliferation, apoptosis, cancer stem-like cells (CSCs) properties, migration and invasion were evaluated under circumstances of treatment with either EZH2 shRNA or EZH2 inhibitor GSK126.,Antitumor activity and frequency of CSCs were determined by xenografted and PDX models with NOD/SCID mice.,Hepatic metastasis was evaluated with NOG mice.,We found that EZH2 overexpressed in UM promoted the growth of UM; EZH2 increased the percentage and self-renewal of CSCs by miR-29c-DVL2-β-catenin signaling; EZH2 facilitates migration and invasion of UM cells via RhoGDIγ-Rac1 axis.,Targeting EZH2 either by genetics or small molecule inhibitor GSK126 decreased CSCs and motility and abrogated the liver metastasis of UM.,These findings validate EZH2 as a druggable target in metastatic UM patients, and may shed light on the understanding and interfering the complicated metastatic process.

Oncogene-driven metabolic rewiring is an adaptation to low nutrient and oxygen conditions in the tumor microenvironment that enables cancer cells of diverse origin to hyperproliferate.,Aerobic glycolysis and enhanced reliance on glutamine utilization are prime examples of such rewiring.,However, tissue of origin as well as specific genetic and epigenetic changes determines gene expression profiles underlying these metabolic alterations in specific cancers.,In melanoma, activation of the MAPK pathway driven by mutant BRAF or NRAS is a primary cause of malignant transformation.,Activity of the MAPK pathway, as well as other factors, such as HIF1α, Myc and MITF, are among those that control the balance between non-oxidative and oxidative branches of central carbon metabolism.,Here, we discuss the nature of metabolic alterations that underlie melanoma development and affect its response to therapy.

Advances in melanoma treatment through targeted inhibition of oncogenic BRAF are limited owing to the development of acquired resistance.,The involvement of BRAFV600E in metabolic reprogramming of melanoma cells provides a rationale for co-targeting metabolism as a therapeutic approach.,We examined the effects of dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, on the growth and metabolic activity of human melanoma cell lines.,The combined effect of DCA and the BRAF inhibitor vemurafenib was investigated in BRAFV600E -mutated melanoma cell lines.,Vemurafenib-resistant cell lines were established in vitro and their sensitivity to DCA was tested.,DCA induced a reduction in glycolytic activity and intracellular ATP levels, and inhibited cellular growth.,Co-treatment of BRAFV600E-mutant melanoma cells with DCA and vemurafenib induced a greater reduction in intracellular ATP levels and cellular growth than either compound alone.,In addition, melanoma cells with in vitro acquired resistance to vemurafenib retained their sensitivity to DCA.,These results suggest that DCA potentiates the effect of vemurafenib through a cooperative attenuation of energy production.,Furthermore, the demonstration of retained sensitivity to DCA in melanoma cells with acquired resistance to vemurafenib could have implications for melanoma treatment.,The online version of this article (doi:10.1186/s12967-014-0247-5) contains supplementary material, which is available to authorized users.

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.

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

The discovery of the role of the RAS/RAF/MEK/ERK pathway in melanomagenesis and its progression have opened a new era in the treatment of this tumor.,Vemurafenib was the first specific kinase inhibitor approved for therapy of advanced melanomas harboring BRAF-activating mutations, followed by dabrafenib and encorafenib.,However, despite the excellent results of first-generation kinase inhibitors in terms of response rate, the average duration of the response was short, due to the onset of genetic and epigenetic resistance mechanisms.,The combination therapy with MEK inhibitors is an excellent strategy to circumvent drug resistance, with the additional advantage of reducing side effects due to the paradoxical reactivation of the MAPK pathway.,The recent development of RAS and extracellular signal-related kinases (ERK) inhibitors promises to add new players for the ultimate suppression of this signaling pathway and the control of pathway-related drug resistance.,In this review, we analyze the pharmacological, preclinical, and clinical trial data of the various MAPK pathway inhibitors, with a keen interest for their clinical applicability in the management of advanced melanoma.

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

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

The non-malignant cells of the tumour stroma have a critical role in tumour biology.,Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention.,For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts.,Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts.,Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively.,Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression.,5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts.,5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma.,The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents.

Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM).,We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions.,Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain.,Retrospective data on gene expression class and patient survival were retrieved.,Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy.,The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively.,PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02).,The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004).,Forty percent of the tumors had heterogeneous BAP-1 expression.,Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01).,PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM.,Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.

Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia.,Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis.,It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.,By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia.,We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis.,In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants.,The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein.,We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions.,Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.,We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.

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

TP53 has been proved to be associated with cytotoxic T-cell induced apoptosis, however, the association between TP53 and the benefit of immunotherapy in melanoma has not been studied.,In the present study, we examined the relationship between TP53 mutation and response to CTLA-4 blockade in metastatic melanoma by analyzing the data from one public cohort consisting of 110 patients with metastatic melanoma.,The sequencing, mRNA and survival data of 368 patients with skin melanoma from The Cancer Genome Atlas (TCGA) was used to explore the underlying mechanism.,TP53 mutation was associated with significant poorer progression-free survival (HR, 2.25; 95% CI, 1.15-4.37; P = 0.014), poorer overall survival (HR, 2.05; 95% CI, 1.02-4.13; P = 0.040) and trend of poorer response (OR, 0.20; 95% CI, 0.02-1.62; P = 0.131).,The correlations were significant in multivariate analysis including lactate dehydrogenase, tumor mutational burden and tumor stage (P < 0.05).,In TCGA, no association was observed between TP53 mutation and survival (P = 0.55).,The mRNA expression of FAS was lower in patients with TP53 mutation than TP53 wild-type.,Our findings suggest that TP53 mutation is a potential negative predictor of metastatic melanoma treated with CTLA-4 blockade.,•TP53 mutation is associated with poorer outcomes in patients with metastatic melanoma receiving anti-CTLA-4 treatment.,•The mRNA expression of FAS was lower in patients with TP53 mutation than TP53 wild-type.,•TP53 is a potential negative predictor of metastatic melanoma treated with CTLA-4 blockade.,TP53 mutation is associated with poorer outcomes in patients with metastatic melanoma receiving anti-CTLA-4 treatment.,The mRNA expression of FAS was lower in patients with TP53 mutation than TP53 wild-type.,TP53 is a potential negative predictor of metastatic melanoma treated with CTLA-4 blockade.,Immune checkpoint blockades by enhancing the anti-tumor activity of immune system have been standard treatment for several advanced tumors, however, only a subset of patients can benefit from them.,In this study, we find patients with melanoma harboring TP53 mutation show poorer survival outcome and response of anti-CTLA-4 treatment.,The mRNA expression of FAS is lower in patients with TP53 mutation, suggesting that TP53 down-regulates the expression of FAS and impede cytotoxic T-cell induced apoptosis in melanoma.,These results suggest that TP53 mutation may serve as a negative predictor of anti-CTLA-4 treatment in melanoma.

