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We have investigated the role of vascular-endothelial (VE)-cadherin in melanoma and breast cancer metastasis.,We found that VE-cadherin is expressed in highly aggressive melanoma and breast cancer cell lines.,Remarkably, inactivation of VE-cadherin triggered a significant loss of malignant traits (proliferation, adhesion, invasion and transendothelial migration) in melanoma and breast cancer cells.,These effects, except transendothelial migration, were induced by the VE-cadherin RGD motifs.,Co-immunoprecipitation experiments demonstrated an interaction between VE-cadherin and α2β1 integrin, with the RGD motifs found to directly affect β1 integrin activation.,VE-cadherin-mediated integrin signaling occurred through specific activation of SRC, ERK and JNK, including AKT in melanoma.,Knocking down VE-cadherin suppressed lung colonization capacity of melanoma or breast cancer cells inoculated in mice, while pre-incubation with VE-cadherin RGD peptides promoted lung metastasis for both cancer types.,Finally, an in silico study revealed the association of high VE-cadherin expression with poor survival in a subset of melanoma patients and breast cancer patients showing low CD34 expression.,These findings support a general role for VE-cadherin and other RGD cadherins as critical regulators of lung and liver metastasis in multiple solid tumours.,These results pave the way for cadherin-specific RGD targeted therapies to control disseminated metastasis in multiple cancers.

Metastatic melanoma is a highly aggressive skin cancer and currently resistant to systemic therapy.,Melanomas may involve genetic, epigenetic and metabolic abnormalities.,Evidence is emerging that epigenetic changes might play a significant role in tumor cell plasticity and metastatic phenotype of melanoma cells.,In this study, we developed a systematic approach to identify genes implicated in melanoma progression.,To do this, we used the Affymetrix GeneChip Arrays to screen 34,000 mouse transcripts in melan-a melanocytes, 4C pre-malignant melanocytes, 4C11− non-metastatic and 4C11+ metastatic melanoma cell lines.,The genome-wide association studies revealed pathways commonly over-represented in the transition from immortalized to pre-malignant stage, and under-represented in the transition from non-metastatic to metastatic stage.,Additionally, the treatment of cells with 10 µM 5-aza-2′-deoxycytidine (5AzaCdR) for 48 hours allowed us to identify genes differentially re-expressed at specific stages of melan-a malignant transformation.,Treatment of human primary melanocytes with the demethylating agent 5AzaCdR in combination to the histone deacetylase inhibitor Trichostatin A (TSA) revealed changes on melanocyte morphology and gene expression which could be an indicator of epigenetic flexibility in normal melanocytes.,Moreover, changes on gene expression recognized by affecting the melanocyte biology (NDRG2 and VDR), phenotype of metastatic melanoma cells (HSPB1 and SERPINE1) and response to cancer therapy (CTCF, NSD1 and SRC) were found when Mel-2 and/or Mel-3-derived patient metastases were exposed to 5AzaCdR plus TSA treatment.,Hierarchical clustering and network analyses in a panel of five patient-derived metastatic melanoma cells showed gene interactions that have never been described in melanomas.,Despite the heterogeneity observed in melanomas, this study demonstrates the utility of our murine melanoma progression model to identify molecular markers commonly perturbed in metastasis.,Additionally, the novel gene expression signature identified here may be useful in the future into a model more closely related to translational research.

Numerous tyrosine kinase inhibitors (TKIs) targeting c-Met are currently in clinical trials for several cancers.,Their efficacy is limited due to the development of resistance.,The present study aims to elucidate this mechanism of c-Met TKI resistance by investigating key mTOR and Wnt signaling proteins in melanoma cell lines resistant to SU11274, a c-Met TKI.,Xenografts from RU melanoma cells treated with c-Met TKIs SU11274 and JNJ38877605 showed a 7- and 6-fold reduction in tumor size, respectively.,Resistant cells displayed upregulation of phosphorylated c-Met, mTOR, p70S6Kinase, 4E-BP1, ERK, LRP6, and active β-catenin.,In addition, GATA-6, a Wnt signaling regulator, was upregulated, and Axin, a negative regulator of the Wnt pathway, was downregulated in resistant cells.,Modulation of these mTOR and Wnt pathway proteins was also prevented by combination treatment with SU11274, everolimus, an mTOR inhibitor, and XAV939, a Wnt inhibitor.,Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells.,The V600E BRAF mutation was found to be positive only in MU cells.,Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability.,These studies indicate activation of mTOR and Wnt signaling pathways in c-Met TKI resistant melanoma cells and suggest that concurrent targeting of c-Met, mTOR, and Wnt pathways and BRAF may improve efficacy over traditional TKI monotherapy in melanoma patients.

E1694 tested GM2-KLH-QS21 vaccine versus high-dose interferon-α2b (HDI) as adjuvant therapy for operable stage IIB-III melanoma.,We tested banked serum specimens from patients in the vaccine arm of E1694 for prognostic biomarkers.,Aushon Multiplex Platform was used to quantitate baseline serum levels of 115 analytes from 40 patients.,Least absolute shrinkage and selection operator proportional hazard regression (Lasso PH) was used to select markers that are most informative for relapse-free survival (RFS) and overall survival (OS).,Regular Cox PH models were then fit with the markers selected by the Lasso PH.,Survival receiver operating characteristic (ROC) analysis was used to evaluate the ability of the models to predict 1-year RFS and 5-year OS.,Four markers that include Tumor Necrosis Factor alpha Receptor II (TNF-RII), Transforming Growth Factor alpha (TGF-α), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), and C-reactive protein (CRP) were found to be most informative for the prediction of OS (high levels correlate with worse prognosis).,The dichotomized risk score based on the four markers could significantly separate the OS curves (p = 0.0005).,When using the four-marker PH model to predict 5-year OS, we achieved an area under the curve (AUC) of 89% (cross validated AUC = 72%).,High baseline TNF-RII was also significantly associated with worse RFS.,The RFS with high (above median) TNF-RII was significantly lower than low TNF-RII (p = 0.01).,The biomarker signature consisting of TNFR-II, TGF-α, TIMP-1 and CRP is significantly prognostic of survival in patients with high-risk melanoma and warrants further investigation.

Non-melanoma skin cancers (NMSCs) are the leading cause of skin cancer-related morbidity and mortality.,Effective strategies are needed to control NMSC occurrence and progression.,Non-toxic, plant-derived extracts have been shown to exert multiple anti-cancer effects.,Graviola (Annona muricata), a tropical fruit-bearing plant, has been used in traditional medicine against multiple human diseases including cancer.,The current study investigated the effects of graviola leaf and stem extract (GLSE) and its solvent-extracted fractions on two human NMSC cell lines, UW-BCC1 and A431.,GLSE was found to: (i) dose-dependently suppress UW-BCC1 and A431 cell growth, motility, wound closure, and clonogenicity; (ii) induce G0/G1 cell cycle arrest by downregulating cyclin/cdk factors while upregulating cdk inhibitors, and (iii) induce apoptosis as evidenced by cleavage of caspases-3, -8 and PARP.,Further, GLSE suppressed levels of activated hedgehog (Hh) pathway components Smo, Gli 1/2, and Shh while inducing SuFu.,GLSE also decreased the expression of pro-apoptotic protein Bax while decreasing the expression of the anti-apoptotic protein Bcl-2.,We determined that these activities were concentrated in an acetogenin/alkaloid-rich dichloromethane subfraction of GLSE.,Our data identify graviola extracts and their constituents as promising sources for new chemopreventive and therapeutic agent(s) to be further developed for the control of NMSCs.

The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models.,This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients.,In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma.,The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%).,An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly.,Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss.,No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease.,The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo.,Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit.,Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ.,This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity.,Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted.

Circulating tumour DNA (ctDNA) may serve as a measure of tumour burden and a useful tool for non-invasive monitoring of cancer.,However, ctDNA is not always detectable in patients at time of diagnosis of metastatic disease.,Therefore, there is a need to understand the correlation between ctDNA levels and the patients’ overall metabolic tumour burden (MTB).,Thirty-two treatment naïve metastatic melanoma patients were included in the study.,MTB and metabolic tumour volume (MTV) was measured by 18F-fluoro-D-glucose positron emission tomography/computed tomography (FDG PET/CT).,Plasma ctDNA was quantified using droplet digital PCR (ddPCR).,CtDNA was detected in 23 of 32 patients.,Overall, a significant correlation was observed between ctDNA levels and MTB (p < 0.001).,CtDNA was not detectable in patients with an MTB of ≤10, defining this value as the lower limit of tumour burden that can be detected through ctDNA analysis by ddPCR.,We showed that ctDNA levels measured by ddPCR correlate with MTB in treatment naïve metastatic melanoma patients and observed a limit in tumour size for which ctDNA cannot be detected in blood.,Nevertheless, our findings support the use of ctDNA as a non-invasive complementary modality to functional imaging for monitoring tumour burden.,The online version of this article (10.1186/s12885-018-4637-6) contains supplementary material, which is available to authorized users.

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene.,With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality.,BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18).,Nineteen patients had tissues available from multiple metastatic sites.,Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR.,The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes.,To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases.,Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors.,Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation.,In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients.,Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.

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

S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation.,Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival.,In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial.,To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas.,The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry.,The Kaplan-Meier method was used to evaluate the patient survival.,The univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up.,Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas.,Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II-IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (P = 0.005).,Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) but not metastatic melanoma (P>0.05).,This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival.,Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease.

Melanoma is an aggressive malignancy with a high metastatic potential. microRNA-17 (miR-17) is a member of the oncogenic miR-17/92 cluster.,Here we study the effect of miR-17 on melanoma cell motility.,Over expression of the mature or pri-microRNA form of miR-17 in WM-266-4 and 624mel melanoma lines enhances cell motility, evident in both wound healing and transwell migration assays.,TargetScan algorithm predicts the PEA3-subfamily member ETV1 as a direct target of miR-17.,Indeed, a 3-4-fold decrease of ETV1 protein levels are observed following miR-17 transfection into the various melanoma lines, with no significant change in ETV1 mRNA expression.,Dual luciferase experiments demonstrate direct binding of miR-17 to the 3′-untranslated region of ETV1, confirmed by abolishing point mutations in the putative binding site.,These combined results suggest regulation of ETV1 by miR-17 by a direct translational repression.,Further, in both melanoma cell lines ETV1 knockdown by selective siRNA successfully pheno-copies the facilitated cell migration, while overexpression of ETV1 inhibits cell motility and migration.,Altered ETV1 expression does not affect melanoma net-proliferation.,In conclusion, we show a new role for miR-17 in melanoma, facilitating cell motility, by targeting the translation of ETV1 protein, which may support the development of metastasis.

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

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

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.

The objective of the study was to summarize the role of DNA methylation in the development and metastasis of uveal melanoma (UM).,The relevant studies in MEDLINE were searched.,In this review, we performed a comprehensive literature search in MEDLINE using “uveal melanoma” AND (“DNA methylation” OR “epigenetics”) for original research/review articles published before February 2018 on the relationship between DNA methylation and UM.,References of the retrieved studies were also examined to search for potentially relevant papers.,Previous studies on the relationship between DNA methylation and UM covered many genes including tumor suppressor genes (TSGs), cyclin-dependent kinase genes, and other genes.,Among them, the TSG genes such as RASSF1A and p16INK4a, which encodes a cyclin-dependent kinase inhibitor, are relatively well-studied genes.,Specifically, a high percentage of promoter methylation of RASSF1A was observed in UM cell lines and/or patients with UM.,Promoter methylation of RASSF1A was also associated with the development of metastasis.,Similarly, a high percentage of promoter hypermethylation of p16INK4a was found in UM cell lines.,DNA promoter methylation can control the expression of p16INK4a, which affect cell growth, migration, and invasion in UM.,Many other genes might also be involved in the pathogenesis of UM such as the Ras and EF-hand domain containing (RASEF) gene, RAB31, hTERT, embryonal fyn-associated substrate, and deleted in split-hand/split-foot 1.,Our review reveals the complex mechanisms underlying the tumorigenesis of UM and highlights the great needs of future studies to discover more genes/5’-C-phosphate-G-3’ sites contributing to the development/metastasis of UM and explore the mechanisms through which epigenetic changes exert their function in UM.

In this review, Pandiani et al. discuss the differences in tumorigenic processes (etiologic factors and genetic alterations) and tumor biology (gene expression and signaling pathways) between cutaneous melanoma and uveal melanoma.,Cutaneous melanoma (CM) and uveal melanoma (UM) derive from cutaneous and uveal melanocytes that share the same embryonic origin and display the same cellular function.,However, the etiopathogenesis and biological behaviors of these melanomas are very different.,CM and UM display distinct landscapes of genetic alterations and show different metastatic routes and tropisms.,Hence, therapeutic improvements achieved in the last few years for the treatment of CM have failed to ameliorate the clinical outcomes of patients with UM.,The scope of this review is to discuss the differences in tumorigenic processes (etiologic factors and genetic alterations) and tumor biology (gene expression and signaling pathways) between CM and UM.,We develop hypotheses to explain these differences, which might provide important clues for research avenues and the identification of actionable vulnerabilities suitable for the development of new therapeutic strategies for metastatic UM.

Malignant melanoma displays a high degree of cellular plasticity during disease progression.,Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation.,Here we uncover a relationship between geometric features at perimeter regions of melanoma aggregates, and reprogramming to a stem cell-like state through histone marks H3K4Me2 and H3K9Ac.,Using an in vitro tumor microengineering approach, we find spatial enrichment of these histone modifications with concurrent expression of stemness markers.,The epigenetic modifier PRDM14 overlaps with H3K9Ac and shows elevated expression in cells along regions of perimeter curvature. siRNA knockdown of PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity.,Our results suggest mechanotransduction at the periphery of melanoma aggregates may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.,Junmin Lee et al. study the role of geometric features at the perimeter regions of melanoma aggregates in programming stem cell-like state through histone marks.,They use a tumor microengineering approach in vitro and report a spatial enrichment of histone modifications with stemness markers.,Their work uncovers a mechanotransduction signaling that regulates epigenetic modifiers to regulate tumorigenicity.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts.,However, functional contributions of glycans to cancer initiation and progression remain poorly understood.,Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes.,This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans.,We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival.,Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death.,More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins.,Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.,Aberrant glycosylation patterns on cancer cells promote several pro-tumorigenic functions, including enhancing tumor cell proliferation.,Here the authors provide data that show melanoma cells downregulate GCNT2 with consequent loss of I-branched glycans; this leads to the formation of extended i-linear glycans and enhances melanoma growth via increases, in part, by IGF-1- and extracellular matrix-induced signaling.

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

SOX10 was identified as a methylated gene in our previous cancer methylome study.,Here we further analyzed its epigenetic inactivation, biological functions and related cell signaling in digestive cancers (colorectal, gastric and esophageal cancers) in detail.,SOX10 expression was decreased in multiple digestive cancer cell lines as well as primary tumors due to its promoter methylation.,Pharmacologic or genetic demethylation reversed SOX10 silencing.,Ectopic expression of SOX10in SOX10-deficient cancer cells inhibits their proliferation, tumorigenicity, and metastatic potentials in vitro and in vivo.,SOX10 also suppressed the epithelial to mesenchymal transition (EMT) and stemness properties of digestive tumor cells.,Mechanistically, SOX10 competes with TCF4 to bind β-catenin and transrepresses its downstream target genes via its own DNA-binding property.,SOX10 mutations that disrupt the SOX10-β-catenin interaction partially prevented tumor suppression.,SOX10is thus a commonly inactivated tumor suppressor that antagonizes Wnt/β-catenin signaling in cancer cells from different digestive tissues.

Plenty of evidence has suggested that long non-coding RNAs (lncRNAs) have played a vital part may act as prognostic biomarkers in a variety of cancers.,The aim of this study was to screen survival-related lncRNAs and to construct a lncRNA-based prognostic model in patients with cutaneous melanoma (CM).,We obtained lncRNAs expression profiles and clinicopathological data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases.,A lncRNA-based prognostic model was established in training set.,The established prognostic model was evaluated, and validated in the validation set.,Then, a prognostic nomogram combining the lncRNA-based risk score and clinicopathological characteristics was developed in training set, and assessed in the validation set.,The accuracy of the model was evaluated by the discrimination and calibration plots.,A total of 212 lncRNAs were identified to be differentially expressed in CM.,After univariate analysis, LASSO penalized regression analysis, and multivariate analysis, 3 lncRNAs were used to construct risk score model.,The proposed risk score model could divide patients into high-risk and low-risk groups with significantly different survival in both training set and validation set.,The ROC curve showed good performance in survival prediction in both sets.,Furthermore, the nomogram for predicting 3-, 5-, and 10-year OS was established based on lncRNA-based risk score and clinicopathologic factors.,The prognostic accuracy of the risk model was confirmed by the discrimination and calibration plots in both training set and validation set.,We established a novel three lncRNA-based risk score model and nomogram to predict overall survival of CM.,The proposed nomogram may provide information for individualized treatment in CM patients.

