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Macrophages are located in essentially all tissues due to their “janitor” function.,Macrophages can exert either anti- or pro-tumor activities depending upon the specific tumor microenvironment they inhabit.,Substantial evidence indicates that macrophages, owing to their plasticity, can be reeducated to adopt a protumoral phenotype within a tumor microenvironment through the help of growth factors in the microenvironment and intercellular interactions.,As the lethality of malignant melanoma is due to its aggressive capacity for metastasis and resistance to therapy, considerable effort has gone toward treatment of metastatic melanoma.,In the present review, we focus on the pro-tumor activities of macrophages in melanoma.,Based upon the information presented in this review it is anticipated that new therapies will soon be developed that target pro-tumor activities of macrophages for use in the treatment of melanoma.

Loss of expression of surface antigens represents a significant problem for cancer immunotherapy.,Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes.,We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels.,Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines.,We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR.,Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma.,Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation.,This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.

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

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

Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones.,Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.,By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib.,We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2.,Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway.,In doing so, miR-211 serves as an effector of vemurafenib's pro-pigmentation activity.,We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib.,In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells.,Our work challenges the widely accepted “same miRNA family = same function” rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.

A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression.,The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis.,Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others.,We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration.,Individual tumor cells can invade in either elongated, “mesenchymal-type” or rounded, “amoeboid-like” modes and these two modes of invasion are inter-convertible [1].,In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion.,MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion.,Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions.,In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology.,Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

Patients with locally/regionally advanced melanoma were treated with neoadjuvant combination immunotherapy with high-dose interferon α-2b (HDI) and ipilimumab in a phase I clinical trial.,Tumor specimens were obtained prior to the initiation of neoadjuvant therapy, at the time of surgery and progression if available.,In this study, gene expression profiles of tumor specimens (N = 27) were investigated using the NanoString nCounter® platform to evaluate associations with clinical outcomes (pathologic response, radiologic response, relapse-free survival (RFS), and overall survival (OS)) and define biomarkers associated with tumor response.,The Tumor Inflammation Signature (TIS), an 18-gene signature that enriches for response to Programmed cell death protein 1 (PD-1) checkpoint blockade, was also evaluated for association with clinical response and survival.,It was observed that neoadjuvant ipilimumab-HDI therapy demonstrated an upregulation of immune-related genes, chemokines, and transcription regulator genes involved in immune cell activation, function, or cell proliferation.,Importantly, increased expression of baseline pro-inflammatory genes CCL19, CD3D, CD8A, CD22, LY9, IL12RB1, C1S, C7, AMICA1, TIAM1, TIGIT, THY1 was associated with longer OS (p < 0.05).,In addition, multiple genes that encode a component or a regulator of the extracellular matrix such as MMP2 and COL1A2 were identified post-treatment as being associated with longer RFS and OS.,In all baseline tissues, high TIS scores were associated with longer OS (p = 0.0166).,Also, downregulated expression of cell proliferation-related genes such as CUL1, CCND1 and AAMP at baseline was associated with pathological and radiological response (unadjusted p < 0.01).,In conclusion, we identified numerous genes that play roles in multiple biological pathways involved in immune activation, immune suppression and cell proliferation correlating with pathological/radiological responses following neoadjuvant immunotherapy highlighting the complexity of immune responses modulated by immunotherapy.,Our observations suggest that TIS may be a useful biomarker for predicting survival outcomes with combination immunotherapy.