CC-115 is a dual inhibitor of the mechanistic target of rapamycin (mTOR) kinase and the DNA-dependent protein kinase (DNA-PK) that is currently being studied in phase I/II clinical trials.,DNA-PK is essential for the repair of DNA-double strand breaks (DSB).,Radiotherapy is frequently used in the palliative treatment of metastatic melanoma patients and induces DSBs.,Melanoma cell lines and healthy-donor skin fibroblast cell lines were treated with CC-115 and ionizing irradiation (IR).,Apoptosis, necrosis, and cell cycle distribution were analyzed.,Colony forming assays were conducted to study radiosensitizing effects.,Immunofluorescence microscopy was performed to determine the activity of homologous recombination (HR).,In most of the malign cell lines, an increasing concentration of CC-115 resulted in increased cell death.,Furthermore, strong cytotoxic effects were only observed in malignant cell lines.,Regarding clonogenicity, all cell lines displayed decreased survival fractions during combined inhibitor and IR treatment and supra-additive effects of the combination were observable in 5 out of 9 melanoma cell lines.,CC-115 showed radiosensitizing potential in 7 out of 9 melanoma cell lines, but not in healthy skin fibroblasts.,Based on our data CC-115 treatment could be a promising approach for patients with metastatic melanoma, particularly in the combination with radiotherapy.

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types.1-5 One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8 T-cells (termed adaptive immune resistance).6,7 Here we show that pre-existing CD8 T-cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy.,We analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next generation sequencing for T-cell receptors (TCR).,In serially sampled tumours, responding patients showed proliferation of intratumoural CD8+ T-cells that directly correlated with radiographic reduction in tumour size.,Pre-treatment samples obtained from responding patients showed higher numbers of CD8, PD1, and PD-L1 expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire.,Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients.,Our findings indicate that tumour regression following therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1 mediated adaptive immune resistance.

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

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

The principles governing evolution of tumors exposed to targeted therapy are poorly understood.,Here we modeled the selection and propagation of BRAF amplification (BRAFamp) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor, alone or in combination with other pathway inhibitors.,Single cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in parallel evolutionary tracts, arising in the same tumor shortly after treatment.,The evolutionary selection of BRAFamp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy.,This differed for ERK signaling inhibitors, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number.,Concurrent targeting of RAF, MEK and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification.,Administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11-lung cancer and melanoma PDX without apparent toxicity in mice.,Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity.,Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and merit testing in patients.

Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors.,However, a limitation to such treatment is the occurrence of resistance.,Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent.,In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results.,However, also in this case resistance inevitably occurs.,It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.,Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D).,Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array.,Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies.,The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting.,The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.,In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT.,Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth.,We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.,On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.

Obesity is a risk factor for malignancy; however, its prognostic role in patients with metastatic melanoma is controversial.,We aim to investigate the prognostic role of body mass index (BMI) in patients with metastatic melanoma receiving mitogen-activated pathway kinase inhibitors (MAPKi), immune checkpoint inhibitors (ICIs) alone or their sequence.,Data on patients with metastatic melanoma receiving ≥1 line of systemic treatment were retrieved from prospectively collected databases.,Progression-free survival (PFS) and overall survival (OS) were analyzed by means of multivariable stratified Cox regression models; disease control rate (DCR) was analyzed by multivariable stratified logistic regression models.,Subgroup analyzes according to the type of treatments received, and in BRAF-mutated patients were pre-planned.,All multivariable models included BMI, age, gender, American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment sequencing strategy as covariates.,Between November 2010 and November 2018, 688 patients from three Italian and two Polish centers were enrolled. 379 (57%) patients had M1c/d disease, 273 (41%) were female and the mean BMI was 27.1 (SD=4.9).,Considering first-line treatment, 446 patients (66.8%) received ICIs and 222 MAPKi.,No impact of BMI on OS was detected either considering the first line of ICIs, or ICIs sequencing (HR=1.02, 95% CI: 0.99 to 1.05, p=0.202, and HR=1.02, 95% CI: 0.99 to 1.04, p=0.237, respectively).,A late effect of BMI on OS was found in patients treated with MAPKi: for five units increment, a 51% of risk reduction at 18 months and a 76% of risk reduction at 30 months were observed.,No significant effect of BMI on PFS and DCR was found in any of the subgroup analyzes.,In patients with metastatic melanoma receiving ICIs, there is no impact of BMI on DCR, PFS and OS.,The late prognostic effect of BMI in patients treated with MAPKi should be considered hypothesis generating and needs to be further investigated.