Previous work identified RMEL3 as a lncRNA with enriched expression in melanoma.,Analysis of The Cancer Genome Atlas (TCGA) data confirmed RMEL3 enriched expression in melanoma and demonstrated its association with the presence of BRAFV600E.,RMEL3 siRNA-mediated silencing markedly reduced (95%) colony formation in different BRAFV600E melanoma cell lines.,Multiple genes of the MAPK and PI3K pathways found to be correlated with RMEL3 in TCGA samples were experimentally confirmed.,RMEL3 knockdown led to downregulation of activators or effectors of these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2.,RMEL3 knockdown induces gain of protein levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1.,Consistently, knockdown resulted in an accumulation of cells in G1 phase and subG0/G1 in an asynchronously growing population.,Thus, TCGA data and functional experiments demonstrate that RMEL3 is required for MAPK and PI3K signaling, and its knockdown decrease BRAFV600E melanoma cell survival and proliferation.

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

Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths.,BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60-70% of malignant melanomas.,The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion.,In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK.,These pathways are potential targets for prevention and treatment of melanoma.,In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism.,Treatment of multiple human malignant melanoma cell lines with fisetin (5-20 µM) resulted in inhibition of cell invasion.,BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2.,In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway.,Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion.,Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein).,Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin.,These results indicate that fisetin inhibits melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways.

Uveal melanoma (UM) represents the most frequent primary tumor of the eye.,Despite the development of new drugs and screening programs, the prognosis of patients with UM remains poor and no effective prognostic biomarkers are yet able to identify high-risk patients.,Therefore, in the present study, microRNA (miRNA or miR) expression data, contained in the TCGA UM (UVM) database, were analyzed in order to identify a set of miRNAs with prognostic significance to be used as biomarkers in clinical practice.,Patients were stratified into 2 groups, including tumor stage (high-grade vs. low-grade) and status (deceased vs. alive); differential analyses of miRNA expression among these groups were performed.,A total of 20 deregulated miRNAs for each group were identified.,In total 7 miRNAs were common between the groups.,The majority of common miRNAs belonged to the miR-506-514 cluster, known to be involved in UM development.,The prognostic value of the 20 selected miRNAs related to tumor stage was assessed.,The deregulation of 12 miRNAs (6 upregulated and 6 downregulated) was associated with a worse prognosis of patients with UM.,Subsequently, miRCancerdb and microRNA Data Integration Portal bioinformatics tools were used to identify a set of genes associated with the 20 miRNAs and to establish their interaction levels.,By this approach, 53 different negatively and positively associated genes were identified.,Finally, DIANA-mirPath prediction pathway and Gene Ontology enrichment analyses were performed on the lists of genes previously generated to establish their functional involvement in biological processes and molecular pathways.,All the miRNAs and genes were involved in molecular pathways usually altered in cancer, including the mitogen-activated protein kinase (MAPK) pathway.,Overall, the findings of the presents study demonstrated that the miRNAs of the miR-506-514 cluster, hsa-miR-592 and hsa-miR-199a-5p were the most deregulated miRNAs in patients with high-grade disease compared to those with low-grade disease and were strictly related to the overall survival (OS) of the patients.,However, further in vitro and translational approaches are required to validate these preliminary findings.

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

The high mortality rate of melanoma is broadly associated with its metastatic potential.,Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis.,Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients.,Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay.,Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P‐value = 0.044) and the lymphangiogenic receptor VEGFR‐3 (P‐value = 0.002) were independent prognostic factors of overall survival in melanoma patients.,Enhanced reduced representation bisulfite sequencing‐based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells.,In patients, VEGFC (P‐value = 0.031), ANGPT2 (P‐value < 0.001), and SIX1 (P‐value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival.,Hence, our data suggest that these angio‐ and lymphangiogenesis factors are potential biomarkers of melanoma prognosis.,Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.

In melanoma, the presence of promoter related hypermethylation has previously been reported, however, no methylation-based distinction has been drawn among the diverse melanoma subtypes.,Here, we investigated DNA methylation changes associated with melanoma progression and links between methylation patterns and other types of somatic alterations, including the most frequent mutations and DNA copy number changes.,Our results revealed that the methylome, presenting in early stage samples and associated with the BRAFV600E mutation, gradually decreased in the medium and late stages of the disease.,An inverse relationship among the other predefined groups and promoter methylation was also revealed except for histologic subtype, whereas the more aggressive, nodular subtype melanomas exhibited hypermethylation as well.,The Breslow thickness, which is a continuous variable, allowed for the most precise insight into how promoter methylation decreases from stage to stage.,Integrating our methylation results with a high-throughput copy number alteration dataset, local correlations were detected in the MYB and EYA4 genes.,With regard to the effects of DNA hypermethylation on melanoma patients' survival, correcting for clinical cofounders, only the KIT gene was associated with a lower overall survival rate.,In this study, we demonstrate the strong influence of promoter localized DNA methylation changes on melanoma initiation and show how hypermethylation decreases in melanomas associated with less favourable clinical outcomes.,Furthermore, we establish the methylation pattern as part of an integrated apparatus of somatic DNA alterations.

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.

Supplemental Digital Content is available in the text.,The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) immune checkpoints are negative regulators of T-cell immune function.,Inhibition of these targets, resulting in increased activation of the immune system, has led to new immunotherapies for melanoma, non-small cell lung cancer, and other cancers.,Ipilimumab, an inhibitor of CTLA-4, is approved for the treatment of advanced or unresectable melanoma.,Nivolumab and pembrolizumab, both PD-1 inhibitors, are approved to treat patients with advanced or metastatic melanoma and patients with metastatic, refractory non-small cell lung cancer.,In addition the combination of ipilimumab and nivolumab has been approved in patients with BRAF WT metastatic or unresectable melanoma.,The roles of CTLA-4 and PD-1 in inhibiting immune responses, including antitumor responses, are largely distinct.,CTLA-4 is thought to regulate T-cell proliferation early in an immune response, primarily in lymph nodes, whereas PD-1 suppresses T cells later in an immune response, primarily in peripheral tissues.,The clinical profiles of immuno-oncology agents inhibiting these 2 checkpoints may vary based on their mechanistic differences.,This article provides an overview of the CTLA-4 and PD-1 pathways and implications of their inhibition in cancer therapy.

Therapies targeting anti-tumor T-cell responses have proven successful in the treatment of a variety of malignancies.,However, as most patients still fail to respond, approaches to augment immunotherapeutic efficacy are needed.,Here, we investigated the ability of histone deacetylase 6 (HDAC6)-selective inhibitors to decrease immunosuppression and enhance immune function of melanoma patient T-cells in ex vivo cultures.,T-cells were harvested from peripheral blood or tumor biopsies of metastatic melanoma patients and cultured in the presence of pan-, class-specific or class-selective histone deacetylase (HDAC) inhibitors.,Changes in cytokine production were evaluated by Luminex and intracellular flow cytometry staining.,Expression of surface markers, transcription factors, protein phosphorylation, and cell viability were assessed by flow cytometry.,Changes in chromatin structure were determined by ATAC-seq.,T-cell viability was impaired with low doses of pan-HDAC inhibitors but not with specific or selective HDAC inhibitors.,The HDAC6-selective inhibitors ACY-1215 (ricolinostat) and ACY-241 (citarinostat) decreased Th2 cytokine production (i.e.,IL-4, IL-5, IL-6, IL-10 and IL-13).,Expansion of peripheral blood T-cells from melanoma patients in the presence of these inhibitors resulted in downregulation of the Th2 transcription factor GATA3, upregulation of the Th1 transcription factor T-BET, accumulation of central memory phenotype T-cells (CD45RA-CD45RO + CD62L + CCR7+), reduced exhaustion-associated phenotypes (i.e.,TIM3 + LAG3 + PD1+ and EOMES+PD1+), and enhanced killing in mixed lymphocyte reactions.,The frequency, FOXP3 expression, and suppressive function of T regulatory cells (Tregs) were decreased after exposure to ACY-1215 or ACY-241.,Higher frequencies of T-cells expressing CD107a + IFNγ+ and central memory markers were observed in melanoma tumor-infiltrating lymphocytes (TIL), which persisted after drug removal and further expansion.,After ACY-1215 treatment, increased chromatin accessibility was observed in regions associated with T-cell effector function and memory phenotypes, while condensed chromatin was found in regions encoding the mTOR downstream molecules AKT, SGK1 and S6K.,Decreased phosphorylation of these proteins was observed in ACY-1215 and ACY-241-treated T-cells.,AKT- and SGK1-specific inhibition recapitulated the increase in central memory frequency and decrease in IL-4 production, respectively, similar to the observed effects of HDAC6-selective inhibition.,HDAC6-selective inhibitors augmented melanoma patient T-cell immune properties, providing a rationale for translational investigation assessing their potential clinical efficacy.,The online version of this article (10.1186/s40425-019-0517-0) contains supplementary material, which is available to authorized users.

Immunotherapy is among the most rapidly evolving treatment strategies in oncology.,The therapeutic potential of immune-checkpoint inhibitors is exemplified by the recent hail of Food and Drug Administration (FDA) approvals for their use in various malignancies.,Continued efforts to enhance outcomes with immunotherapy agents have led to the formulation of advanced treatment strategies.,Recent evidence from pre-clinical studies evaluating immune-checkpoint inhibitors in various cancer cell-lines has suggested that combinatorial approaches may have superior survival outcomes compared to single-agent immunotherapy regimens.,Preliminary trials assessing combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 immune-checkpoint inhibitors have documented considerable advantages in survival indices over single-agent immunotherapy.,The therapeutic potential of combinatorial approaches is highlighted by the recent FDA approval of nivolumab plus ipilimumab for patients with advanced melanoma.,Presently, dual-immune checkpoint inhibition with anti-programmed death receptor-1/programmed cell death receptor- ligand-1 (anti-PD-1/PD-L1) plus anti-cytotoxic T lymphocyte associated antigen-4 (anti-CTLA-4) monoclonal antibodies (MoAbs) is being evaluated for a wide range of tumor histologies.,Furthermore, several ongoing clinical trials are investigating combination checkpoint inhibition in association with traditional treatment modalities such as chemotherapy, surgery, and radiation.,In this review, we summarize the current landscape of combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 MoAbs for patients with melanoma and non-small cell lung cancer (NSCLC).,We present a synopsis of the prospects for expanding the indications of dual immune-checkpoint inhibition therapy to a more diverse set of tumor histologies.

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

Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.

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

Quantitative assessment of key proteins that control the tumor-immune interface is one of the most formidable analytical challenges in immunotherapeutics.,We developed a targeted MS platform to quantify programmed cell death-1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death 1 ligand 2 (PD-L2) at fmol/microgram protein levels in formalin fixed, paraffin-embedded sections from 22 human melanomas.,PD-L1 abundance ranged 50-fold, from ∼0.03 to 1.5 fmol/microgram protein and the parallel reaction monitoring (PRM) data were largely concordant with total PD-L1-positive cell content, as analyzed by immunohistochemistry (IHC) with the E1L3N antibody.,PD-1 was measured at levels up to 20-fold lower than PD-L1, but the abundances were not significantly correlated (r2 = 0.062, p = 0.264).,PD-1 abundance was weakly correlated (r2 = 0.3057, p = 0.009) with the fraction of lymphocytes and histiocytes in sections.,PD-L2 was measured from 0.03 to 1.90 fmol/microgram protein and the ratio of PD-L2 to PD-L1 abundance ranged from 0.03 to 2.58.,In 10 samples, PD-L2 was present at more than half the level of PD-L1, which suggests that PD-L2, a higher affinity PD-1 ligand, is sufficiently abundant to contribute to T-cell downregulation.,We also identified five branched mannose and N-acetylglucosamine glycans at PD-L1 position N192 in all 22 samples.,Extent of PD-L1 glycan modification varied by ∼10-fold and the melanoma with the highest PD-L1 protein abundance and most abundant glycan modification yielded a very low PD-L1 IHC estimate, thus suggesting that N-glycosylation may affect IHC measurement and PD-L1 function.,Additional PRM analyses quantified immune checkpoint/co-regulator proteins LAG3, IDO1, TIM-3, VISTA, and CD40, which all displayed distinct expression independent of PD-1, PD-L1, and PD-L2.,Targeted MS can provide a next-generation analysis platform to advance cancer immuno-therapeutic research and diagnostics.

The indoleamine 2,3-dioxygenase (IDO) pathway is a key counter-regulatory mechanism that, in cancer, is exploited by tumors to evade antitumor immunity.,Indoximod is a small-molecule IDO pathway inhibitor that reverses the immunosuppressive effects of low tryptophan (Trp) and high kynurenine (Kyn) that result from IDO activity.,In this study, indoximod was used in combination with a checkpoint inhibitor (CPI) pembrolizumab for the treatment for advanced melanoma.,Patients with advanced melanoma were enrolled in a single-arm phase II clinical trial evaluating the addition of indoximod to standard of care CPI approved for melanoma.,Investigators administered their choice of CPI including pembrolizumab (P), nivolumab (N), or ipilimumab (I).,Indoximod was administered continuously (1200 mg orally two times per day), with concurrent CPI dosed per US Food and Drug Administration (FDA)-approved label.,Between July 2014 and July 2017, 131 patients were enrolled.,(P) was used more frequently (n=114, 87%) per investigator’s choice.,The efficacy evaluable population consisted of 89 patients from the phase II cohort with non-ocular melanoma who received indoximod combined with (P).,The objective response rate (ORR) for the evaluable population was 51% with confirmed complete response of 20% and disease control rate of 70%.,Median progression-free survival was 12.4 months (95% CI 6.4 to 24.9).,The ORR for Programmed Death-Ligand 1 (PD-L1)-positive patients was 70% compared with 46% for PD-L1-negative patients.,The combination was well tolerated, and side effects were similar to what was expected from single agent (P).,In this study, the combination of indoximod and (P) was well tolerated and showed antitumor efficacy that is worth further evaluation in selected patients with advanced melanoma.

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

Melanoma rates continue to increase; however, few risk factors other than sun sensitivity and ultraviolet radiation (including sun exposure) have been identified.,Although studies of farmers have shown an excess risk of melanoma and other skin cancers, it is unclear how much of this is related to sun exposure compared with other agricultural exposures.,We examined dose-response relationships for 50 agricultural pesticides and cutaneous melanoma incidence in the Agricultural Health Study cohort of licensed pesticide applicators, along with ever use of older pesticides that contain arsenic.,Logistic regression was used to examine odds ratios (ORs) and 95% confidence intervals (CIs) associated with pesticide exposure adjusted for age, sex, and other potential confounders.,We found significant associations between cutaneous melanoma and maneb/mancozeb (63 exposure days: OR = 2.4; 95% CI, 1.2-4.9; trend p = 0.006), parathion (≥ 56 exposure days: OR = 2.4; 95% CI, 1.3-4.4; trend p = 0.003), and carbaryl (≥ 56 exposure days: OR = 1.7; 95% CI, 1.1-2.5; trend p = 0.013).,Other associations with benomyl and ever use of arsenical pesticides were also suggested.,Most previous melanoma literature has focused on host factors and sun exposure.,Our research shows an association between several pesticides and melanoma, providing support for the hypotheses that agricultural chemicals may be another important source of melanoma risk.