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice.,We have now turned this idea to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases.,We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds.,We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage.,In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15.,Ribosome biogenesis was upregulated in skin shortly after UVR exposure.,Mechanistically, variation in the ‘usual suspects’ by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC.,Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important.,Melanoma is a type of skin cancer.,Melanoma tumors form in the skin’s pigment-producing cells or melanocytes.,Growing evidence points to complex interactions between genetics and environmental exposures that contribute to the risk of developing melanoma.,Ultraviolet (UV) radiation from the sun causes genetic mutations in melanocytes.,This sun exposure interacts with genetic variations that may make people more or less vulnerable to such DNA damage.,For example, genetic variations that control skin color or the cell’s ability to repair DNA, and that influence how easily people develop sunburn, all affect whether UV damage leads to melanoma.,However, some forms of melanoma are not caused by sun exposure at all.,Most of the genetic variations linked to melanoma have a small effect on the risk of developing the disease.,So, it is unlikely that these genes alone cause melanoma.,Few studies have been able to map the complex interactions between genes and the environment that lead to melanoma.,So far, it has been unclear if there are different genetic mechanisms that lead to an increased risk for sun-exposure linked melanoma and non-sun linked melanoma.,Now, Ferguson et al. show that variations in the genes involved in DNA repair during normal cell growth are linked to non-sun linked melanoma.,Sun-linked melanoma, on the other hand, was associated with genes involved in the production of proteins in part of the cell called ribosomes.,In the experiments, the effects of both UV light and various genetic variations were assessed across many different strains of mice.,Mutations that impair the cell’s ability to repair UV-induced DNA damage or that contribute to excessive inflammation in response to sunburn did not increase melanoma susceptibility in these experiments.,Ferguson et al. show that the amount of UV-induced DNA damage alone does not explain melanoma risk, which may not always depend on skin pigmentation.,The experiments also suggest that non-UV linked melanoma is caused by a different mechanism than sun exposure-linked melanoma.,Learning more about different genetic factors that affect the risk of developing different types of melanoma may help scientists develop more specific treatments.

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

Malignant melanoma is a rare disease in Asia, and knowledge on its characteristics and clinical outcome in Asian patients is limited.,The purpose of this observational study was to determine the clinical presentation and outcome of patients with melanoma in China.,A database was prospectively established for the purpose of this analysis.,The elements of the database included basic demographic data of patients and prognosticators previously reported in literature, as well as follow-up data including clinical outcome after treatment.,Medical record of all patients with pathologically diagnosed malignant melanoma consulted in our center since 2006 were retrieved and reviewed.,No patient was excluded in this study.,Statistical analyses including survival and multivariate analyses of factors associated with survival were respectively performed by Kaplan-Meier method and Cox proportional hazard model.,A total of 522 consecutive and nonselected cases were evaluated.,There were 218 cases (41.8%) of acral lentiginous melanoma (ALM), 118 (22.6%) of mucosal melanoma (MCM), 103 (19.7%) of nodular melanoma (NM), 33 (6.3%) of superficial spreading melanoma (SSM), and others were Lentigo maligna melanoma or unclassifiable disease.,The proportion of patients with clinical stage I, II, III, and IV diseases were 6.1%, 55.9%, 25.1%, and 12.8%, respectively.,Among the 357 cases of cutaneous melanoma, 234 patients (65.5%) had ulceration.,The 5-year overall survival rate of all 522 patients was 41.6%, and the median survival time was 3.92 years (95% CI, 3.282 to 4.558).,Five-year survival rates of patients with stage I, II, III, and IV diseases were 94.1%, 44.0%, 38.4% and 4.6% respectively (P < 0.001).,Multivariate analysis revealed that clinical stage and the ulceration were two significant prognosticators for OS.,In addition, extent of surgery and use of adjuvant therapy were significant prognosticators for DFS in patients with non-metastatic disease after definitive treatment.,Pathological subtype was not a significant prognostic factor to predict wither OS or DFS.,Prognoses of patients with malignant melanoma diagnosed in China were suboptimal, and most patients were diagnosed with locally advanced disease (i.e., stage II or above).,ALM and MCM are the two most commonly diagnosed pathological subtypes.,Clinical staging and presence of ulceration was significantly associated with clinical outcome in terms of OS, while treatment strategy including extent of surgery and use of adjuvant therapy were significant predictors of DFS.