Vitamin D deficiency (≤20 ng/mL) is associated with an increased incidence and worse prognosis of various types of cancer including melanoma.,A retrospective, single-center study of individuals diagnosed with melanoma from January 2007 through June 2013 who had a vitamin D (25(OH)D3) level measured within one year of diagnosis was performed to determine whether vitamin D deficiency and repletion are associated with melanoma outcome.,A total of 409 individuals diagnosed with histopathology-confirmed melanoma who had an ever measured serum 25(OH)D3 level were identified. 252 individuals with a 25(OH)D3 level recorded within one year after diagnosis were included in the study and the individual and melanoma characteristics such as age, sex, Breslow thickness, ulceration, stage, mitotic rate, and LDH were obtained from the medical record.,A worse melanoma prognosis was associated with vitamin D deficiency (P=0.012), higher stage (P<0.001), ulceration (P=0.001), and higher mitotic rate (P=0.001) (HR 1.93, 95% CI 1.15-3.22).,In patients with stage IV metastatic melanoma, vitamin D deficiency was associated with significantly worse melanoma-specific mortality (adjusted HR 2.06, 95% CI 1.10-3.87).,Patients with metastatic melanoma who were initially vitamin D deficient and subsequently had a decrease or ≤20 ng/mL increase in their 25(OH)D3 concentration had significantly worse outcomes (HR 4.68, 95% CI 1.05-20.88) compared to non-deficient patients who had a >20 ng/mL increase.,Our results suggest that initial vitamin D deficiency and insufficient repletion is associated with a worse prognosis in patients with metastatic melanoma.

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

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

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

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis.,Merkel cell polyomavirus (MCPyV) T‐antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC).,Yet, how MCPyV induces tumorigenesis is not fully understood.,Herein, we show that MCPyV T‐antigens induce miR‐375, miR‐30a‐3p and miR‐30a‐5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1.,In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV‐positive tumors.,Ectopic expression of MCPyV small T‐antigen and truncated large T‐antigen (LT), but not the wild‐type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV‐mediated tumorigenesis.,Torin‐1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan‐caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC.,Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV‐mediated tumorigenesis and potential MCC treatment.,What's new?,About four‐fifths of Merkel cell carcinomas harbor the Merkel cell polyomavirus (MCPyV) genome, though mutations in viral T antigens generally render MCPyV replication‐deficient.,Here, the authors describe a network by which MCPyV oncoproteins and viral‐regulated miRNAs hijack autophagy machinery in MCC.,In cells, expression of MCPyV T‐antigens induced miR‐375, miR‐30a‐3p, and miR‐30a‐5p expression.,Subsequent experiments validated these miRNAs as autophagy regulators that specifically target the genes ATG7, SQSTM1, and BECN1.,Autophagy suppression protected MCC cell survival.,This novel insight into the role of MCPyV and autophagy regulation in MCC may be relevant in the generation of future therapeutic strategies.

The discovery of BRAF mutations in the majority of patients with metastatic melanoma combined with the identification of highly selective BRAF inhibitors have revolutionized the treatment of patients with metastatic melanoma.,The first highly specific BRAF inhibitor, vemurafenib, began clinical testing in 2008 and moved towards a rapid approval in 2011.,Vemurafenib induced responses in ~50% of patients with metastatic BRAF-mutant melanoma and demonstrated improved overall survival in a randomized Phase III trial.,Furthermore, vemurafenib is well-tolerated with a low toxicity profile and rapid onset of action.,Finally, vemurafenib is active even in patients with widely metastatic disease.,Despite the success of vemurafenib in treating patients with BRAF-mutant metastatic melanoma, most, if not all, patients ultimately develop resistance resulting in disease progression at a median time of ~6 months.,Multiple mechanisms of resistance have been described and rationale strategies are underway to combat resistance.,This review highlights the development, clinical utility, resistance mechanisms, and future use of vemurafenib both in melanoma and other malignancies.,We consulted PubMed, Scopus, MEDLINE, ASCO annual symposium abstracts, and http://clinicaltrials.gov/ for the purpose of this review.

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway.,About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E).,BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors.,The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3).,The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression.,Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment.,Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation.,In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations.,Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD).,Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy.,For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months.,A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab.,In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2.,Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations.,Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

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.

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.

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

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

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

Real‐world data on treatment patterns/outcomes in patients with advanced melanoma, while scarce, are useful for health technology assessments that govern patient access in many countries.,We collected retrospective data on treatment patterns among patients in France, Germany and the UK with Stage IIIB/IIIC melanoma with macroscopic lymph node involvement, whose primary melanoma and regional lymph node metastases had been completely resected.,Patients ≥18 years were diagnosed between 1 January 2009 and 31 December 2011.,Data were obtained from patients’ medical records and a patient survey.,Forty‐nine centres provided data on 558 patients: 53.6% had Stage IIIB disease; 58.2% were of working age (<65 years), 22.5% reported a change in employment status due to melanoma, 8% were on long‐term sick leave; and 35.1% were deceased over the study period.,Overall median distant metastases‐free survival was 23.4 months and median disease‐free survival was 13.3 months.,Hospitalisation frequency increased during distant metastatic/terminal disease phases.,Adjuvant therapy was received by 7.0% (14/199) of patients in France, 2.6% (5/195) in the UK, and 33.5% (55/164) in Germany.,Low‐dose interferon was used more frequently than other regimens.,High‐dose interferon was associated with discontinuation in 28.6% and dose delay/reduction in 33.3% of patients.,Rapid disease progression combined with increased use of healthcare resources in later phases of disease result in a high burden‐of‐illness for patients and healthcare providers.,The use of adjuvant interferon therapy varies considerably in this population in the countries studied, highlighting the need for improved treatments for melanoma.

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.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy for which there is still no effective therapy.,In order to identify genetic alterations useful for a new treatment design, we used whole-exome sequencing to analyze 14 BPDCN patients and the patient-derived CAL-1 cell line.,The functional enrichment analysis of mutational data reported the epigenetic regulatory program to be the most significantly undermined (P<0.0001).,In particular, twenty-five epigenetic modifiers were found mutated (e.g.,ASXL1, TET2, SUZ12, ARID1A, PHF2, CHD8); ASXL1 was the most frequently affected (28.6% of cases).,To evaluate the impact of the identified epigenetic mutations at the gene-expression and Histone H3 lysine 27 trimethylation/acetylation levels, we performed additional RNA and pathology tissue-chromatin immunoprecipitation sequencing experiments.,The patients displayed enrichment in gene signatures regulated by methylation and modifiable by decitabine administration, shared common H3K27-acetylated regions, and had a set of cell-cycle genes aberrantly up-regulated and marked by promoter acetylation.,Collectively, the integration of sequencing data showed the potential of a therapy based on epigenetic agents.,Through the adoption of a preclinical BPDCN mouse model, established by CAL-1 cell line xenografting, we demonstrated the efficacy of the combination of the epigenetic drugs 5’-azacytidine and decitabine in controlling disease progression in vivo.