Primary cutaneous melanoma still constitutes the main cause of skin cancer death in developed countries, and its incidence in recent years has been increasing in a steady, worrisome manner.,This study evaluated the clinical, epidemiological and demographic aspects of this disease, and correlated them with patient prognosis.,Using epidemiologic and clinical data, we analyzed 84 patients with mild to severe primary cutaneous melanoma treated between 1990 and 2007.,Slides containing surgical specimens were analyzed, and new slides were made from archived paraffin sections when necessary.,The melanoma incidence was higher in areas of sun exposure, with lesions commonly observed in the trunk for males, and lower limbs for females.,In addition to Breslow’s thickness and ulceration (p = 0.043 and p < 0.001, respectively), the mitotic rate per mm2 also correlated with worse patient outcome (p = 0.0007).,The sum of ulceration (0 when absent or 1 when present), the Breslow index (1 when <1 mm, 2 when >1 mm and <4 mm, 3 when >4 mm) and the mitotic index (0 when absent or 1 when ≥1 per mm2) allowed the establishment of a prognostic score: if the sum was equal to or over three, nearly all (91.7%) patients had systemic disease.,The 5-year survival was approximately seventy percent.,Because American Join Committee of Cancer Staging will update the classification of malignant tumors (TNM) staging in the near future, and introduce mitosis as a prognostic factor, our results show the importance of such a feature.,Additional studies are necessary to confirm the importance of a prognostic score as proposed herein.

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

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

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.

microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway.,While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma.,Here, we investigated the biological and clinical significance of miR-7-5p in melanoma.,We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest.,Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo.,We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis.,Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1β, IL-6 and IL-8.,Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown.,Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors.,Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling.,Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease.

Chapuis et al. demonstrate that the combination of adoptive cellular therapy with CTLA4 blockade induces long-term remission in a melanoma patient resistant to both modalities administered serially and individually.,Adoptive transfer of peripheral blood-derived, melanoma-reactive CD8+ cytotoxic T lymphocytes (CTLs) alone is generally insufficient to eliminate bulky tumors.,Similarly, monotherapy with anti-CTLA4 infrequently yields sustained remissions in patients with metastatic melanoma.,We postulated that a bolus of enhanced IL-21-primed polyclonal antigen-specific CTL combined with CTLA4 blockade might boost antitumor efficacy.,In this first-in-human case study, the combination successfully led to a durable complete remission (CR) in a patient whose disease was refractory to both monoclonal CTL and anti-CTLA4.,Long-term persistence and sustained anti-tumor activity of transferred CTL, as well as responses to nontargeted antigens, confirmed mutually beneficial effects of the combined treatment.,In this first-in-human study, Chapuis et al. demonstrate that the combination of adoptive cellular therapy with CTLA4 blockade induces long-term remission in a melanoma patient resistant to both modalities administered serially and individually.

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.

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

Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis.,Here, we describe mutations occurring exclusively at arginine-625 in splicing factor 3B subunit 1 (SF3B1) in low-grade uveal melanomas with good prognosis.,Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutation, and these mutations denote a distinct molecular subset of uveal melanomas.

Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP,POT1), have also been implicated in melanomagenesis.,Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.,−57 T>G variant) has been reported in one family.,We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls.,Germline lymphocyte telomere length was estimated in carriers.,The c.,−57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls.,One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour.,The blood leukocyte telomere length of a carrier was similar to wild-type cases.,We provide evidence confirming that a rare promoter variant of TERT (c.,−57 T>G) is associated with high penetrance, early onset melanoma and potentially other cancers, and explains <1 % of UK melanoma multicase families.,The identification of POT1 and TERT germline mutations highlights the importance of telomere integrity in melanoma biology.,The online version of this article (doi:10.1007/s10689-015-9841-9) contains supplementary material, which is available to authorized users.

Whole-genome sequencing identifies a novel germline variant in the oncogene MITF, which is associated with the development of melanoma.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.,Two papers in this issue of Nature demonstrate that missense substitutions in the gene encoding for microphthalmia-associated transcription factor (MITF) are associated with susceptibility to melanoma and renal cell carcinoma.,Functional analysis shows that the variant has impaired sumoylation that leads to differential regulation of several MITF targets, and promotes tumour cell clonogenicity, migration and invasion.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.,So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families.,Mutations in CDKN2A account for approximately 40% of familial cases1, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds2.,Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma.,We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF).,Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant.,Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample.,Likewise, it was similarly associated in an independent case-control sample from the United Kingdom.,In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both.,The variant allele was also associated with increased naevus count and non-blue eye colour.,Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets.,These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.

Despite significant development of melanoma therapies, death rates remain high.,MicroRNAs, controlling posttranscriptionally gene expression, play role in development of resistance to BRAF inhibitors.,The aim of the study was to assess the role of miR-410-3p in response to vemurafenib-BRAF inhibitor.,FFPE tissue samples of 12 primary nodular melanomas were analyzed.,With the use of Laser Capture Microdissection, parts of tumor, transient tissue, and adjacent healthy tissue were separated.,In vitro experiments were conducted on human melanoma cell lines A375, G361, and SK-MEL1.,IC50s of vemurafenib were determined using MTT method.,Cells were transfected with miR-410-3p mimic, anti-miR-410-3p and their non-targeting controls.,ER stress was induced by thapsigargin.,Expression of isolated RNA was determined using qRT-PCR.,We have found miR-410-3p is downregulated in melanoma tissues.,Its expression is induced by vemurafenib in melanoma cells.,Upregulation of miR-410-3p level increased melanoma cells resistance to vemurafenib, while its inhibition led to the decrease of resistance.,Induction of ER stress increased the level of miR-410-3p. miR-410-3p upregulated the expression of AXL in vitro and correlated with markers of invasive phenotype in starBase.,The study shows a novel mechanism of melanoma resistance. miR-410-3p is induced by vemurafenib in melanoma cells via ER stress.,It drives switching to the invasive phenotype that leads to the response and resistance to BRAF inhibition.

Molecular diagnostics are increasingly performed routinely in the diagnosis and management of patients with melanoma due to the development of novel therapies that target specific genetic mutations.,The development of next-generation sequencing (NGS) technologies has enabled to sequence multiple cancer-driving genes in a single assay, with improved sensitivity in mutation detection.,The main objective of this study was the design and implementation of a melanoma-specific sequencing panel, and the identification of the spectrum of somatic mutations in a series of primary melanoma samples.,A custom panel was designed to cover the coding regions of 35 melanoma-related genes.,Panel average coverage was 2,575.5 reads per amplicon, with 92,8% of targeted bases covered ≥500×.,Deep coverage enabled sensitive discovery of mutations in as low as 0.5% mutant allele frequency.,Eighty-five percent (85/100) of the melanomas had at least one somatic mutation.,The most prevalent mutated genes were BRAF (50%;50/199), NRAS (15%;15/100), PREX2 (14%;14/100), GRIN2A (13%;13/100), and ERBB4 (12%;12/100).,Turn-around-time and costs for NGS-based analysis was reduced in comparison to conventional molecular approaches.,The results of this study demonstrate the cost-effectiveness and feasibility of a custom-designed targeted NGS panel, and suggest the implementation of targeted NGS into daily routine practice.

Melanoma is a malignant tumor deriving from neoplastic transformation of melanocytes.,The incidence of melanoma has increased dramatically over the last 50 years.,It accounts for most cases of skin cancer deaths.,Early diagnosis leads to remission in 90% of cases of melanoma; conversely, for melanoma at more advanced stages, prognosis becomes more unfavorable also because dvanced melanoma is often resistant to pharmacological and radiological therapies due to genetic plasticity, presence of cancer stem cells that regenerate the tumor, and efficient elimination of drugs.,This review illustrates the role of autophagy in tumor progression and resistance to therapy, focusing on molecular targets for future drugs.

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

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

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.

Vulvar melanoma (VuM) and vaginal melanoma (VaM) represent a unique subgroup of malignant melanomas with important differences in biology and treatment.,The objective of this study was to describe the epidemiology and prognosis of VuM and VaM in a large representative cohort.,Women with invasive VuM or VaM were identified from the Surveillance, Epidemiology and End Results-18 population representing 27.8% of the US population.,Data on age, ethnicity, stage, location, histopathology, primary surgery, and lymphadenectomy were collected.,The Kaplan-Meier method was used to analyze disease-specific and overall survival.,Univariate and multivariate regression models were used to identify factors with a significant association with disease-specific survival.,A total of 1400 VuM and 463 VaM were included for further analysis; 78.6% and 49.7% of women with VuM and VaM underwent surgery, but only 52.9% of women with non-metastatic VuM and 42.9% of women with non-metastatic VaM undergoing surgery had lymph node assessment; one third of these had positive nodes.,Superficial spreading was the most common subtype in VuM, and nodular melanoma in VaM (p < 0.001).,The median disease-specific survival was 99 months (95% confidence interval 60-138) and 19 months (95% confidence interval 16-22), respectively.,Survival was significantly associated with age at diagnosis, ethnicity, stage, surgery, lymph node metastases, histologic subtype, ulceration, mitotic count, and tumor thickness in VuM, and stage, surgery, and lymph node involvement in VaM.,In the Cox model, lymph node status and number of mitoses remained independent predictors of outcome in VuM; in VaM, only lymph node status remained significant.,The overall prognosis of VuM and VaM remains poor.,The American Joint Committee on Cancer staging system is applicable and should be used for VuM; however, lymph node status and mitotic rate are the most important predictors of survival.,Lymph node status should be assessed and patients with positive nodes may be candidates for adjuvant treatment.

Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-MET) signaling is involved in complex cellular programs that are important for embryonic development and tissue regeneration, but its activity is also utilized by cancer cells during tumor progression.,HGF and c-MET usually mediate heterotypic cell-cell interactions, such as epithelial-mesenchymal, including tumor-stroma interactions.,In the skin, dermal fibroblasts are the main source of HGF.,The presence of c-MET on keratinocytes is crucial for wound healing in the skin.,HGF is not released by normal melanocytes, but as melanocytes express c-MET, they are receptive to HGF, which protects them from apoptosis and stimulates their proliferation and motility.,Dissimilar to melanocytes, melanoma cells not only express c-MET, but also release HGF, thus activating c-MET in an autocrine manner.,Stimulation of the HGF/c-MET pathways contributes to several processes that are crucial for melanoma development, such as proliferation, survival, motility, and invasiveness, including distant metastatic niche formation.,HGF might be a factor in the innate and acquired resistance of melanoma to oncoprotein-targeted drugs.,It is not entirely clear whether elevated serum HGF level is associated with low progression-free survival and overall survival after treatment with targeted therapies.,This review focuses on the role of HGF/c-MET signaling in melanoma with some introductory information on its function in skin and melanocytes.

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.

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

Tumor cells are subjected to evolutionary selection pressures during progression from initiation to metastasis.,We analyzed the clonal evolution of squamous skin carcinomas induced by DMBA/TPA treatment using the K5CreER-Confetti mouse and stage-specific lineage tracing.,We show that benign tumors are polyclonal, but only one population contains the Hras driver mutation.,Benign papillomas are therefore monoclonal in origin, but recruit neighboring epithelial cells during growth.,Papillomas that never progress to malignancy retain several distinct clones, whereas progression to carcinoma is associated with a clonal sweep.,Newly generated clones within carcinomas demonstrate intratumoral invasion and clonal intermixing, often giving rise to metastases containing two or more distinct clones derived from the matched primary tumor.,These data demonstrate that late stage tumor progression and dissemination are governed by evolutionary selection pressures that operate at a multicellular level, and thus differ from the clonal events that drive initiation and the benign-malignant transition.

Cutaneous human papillomaviruses (HPVs) are considered as cofactors for non-melanoma skin cancer (NMSC) development, especially in association with UVB.,Extensively studied transgenic mouse models failed to mimic all aspects of virus-host interactions starting from primary infection to the appearance of a tumor.,Using the natural model Mastomys coucha, which reflects the human situation in many aspects, we provide the first evidence that only UVB and Mastomys natalensis papillomavirus (MnPV) infection strongly promote NMSC formation.,Using UVB exposures that correspond to UV indices of different geographical regions, irradiated animals developed either well-differentiated keratinizing squamous cell carcinomas (SCCs), still supporting productive infections with high viral loads and transcriptional activity, or poorly differentiated non-keratinizing SCCs almost lacking MnPV DNA and in turn, early and late viral transcription.,Intriguingly, animals with the latter phenotype, however, still showed strong seropositivity, clearly verifying a preceding MnPV infection.,Of note, the mere presence of MnPV could induce γH2AX foci, indicating that viral infection without prior UVB exposure can already perturb genome stability of the host cell.,Moreover, as shown both under in vitro and in vivo conditions, MnPV E6/E7 expression also attenuates the excision repair of cyclobutane pyrimidine dimers upon UVB irradiation, suggesting a viral impact on the DNA damage response.,While mutations of Ras family members (e.g.,Hras, Kras, and Nras) were absent, the majority of SCCs harbored-like in humans-Trp53 mutations especially at two hot-spots in the DNA-binding domain, resulting in a loss of function that favored tumor dedifferentiation, counter-selective for viral maintenance.,Such a constellation provides a reasonable explanation for making continuous viral presence dispensable during skin carcinogenesis as observed in patients with NMSC.

squamous cell carcinoma is the second most common type of skin malignancy and may evolve to regional lymph node and distant metastases.,The objective of this study was to evaluate patients with head and neck cutaneous squamous cell carcinoma to identify its clinical and histopathological characteristics, as well as the frequency of local recurrence and metastasis.,A retrospective cohort of patients with head and neck cutaneous squamous cell carcinoma.,Inclusion criteria: histopathological confirmation, follow-up for longer than one year after diagnosis.,Exclusion criteria: immunosuppression; lip and oral cavity squamous cell carcinoma; and non-surgical resection of the lesion.,We evaluated demographic, clinical and anatomopathologic findings and explored their associations.,Sixty-one patients with 79 tumors and followed by 4.8±3.0 years were selected.,The average age was 67.1 years, and 63% of tumors had up to two centimeters.,Seven tumors (8.9%) recurred and two of them had positive margins.,Recurrence was associated with higher Broders' grade (p<0.01).,Two patients (3.3%) had regional lymph node metastases.,There were no distant metastases.,Seventy tumors were considered to be usual tumors (89.7%), and 68 (87.2%) were classified as Broders' grade 1 and 2.,Additionally, 64.1% of tumors had a depth of invasion below four millimeters.,Thirteen tumors (16.7%) had positive histological margins.,Most patients had good prognosis in the first year of follow-up, confirming that head and neck cutaneous squamous cell carcinoma has a better prognosis than squamous cell carcinoma of other regions such as mucosa, oral cavity, and internal organs.

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

Although long non-coding RNAs (lncRNAs) have been implicated in various human cancer types, the role of lncRNA ezrin antisense RNA 1 (EZR-AS1) in cutaneous squamous cell carcinoma (cSCC) remains unclear.,The present study aimed to investigate the effect of lncRNAEZR-AS1 on cSCC and identify the underlying molecular mechanisms.,EZR-AS1 expression was measured in cSCC tissue and cells detected using reverse transcription-quantitative PCR.,Gain-of-function assays were performed in A431 cells, which have a relatively low expression of EZR-AS1, while loss-of-function assays were performed in SCC13 and SCL-1 colon cancer cells, which have a relatively high expression of EZR-AS1.,Cell viability, proliferation, migration, invasion and apoptosis were assessed using MTT, plate cloning, wound healing, Transwell and flow cytometry assays, respectively.,EZR-AS1 mRNA expression levels were significantly upregulated in cSCC tissues and cells compared with adjacent healthy tissues and HaCaT cells, respectively.,Compared with the small interfering RNA (si)-negative control (NC) group, si-EZR-AS1 significantly inhibited SCC13 and SCL-1 cell proliferation, migration and invasion, but promoted cell apoptosis.,By contrast, compared with the pc-NC group, EZR-AS1 overexpression significantly enhanced A431 cell proliferation, migration and invasion, but inhibited cell apoptosis.,Moreover, focal adhesion kinase (FAK) was identified as a target of EZR-AS1, and EZR-AS1 knockdown significantly decreased FAK expression compared with the si-NC group.,Moreover, EZR-AS1 knockdown significantly downregulated the protein expression levels of phosphorylated (p)-PI3K/PI3K and p-AKT/AKT in cSCC cells compared with the si-NC group.,The PI3K agonist 740Y-P significantly reversed si-EZR-AS1-mediated effects on SCC13 and SCL-1 cell proliferation, migration, invasion and apoptosis.,In conclusion, the present study demonstrated that si-EZR-AS1 inhibited cSCC cell proliferation, migration and invasion, and promoted cell apoptosis, potentially via regulating the PI3K/AKT signaling pathway.,Therefore, the present study provided novel insights into the diagnosis and treatment of cSCC.

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

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

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.