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.

MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs.,Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes.,In the present study, we investigated the role of miRNA-34b/c in uveal melanoma.,Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples.,Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry.,Cell apoptosis was measured by caspase3/7 assay.,Cell migration was evaluated by transwell migration assay.,The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay.,In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting.,miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs.,The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G1 arrest rather than the induction of apoptosis.,Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells.,Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.,Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.

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

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

Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties.,Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy.,One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages.,Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching.,Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state.,We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching.,We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states.,Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4).,A gene set related to cancer cell motility and invasiveness was concomitantly downregulated.,Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein.,SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology.,We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity.,ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state.,This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development.,The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.

The question whether tumorigenic cancer stem cells exist in human melanomas has arisen recently1.,Here we show that in melanomas, tumor stem cells (MTSC) can be isolated prospectively as a highly enriched CD271+ MTSC population using a process that maximizes viable cell transplantation1,6.,In this study the tumors sampled were taken from a broad spectrum of sites and stages.,High viability FACS isolated cells resuspended in a matrigel vehicle were implanted into T, B, and NK deficient Rag2−/− γc−/− mice (RG) mice.,The CD271+ subset of cells was the tumor initiating population in 9/10 melanomas tested.,Transplantation of isolated melanoma cells into engrafted human skin or bone in RG mice resulted in melanoma from CD271+ but not CD271− cells.,We also showed that tumors transplanted by CD271+ patient cells were capable of metastasis in-vivo.,Importantly, CD271+ melanoma cells lacked expression of TYR, MART and MAGE in 86%, 69% and 68% of melanoma patients respectively suggesting why T cell therapies directed at these antigens usually result in only temporary tumor shrinkage.

The incidence of melanoma, one of the most aggressive of the skin cancers, has been increasing worldwide in the last few decades.,Data from Latin America and Brazil remain scarce.,We aimed to describe the demographic, clinical, and histopathological data; therapy characteristics; and survival rates of the Brazilian melanoma patient population.,We collected and analysed retrospective data from 15 years at a tertiary cancer centre.,We describe patient characteristics and treatment.,We calculated survival, and identified the main prognostic factors through univariate and multivariate analysis.,We analysed a total of 1073 patients, with a mean age of 56.7 years.,Men and women experienced similar prevalence, and 91.2% of patients had white skin.,The most prevalent subtype was superficial spreading, and the most prevalent anatomic location was the trunk (32.2%), followed by the lower extremities (28%).,Of all cases, 567 (52.9%) were assigned to clinical stages I and II, while 382 (32.6%) were stages III and IV.,Surgery was the main treatment.,Sentinel node biopsy was performed in 373 patients, with 23.8% positivity.,Overall actuarial 5-year survival was 67.6%.,Multivariate analysis showed that gender, serum lactate dehydrogenase (LDH) levels at diagnosis; anatomic location, TNM stage, and local recurrence were significant prognostic factors.,Overall survival was lower than worldwide rates.,The main factors influencing survival were similar to those in other populations.,Local recurrence was independently associated with lower survival rates.,The high prevalence of advanced cases reinforces the importance of strategies to diagnose melanomas in the early stages.,There is a need for future multi-institutional prospective studies to attain a better understanding of possible socioeconomic and other influences on survival among melanoma populations in Brazil and Latin America.