The incidence of melanoma continues to rise globally and is increasing at a rate greater than any other cancer.,To systematically search for new genes involved in melanomagenesis, we collated exome sequencing data from independent melanoma cohort datasets, including those in the public domain.,We identified recurrent mutations that may drive melanoma growth, survival or metastasis, and which may hold promise for the design of novel therapies to treat melanoma.,These included a frequent recurrent (i.e. hotspot) mutation in the 5' untranslated region of RPS27 in ~10% of samples.,We show that the mutation expands the 5'TOP element, a motif known to regulate the expression of most of the ribosomal protein family, to which RPS27 belongs, and thus might sensitize the mutated transcript to growth-mediated regulation.,This finding highlights not only the important role of non-protein coding genetic aberrations in cancer development but also their potential as novel therapeutic targets.

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer.,BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma.,In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436).,Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death.,In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition.,However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors.,In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models.,Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Combination concepts of radiotherapy and immune checkpoint inhibition are currently of high interest.,We examined imaging findings, acute toxicity, and local control in patients with melanoma brain metastases receiving programmed death 1 (PD-1) inhibitors and/or robotic stereotactic radiosurgery (SRS).,Twenty-six patients treated with SRS alone (n = 13; 20 lesions) or in combination with anti-PD-1 therapy (n = 13; 28 lesions) were analyzed.,Lesion size was evaluated three and six months after SRS using a volumetric assessment based on cranial magnetic resonance imaging (cMRI) and acute toxicity after 12 weeks according to the Common Terminology Criteria for Adverse Events (CTCAE).,Local control after six months was comparable (86%, SRS + anti-PD-1, and 80%, SRS).,All toxicities reported were less than or equal to grade 2.,One metastasis (5%) in the SRS group and six (21%) in the SRS + anti-PD-1 group increased after three months, whereas four (14%) of the six regressed during further follow-ups.,This was rated as pseudoprogression (PsP).,Three patients (23%) in the SRS + anti-PD-1 group showed characteristics of PsP.,Treatment with SRS and anti-PD-1 antibodies can be combined safely in melanoma patients with cerebral metastases.,Early volumetric progression of lesions under simultaneous treatment may be related to PsP; thus, the evaluation of combined radioimmunotherapy remains challenging and requires experienced teams.

Melanoma brain metastasis is associated with an extremely poor prognosis, with a median overall survival of 4-5 months.,Since 2011, the overall survival of patients with stage IV melanoma has been significantly improved with the advent of new targeted therapies and checkpoint inhibitors.,We analyze the survival outcomes of patients diagnosed with brain metastasis after the introduction of these novel drugs.,We performed a retrospective analysis of our melanoma center database and identified 79 patients with brain metastasis between 2011 and 2015.,The median time from primary melanoma diagnosis to brain metastasis was 3.2 years.,The median overall survival duration from the time of initial brain metastasis was 12.8 months.,Following a diagnosis of brain metastasis, 39 (49.4%), 28 (35.4%), and 24 (30.4%) patients were treated with anti-CTLA-4 antibody, anti-PD-1 antibody, or BRAF inhibitors (with or without a MEK inhibitor), with a median overall survival of 19.2 months, 37.9 months and 12.7 months, respectively.,Factors associated with significantly reduced overall survival included male sex, cerebellar metastasis, higher number of brain lesions, and treatment with whole-brain radiation therapy.,Factors associated with significantly longer overall survival included treatment with craniotomy, stereotactic radiosurgery, or with anti-PD-1 antibody after initial diagnosis of brain metastasis.,These results show a significant improvement in the overall survival of patients with melanoma brain metastasis in the era of novel therapies.,In addition, they suggest the activity of anti-PD-1 therapy specifically in the setting of brain metastasis.

The therapeutic landscape for advanced melanoma has recently been transformed by several novel agents (immune checkpoint inhibitors and molecular‐targeted agents).,The prospective, multi‐site, observational study IMAGE (ipilimumab: management of advanced melanoma in real practice) included a retrospective cohort to describe real‐world treatment prior to approval of the immune checkpoint inhibitor ipilimumab.,This retrospective cohort of patients, who started second‐line/subsequent treatment (index therapy) for advanced melanoma within 3 years before ipilimumab approval, was selected randomly by chart review.,Collected data included treatment history, patient outcomes, and healthcare resource utilization.,All patients had ≥1 year of follow‐up data.,This analysis included 177 patients from Europe (69%) and North America (31%).,The most common index therapies (used alone or in combination) were fotemustine (23%), dacarbazine (21%), temozolomide (14%), and platinum‐based chemotherapy (14%).,Most patients (89%) discontinued index treatment during the study period; the most common reason was disease progression (59%).,Among patients with tumor assessment (153/177; 86%), 2% had complete response, 5% had partial response, and 12% had stable disease on last tumor assessment.,At 1‐year study follow‐up, median progression‐free survival was 2.6 months (95% confidence interval [CI], 2.1-2.9) and median overall survival was 8.8 months (95% CI, 6.5-9.7).,During follow‐up, 95% of the patients had healthcare visits for advanced melanoma, 74% of whom were hospitalized or admitted to a hospice facility.,These results provide insights into patient care with advanced melanoma in the era before ipilimumab and may serve as a benchmark for new agents in future real‐world studies.