Melanoma has several clinically and pathologically distinguishable subtypes, which also differ genetically.,Mutation patterns vary among different melanoma subtypes, and efficacy of immune-checkpoint inhibitors differs depending on the subtype of melanoma.,In spite of the recent revolution of systemic therapies for advanced melanoma, access to innovative agents is still restricted in many countries.,This review article aimed to describe the epidemiology and current status of systemic therapies for melanoma in Japan, where melanoma is rare, but access to innovative agents is available.,Acral and mucosal melanomas, which are common in Asian populations, predominantly occur in sun-protected areas and share several biological features.,Both the melanomas harbor KIT mutation in approximately 15% of the cases; BRAF or NRAS mutation is found in approximately 10-15% of acral melanoma, but these mutations are less frequent in mucosal melanoma.,Combined use of BRAF and MEK inhibitors is one of the standards of care for patients with advanced BRAF-mutant melanoma.,In patients with melanoma harboring KIT mutation in exon 11 or 13, KIT inhibitors can be a treatment option; however, none of them have been approved in Japan.,Immune-checkpoint inhibitors are expected to be less effective against acral and mucosal melanomas because their somatic mutation burden is lower than those in non-acral cutaneous melanomas.,A recently completed phase II trial of nivolumab and ipilimumab combination therapy in 30 Japanese patients with melanoma, including seven with acral and 12 with mucosal melanoma, demonstrated an objective response rate of 43%.,Regarding oncolytic viruses, canerpaturev (C-REV, also known as HF10) and talimogene laherparepvec (T-VEC) are currently under review in early phase trials.,In the adjuvant setting, dabrafenib plus trametinb combination, nivolumab monotherapy, and pembrolizumab monotherapy were approved in July, August, and December 2018 in Japan, respectively.,However, most of the adjuvant phase III trials excluded patients with mucosal melanoma.,A phase III trial of adjuvant therapy with locoregional interferon (IFN)-β versus surgery alone is ongoing in Japan (JCOG1309, J-FERON), in which IFN-β is injected directly into the site of the primary tumor postoperatively, so that it would be drained through the untreated lymphatic route to the regional node basin.,After the recent approval of these new agents, the JCOG1309 trial will be revised to focus on patients with stage II disease.,In conclusion, acral and mucosal melanomas have been treated based on the available medical evidence for the treatment of non-acral cutaneous melanomas.,Considering the differences in genetic backgrounds and therapeutic efficacy of immunotherapy, specialized therapeutic strategies for these subtypes of melanoma should be established in the future.

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

The antisense transcript, emanating from the opposite strand to a protein-coding or sense strand, has been reported to have critical roles in gene regulation.,The perturbation of an antisense RNA can alter the expression of sense messenger RNAs.,In this study, a long noncoding RNA TTN-AS1 (lncRNA-TTN-AS1), which is transcribed in the opposite direction of the human titin (TTN) gene, has been identified and explored in skin cutaneous melanoma (SKCM).,We found that the expression of TTN and lncRNA-TTN-AS1 had a significantly positive correlation in SKCM cells.,Functionally, ectopic expression of TTN and lncRNA-TTN-AS1 promoted SKCM tumorigenesis and metastasis both in vitro and in vivo.,Moreover, knockdown of TTN partially abrogated lncRNA-TTN-AS1 induced SKCM tumorigenesis.,Mechanistically, hypomethylation of transcription initiation site was responsible for lncRNA-TTN-AS1 high expression levels.,LncRNA-TTN-AS1 facilitated SKCM progression by promoting TTN expression at both transcriptional and posttranscriptional levels.,As detailed, lncRNA-TTN-AS1 had a significant effect on the increase of TTN promoter activity.,Besides, lncRNA-TTN-AS1 also induced the accumulation of TTN in cytoplasm by increasing the stability of TTN mRNA.,Clinically, we found that high TTN and lncRNA-TTN-AS1 expression were positively correlated with poor overall survival of SKCM patients, and may be considered as novel biomarkers and drug targets for SKCM patients.

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

The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC).,The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway.,Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin.,Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway.,SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes.,We sequenced tumor-normal pairs from patients with early sporadic BCCs.,This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation.,Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development.,Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation.,Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

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

Despite recent progresses in its treatment, malignant cutaneous melanoma remains a cancer with very poor prognosis.,Emerging evidences suggest that the receptor for advance glycation end products (RAGE) plays a key role in melanoma progression through its activation in both cancer and stromal cells.,In tumors, RAGE activation is fueled by numerous ligands, S100B and HMGB1 being the most notable, but the role of many other ligands is not well understood and should not be underappreciated.,Here, we provide a review of the current role of RAGE in melanoma and conclude that targeting RAGE in melanoma could be an approach to improve the outcomes of melanoma patients.

It has been suggested that diet may influence the risk of melanoma, but few studies are available on this topic.,We assessed the relation between food consumption and the risk of cutaneous melanoma in a Northern Italy population.,We carried out a population-based case-control study involving 380 cases of melanoma and 719 age- and sex-matched controls.,Dietary habits were established through a self-administered semi-quantitative food frequency questionnaire.,We computed the odds ratios (ORs) of melanoma and the corresponding 95% confidence intervals (CIs) according to tertiles of daily intake of each food item, using multiple logistic regression models adjusted for major confounding factors.,We observed an indication of a positive association between melanoma risk and consumption of cereals and cereal products (OR = 1.32; 95% CI 0.89-1.96, higher vs. lowest tertile), sweets (OR = 1.22; 95% CI 0.84-1.76), chocolate, candy bars. etc., (OR = 1.51; 95% CI 1.09-2.09) and cabbages (OR = 1.51; 95% CI 1.09-2.09).,Conversely, an inverse association with disease risk was found for the intake of legumes (OR = 0.77; 95% CI 0.52-1.13), olive oil (OR = 0.77; 95% CI 0.51-1.16), eggs (OR = 0.58; 95% CI 0.41-0.82), and onion and garlic (OR = 0.80; 95% CI 0.52-1.14).,No relationship was observed with beverage consumption.,Our results suggest potentially adverse effects on melanoma risk of foods characterized by high contents of refined flours and sugars, while suggesting a protective role for eggs and two key components of the Mediterranean diet, legumes and olive oil.,These associations warrant further investigation and, if confirmed, they might have important public health implications for the reduction of melanoma incidence through dietary modification.

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma.,Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms.,In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma.,Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors.,Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3.,Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion.,Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling.,Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present.,Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis.,Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.

Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined.,Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy.,While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy.,Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death.,Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress.,Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

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.

Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined.,Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy.,While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy.,Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death.,Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress.,Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

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

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

The discovery of the BRAFV600E mutation led to the development of vemurafenib (PLX4032), a selective BRAF inhibitor specific to the kinase, for the treatment of metastatic melanomas.,However, initial success of the drug was dampened by the development of acquired resistance.,Melanoma was shown to relapse in patients following treatment with vemurafenib which eventually led to patients’ deaths.,It has been proposed that mechanisms of resistance can be due to (1) reactivation of the mitogen-activated protein kinase (MAPK) signalling pathway via secondary mutations, amplification or activation of target kinase(s), (2) the bypass of oncogenic pathway via activation of alternative signalling pathways, (3) other uncharacterized mechanisms.,Studies showed that receptor tyrosine kinases (RTK) such as PDGFRβ, IGF1R, EGFR and c-Met were overexpressed in melanoma cells.,Along with increased secretion of growth factors such as HGF and TGF-α, this will trigger intracellular signalling cascades.,This review discusses the role MAPK and Phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K-AKT-mTOR) pathways play in the mechanism of resistance of melanomas.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012.,This Registered Report describes the proposed replication plan of key experiments from ‘Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion’ by Straussman and colleagues, published in Nature in 2012 (Straussman et al., 2012).,The key experiments being replicated in this study are from Figure 2A, C, and D (and Supplemental Figure 11) and Figure 4C (and Supplemental Figure 19) (Straussman et al., 2012).,Figure 2 demonstrates resistance to drug sensitivity conferred by co-culture with some stromal cell lines and identifies the secreted factor responsible as HGF.,In Figure 4, Straussman and colleagues show that blocking the HGF receptor MET abrogates HGF’s rescue of drug sensitivity.,The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.,DOI:http://dx.doi.org/10.7554/eLife.04034.001

This international, multicenter, single-arm trial assessed efficacy and safety of intralesional rose bengal (PV-10) in 80 patients with refractory cutaneous or subcutaneous metastatic melanoma.,Sixty-two stage III and 18 stage IV melanoma patients with disease refractory to a median of six prior interventions received intralesional PV-10 into up to 20 cutaneous and subcutaneous lesions up to four times over a 16-week period and were followed for 52 weeks.,Objectives were to determine best overall response rate in injected target lesions and uninjected bystander lesions, assess durability of response, and characterize adverse events.,For target lesions, the best overall response rate was 51 %, and the complete response rate was 26 %.,Median time to response was 1.9 months, and median duration of response was 4.0 months, with 8 % of patients having no evidence of disease after 52 weeks.,Response was dependent on untreated disease burden, with complete response achieved in 50 % of patients receiving PV-10 to all of their disease.,Response of target lesions correlated with bystander lesion regression and the occurrence of locoregional blistering.,Adverse events were predominantly mild to moderate and locoregional to the treatment site, with no treatment-associated grade 4 or 5 adverse events.,Intralesional PV-10 yielded durable local control with high rates of complete response.,Toxicity was confined predominantly to the injection site.,Cutaneous bystander tumor regression is consistent with an immunologic response secondary to ablation.,This intralesional approach for local disease control could be complementary to current and investigational treatments for melanoma.

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma.,To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized.,In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions.,In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions.,Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases.,Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10.,Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models.,In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed.,Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma.,These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies.

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

In this study, Falletta et al. show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,Their results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.,The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance.,Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown.,In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance.,However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood.,Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors.,However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion.,Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance.,Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Circulating tumour DNA (ctDNA) may serve as a measure of tumour burden and a useful tool for non-invasive monitoring of cancer.,However, ctDNA is not always detectable in patients at time of diagnosis of metastatic disease.,Therefore, there is a need to understand the correlation between ctDNA levels and the patients’ overall metabolic tumour burden (MTB).,Thirty-two treatment naïve metastatic melanoma patients were included in the study.,MTB and metabolic tumour volume (MTV) was measured by 18F-fluoro-D-glucose positron emission tomography/computed tomography (FDG PET/CT).,Plasma ctDNA was quantified using droplet digital PCR (ddPCR).,CtDNA was detected in 23 of 32 patients.,Overall, a significant correlation was observed between ctDNA levels and MTB (p < 0.001).,CtDNA was not detectable in patients with an MTB of ≤10, defining this value as the lower limit of tumour burden that can be detected through ctDNA analysis by ddPCR.,We showed that ctDNA levels measured by ddPCR correlate with MTB in treatment naïve metastatic melanoma patients and observed a limit in tumour size for which ctDNA cannot be detected in blood.,Nevertheless, our findings support the use of ctDNA as a non-invasive complementary modality to functional imaging for monitoring tumour burden.,The online version of this article (10.1186/s12885-018-4637-6) contains supplementary material, which is available to authorized users.

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

Skin cancer is believed to impose a heavy burden on healthcare services, but the burden of skin lesions suspected of malignancy on primary healthcare has never been evaluated.,Therefore the aim of this study was to determine the demand for care in general practice due to these suspected skin lesions (i.e. lesions that are suspected of malignancy by either the patient or the GP).,Registry study based on data (2001-2010) from the Registration Network Groningen.,This is a general practice registration network in the northern part of the Netherlands with an average annual population of approximately 30,000 patients.,All patient contacts are coded according to the International Classification of Primary Care (ICPC).,Consultations for skin lesions suspected of malignancy were selected according to the assigned ICPC codes.,Subsequently, the number of consultations per year and the annual percent change in number of contacts (using the JoinPoint regression program) were calculated and analysed.,Additionally, the percentage of patients referred to secondary care or receiving minor surgery within one year after the first contact were calculated.,From 2001 onwards we found an annual increase in demand for care due to skin lesions suspected of malignancy of 7.3% (p < 0.01) and in 2010 the benign:malignant ratio was 10:1.,In total 13.0% of the patients were referred and after 2006, minor surgery was performed on 31.2% of the patients.,Most surgeries and referrals took place within 30 days.,Suspected skin lesions impose an increasing burden on primary healthcare and most likely on healthcare costs as well.,General practitioners should therefore be trained in diagnosing skin lesions suspected of malignancy, as a high diagnostic accuracy can save lives in the case of melanoma, and may also prevent unnecessary, costly, excisions and referrals to secondary healthcare.

Objectives To assess whether adding a novel computerised diagnostic tool, the MoleMate system (SIAscopy with primary care scoring algorithm), to current best practice results in more appropriate referrals of suspicious pigmented lesions to secondary care, and to assess its impact on clinicians and patients.,Design Randomised controlled trial.,Setting 15 general practices in eastern England.,Participants 1297 adults with pigmented skin lesions not immediately diagnosed as benign.,Interventions Patients were assessed by trained primary care clinicians using best practice (clinical history, naked eye examination, seven point checklist) either alone (control group) or with the MoleMate system (intervention group).,Main outcome measures Appropriateness of referral, defined as the proportion of referred lesions that were biopsied or monitored.,Secondary outcomes related to the clinicians (diagnostic performance, confidence, learning effects) and patients (satisfaction, anxiety).,Economic evaluation, diagnostic performance of the seven point checklist, and five year follow-up of melanoma incidence were also secondary outcomes and will be reported later.,Results 1297 participants with 1580 lesions were randomised: 643 participants with 788 lesions to the intervention group and 654 participants with 792 lesions to the control group.,The appropriateness of referral did not differ significantly between the intervention or control groups: 56.8% (130/229) v 64.5% (111/172); difference −8.1% (95% confidence interval −18.0% to 1.8%).,The proportion of benign lesions appropriately managed in primary care did not differ (intervention 99.6% v control 99.2%, P=0.46), neither did the percentage agreement with an expert decision to biopsy or monitor (intervention 98.5% v control 95.7%, P=0.26).,The percentage agreement with expert assessment that the lesion was benign was significantly lower with MoleMate (intervention 84.4% v control 90.6%, P<0.001), and a higher proportion of lesions were referred (intervention 29.8% v control 22.4%, P=0.001).,Thirty six histologically confirmed melanomas were diagnosed: 18/18 were appropriately referred in the intervention group and 17/18 in the control group.,Clinicians in both groups were confident, and there was no evidence of learning effects, and therefore contamination, between groups.,Patients in the intervention group ranked their consultations higher for thoroughness and reassuring care, although anxiety scores were similar between the groups.,Conclusions We found no evidence that the MoleMate system improved appropriateness of referral.,The systematic application of best practice guidelines alone was more accurate than the MoleMate system, and both performed better than reports of current practice.,Therefore the systematic application of best practice guidelines (including the seven point checklist) should be the paradigm for management of suspicious skin lesions in primary care.,Trial registration Current Controlled Trials ISRCTN79932379.

Acral melanoma, the most common melanoma subtype among non-White individuals, is associated with poor prognosis.,However, its key molecular drivers remain obscure.,Here, we perform integrative genomic and clinical profiling of acral melanomas from 104 patients treated in North America (n = 37) or China (n = 67).,We find that recurrent, late-arising focal amplifications of cytoband 22q11.21 are a leading determinant of inferior survival, strongly associated with metastasis, and linked to downregulation of immunomodulatory genes associated with response to immune checkpoint blockade.,Unexpectedly, LZTR1 - a known tumor suppressor in other cancers - is a key candidate oncogene in this cytoband.,Silencing of LZTR1 in melanoma cell lines causes apoptotic cell death independent of major hotspot mutations or melanoma subtypes.,Conversely, overexpression of LZTR1 in normal human melanocytes initiates processes associated with metastasis, including anchorage-independent growth, formation of spheroids, and an increase in MAPK and SRC activities.,Our results provide insights into the etiology of acral melanoma and implicate LZTR1 as a key tumor promoter and therapeutic target.,Despite acral melanoma being the most common melanoma subtype in non-White individuals, its molecular drivers remain unknown.,Here, the authors integrate genomic and clinical data from 104 patients and identify late-arising focal amplifications of chr22q11.21 and LZTR1 as a key tumour promoter in this region.

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

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.