Ipilimumab improves survival in patients with advanced melanoma.,The activity and safety of ipilimumab outside of a clinical trial was assessed in an expanded access programme (EAP).,Ipilimumab was available upon physician request for patients aged 16 or over with pretreated stage III (unresectable)/IV melanoma, for whom no other therapeutic option was available.,Patients received ipilimumab 3 mg/kg every 3 weeks for four doses.,Patients with stable disease or an objective response to ipilimumab were eligible for retreatment upon disease progression.,Tumour assessments were conducted at baseline and week 12.,Patients were monitored for adverse events (AEs) within 3 to 4 days of each scheduled visit.,Of 855 patients participating in the EAP in Italy, 833 were evaluable for response.,Of these, 13% had an objective immune response, and the immune-related disease control rate was 34%.,Median progression-free survival and overall survival were 3.7 and 7.2 months, respectively.,Efficacy was independent of BRAF and NRAS mutational status.,Overall, 33% of patients reported an immune-related AE (irAE).,The frequency of irAEs was not associated with response to ipilimumab.,Outside of a clinical trial setting, ipilimumab is a feasible treatment option in patients with pretreated metastatic melanoma, regardless of BRAF and NRAS mutational status.,Data from this large cohort of patients support clinical trial evidence that ipilimumab can induce durable disease control and long-term survival in patients who have failed to respond to prior treatment.

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

Checkpoint inhibitors are revolutionizing treatment options and expectations for patients with melanoma.,Ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), was the first approved checkpoint inhibitor.,Emerging long-term data indicate that approximately 20% of ipilimumab-treated patients achieve long-term survival.,The first programmed death 1 (PD-1) inhibitor, pembrolizumab, was recently approved by the United States Food and Drug Administration for the treatment of melanoma; nivolumab was previously approved in Japan.,PD-1 inhibitors are also poised to become standard of care treatment for other cancers, including non-small cell lung cancer, renal cell carcinoma and Hodgkin's lymphoma.,Immunotherapy using checkpoint inhibition is a different treatment approach to chemotherapy and targeted agents: instead of directly acting on the tumor to induce tumor cell death, checkpoint inhibitors enhance or de novo stimulate antitumor immune responses to eliminate cancer cells.,Initial data suggest that objective anti-tumor response rates may be higher with anti-PD-1 agents compared with ipilimumab and the safety profile may be more tolerable.,This review explores the development and next steps for PD-1 pathway inhibitors, including discussion of their novel mechanism of action and clinical data to-date, with a focus on melanoma.

Herein, we describe the use of systemic immunotherapy for both locally advanced and metastatic conjunctival melanoma.,Current treatments for advanced conjunctival melanoma typically result in poor local control leading to disfiguring orbital exenteration surgery.,Locoregional spread of conjunctival malignant melanoma typically requires pre-auricular and cervical lymph node dissection with post-operative adjuvant radiation therapy.,In addition, classic systemic chemotherapy has been unsuccessful in the treatment of metastatic disease.,This is a retrospectively analyzed clinical case series of 5 patients with biopsy proven conjunctival melanoma who were treated with checkpoint inhibition therapy.,Of these, 3 patients were treated for residual ocular disease present after failing multiple local therapies and refusing orbital exenteration surgery and two (with local ocular control) for metastatic conjunctival melanoma.,Both those with locally advanced disease and patients with metastatic disease received an anti-PD1 agent in combination with another immunotherapeutic agent.,All 5 were given multiple cycles of systemic anti-PD1 therapy, 1 was initially treated with single agent ipilimumab (3 mg/kg) prior to approval of anti-PD1 agents and two received interferon eye drops.,As part of each ophthalmic examination, photographs of all conjunctival and eyelid surfaces were obtained.,Systemic evaluations involved initial staging scans as well as periodic re-imaging.,All cases have shown responses.,Of the 2 complete responses, 1 was a patient with systemic disease.,No patients developed ocular toxicity or loss of vision.,However, systemic adverse effects included adrenal insufficiency, Grade-III colitis, Grade-II dermatitis, Grade-II hepatotoxicity and Grade-II pneumonitis.,This report suggests that systemic immunotherapy with or without topical interferon is effective in treatment of malignant melanoma of the conjunctiva.,Therefore, it can be considered for patients with advanced local conjunctival melanoma, those who refuse orbital exenteration surgery and those with systemic metastasis.

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

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

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors.,However, a limitation to such treatment is the occurrence of resistance.,Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities.,Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma.,In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors.,This strongly correlates with increased transcriptional activation of its ligand neuregulin.,Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro.,In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain.,These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model.,Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth.,Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

Epidermal squamous cell carcinoma (SCC) is a common and highly invasive form of cancer.,SCC arises due to ultraviolet light exposure and is associated with increased expression of pro-cancer genes and reduced expression of cancer suppressors.,Actin-Like Protein 6A (ACTL6A, BAF53a) is an important protein subunit of the SWI/SNF epigenetic chromatin regulatory complex.,ACTL6A is elevated in cancer cells and has been implicated as a driver of cancer cell proliferation and tumor growth.,In the present study, we show that ACTL6A drives SCC cell proliferation, spheroid formation, invasion and migration, and that these activities are markedly reduced by ACTL6A knockdown.,We further show that ACTL6A expression is associated with reduced levels of the p21Cip1 cyclin-dependent kinase inhibitor and tumor suppressor protein.,Molecular studies show that ACTL6A interacts with p53 DNA response elements in the p21Cip1 gene promoter to suppress p21Cip1 promoter activity and mRNA and protein level.,Additional studies show that an increase in p21Cip1 expression in ACTL6A knockdown cells is required for suppression of the SCC cell phenotype, suggesting that p21Cip1 is a mediator of ACTL6A action.,We further show that this regulation is p53 independent.,These findings suggest that ACTL6A suppresses p21Cip1 promoter activity to reduce p21Cip1 protein as a mechanism to maintain the aggressive epidermal squamous cell carcinoma phenotype.

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.

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

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

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

Dysregulation of miR-183 has been recently elucidated in several carcinomas.,However, the expression patterns and mechanisms of miR-183 involved in malignant melanoma remain unidentified.,Here, we found down-regulation of miR-183 in melanoma tissues and cells.,Decreased level of miR-183 was relevant to poor overall survival, while miR-183 up-regulation resulted in a marked suppression of cell growth in vitro and in vivo.,We further found that the expression and function of miR-183 were suppressed by MALAT1.,Integrin β1 (ITGB1) was then speculated and confirmed as a direct target of miR-183.,We also illustrated that MALAT1 may function as a sponge competitive endogenous RNA (ceRNA) for miR-183, and thus regulate the molecular expression of ITGB1.,Collectively, we found a new signaling pathway promoting melanoma development by MALAT1-miR-183-ITGB1 axis, which may be clinically valuable as new targets for malignant melanoma therapy.

Although clinical guidelines exist, the management of patients with cutaneous melanoma (CM) is a complex process that may vary between different care providers with potential dysfunctions ultimately mirrored in the overall patient satisfaction.,The aim of the present study was to investigate the CM management as related to lead times, surgical quality and diagnosis communication with the hypothesis that the care may differ between providers and disparities may impact patient satisfaction.,Medical records of 181 patients were retrospectively analyzed with parallel patient satisfaction evaluation by telephone interviews.,Overall mean lead times from initial diagnosis until completion of all surgery and histopathology reports were 80-100 days and delays occurred at every step of the process.,General practitioners performed excision biopsies faster however this was mitigated by slower histopathology processing.,University level CM care showed less lag time between excision biopsy, wide local excision for thick melanomas and histopathology confirmation.,University level care operated with twice the surgical margin as compared to general practitioners and non-university level specialists.,Male patients had larger excision biopsy margins and significantly shorter lead times than female patients.,Patient satisfaction rates were generally higher in the academic hospitals as compared to general practitioners and non-university dermatology clinics.,Surprisingly, there was no correlation between lead times and patient satisfaction.,Taken together, CM show substantial variation and caution should be practiced when using patient satisfaction as a quality indicator.

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

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

Melanoma arises from neural crest‐derived melanocytes which reside mostly in the skin in an adult organism.,Epithelial-mesenchymal transition (EMT) is a tumorigenic programme through which cells acquire mesenchymal, more pro‐oncogenic phenotype.,The reversible phenotype switching is an event still not completely understood in melanoma.,The EMT features and increased invasiveness are associated with lower levels of the pivotal lineage identity maintaining and melanoma‐specific transcription factor MITF (microphthalmia‐associated transcription factor), whereas increased proliferation is linked to higher MITF levels.,However, the precise role of MITF in phenotype switching is still loosely characterized.,To exclude the changes occurring upstream of MITF during MITF regulation in vivo, we employed a model whereby MITF expression was inducibly regulated by shRNA in melanoma cell lines.,We found that the decrease in MITF caused only moderate attenuation of proliferation of the whole cell line population.,Proliferation was decreased in five of 15 isolated clones, in three of them profoundly.,Reduction in MITF levels alone did not generally produce EMT‐like characteristics.,The stem cell marker levels also did not change appreciably, only a sharp increase in SOX2 accompanied MITF down‐regulation.,Oppositely, the downstream differentiation markers and the MITF transcriptional targets melastatin and tyrosinase were profoundly decreased, as well as the downstream target livin.,Surprisingly, after the MITF decline, invasiveness was not appreciably affected, independently of proliferation.,The results suggest that low levels of MITF may still maintain relatively high proliferation and might reflect, rather than cause, the EMT‐like changes occurring in melanoma.

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas.,In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS.,Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects.,We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling.,Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164).,We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C.,Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C.,Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma.,DOI:http://dx.doi.org/10.7554/eLife.01460.001,Cancer is a leading cause of death worldwide, and although cancer can have many causes, mutations to a small number of genes are often responsible for a large number of cancer cases.,For example, about 70% of cases of the deadliest form of skin cancer have mutations in two specific genes.,Many cancer-causing genes are regulated by small RNA molecules that impede the function of other genes by blocking the machinery that turns a gene into a functional protein.,However, until recently there was a limited understanding of the role of these microRNAs in the skin cancers caused by the most common mutations.,Now, Forloni et al. have looked at all the microRNAs present in cells carrying a mutated form of the one of these genes, and compared these with the microRNAs found in healthy cells.,A microRNA, called miR-146a, was discovered to be much more common in the cancer cells.,Moreover, production of this microRNA was increased by the two cancer-causing mutations.,Forloni et al. found that increasing production of this microRNA caused human skin cancer cells to grow faster, and also caused tumors to develop in mice.,Reducing the level of miR-146a had the opposite effect.,Forloni et al. also looked at cancer cells taken from individuals at different stages of skin cancer and found that, as the disease progresses, an unprocessed form of miR-146a tends to acquire a mutation, which leads to much higher levels of the active, processed form of miR-146a in the cancer cells.,High levels of miR-146a also switched off a gene that encodes for a protein that, in turn, switches off a protein called NOTCH that is linked to skin cancer.,As such, excess miR-146a actually increases the activation of the NOTCH protein.,These results led Forloni et al. to test if delivering drugs that block the production of miR-146a by inhibiting skin cancer-causing genes, along with other drugs that inhibit the Notch signaling pathway, could be more effective than treatment with either drug on its own.,The combined treatment was very effective against human skin cancer cells and could represent a promising development in the treatment of this disease.,DOI:http://dx.doi.org/10.7554/eLife.01460.002