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

The keratinocyte cancers (KC), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most common cancers in fair-skinned people.,KC treatment represents the second highest cancer healthcare expenditure in Australia.,Increasing our understanding of the genetic architecture of KC may provide new avenues for prevention and treatment.,We first conducted a series of genome-wide association studies (GWAS) of KC across three European ancestry datasets from Australia, Europe and USA, and used linkage disequilibrium (LD) Score regression (LDSC) to estimate their pairwise genetic correlations.,We employed a multiple-trait approach to map genes across the combined set of KC GWAS (total N = 47 742 cases, 634 413 controls).,We also performed meta-analyses of BCC and SCC separately to identify trait specific loci.,We found substantial genetic correlations (generally 0.5-1) between BCC and SCC suggesting overlapping genetic risk variants.,The multiple trait combined KC GWAS identified 63 independent genome-wide significant loci, 29 of which were novel.,Individual separate meta-analyses of BCC and SCC identified an additional 13 novel loci not found in the combined KC analysis.,Three new loci were implicated using gene-based tests.,New loci included common variants in BRCA2 (distinct to known rare high penetrance cancer risk variants), and in CTLA4, a target of immunotherapy in melanoma.,We found shared and trait specific genetic contributions to BCC and SCC.,Considering both, we identified a total of 79 independent risk loci, 45 of which are novel.

To search for new sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conducted a genome-wide association study of 38.5 million single nucleotide polymorphisms (SNPs) and small indels identified through whole-genome sequencing of 2230 Icelanders.,We imputed genotypes for 4208 BCC patients and 109 408 controls using Illumina SNP chip typing data, carried out association tests and replicated the findings in independent population samples.,We found new BCC susceptibility loci at TGM3 (rs214782[G], P = 5.5 × 10−17, OR = 1.29) and RGS22 (rs7006527[C], P = 8.7 × 10−13, OR = 0.77).,TGM3 encodes transglutaminase type 3, which plays a key role in production of the cornified envelope during epidermal differentiation.

Uveal melanoma is characterised by mutations in GNAQ and GNA11, resulting in Ras/Raf/MEK/ERK pathway activation.,Treatment with selumetinib (AZD6244, ARRY-142886), a MEK1/2 inhibitor, results in antitumour effects in uveal melanoma pre-clinical models.,A randomised phase II trial demonstrated improved progression-free survival (PFS) and response rate (RR) with selumetinib monotherapy versus chemotherapy with temozolomide or dacarbazine in patients with metastatic uveal melanoma.,Pre-clinically, selumetinib in combination with alkylating agents enhanced antitumour activity compared with chemotherapy alone.,We hypothesise that selumetinib in combination with dacarbazine will result in improved clinical outcomes in patients with metastatic uveal melanoma versus dacarbazine alone.,SUMIT is a randomised, international, double-blind, placebo-controlled, phase III study assessing the efficacy and safety of selumetinib in combination with dacarbazine in patients with metastatic uveal melanoma who have not received prior systemic therapy.,Primary endpoint is PFS.,Secondary endpoints include objective RR, duration of response, change in tumour size at Week 6, overall survival, safety and tolerability.,Exploratory endpoints include efficacy in tumours with GNAQ or GNA11 mutations.,Eligible patients must have: ≥1 lesion that can be accurately measured at baseline, and is suitable for accurate repeated measurements; ECOG performance status 0-1; life expectancy >12 weeks.,Mutation status for GNAQ/GNA11 will be assessed retrospectively.,An estimated 128 patients from approximately 50 sites globally will be randomised (3:1) to selumetinib 75 mg twice daily or placebo in combination with dacarbazine 1000 mg/m2 on Day 1 of every 21-day cycle until objective disease progression, intolerable toxicity or occurrence of another discontinuation criterion.,Randomisation will be stratified by the presence/absence of liver metastases.,Tumours will be evaluated by RECIST v1.1 every 6 weeks.,All patients have the option of receiving selumetinib with or without dacarbazine at disease progression.,Study enrolment began in April 2014 and is expected to complete in early 2015.,Treatment of patients with metastatic uveal melanoma represents an area of high unmet medical need.,This study evaluating selumetinib in combination with dacarbazine was designed with input from the US FDA, and is the first potential registration trial to be conducted in patients with metastatic uveal melanoma.,Clinicaltrials.gov (Date of registration, October 10, 2013),Registration number: NCT01974752,Trial abbreviation: SUMIT

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

Melanoma is a heterogeneous tumor with different subpopulations showing different proliferation rates.,Slow-cycling cells were previously identified in melanoma, but not fully biologically characterized.,Using the label-retention method, we identified a subpopulation of slow-cycling cells, defined as label-retaining cells (LRC), with strong invasive properties.,We demonstrate through live imaging that LRC are leaving the primary tumor mass at a very early stage and disseminate to peripheral organs.,Through global proteome analyses, we identified the secreted protein SerpinE2/protease nexin-1 as causative for the highly invasive potential of LRC in melanomas.

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

Multiple primary melanoma (MPM) is a rare condition, whose genetic basis has not yet been clarified.,Only 8-12% of MPM are due to germline mutations of CDKN2A.,However, other genes (POT1, BRCA1/2, MC1R, MGMT) have been demonstrated to be involved in predisposition to this pathology.,To our knowledge, this is the first family study based on two siblings with the rare coexistence of MPM and oculocutaneous albinism (OCA), an autosomal recessive disease characterized by the absence or decrease in pigmentation in the skin, hair, and eyes.,In this study, we evaluated genes involved in melanoma predisposition (CDKN2A, CDK4, MC1R, MITF, POT1, RB1, MGMT, BRCA1, BRCA2), pathogenesis (BRAF, NRAS, PIK3CA, KIT, PTEN), skin/hair pigmentation (MC1R, MITF) and in immune pathways (CTLA4) to individuate alterations able to explain the rare onset of MPM and OCA in indexes and the transmission in their pedigree.,From the analysis of the pedigree, we were able to identify a “protective” haplotype with respect to MPM, including MGMT p.I174V alteration.,The second generation offspring is under strict follow up as some of them have a higher risk of developing MPM according to our model.

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

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts.,By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease.,In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival.,Loss of both copies of B2M is found only in non-responders.,B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.,Resistance to immune-checkpoint blockade often occurs in treated patients.,Here, the authors demonstrate that B2M loss is a mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1 in melanoma patients.

The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance.,Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity.,Here we investigate the role of ADAR1 in melanoma immune resistance.,Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism.,We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance.,ADAR1 thus has a novel, pivotal, role in cancer immune resistance.,Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab.,These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment.

Introduction: Mucosal melanoma is rare and associated with poorer prognosis in comparison to conventional melanoma subtypes.,Little is known about the prognostic significance as well as possible associations between PARP1 and immunologic response in mucosal melanoma.,Methods: PARP1, PD-L1 and IDO1 immunostains were performed on 192 mucosal melanomas including 86 vulvar, 89 sinonasal, and 17 anorectal melanomas.,Results: By Kaplan-Meier analyses, high PARP1 expression correlated with worse overall and melanoma-specific survival (log-rank p values = 0.026 and 0.047, respectively).,Tumors with combined PARP1 and IDO1 high expression correlated with worse overall and melanoma-specific survival (p = 0.015, 0.0034 respectively).,By multivariate analyses, high PARP1 expression remained a predictor of worse survival independent of stage.,By Fisher’s exact test, high PARP1 expression correlated with highly mitogenic tumors (p = 0.02).,High tumoral PD-L1 and IDO1 expression were associated with ulcerated primary tumors (p = 0.019, 0.0019, respectively).,By linear regression analyses, correlations between PARP1 expression versus IDO1 expression (p = 0.0001) and mitotic index (p = 0.0052) were observed.,Conclusion: Increased expression of PARP1 is an independent negative prognostic marker in mucosal melanomas.,The association between PARP1 and IDO1 and their combined adverse prognostic role raise the potential of combined therapy in mucosal melanoma.

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.

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.

Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity.,Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness.,As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.,Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters.,SOX9 is methylated and lowly expressed in the highly proliferative group.,SOX9 overexpression results in decreased proliferation but increased invasion in vitro.,In a B16 mouse model, sox9 overexpression increases the number of lung metastases.,Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways.,Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates.,Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.,Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups.,One of the genes regulated by DNA methylation between the two groups is SOX9.,SOX9 induces melanoma cell invasion and metastasis and decreases patient survival.,A number of genes downregulated by SOX9 have a negative impact on patient survival.,In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.,The online version of this article (doi:10.1186/s13059-015-0594-4) contains supplementary material, which is available to authorized users.

The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood.,Here, we show that the growth of tumors formed by mutant BrafV600E mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation.,Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in BrafV600E mouse melanoma cells, as well as in NrasG12D melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways.,This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species.,Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.,•Cyclooxygenase in tumors induces PGE2 that subverts myeloid cell function•COX ablation in tumors enables immune control•COX inhibition synergizes with checkpoint blockade therapy•A COX inflammatory signature is conserved across mouse and human cancer biopsies,Cyclooxygenase in tumors induces PGE2 that subverts myeloid cell function,COX ablation in tumors enables immune control,COX inhibition synergizes with checkpoint blockade therapy,A COX inflammatory signature is conserved across mouse and human cancer biopsies,Cyclooxygenase-driven prostaglandin E2, produced by a variety of tumors, drives malignant growth through successful evasion of type I interferon and/or T-cell-dependent tumor elimination.,A remarkable synergy between cyclooxygenase inhibitors and checkpoint blockade immunotherapy results in tumor eradication.

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

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

Group 2 innate lymphoid cells (ILC2) are essential to maintain tissue homeostasis.,In cancer, ILC2 can harbor both pro- and anti-tumorigenic functions but we know very little about their underlying mechanisms, nor whether they could be clinically relevant or targeted to improve patient outcomes.,Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis.,ILC2 are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) which coordinate the recruitment and activation of eosinophils to enhance anti-tumor responses.,Tumor-infiltrating ILC2 expressed programmed cell death protein-1 (PD-1), which limited their intratumoral accumulation, proliferation and anti-tumor effector functions.,This inhibition could be overcome in vivo by combining IL-33-driven ILC2 activation with PD-1 blockade to significantly increase anti-tumor responses.,Together, our results identified ILC2 as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for anti-tumor immunotherapies.

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

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

BAP1 is a histone deubiquitinase that acts as a tumor and metastasis suppressor associated with disease progression in human cancer.,We have used the “Calling Card System” of transposase-directed transposon insertion mapping to identify the genomic targets of BAP1 in uveal melanoma (UM).,This system was developed to identify the genomic loci visited by transcription factors that bind directly to DNA; our study is the first use of the system with a chromatin-remodeling factor that binds to histones but does not interact directly with DNA.,The transposase piggyBac (PBase) was fused to BAP1 and expressed in OCM-1A UM cells.,The insertion of transposons near BAP1 binding sites in UM cells were identified by genomic sequencing.,We also examined RNA expression in the same OCM-1A UM cells after BAP1 depletion to identify BAP1 binding sites associated with BAP1-responsive genes.,Sets of significant genes were analyzed for common pathways, transcription factor binding sites, and ability to identify molecular tumor classes.,We found a strong correlation between multiple calling-card transposon insertions targeted by BAP1-PBase and BAP1-responsive expression of adjacent genes.,BAP1-bound genomic loci showed narrow distributions of insertions and were near transcription start sites, consistent with recruitment of BAP1 to these sites by specific DNA-binding proteins.,Sequence consensus analysis of BAP1-bound sites showed enrichment of motifs specific for YY1, NRF1 and Ets transcription factors, which have been shown to interact with BAP1 in other cell types.,Further, a subset of the BAP1 genomic target genes was able to discriminate aggressive tumors in published gene expression data from primary UM tumors.,The calling card methodology works equally well for chromatin regulatory factors that do not interact directly with DNA as for transcription factors.,This technique has generated a new and expanded list of BAP1 targets in UM that provides important insight into metastasis pathways and identifies novel potential therapeutic targets.,The online version of this article (10.1186/s12920-018-0424-0) contains supplementary material, which is available to authorized users.

Quantitative imaging of single living tumor cells invading three-dimensional collagen matrices is used in tandem with unsupervised computational analysis to characterize melanoma-cell shape space.,Melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space-an apolar and a polar route.,Melanoma cells can adopt two functionally distinct forms, amoeboid and mesenchymal, which facilitates their ability to invade and colonize diverse environments during the metastatic process.,Using quantitative imaging of single living tumor cells invading three-dimensional collagen matrices, in tandem with unsupervised computational analysis, we found that melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space-an apolar and polar route.,We show that whereas particular Rho-family GTPases are required for the morphogenesis of amoeboid and mesenchymal forms, others are required for transitions via the apolar or polar route and not amoeboid or mesenchymal morphogenesis per se.,Altering the transition rates between particular routes by depleting Rho-family GTPases can change the morphological heterogeneity of cell populations.,The apolar and polar routes may have evolved in order to facilitate conversion between amoeboid and mesenchymal forms, as cells are either searching for, or attracted to, particular migratory cues, respectively.

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

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

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

Because only a small fraction of asbestos-exposed individuals develop malignant mesothelioma1, and because mesothelioma clustering is observed in some families1, we searched for genetic predisposing factors.,We discovered germline mutations in BAP1 (BRCA1-associated protein 1) in two families with a high incidence of mesothelioma.,Somatic alterations affecting BAP1 were observed in familial mesotheliomas, indicating biallelic inactivation.,Besides mesothelioma, some BAP1 mutation carriers developed uveal melanoma.,Germline BAP1 mutations were also found in two of 26 sporadic mesotheliomas: both patients with mutant BAP1 were previously diagnosed with uveal melanoma.,Truncating mutations and aberrant BAP1 expression were common in sporadic mesotheliomas without germline mutations.,These results reveal a BAP1-related cancer syndrome characterized by mesothelioma and uveal melanoma.,We hypothesize that other cancers may also be involved, and that mesothelioma predominates upon asbestos exposure.,These findings will help identify individuals at high risk of mesothelioma who could be targeted for early intervention.

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops.,Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity.,Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1.,Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo.,Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors.,Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance.,Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.,BRAF or MEK1/2 inhibitors are cytostatic in melanoma and the surviving cells develop drug resistance.,This study shows that the pro-survival pool is biased towards MCL1 in melanoma so that BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, improving tumour growth inhibition.

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment.,Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance.,Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1.,Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis.,Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines.,Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors.,In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA.,Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination.,Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1.,These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors.

Background: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood.,Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes.,Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance.,Methods: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs.,The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions.,Results: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs.,Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced.,TGF-β transported by sEVs contributed to the promotion of a suppressive phenotype of APCs.,Conclusion: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.

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

Talimogene laherparepvec is an oncolytic immunotherapy approved in the US, Europe, Australia and Switzerland.,We report the final planned analysis of OPTiM, a randomized open-label phase III trial in patients with unresectable stage IIIB-IVM1c melanoma.,Patients were randomized 2:1 to receive intratumoral talimogene laherparepvec or subcutaneous recombinant GM-CSF.,In addition to overall survival (OS), durable response rate (DRR), objective response rate (ORR), complete responses (CR), and safety are also reported.,All final analyses are considered to be descriptive and treatment responses were assessed by the investigators.,Of 436 patients in the intent-to-treat population, 295 were allocated to talimogene laherparepvec and 141 to GM-CSF.,Median follow-up in the final OS analysis was 49 months.,Median OS was 23.3 months (95% confidence interval [CI], 19.5-29.6) and 18.9 months (95% CI, 16.0-23.7) in the talimogene laherparepvec and GM-CSF arms, respectively (unstratified hazard ratio, 0.79; 95% CI, 0.62-1.00; p = 0.0494 [descriptive]).,DRR was 19.0 and 1.4% (unadjusted odds ratio, 16.6; 95% CI, 4.0-69.2; p < 0.0001); ORR was 31.5 and 6.4%.,Fifty (16.9%) and 1 (0.7%) patient in the talimogene laherparepvec and GM-CSF arms, respectively, achieved CR.,In talimogene laherparepvec-treated patients, median time to CR was 8.6 months; median CR duration was not reached.,Among patients with a CR, 88.5% were estimated to survive at a 5-year landmark analysis.,Talimogene laherparepvec efficacy was more pronounced in stage IIIB-IVM1a melanoma as already described in the primary analysis.,The safety reporting was consistent with the primary OPTiM analysis.,In this final planned OPTiM analysis, talimogene laherparepvec continued to result in improved longer-term efficacy versus GM-CSF and remained well tolerated.,The final analysis also confirms that talimogene laherparepvec was associated with durable CRs that were associated with prolonged survival.,ClinicalTrials.gov identifier: NCT00769704.,The online version of this article (10.1186/s40425-019-0623-z) contains supplementary material, which is available to authorized users.

Cutaneous head and neck melanoma has poor outcomes and limited treatment options.,In OPTiM, a phase 3 study in patients with unresectable stage IIIB/IIIC/IV melanoma, intralesional administration of the oncolytic virus talimogene laherparepvec improved durable response rate (DRR; continuous response ≥6 months) compared with subcutaneous granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).,Retrospective review of OPTiM identified patients with cutaneous head and neck melanoma given talimogene laherparepvec (n = 61) or GM‐CSF (n = 26).,Outcomes were compared between talimogene laherparepvec and GM‐CSF treated patients with cutaneous head and neck melanoma.,DRR was higher for talimogene laherparepvec-treated patients than for GM‐CSF treated patients (36.1% vs 3.8%; p = .001).,A total of 29.5% of patients had a complete response with talimogene laherparepvec versus 0% with GM‐CSF.,Among talimogene laherparepvec-treated patients with a response, the probability of still being in response after 12 months was 73%.,Median overall survival (OS) was 25.2 months for GM‐CSF and had not been reached with talimogene laherparepvec.,Treatment with talimogene laherparepvec was associated with improved response and survival compared with GM‐CSF in patients with cutaneous head and neck melanoma.,© 2016 Wiley Periodicals, Inc.,Head Neck 38: 1752-1758, 2016

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

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) homeostasis.,Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities.,Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas.,Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases.,In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis.,The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas.,With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases.,Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity.,Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes.,Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival.,In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.

Angiogenesis is important for the progression of cutaneous melanoma.,Here, we analyzed the prognostic impact of the angiogenic factor urokinase plasminogen activator resecptor (uPAR), vascular proliferation index (VPI) and tumor necrosis as a measure of hypoxia in a patient series of nodular melanomas (n = 255) and matched loco-regional metastases (n = 78).,Expression of uPAR was determined by immunohistochemistry and VPI was assessed by dual immunohistochemistry using Factor-VIII/Ki67 staining.,Necrosis was recorded based on HE-slides.,As novel findings, high uPAR expression and high VPI were associated with each other, and with increased tumor thickness, presence of tumor necrosis, tumor ulceration, increased mitotic count and reduced cancer specific survival in primary melanoma.,In matched cases, VPI was decreased in metastases, whereas the frequency of necrosis was increased.,Our findings demonstrate for the first time the impact on melanoma specific survival of uPAR expression and VPI in primary tumors, and of increased necrosis as an indicator of tumor hypoxia in loco-regional metastases.,These findings support the importance of tumor angiogenesis in melanoma aggressiveness, and suggest uPAR as an indicator of vascular proliferation and a potential biomarker in melanoma.

Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated.,In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells.,Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR).,It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis.,To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed.,Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A.,Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment.,In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway.

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

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

Recent studies have shown that vanillin has anti-cancer, anti-mutagenic, and anti-metastatic activity; however, the precise molecular mechanism whereby vanillin inhibits metastasis and cancer progression is not fully elucidated.,In this study, we examined whether vanillin has anti-cancer and anti-metastatic activities via inhibition of hypoxia-inducible factor-1α (HIF-1α) in A2058 and A375 human malignant melanoma cells.,Immunoblotting and quantitative real time (RT)-PCR analysis revealed that vanillin down-regulates HIF-1α protein accumulation and the transcripts of HIF-1α target genes related to cancer metastasis including fibronectin 1 (FN1), lysyl oxidase-like 2 (LOXL2), and urokinase plasminogen activator receptor (uPAR).,It was also found that vanillin significantly suppresses HIF-1α mRNA expression and de novo HIF-1α protein synthesis.,To understand the suppressive mechanism of vanillin on HIF-1α expression, chromatin immunoprecipitation was performed.,Consequently, it was found that vanillin causes inhibition of promoter occupancy by signal transducer and activator of transcription 3 (STAT3), but not nuclear factor-κB (NF-κB), on HIF1A.,Furthermore, an in vitro migration assay revealed that the motility of melanoma cells stimulated by hypoxia was attenuated by vanillin treatment.,In conclusion, we demonstrate that vanillin might be a potential anti-metastatic agent that suppresses metastatic gene expression and migration activity under hypoxia via the STAT3-HIF-1α signaling pathway.

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

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide.,Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition.,In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions.,In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients.,On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

This study represents the first attempt to perform a profiling analysis of the intergenerational differences in the microRNAs (miRNAs) of primary cutaneous melanocytic neoplasms in young adult and older age groups.,The data emphasize the importance of these master regulators in the transcriptional machinery of melanocytic neoplasms and suggest that differential levels of expressions of these miRs may contribute to differences in phenotypic and pathologic presentation of melanocytic neoplasms at different ages.,An exploratory miRNA analysis of 666 miRs by low density microRNA arrays was conducted on formalin fixed and paraffin embedded tissues (FFPE) from 10 older adults and 10 young adults including conventional melanoma and melanocytic neoplasms of uncertain biological significance.,Age-matched benign melanocytic nevi were used as controls.,Primary melanoma in patients greater than 60 years old was characterized by the increased expression of miRs regulating TLR-MyD88-NF-kappaB pathway (hsa-miR-199a), RAS/RAB22A pathway (hsa-miR-204); growth differentiation and migration (hsa-miR337), epithelial mesenchymal transition (EMT) (let-7b, hsa-miR-10b/10b*), invasion and metastasis (hsa-miR-10b/10b*), hsa-miR-30a/e*, hsa-miR-29c*; cellular matrix components (hsa-miR-29c*); invasion-cytokinesis (hsa-miR-99b*) compared to melanoma of younger patients.,MiR-211 was dramatically downregulated compared to nevi controls, decreased with increasing age and was among the miRs linked to metastatic processes.,Melanoma in young adult patients had increased expression of hsa-miR-449a and decreased expression of hsa-miR-146b, hsa-miR-214*.,MiR-30a* in clinical stages I-II adult and pediatric melanoma could predict classification of melanoma tissue in the two extremes of age groups.,Although the number of cases is small, positive lymph node status in the two age groups was characterized by the statistically significant expression of hsa-miR-30a* and hsa-miR-204 (F-test, p-value < 0.001).,Our findings, although preliminary, support the notion that the differential biology of melanoma at the extremes of age is driven, in part, by deregulation of microRNA expression and by fine tuning of miRs that are already known to regulate cell cycle, inflammation, Epithelial-Mesenchymal Transition (EMT)/stroma and more specifically genes known to be altered in melanoma.,Our analysis reveals that miR expression differences create unique patterns of frequently affected biological processes that clearly distinguish old age from young age melanomas.,This is a novel characterization of the miRnomes of melanocytic neoplasms at two extremes of age and identifies potential diagnostic and clinico-pathologic biomarkers that may serve as novel miR-based targeted modalities in melanoma diagnosis and treatment.

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

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

Outcomes of melanoma patient treatment remain unsatisfactory despite accessibility of oncoprotein-targeting drugs and immunotherapy.,Here, we reported that 17-aminogeldanamycin more potently activated caspase-3/7 in BRAFV600E melanoma cells than geldanamycin, another inhibitor of heat shock protein 90 (HSP90). 17-aminogeldanamycin alleviated self-triggered compensatory increase in HSP70 mRNA level and induced endoplasmic reticulum (ER) stress, which was followed by selective diminution of cytoprotective IRE1α-XBP1s pathway activity of unfolded protein response (UPR), inhibition of ERK1/2 activity and induction of apoptosis.,Concomitantly, ATF6/p50 level and expression of PERK-dependent genes, CHOP and BIM, remained unaltered.,This might result from an inframe deletion in EIF2AK3 leading to a PERKL21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRASQ61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1P187S).,In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAFV600E) in induction of apoptosis in melanoma cell lines as evidenced by in-cell caspase-3/7 activation and PARP cleavage that occurred earlier compared with either drug used alone.,As trametinib and vemurafenib did not significantly affect HSP70 and GRP78 transcript levels, cooperation of MEK/BRAFV600E inhibitors and 17-aminogeldanamycin might result from a concurrent inhibition of the RAS/RAF/MEK/ERK cascade and IRE1α-dependent signaling, and cell-intrinsic ER homeostasis can determine the extent of the drug cooperation.,Our study indicates that 17-aminogeldanamycin takes several advantages compared with other HSP90-targeting compounds, and can complement activity of BRAF/MEK inhibitors in melanoma cells of different genetic subtypes.,The online version of this article (10.1007/s10495-019-01542-y) contains supplementary material, which is available to authorized users.

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

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

In this study, Falletta et al. show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,Their results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.,The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance.,Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown.,In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance.,However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood.,Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors.,However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion.,Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance.,Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Treatment of BRAF mutant melanoma with kinase inhibitors has been associated with rapid tumor regression; however, this clinical benefit is short-lived, and most patients relapse.,A number of studies suggest that the extracellular environment promotes BRAF inhibitor resistance and tumor progression.,Extracellular vesicles, such as exosomes, are functional mediators in the extracellular environment.,They are small vesicles known to carry a concentrated group of functional cargo and serve as intercellular communicators not only locally but also systemically.,Increasingly, it is reported that extracellular vesicles facilitate the development of drug resistance in cancer; however, their role in BRAF inhibitor resistance in melanoma is unclear.,Here we investigated if extracellular vesicles from BRAF inhibitor-resistant melanoma could influence drug sensitivity in recipient melanoma cells.,We demonstrate that the resistance driver, PDGFRβ, can be transferred to recipient melanoma cells via extracellular vesicles, resulting in a dose-dependent activation of PI3K/AKT signaling and escape from MAPK pathway BRAF inhibition.,These data suggest that the BRAF inhibitor-sensitive phenotype of metastatic melanoma can be altered by delivery of PDGFRβ by extracellular vesicles derived from neighboring drug-resistant melanoma cells.

Exosomes are extracellular vesicles released by various cell types and play roles in cell-cell communication.,Several studies indicate that cancer cell‐derived exosomes play important pathophysiological roles in tumor progression.,Biodistribution of cancer cell‐derived exosomes in tumor tissue is an important factor for determining their role in tumor proliferation; however, limited studies have assessed the biodistribution of exosomes in tumor tissues.,In the present study, we examined the effect of cancer‐cell derived exosomes on tumor growth by analyzing their biodistribution.,Murine melanoma B16BL6‐derived exosomes increased the proliferation and inhibited the apoptosis of B16BL6 cells, which was associated with an increase and decrease in the levels of proliferation‐ and apoptosis‐related proteins, respectively.,GW4869‐induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells, and treatment of GW4869‐treated cells with B16BL6‐derived exosomes restored their proliferation.,Next, we treated B16BL6 tumors in mice with B16BL6‐derived exosomes and examined the biodistribution and cellular uptake of these exosomes.,After the intratumoral injection of radiolabeled B16BL6‐derived exosomes, most radioactivity was detected within the tumor tissues of mice.,Fractionation of cells present in the tumor tissue showed that fluorescently labeled exosomes were mainly taken up by B16BL6 cells.,Moreover, intratumoral injection of B16BL6‐derived exosomes promoted tumor growth, whereas intratumoral injection of GW4869 suppressed tumor growth.,These results indicate that B16BL6 cells secrete and take up their own exosomes to induce their proliferation and inhibit their apoptosis, which promotes tumor progression.

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.

Treatment of BRAF mutant melanoma with kinase inhibitors has been associated with rapid tumor regression; however, this clinical benefit is short-lived, and most patients relapse.,A number of studies suggest that the extracellular environment promotes BRAF inhibitor resistance and tumor progression.,Extracellular vesicles, such as exosomes, are functional mediators in the extracellular environment.,They are small vesicles known to carry a concentrated group of functional cargo and serve as intercellular communicators not only locally but also systemically.,Increasingly, it is reported that extracellular vesicles facilitate the development of drug resistance in cancer; however, their role in BRAF inhibitor resistance in melanoma is unclear.,Here we investigated if extracellular vesicles from BRAF inhibitor-resistant melanoma could influence drug sensitivity in recipient melanoma cells.,We demonstrate that the resistance driver, PDGFRβ, can be transferred to recipient melanoma cells via extracellular vesicles, resulting in a dose-dependent activation of PI3K/AKT signaling and escape from MAPK pathway BRAF inhibition.,These data suggest that the BRAF inhibitor-sensitive phenotype of metastatic melanoma can be altered by delivery of PDGFRβ by extracellular vesicles derived from neighboring drug-resistant melanoma cells.

Squamous cell carcinoma (SCC) is a common skin cancer, and its treatment is still difficult.,The aim of this study was to evaluate the effectiveness of nanoparticle (NP)-assisted 5-aminolevulinic acid (ALA) delivery for topical photodynamic therapy (PDT) of cutaneous SCC.,Ultraviolet-induced cutaneous SCCs were established in hairless mice.,ALA-loaded polylactic-co-glycolic acid (PLGA) NPs were prepared and characterized.,The kinetics of ALA PLGA NP-induced protoporphyrin IX fluorescence in SCCs, therapeutic efficacy of ALA NP-mediated PDT, and immune responses were examined.,PLGA NPs enhanced protoporphyrin IX production in SCC.,ALA PLGA NP-mediated topical PDT was more effective than free ALA of the same concentration in treating cutaneous SCC.,PLGA NPs provide a promising strategy for delivering ALA in topical PDT of cutaneous SCC.

Objectives To assess the effects of treatments for non-metastatic invasive squamous cell carcinoma (SCC) of the skin using evidence from observational studies, given the paucity of evidence from randomised controlled trials.,Design Systematic review of observational studies.,Data sources Medline, Embase, to December 2012.,Review methods Observational studies of interventions for primary, non-metastatic, invasive, SCC of the skin that reported recurrence during follow-up, quality of life, initial response to treatment, adverse events, cosmetic appearance, or death from disease.,Studies were excluded if data for primary cutaneous SCC was not separable from other data.,Data were extracted independently by two reviewers.,Meta-analysis was performed where appropriate using a random effects model to estimate the pooled proportion of an event with 95% confidence intervals.,Results 118 publications were included, covering seven treatment modalities.,Pooled estimates of recurrence of SCCs were lowest after cryotherapy (0.8% (95% confidence interval 0.1% to 2%)) and curettage and electrodesiccation (1.7% (0.5% to 3.4%)), but most treated SCCs were small, low risk lesions.,After Mohs micrographic surgery, the pooled estimate of local recurrence during variable follow-up periods from 10 studies was 3.0% (2.2% to 3.9%), which was non-significantly lower than the pooled average local recurrence of 5.4% (2.5% to 9.1%) after standard surgical excision (12 studies), and 6.4% (3.0% to 11.0%) after external radiotherapy (7 studies).,After an apparently successful initial response of SCCs to photodynamic therapy, pooled average recurrence of 26.4% (12.3% to 43.7%; 8 studies) was significantly higher than other treatments.,Evidence was limited for laser treatment (1 study) and for topical and systemic treatments (mostly single case reports or small non-comparative series with limited follow-up).,Conclusions Many observational studies have looked at different treatment modalities for SCC, but the evidence base for the effectiveness of these interventions is poor.,Comparison of outcomes after different treatments should be interpreted cautiously owing to biases inherent in the types of study included, and lack of direct comparisons to enable the estimation of relative treatment effect.,Further evidence is needed to develop a prognostic model and stratify individuals at high risk of developing SCC, to improve the evidence base for this common cancer and to optimise clinical management.,Protocol registration International Prospective Register of Systematic Reviews (PROSPERO) registration number CRD42011001450.

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.

Melanoma is one of the most aggressive cancers with extremely poor prognosis, and the median survival time for stage IV patients is approximately 6 to 8 months.,Unlike cutaneous melanoma, mucosal melanoma is a rare melanoma subtype among Caucasian patients but its incidence remains as high as 22.6% among Chinese patients.,Screening specific genetic variations is the guideline to select targeted drugs for the treatment of advanced melanoma, whereas the genetic variation spectrum and potential therapeutic targets for mucosal melanoma are largely unclear.,It is urgent to identify promising genetic variants for mucosal melanoma so as to develop effective targeted therapies for this disease.,Tumor samples from 213 Chinese mucosal melanoma patients were involved in this study.,P16INK4a/Cyclin D1/CDK4 copy number was examined using the QuantiGene Plex DNA assay and the correlation between abnormal copy number and clinicopathological parameters was analyzed.,Patient-derived xenograft models (PDX) were performed to detect the effects of CDK4/6 inhibitors on the proliferation of mucosal melanoma cells with altered copy number of CDK4 pathway (CDK4, Cyclin D1 and P16INK4a).,The molecular mechanisms of CDK4/6 inhibitors on the proliferation of mucosal melanoma were analyzed by RNAseq.,Among the 213 samples, the amplification rate of CDK4 and CCND1 was 47.0% and 27.7%, respectively, and the deletion rate of P16INK4a was 57.7%.,Patients with more than one genetic abnormality were up to 81.7%.,CDK4 pathway gene copy number variation was not associated with the prognosis of patients with mucosal melanoma (P > 0.05).,Drug sensitivity tests showed that AT7519, a broad-spectrum CDK inhibitor, and PD0332991, a specific CDK4/6 inhibitor, exhibited higher inhibitory effect on CDK4 signaling pathway abnormal mucosal melanoma cells-derived PDX tumors growth than CDK4 signaling pathway normal ones.,RNA-seq analysis showed that CDK4 inhibitors may affect tumor proliferation through multiple signaling pathways.,Abnormal copy number of cell cycle related genes is frequently found in mucosal melanoma.,CDK4/6 inhibitors significantly suppress the PDX tumor growth with abnormal CDK4 pathway.,CDK4 signaling variations predict the effectiveness of CDK4 inhibitors in mucosal melanoma.,The online version of this article (10.1186/s12967-019-1987-z) contains supplementary material, which is available to authorized users.