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns.,They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes.,Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context.,Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined.,MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF).,By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211.,Expression of this miRNA is often reduced in melanoma samples.,Here, we investigated functional roles of miR-211 by identifying and studying new target genes.,We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively.,MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion.,These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range.,Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells.,When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses.,In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells.,Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca2+-modulated activities with tailored function.,DOI:http://dx.doi.org/10.7554/eLife.10024.001,Optogenetics is a technique that has been used to study nerve cells for several years.,It involves genetically engineering these cells to produce proteins from light-sensitive bacteria, and results in nerve cells that will either send, or stop sending, nerve impulses when they are exposed to a particular color of light.,Neuroscientists have learned a lot about brain circuits using the technique, and now researchers in many other fields are giving it a try.,There are, however, several challenges to using optogenetics in other types of cells.,Nerve cells create a tiny electrical impulses when they are activated, which helps them quickly transmit messages.,But other types of cells use more diverse means to communicate and transmit signals.,This means that optogenetics techniques must be adapted.,Additionally, many cells are located deep in the body and so getting the light to them can be difficult.,He, Zhang et al. have now developed an optogenetic system (termed “Opto-CRAC”) that can control immune cells buried deep in tissue.,The action of immune cells can be tuned by controlling the flow of calcium ions through gate-like proteins in their membranes.,He, Zhang et al. genetically engineered immune cells so that a calcium gate-controlling protein became light sensitive.,When the cells were exposed to a blue light the calcium ion gates opened.,When the light was turned off, the gates closed.,More intense light caused more calcium to enter into the cells.,Further experiments then revealed that exposing these engineered immune cells to blue light in the laboratory could trigger an immune response.,The next obstacle was getting light to immune cells in a live animal.,So, He, Zhang et al. used specific nanoparticles that have been shown to help transmit light deep within tissue.,In these experiments, mice were injected with the light-sensitive immune cells and the nanoparticles.,Then, a near-infrared laser beam that can transmit into the tissues was pointed at the mice.,This caused calcium channels to open in the engineered cells deep in the mice.,Finally, further experiments were used to show that this light-based stimulation could boost an immune response to aid the killing of cancer cells.,Other scientists will likely use the technique to help them study immune, heart, and other types of cells that use calcium to communicate.,DOI:http://dx.doi.org/10.7554/eLife.10024.002

Calcium signaling mediated by STIM1 and Orai1 activates Src to promote invadopodium assembly while simultaneously promoting MT1-MMP recycling to the plasma membrane to promote ECM degradation.,Ca2+ signaling has been increasingly implicated in cancer invasion and metastasis, and yet, the underlying mechanisms remained largely unknown.,In this paper, we report that STIM1- and Orai1-mediated Ca2+ oscillations promote melanoma invasion by orchestrating invadopodium assembly and extracellular matrix (ECM) degradation.,Ca2+ oscillation signals facilitate invadopodial precursor assembly by activating Src.,Disruption of Ca2+ oscillations inhibited invadopodium assembly.,Furthermore, STIM1 and Orai1 regulate the proteolysis activity of individual invadopodia.,Mechanistically, Orai1 blockade inhibited the recycling of MT1-matrix metalloproteinase (MMP) to the plasma membrane and entrapped MT1-MMP in the endocytic compartment to inhibit ECM degradation.,STIM1 knockdown significantly inhibited melanoma lung metastasis in a xenograft mouse model, implicating the importance of this pathway in metastatic dissemination.,Our findings provide a novel mechanism for Ca2+-mediated cancer cell invasion and shed new light on the spatiotemporal organization of store-operated Ca2+ signals during melanoma invasion and metastasis.

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

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.

Metastasis development represents an important threat for melanoma patients, even when diagnosed at early stages and upon removal of the primary tumor.,In this scenario, determination of prognostic biomarkers would be of great interest.,Serum contains information about the general status of the organism and therefore represents a valuable source for biomarkers.,Thus, we aimed to define serological biomarkers that could be used along with clinical and histopathological features of the disease to predict metastatic events on the early‐stage population of patients.,We previously demonstrated that in stage II melanoma patients, serum levels of dermcidin (DCD) were associated with metastatic progression.,Based on the relevance of the immune response on the cancer progression and the recent association of DCD with local and systemic immune response against cancer cells, serum DCD was analyzed in a new cohort of patients along with interleukin 4 (IL‐4), IL‐6, IL‐10, IL‐17A, interferon γ (IFN‐γ), transforming growth factor‐β (TGF‐ β), and granulocyte-macrophage colony‐stimulating factor (GM‐CSF).,We initially recruited 448 melanoma patients, 323 of whom were diagnosed as stages I‐II according to AJCC.,Levels of selected cytokines were determined by ELISA and Luminex, and obtained data were analyzed employing machine learning and Kaplan-Meier techniques to define an algorithm capable of accurately classifying early‐stage melanoma patients with a high and low risk of developing metastasis.,The results show that in early‐stage melanoma patients, serum levels of the cytokines IL‐4, GM‐CSF, and DCD together with the Breslow thickness are those that best predict melanoma metastasis.,Moreover, resulting algorithm represents a new tool to discriminate subjects with good prognosis from those with high risk for a future metastasis.,Melanoma displays a remarkable capacity for dissemination even when detected at early stages.,We developed a decision rule that considers Breslow thickness of the removed malignant lesion and IL‐4, GM‐CSF, and DCD serum levels in order to foresee the risk for future metastasis development of those stage I‐II patients.,This algorithm may represent a tool to design more personalized follow‐up strategies.

Over the past decade, several immunotherapies have been approved for the treatment of melanoma.,The most prominent of these are the immune checkpoint inhibitors, which are antibodies that block the inhibitory effects on the immune system by checkpoint receptors, such as CTLA-4, PD-1 and PD-L1.,Preclinically, blocking these receptors has led to increased activation and proliferation of effector cells following stimulation and antigen recognition, and subsequently, more effective elimination of cancer cells.,Translation from preclinical to clinical outcomes in solid tumors has shown the existence of a wide diversity of individual patient responses, linked to several patient-specific parameters.,We developed a quantitative systems pharmacology (QSP) model that looks at the mentioned checkpoint blockade therapies administered as mono-, combo- and sequential therapies, to show how different combinations of specific patient parameters defined within physiological ranges distinguish different types of virtual patient responders to these therapies for melanoma.,Further validation by fitting and subsequent simulations of virtual clinical trials mimicking actual patient trials demonstrated that the model can capture a wide variety of tumor dynamics that are observed in the clinic and can predict median clinical responses.,Our aim here is to present a QSP model for combination immunotherapy specific to melanoma.

Micro RNAs (miRs) have emerged as key regulators during oncogenesis.,They have been found to regulate cell proliferation, differentiation, and apoptosis.,Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma.,Our goal was therefore to further examine this theory.,We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis.,Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining.,The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.,In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm.,A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53.,Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.,Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.

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

Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis.,Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo.,Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines.,Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model.,Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth.,Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo.,Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols.