Oncolytic virotherapies, including the modified herpes simplex virus talimogene laherparepvec (T-VEC), have shown great promise as potent instigators of anti-tumour immune effects.,The OPTiM trial, in particular, demonstrated the superior anti-cancer effects of T-VEC as compared to systemic immunotherapy treatment using exogenous administration of granulocyte-macrophage colony-stimulating factor (GM-CSF).,Theoretically, a combined approach leveraging exogenous cytokine immunotherapy and oncolytic virotherapy would elicit an even greater immune response and improve patient outcomes.,However, regimen scheduling of combination immunostimulation and T-VEC therapy has yet to be established.,Here, we calibrate a computational biology model of sensitive and resistant tumour cells and immune interactions for implementation into an in silico clinical trial to test and individualize combination immuno- and virotherapy.,By personalizing and optimizing combination oncolytic virotherapy and immunostimulatory therapy, we show improved simulated patient outcomes for individuals with late-stage melanoma.,More crucially, through evaluation of individualized regimens, we identified determinants of combination GM-CSF and T-VEC therapy that can be translated into clinically-actionable dosing strategies without further personalization.,Our results serve as a proof-of-concept for interdisciplinary approaches to determining combination therapy, and suggest promising avenues of investigation towards tailored combination immunotherapy/oncolytic virotherapy.

Cutaneous head and neck melanoma has poor outcomes and limited treatment options.,In OPTiM, a phase 3 study in patients with unresectable stage IIIB/IIIC/IV melanoma, intralesional administration of the oncolytic virus talimogene laherparepvec improved durable response rate (DRR; continuous response ≥6 months) compared with subcutaneous granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).,Retrospective review of OPTiM identified patients with cutaneous head and neck melanoma given talimogene laherparepvec (n = 61) or GM‐CSF (n = 26).,Outcomes were compared between talimogene laherparepvec and GM‐CSF treated patients with cutaneous head and neck melanoma.,DRR was higher for talimogene laherparepvec-treated patients than for GM‐CSF treated patients (36.1% vs 3.8%; p = .001).,A total of 29.5% of patients had a complete response with talimogene laherparepvec versus 0% with GM‐CSF.,Among talimogene laherparepvec-treated patients with a response, the probability of still being in response after 12 months was 73%.,Median overall survival (OS) was 25.2 months for GM‐CSF and had not been reached with talimogene laherparepvec.,Treatment with talimogene laherparepvec was associated with improved response and survival compared with GM‐CSF in patients with cutaneous head and neck melanoma.,© 2016 Wiley Periodicals, Inc.,Head Neck 38: 1752-1758, 2016

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

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

Immune checkpoint blockades (ICBs) are a promising treatment for cancers such as melanoma by blocking important inhibitory pathways that enable tumor cells to evade immune attack.,Programmed death ligand 1 monoclonal antibodies (aPDL1s) can be used as an ICB to significantly enhance the effectiveness of tumor immunotherapy by blocking the PD-1/PD-L1 inhibitory pathway.,However, the effectiveness of aPDL1s may be limited by low selectivity in vivo and immunosuppressed tumor microenvironment including hypoxia.,To overcome the limitations, we develop a multifunctional immunoliposome, called CAT@aPDL1-SSL, with catalase (CAT) encapsulated inside to overcome tumor hypoxia and aPDL1s modified on the surface to enhance immunotherapeutic effects against melanoma.,The multifunctional immunoliposomes (CAT@aPDL1-SSLs) are prepared using the film dispersion/post-insertion method.,The efficacy of CAT@aPDL1-SSLs is verified by multiple experiments in vivo and in vitro.,The results of this study suggest that the multifunctional immunoliposomes preserve and protect the enzyme activity of CAT and ameliorate tumor hypoxia.,Moreover, the enhanced cellular uptake of CAT@aPDL1-SSLs in vitro and their in vivo biodistribution suggest that CAT@aPDL1-SSLs have great targeting ability,resulting in improved delivery and accumulation of immunoliposomes in tumor tissue.Finally, by activating and increasing the infiltration of CD8+ T cells at the tumor site, CAT@aPDL1-SSLs inhibit the growth of tumor and prolong survival time of mice,with low systemic toxicity.,In conclusion, the multifunctional immunoliposomes developed and proposed in this study are a promising candidate for melanoma immunotherapy, and could potentially be combined with other cancer therapies like radiotherapy and chemotherapy to produce positive outcomes.

Intratumoral hypoxia is a well-known feature of solid cancers and constitutes a major contributor to cancer metastasis and poor outcomes including melanoma.,Leucine-rich repeats and Ig-like domains 1 (LRIG1) participate in the aggressive progression of several tumors, where its expression is frequently decreased.,In the present study, hypoxia exposure aggravated melanoma cell invasion, migration, vasculogenic mimicry (VM), and epithelial-mesenchymal transition (EMT).,During this process, LRIG1 expression was also decreased.,Importantly, overexpression of LRIG1 notably counteracted hypoxia-induced invasion, migration, and VM, which was further augmented after LRIG1 inhibition.,Mechanism analysis corroborated that LRIG1 elevation muted hypoxia-induced EMT by suppressing E-cadherin expression and increasing N-cadherin expression.,Conversely, cessation of LRIG1 further potentiated hypoxia-triggered EMT.,Additionally, hypoxia stimulation activated the epidermal growth factor receptor (EGFR)/ERK pathway, which was dampened by LRIG1 up-regulation but further activated by LRIG1 inhibition.,More important, blocking this pathway with its antagonist erlotinib abrogated LRIG1 suppression-induced EMT, and subsequently cell invasion, migration, and VM of melanoma cells under hypoxia.,Together, these findings suggest that LRIG1 overexpression can antagonize hypoxia-evoked aggressive metastatic phenotype by suppressing cell invasion, migration, and VM via regulating EGFR/ERK-mediated EMT process.,Therefore, these findings may provide a promising target for melanoma therapy.

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

Adoptive T-cell therapy (ACT) is a highly intensive immunotherapy regime that has yielded remarkable response rates and many durable responses in clinical trials in melanoma; however, 50-60% of the patients have no clinical benefit.,Here, we searched for predictive biomarkers to ACT in melanoma.,Whole exome- and transcriptome sequencing and neoantigen prediction were applied to pre-treatment samples from 27 patients recruited to a clinical phase I/II trial of ACT in stage IV melanoma.,All patients had previously progressed on other immunotherapies.,We report that clinical benefit is associated with significantly higher predicted neoantigen load.,High mutation and predicted neoantigen load are significantly associated with improved progression-free and overall survival.,Further, clinical benefit is associated with the expression of immune activation signatures including a high MHC-I antigen processing and presentation score.,These results improve our understanding of mechanisms behind clinical benefit of ACT in melanoma.,Adoptive T cell therapy (ACT) has yielded high response rates in melanoma, however 50-60% of patients experience no clinical benefit.,Here, the authors identify predictive biomarkers, high non-synonymous mutation and high expressed neoantigen load, that associate with clinical benefit in ACT melanoma patients.

Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans and it can be locally invasive and metastatic to distant sites.,MicroRNAs (miRNAs or miRs) are endogenous, small, non-coding RNAs of 19-25 nucleotides in length, that are involved in regulating gene expression at a post-transcriptional level.,MicroRNAs have been implicated in diverse biological functions and diseases.,In cancer, miRNAs can proceed either as oncogenic miRNAs (onco-miRs) or as tumor suppressor miRNAs (oncosuppressor-miRs), depending on the pathway in which they are involved.,Dysregulation of miRNA expression has been shown in most of the tumors evaluated.,MiRNA dysregulation is known to be involved in the development of cutaneous squamous cell carcinoma (CSCC).,In this review, we focus on the recent evidence about the role of miRNAs in the development of CSCC and in the prognosis of this form of skin cancer.

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

New therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors.,Drug screening, IC50 determinations as well as synergy assays were detected by the MTT assay.,Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry.,TUNEL staining was performed using immunocytochemistry.,Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry.,To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used.,Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors.,Evaluation of immune infiltrates was carried out by flow cytometry.,Using a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines.,Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS).,TM’s anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition.,Specifically, TM blunted S6 phosphorylation in both BRAFV600E and BRAF wildtype (WT) melanoma cell lines.,TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model.,In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation.,Furthermore TM decreased the infiltration of immune suppressive regulatory CD4+CD25+ T cells and FOXP3 and ROR-γt positive CD4+ T cells.,Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAFV600E mutation and could be additively or synergistically combined with Cobimetinib in both BRAFV600E and BRAF WT melanoma cell lines in inducing anti-cancer effects.,Taken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAFV600E and BRAF WT melanoma.

We have recently shown that radiotherapy may not only be a successful local and regional treatment but, when combined with MSCs, may also be a novel systemic cancer therapy.,This study aimed to investigate the role of exosomes derived from irradiated MSCs in the delay of tumor growth and metastasis after treatment with MSC + radiotherapy (RT).,We have measured tumor growth and metastasis formation, of subcutaneous human melanoma A375 xenografts on NOD/SCID-gamma mice, and the response of tumors to treatment with radiotherapy (2 Gy), mesenchymal cells (MSC), mesenchymal cells plus radiotherapy, and without any treatment.,Using proteomic analysis, we studied the cargo of the exosomes released by the MSC treated with 2 Gy, compared with the cargo of exosomes released by MSC without treatment.,The tumor cell loss rates found after treatment with the combination of MSC and RT and for exclusive RT, were: 44.4% % and 12,1%, respectively.,Concomitant and adjuvant use of RT and MSC, increased the mice surviving time 22,5% in this group, with regard to the group of mice treated with exclusive RT and in a 45,3% respect control group.,Moreover, the number of metastatic foci found in the internal organs of the mice treated with MSC + RT was 60% less than the mice group treated with RT alone.,We reasoned that the exosome secreted by the MSC, could be implicated in tumor growth delay and metastasis control after treatment.,Our results show that exosomes derived form MSCs, combined with radiotherapy, are determinant in the enhancement of radiation effects observed in the control of metastatic spread of melanoma cells and suggest that exosome-derived factors could be involved in the bystander, and abscopal effects found after treatment of the tumors with RT plus MSC.,Radiotherapy itself may not be systemic, although it might contribute to a systemic effect when used in combination with mesenchymal stem cells owing the ability of irradiated MSCs-derived exosomes to increase the control of tumor growth and metastasis.,The online version of this article (10.1186/s12943-018-0867-0) contains supplementary material, which is available to authorized users.

Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising.,We describe the clinical course of a 15‐year‐old female diagnosed with AJCC stage IB non‐ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy.,We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas.,A somatic BRAFV 600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed.,A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma‐predisposing genes.,Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable.

Melanoma is the most deadly form of cutaneous malignancy, and its incidence rates are rising worldwide.,In melanoma, constitutive activation of the BRAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR (PI3K) signaling pathways plays a pivotal role in cell proliferation, survival and tumorigenesis.,A combination of compounds that lead to an optimal blockade of these critical signaling pathways may provide an effective strategy for prevention and treatment of melanoma.,The phytochemical fisetin is known to possess anti-proliferative and pro-apoptotic activities.,We found that fisetin treatment inhibited PI3K signaling pathway in melanoma cells.,Therefore, we investigated the effect of fisetin and sorafenib (an RAF inhibitor) alone and in combination on cell proliferation, apoptosis and tumor growth.,Combination treatment (fisetin + sorafenib) more effectively reduced the growth of BRAF-mutated human melanoma cells at lower doses when compared to individual agents.,In addition, combination treatment resulted in enhanced (i) apoptosis, (ii) cleavage of caspase-3 and PARP, (iii) expression of Bax and Bak, (iv) inhibition of Bcl2 and Mcl-1, and (v) inhibition of expression of PI3K, phosphorylation of MEK1/2, ERK1/2, AKT and mTOR.,In athymic nude mice subcutaneously implanted with melanoma cells (A375 and SK-MEL-28), we found that combination therapy resulted in greater reduction of tumor growth when compared to individual agents.,Furthermore, combination therapy was more effective than monotherapy in: (i) inhibition of proliferation and angiogenesis, (ii) induction of apoptosis, and (iii) inhibition of the MAPK and PI3K pathways in xenograft tumors.,These data suggest that simultaneous inhibition of both these signaling pathways using combination of fisetin and sorafenib may serve as a therapeutic option for the management of melanoma.

Melanoma is a highly aggressive cancer that exhibits metastasis to various critical organs.,Unlike any other cancer cells, melanoma cells can synthesize melanin in large amounts, becoming heavily pigmented.,Until now the role of melanin in melanoma, particularly the effect of melanin presence on the abilities of melanoma cells to spread and metastasize remains unknown.,Recently, we have shown that melanin dramatically modified elastic properties of melanoma cells and inhibited the cells invasive abilities in vitro.,Here, we inoculated human melanoma cells with different melanin content into nude mice and tested the hypothesis that cell elasticity is an important property of cancer cells for their efficient spread in vivo.,The obtained results clearly showed that cells containing melanin were less capable to spread in mice than cells without the pigment.,Our findings indicate that the presence of melanin inhibits melanoma metastasis, emphasizing possible clinical implications of such an inhibitory effect.

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

Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.

In uveal melanoma (UM), the most frequent primary intraocular tumour in adults, loss of one entire chromosome 3 (monosomy 3 (M3)) is observed in ∼50% of tumours and is significantly associated with metastatic disease.,The strong association of metastatic disease with M3 offers the opportunity for molecular prognostic testing of UM patients.,To re-evaluate M3 as prognostic marker in our clinical and laboratory setting and to determine the metastatic potential of rare tumours with partial M3, we performed a comprehensive study on 374 UM patients treated by enucleation in our clinic within 10 consecutive years, starting in 1998.,Genotyping of all tumours was performed by microsatellite analysis.,Median follow-up time was 5.2 years.,The disease-specific mortality rates (death by UM metastases) for tumours with disomy 3 (D3) and M3 were 13.2% and 75.1%, respectively.,The disease-specific survival was worse when M3 was observed together with chromosome 8 alterations (P=0.020).,Death of UM metastases was also observed in 12 patients (9%) with D3 tumours.,The metastasising D3 tumours showed a larger basal tumour diameter (P=0.007), and were more frequently of mixed or epitheloid cell type (P<0.0001) than D3 tumours that did not metastasise.,Mortality rate of tumours showing partial M3 (8.3%) was as low as that for tumours with D3.,This shows that large tumours with disomy 3 have an increased risk to develop metastases.,On the basis of these results, our clinic offers routine prognostic testing of UM patients by chromosome 3 typing.

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

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

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

NRAS and its effector BRAF are frequently mutated in melanoma.,Paradoxically, CRAF but not BRAF was shown to be critical for various RAS-driven cancers, raising the question of the role of RAF proteins in NRAS-induced melanoma.,Here, using conditional ablation of Raf genes in NRAS-induced mouse melanoma models, we investigate their contribution in tumour progression, from the onset of benign tumours to malignant tumour maintenance.,We show that BRAF expression is required for ERK activation and nevi development, demonstrating a critical role in the early stages of NRAS-driven melanoma.,After melanoma formation, single Braf or Craf ablation is not sufficient to block tumour growth, showing redundant functions for RAF kinases.,Finally, proliferation of resistant cells emerging in the absence of BRAF and CRAF remains dependent on ARAF-mediated ERK activation.,These results reveal specific and compensatory functions for BRAF and CRAF and highlight an addiction to RAF signalling in NRAS-driven melanoma.,The melanoma-driver mutations in NRAS and BRAF are mutually exclusive but the contribution of RAF signalling downstream of NRAS remains to be clarified.,Here, using mouse models, the authors show specific roles of each member of the RAF family at different stages of melanomagenesis.

While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly.,One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis.,We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination.,The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome.,We tested this hypothesis in cell lines and in mice.,Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression.,Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A.,No significant changes were observed with BCL-2.,Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID.,No changes in mRNA or protein correlated with response.,Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment.,In xenograft models, navitoclax enhanced the efficacy of PLX4720.,The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance.,Trial Registrations: ClinicalTrials.gov NCT01006980;,ClinicalTrials.gov NCT01107418;,ClinicalTrials.gov NCT01264380;,ClinicalTrials.gov NCT01248936;,ClinicalTrials.gov NCT00949702;,ClinicalTrials.gov NCT01072175

The generation of antitumor CD8+ T cell responses requires type I interferon responsiveness in host antigen-presenting cells,Despite lack of tumor control in many models, spontaneous T cell priming occurs frequently in response to a growing tumor.,However, the innate immune mechanisms that promote natural antitumor T cell responses are undefined.,In human metastatic melanoma, there was a correlation between a type I interferon (IFN) transcriptional profile and T cell markers in metastatic tumor tissue.,In mice, IFN-β was produced by CD11c+ cells after tumor implantation, and tumor-induced T cell priming was defective in mice lacking IFN-α/βR or Stat1.,IFN signaling was required in the hematopoietic compartment at the level of host antigen-presenting cells, and selectively for intratumoral accumulation of CD8α+ dendritic cells, which were demonstrated to be essential using Batf3−/− mice.,Thus, host type I IFNs are critical for the innate immune recognition of a growing tumor through signaling on CD8α+ DCs.

The objective of the study was to summarize the role of DNA methylation in the development and metastasis of uveal melanoma (UM).,The relevant studies in MEDLINE were searched.,In this review, we performed a comprehensive literature search in MEDLINE using “uveal melanoma” AND (“DNA methylation” OR “epigenetics”) for original research/review articles published before February 2018 on the relationship between DNA methylation and UM.,References of the retrieved studies were also examined to search for potentially relevant papers.,Previous studies on the relationship between DNA methylation and UM covered many genes including tumor suppressor genes (TSGs), cyclin-dependent kinase genes, and other genes.,Among them, the TSG genes such as RASSF1A and p16INK4a, which encodes a cyclin-dependent kinase inhibitor, are relatively well-studied genes.,Specifically, a high percentage of promoter methylation of RASSF1A was observed in UM cell lines and/or patients with UM.,Promoter methylation of RASSF1A was also associated with the development of metastasis.,Similarly, a high percentage of promoter hypermethylation of p16INK4a was found in UM cell lines.,DNA promoter methylation can control the expression of p16INK4a, which affect cell growth, migration, and invasion in UM.,Many other genes might also be involved in the pathogenesis of UM such as the Ras and EF-hand domain containing (RASEF) gene, RAB31, hTERT, embryonal fyn-associated substrate, and deleted in split-hand/split-foot 1.,Our review reveals the complex mechanisms underlying the tumorigenesis of UM and highlights the great needs of future studies to discover more genes/5’-C-phosphate-G-3’ sites contributing to the development/metastasis of UM and explore the mechanisms through which epigenetic changes exert their function in UM.

We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas.,In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter.,Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+.,Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001).,PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively).,In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004).,PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter.,Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status.,Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA.,PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes.,Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Oral malignant melanoma (OMM) is the most common canine melanocytic neoplasm.,Overlap between the somatic mutation profiles of canine OMM and human mucosal melanomas suggest a shared UV-independent molecular aetiology.,In common with human mucosal melanomas, most canine OMM metastasise.,There is no reliable means of predicting canine OMM metastasis, and systemic therapies for metastatic disease are largely palliative.,Herein, we employed exon microarrays for comparative expression profiling of FFPE biopsies of 18 primary canine OMM that metastasised and 10 primary OMM that did not metastasise.,Genes displaying metastasis-associated expression may be targets for anti-metastasis treatments, and biomarkers of OMM metastasis.,Reduced expression of CXCL12 in the metastasising OMMs implies that the CXCR4/CXCL12 axis may be involved in OMM metastasis.,Increased expression of APOBEC3A in the metastasising OMMs may indicate APOBEC3A-induced double-strand DNA breaks and pro-metastatic hypermutation.,DNA double strand breakage triggers the DNA damage response network and two Fanconi anaemia DNA repair pathway members showed elevated expression in the metastasising OMMs.,Cross-validation was employed to test a Linear Discriminant Analysis classifier based upon the RT-qPCR-measured expression levels of CXCL12, APOBEC3A and RPL29.,Classification accuracies of 94% (metastasising OMMs) and 86% (non-metastasising OMMs) were estimated.

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

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

The absence of BRCA1-associated protein 1 (BAP1) expression in uveal melanoma (UM) is associated with metastatic progression and reduced survival.,In this study, we examine nuclear BAP1 (nBAP1) protein expression in primary UMs (PUMs) that show both ‘typical' and ‘atypical' clinical courses according to their chromosome 3 status, and secondary hepatic metastatic UM (MUM), correlating the results with histological, clinical and survival data.,Nuclear BAP1 expression was immunohistochemically assessed in tissue microarrays (TMAs) of: (a) 68 PUM patients, who had been treated surgically; and (b) 13 MUM patients, with 5 cases being paired with primary tumour tissue.,All cases were fully annotated.,The percentage of tumour cell nuclei staining positively for BAP1 was scored by independent observers.,Nuclear BAP1 protein expression was absent in 35 out of 68 (51%) PUM patients, correlating strongly with poor prognostic clinicopathological and genetic parameters and reduced survival (Log rank, P<0.001).,Lack of nBAP1 expression importantly identified a subset of ‘atypical' PUM patients with disomy of chromosome 3 but with unexpected metastatic relapse.,Nuclear BAP1 expression was absent in 10 out of 13 (77%) MUM and expression was concordant in all paired PUM and MUM patients.,Absent nBAP1 protein expression is an independent survival predictor for UM patients, easily examined using immunohistochemistry.

N6-methylation of adenosine (m6A) RNA modification plays important roles in development and tumorigenesis.,The functions and mechanisms of m6A demethylases during cancer immunotherapy is still unclear.,Here we employed melanoma and colon syngeneic mouse models to study the roles of m6A demethylases ALKBH5 and FTO during anti-PD-1 antibody and GVAX vaccination therapy.,We found that ALKBH5 knockout in tumor cells enhances efficacy of immunotherapy and prolonged mouse survival.,ALKBH5 modulates target gene expression and gene splicing, leading to changes of metabolite contents, such as lactate in tumor microenvironment, which regulates suppressive lymphocytes Treg and myeloid-derived suppressor cell accumulations.,Importantly, by using ALKBH5-specific inhibitor, we observed the similar phenotype, indicating future translational application of our findings.,Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB.,N6-methylation of adenosine (m6A) in RNA regulates many pathophysiological processes.,Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy.,Alkbh5 has effects on m6A density and splicing events in tumors during ICB.,Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells.,Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy.,Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy.,Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.

The presence of T cells in tumors predicts overall survival for cancer patients.,However, why most tumors are poorly infiltrated by T cells is barely understood.,T-cell recruitment towards the tumor requires a chemokine gradient of the critical IFNγ-induced chemokines CXCL9/10/11.,Here, we describe how tumors can abolish IFNγ-induced chemokines, thereby reducing T-cell attraction.,This mechanism requires extracellular galectin-3, a lectin secreted by tumors.,Galectins bind the glycans of glycoproteins and form lattices by oligomerization.,We demonstrate that galectin-3 binds the glycans of the extracellular matrix and those decorating IFNγ.,In mice bearing human tumors, galectin-3 reduces IFNγ diffusion through the tumor matrix.,Galectin antagonists increase intratumoral IFNγ diffusion, CXCL9 gradient and tumor recruitment of adoptively transferred human CD8+ T cells specific for a tumor antigen.,Transfer of T cells reduces tumor growth only if galectin antagonists are injected.,Considering that most human cytokines are glycosylated, galectin secretion could be a general strategy for tumor immune evasion.,Most tumours are poorly infiltrated by T cells.,Here the authors show that galectin-3 secreted by tumours binds both glycosylated IFNγ and glycoproteins of the tumour extracellular matrix, thus avoiding IFNγ diffusion and the formation of an IFNγ-induced chemokine gradient required for T cell infiltration.

Extracellular matrix 1 (ECM1) is over-expressed in multiple epithelial malignancies.,However, knowledge regarding the expression of ECM1 in melanomas and the mechanisms of ECM1 regulation is limited.,In this study, we found that ECM1 is over-expressed in several melanoma cell lines, when compared to primary melanocytes, and furthermore, that ECM1 expression paralleled that of TFAP2C levels in multiple cell lines.,Knockdown of TFAP2C in the A375 cell line with siRNA led to a reduction in ECM1 expression, and upregulation of TFAP2C with adenoviral vectors in the WM793 cell line resulted in ECM1 upregulation.,Utilizing 5’ RACE to identify transcription start sites (TSS) and luciferase reporter assays in the ECM1-overexpressing A375 cell line, we identified the minimal promoter region of human ECM1 and demonstrate that an approximately 100bp fragment upstream of the TSS containing a TATA box and binding sites for AP1, SP1 and Ets is sufficient for promoter activity.,Chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in the A375 cell line identified an AP2 regulatory region in the promoter of the ECM1 gene.,Gelshift assays further confirmed binding of TFAP2C to this site.,ECM1 knockdown reduces melanoma cell attachment and is consistent with findings that ECM1 overexpression has been associated with a poor prognosis.,Our investigations show an as yet unrecognized role for TFAP2C in melanoma via its regulation of ECM1.

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.

Development and validation of robust molecular biomarkers has so far been limited in melanoma research.,In this paper we used a large population-based cohort to replicate two published gene signatures for melanoma classification.,We assessed the signatures prognostic value and explored their biological significance by correlating them with factors known to be associated with survival (vitamin D) or etiological routes (nevi, sun sensitivity and telomere length).,Genomewide microarray gene expressions were profiled in 300 archived tumors (224 primaries, 76 secondaries).,The two gene signatures classified up to 96% of our samples and showed strong correlation with melanoma specific survival (P=3×10−4), Breslow thickness (P=5×10−10), ulceration (P=9.x10−8) and mitotic rate (P=3×10−7), adding prognostic value over AJCC stage (adjusted hazard ratio 1.79, 95%CI 1.13-2.83), as previously reported.,Furthermore, molecular subtypes were associated with season-adjusted serum vitamin D at diagnosis (P=0.04) and genetically predicted telomere length (P=0.03).,Specifically, molecular high-grade tumors were more frequent in patients with lower vitamin D levels whereas high immune tumors came from patients with predicted shorter telomeres.,Our data confirm the utility of molecular biomarkers in melanoma prognostic estimation using tiny archived specimens and shed light on biological mechanisms likely to impact on cancer initiation and progression.

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.

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

MELOE-1 and MELOE-2, two highly specific melanoma antigens involved in T cell immunosurveillance are produced by IRES-dependent translation of the long « non coding » and polycistronic RNA, meloe.,In the present study, we document the expression of an additional ORF, MELOE-3, located in the 5′ region of meloe.,Data from in vitro translation experiments and transfection of melanoma cells with bicistronic vectors documented that MELOE-3 is exclusively translated by the classical cap-dependent pathway.,Using a sensitive tandem mass spectrometry technique, we detected the presence of MELOE-3 in total lysates of both melanoma cells and normal melanocytes.,This contrasts with our previous observation of the melanoma-restricted expression of MELOE-1 and MELOE-2.,Furthermore, in vitro stimulation of PBMC from 6 healthy donors with overlapping peptides from MELOE-1 or MELOE-3 revealed a very scarce MELOE-3 specific T cell repertoire as compared to the abundant repertoire observed against MELOE-1.,The poor immunogenicity of MELOE-3 and its expression in melanocytes is consistent with an immune tolerance towards a physiologically expressed protein.,In contrast, melanoma-restricted expression of IRES-dependent MELOE-1 may explain its high immunogenicity.,In conclusion, within the MELOE family, IRES-dependent antigens represent the best T cell targets for immunotherapy of melanoma.

Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons.,We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice.,All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers.,Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours.,Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway.,Anti-oxidants promoted distant metastasis in NSG mice.,Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice.,Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

V600BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi).,Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits.,Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine.,Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells.,Isogenic BRAFi sensitive parental V600BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation.,To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied.,Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-l-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo.,We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts.,In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro.,We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance.,When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells.,Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

Melanin is major factor that determines skin color as well as one of the defense systems that prevent the UV-induced damage.,In case of abnormal concentration of melanin, skin diseases or problems occur such as albinism, leukoplakia, melasma, freckles, moles, and lentigo.,With the lifespan of humans has been extended, importance of ‘life quality’ has been increased.,White and clean skin is very important part of the satisfaction of appearance, especially for Asia women.,The aim of this study was to find an anti-melanogenesis activity for which the aerial part of Pueraria thunbergiana can be utilized based on the increase in demands for cosmetics, particularly natural products.,We demonstrated anti-pigmentation effects of aerial part of P. thunbergiana by measuring melanin content and through staining in the B16F10 melanoma cell line.,The aerial part of P. thunbergiana decreased tyrosinase activity significantly in B16F10 cell cultures, while there is no direct effect on enzyme in cell-free conditions.,To define the mechanisms, real-time PCR, western blot, glucosidase activity and antioxidant activity assay were implemented.,As results, we demonstrated that aerial part of P. thunbergiana has anti-melanogenesis activity via two mechanisms.,One is downgrading microphthalmia-associated transcription factor by activating Akt/GSK-3β.,Consequently, transcription of tyrosinase and tyrosinase-related protein 1 is decreased.,Another is interrupting maturation of tyrosinase through inhibiting α-glucosidase.,Furthermore, aerial part of P. thunbergiana showed great efficacy on pigmentation in vivo.,These results suggest that aerial part of P. thunbergiana can be used as an anti-melanogenic agent.

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.

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

While recent years have seen a revolution in the treatment of metastatic cutaneous melanoma, no treatment has yet been able to demonstrate any prolonged survival in metastatic uveal melanoma.,Thus, metastatic uveal melanoma remains a disease with an urgent unmet medical need.,Reports of treatment with immune checkpoint inhibitors have thus far been disappointing.,Based on animal experiments, it is reasonable to hypothesize that the effect of immunotherapy may be augmented by epigenetic therapy.,Proposed mechanisms include enhanced expression of HLA class I and cancer antigens on cancer cells, as well as suppression of myeloid suppressor cells.,The PEMDAC study is a multicenter, open label phase II study assessing the efficacy of concomitant use of the PD1 inhibitor pembrolizumab and the class I HDAC inhibitor entinostat in adult patients with metastatic uveal melanoma.,Primary endpoint is objective response rate.,Eligible patients have histologically confirmed metastatic uveal melanoma, ECOG performance status 0-1, measurable disease as per RECIST 1.1 and may have received any number of prior therapies, with the exception of anticancer immunotherapy.,Twenty nine patients will be enrolled.,Patients receive pembrolizumab 200 mg intravenously every third week in combination with entinostat 5 mg orally once weekly.,Treatment will continue until progression of disease or intolerable toxicity or for a maximum of 24 months.,The PEMDAC study is the first trial to assess whether the addition of an HDAC inhibitor to anti-PD1 therapy can yield objective anti-tumoral responses in metastatic UM.,ClinicalTrials.gov registration number: NCT02697630.,(Registered 3 March 2016).,EudraCT registration number: 2016-002114-50.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease.,Therapeutic options for metastatic UM are limited, with clinical trials having little impact.,Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris).,While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM.,All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX).,We identify three other significantly mutated genes (TP53, RPL5 and CENPE).,Uveal melanoma has a propensity to metastasise.,Here, the authors report the whole genome sequence of 103 uveal melanomas and find that the tumour mutational burden is variable and that two subsets of tumours are characterised by MBD4 mutations and a UV exposure signature.

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

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis.,Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development.,Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma.,Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation.,Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR.,Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER).,This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.,The interaction of DOT1L with MLL oncogenic fusion proteins has been implicated in leukemogenesis.,Here, the authors show a contrasting role for DOT1L in protecting UVR-induced melanomagenesis by facilitating DNA repair through interaction with XPC.

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.