Ipilimumab, an antibody that enhances T-cell activation, may augment immunogenicity of tumor cells that are injured by radiation therapy.,We hypothesized that patients with melanoma brain metastasis treated with both ipilimumab and radiotherapy would have improved overall survival, and that the sequence of treatments may affect disease control in the brain.,We analyzed the clinical and radiographic records of melanoma patients with brain metastases who were treated with whole brain radiation therapy or stereotactic radiosurgery between 2005 and 2012.,The hazard ratios for survival were estimated to assess outcomes as a function of ipilimumab use and radiation type.,Seventy patients were identified, 33 of whom received ipilimumab and 37 who did not.,The patients who received ipilimumab had a censored median survival of 18.3 months (95% confidence interval 8.1-25.5), compared with 5.3 months (95% confidence interval 4.0-7.6) for patients who did not receive ipilimumab.,Ipilimumab and stereotactic radiosurgery were each significant predictors of improved overall survival (hazard ratio = 0.43 and 0.45, with P = 0.005 and 0.008, respectively).,Four of 10 evaluable patients (40.0%) who received ipilimumab prior to radiotherapy demonstrated a partial response to radiotherapy, compared with two of 22 evaluable patients (9.1%) who did not receive ipilimumab.,Ipilimumab is associated with a significantly reduced risk of death in patients with melanoma brain metastases who underwent radiotherapy, and this finding supports the need for multimodality therapy to optimize patient outcomes.,Prospective studies are needed and are underway.

Immunotherapy is regarded as one of the major breakthroughs in cancer treatment.,Despite its success, only a subset of patients responds-urging the quest for predictive biomarkers.,We hypothesize that artificial intelligence (AI) algorithms can automatically quantify radiographic characteristics that are related to and may therefore act as noninvasive radiomic biomarkers for immunotherapy response.,In this study, we analyzed 1055 primary and metastatic lesions from 203 patients with advanced melanoma and non-small-cell lung cancer (NSCLC) undergoing anti-PD1 therapy.,We carried out an AI-based characterization of each lesion on the pretreatment contrast-enhanced CT imaging data to develop and validate a noninvasive machine learning biomarker capable of distinguishing between immunotherapy responding and nonresponding.,To define the biological basis of the radiographic biomarker, we carried out gene set enrichment analysis in an independent dataset of 262 NSCLC patients.,The biomarker reached significant performance on NSCLC lesions (up to 0.83 AUC, P < 0.001) and borderline significant for melanoma lymph nodes (0.64 AUC, P = 0.05).,Combining these lesion-wide predictions on a patient level, immunotherapy response could be predicted with an AUC of up to 0.76 for both cancer types (P < 0.001), resulting in a 1-year survival difference of 24% (P = 0.02).,We found highly significant associations with pathways involved in mitosis, indicating a relationship between increased proliferative potential and preferential response to immunotherapy.,These results indicate that radiographic characteristics of lesions on standard-of-care imaging may function as noninvasive biomarkers for response to immunotherapy, and may show utility for improved patient stratification in both neoadjuvant and palliative settings.

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

Adaptive responses have been demonstrated to limit activity to targeted therapies.,Saei et al. show that loss of USP28/FBW7-mediated BRAF degradation is observed in a proportion of melanoma patients and can be responsible for resistance through upregulation of MAPK signaling pathway.,RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma.,However, rarely do tumors regress completely, with the majority of responses being short-lived.,This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling.,Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members.,Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models.,Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients.,Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors.,Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

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

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

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

Oncogene-driven metabolic rewiring is an adaptation to low nutrient and oxygen conditions in the tumor microenvironment that enables cancer cells of diverse origin to hyperproliferate.,Aerobic glycolysis and enhanced reliance on glutamine utilization are prime examples of such rewiring.,However, tissue of origin as well as specific genetic and epigenetic changes determines gene expression profiles underlying these metabolic alterations in specific cancers.,In melanoma, activation of the MAPK pathway driven by mutant BRAF or NRAS is a primary cause of malignant transformation.,Activity of the MAPK pathway, as well as other factors, such as HIF1α, Myc and MITF, are among those that control the balance between non-oxidative and oxidative branches of central carbon metabolism.,Here, we discuss the nature of metabolic alterations that underlie melanoma development and affect its response to therapy.

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

We recently demonstrated that upregulation of a chemokine receptor CCR6 and its ligand CCL20 led to metastasis of advanced cutaneous T-cell lymphoma (CTCL) cells, suggesting the involvement of CCL20-CCR6 interaction in initiating CTCL cell metastasis.,In this study, we determined whether this interaction is functional in metastatic CTCL cells.,We first demonstrated increased STAT3 expression during the progression of primary CTCL.,STAT3 was spontaneously activated and mediated the transcription of CCL20 in CTCL cell lines.,Next, to determine whether the transient knockdown of STAT3, CCL20, or CCR6 or treatment with neutralizing antibody against CCL20 (neutralizing CCL20 antibody) could reduce the migration ability of CTCL cells, we conducted an in vitro migration assay.,All treatments reduced the nutrition-dependent migration activity of CTCL cells.,Notably, treatment with neutralizing CCL20 antibody reduced the migration ability of the cells without decreasing the expression of CCL20 and CCR6.,This demonstrated that the CCL20-CCR6 interaction is actually functional in metastatic CTCL cells.,Finally, to examine the in vivo effect of neutralizing CCL20 antibody, we used NOD/Shi-scid IL-2γnul mice inoculated with CTCL cells.,These mice were expected to die due to metastasis of CTCL cells into multiple organs.,However, administration of neutralizing CCL20 antibody significantly prolonged the survival of the xenografted mice.,These findings suggested that automatic activation of the STAT3/CCL20/CCR6 cascade was involved in CTCL lymphomagenesis and that disruption of CCL20-CCR6 interaction could be a key therapeutic strategy against advanced CTCL.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes.,We performed exome and whole genome DNA sequence and RNA sequencing on purified CTCL and matched normal cells.,The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NFκB signaling, chromatin remodeling, and DNA damage response.,CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs vs.,1.0 somatic single nucleotide variants per CTCL).,These findings have implications for novel therapeutics.