Cutaneous melanoma is a skin cancer with low incidence but high mortality rates.,Several factors are associated with increased risk of melanoma, such as excessive sun exposure, fair skin, and family history, among others.,Little is known about the spatial distribution of this cancer in Brazil.,To identify, through the use of geostatistical tools, spatial clusters of municipalities in the state of São Paulo based on their incidence of cutaneous melanoma.,This was an ecological and exploratory study of data on new cases obtained from Fundação Oncocentro for the period 1 January 2006-31 December 2011.,Cases were separated by gender and rates per 100,000 inhabitants were calculated and used to compile thematic maps, Moran maps and kernel maps, using TerraView software.,There were 3,172 new cases of cutaneous melanoma in the study period.,High rates were identified in the North, Northwest, Southwest, and Southeast regions of São Paulo state.,Global Moran's I values were statistically significant (p<0.05) at 0.12, 0.08, and 0.16, respectively, for males, females, and all cases.,Areas such as the Southeast, North, and Northwest of São Paulo were identified as being of high priority for intervention.,Spatial clusters of municipalities with high incidence rates of cutaneous melanoma in the state of São Paulo were identified.,These data can serve as an important input for public health agencies.

PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome.,Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi).,However, the function and regulation of expression of PD-L1 remain incompletely understood.,After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression.,These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement.,Consistently, in vitro they showed significantly increased invasive properties.,This phenotype was controlled in part by PD-L1, as determined after silencing the molecule.,Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA.,Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays.,In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis.,Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi.,Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions.,In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells.,We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.

Cutaneous malignant melanoma is among the deadliest human cancers, broadly resistant to most clinical therapies.,A majority of patients with BRAFV600E melanomas respond well to inhibitors such as vemurafenib, but all ultimately relapse.,Moreover, there are no viable treatment options available for other non-BRAF melanoma subtypes in the clinic.,A key to improving treatment options lies in a better understanding of mechanisms underlying melanoma progression, which are complex and heterogeneous.,In this study we integrated gene and microRNA (miRNA) expression data from genetically engineered mouse models of highly and poorly malignant melanocytic tumors, as well as available human melanoma databases, and discovered an important role for a pathway centered on a tumor suppressor miRNA, miR-32.,Malignant tumors frequently exhibited poor expression of miR-32, whose targets include NRAS, PI3K and notably, MCL-1.,Accordingly, MCL-1 was often highly expressed in melanomas, and when knocked down diminished oncogenic potential.,Forced MCL-1 overexpression transformed immortalized primary mouse melanocytes, but only when also expressing activating mutations in BRAF, CRAF or PI3K.,Importantly, both miR-32 replacement therapy and the MCL-1-specific antagonist sabutoclax demonstrated single-agent efficacy, and acted synergistically in combination with vemurafenib in preclinical melanoma models.,We here identify miR-32/MCL-1 pathway members as key early genetic events driving melanoma progression, and suggest that their inhibition may be an effective anti-melanoma strategy irrespective of NRAS, BRAF, and PTEN status.

Randomized trials evaluating programmed cell death protein 1 (PD-1) inhibitors in metastatic melanoma either permitted treatment for 2 years (pembrolizumab) or more (nivolumab).,The optimal duration of therapy is currently unknown due to limited data, and shorter therapies may be effective.,Data of patients with metastatic cutaneous melanoma treated with single-agent PD-1 inhibitors at Huntsman Cancer Institute from January 1, 2015, to December 31, 2018, was reviewed to identify a continuous series of patients who made the joint decision with their provider to electively discontinue therapy at 1 year (>6 months and <18 months) in the setting of ongoing treatment response or disease stability.,Patients were excluded if they received PD-1 inhibitors with other systemic therapy, had prior exposure to PD-1 therapy, or discontinued treatment due to disease progression or immune-related adverse event.,Best objective response (BOR) per RECIST V.1.1 at treatment discontinuation, progression-free survival (PFS), and retreatment characteristics was analyzed.,Of 480 patients who received PD-1 inhibitors, 52 met the inclusion criteria.,The median treatment duration from first to the last dose was 11.1 months (95% CI 10.5 to 11.4).,BOR was complete response in 13 (25%), partial response in 28 (53.8%), and stable disease in 11 (21.2%) patients.,After a median follow-up of 20.5 months (range 3-49.2) from treatment discontinuation, 39 (75%) patients remained without disease progression, while 13 (25%) had progression (median PFS 3.9 months; range 0.7-30.9).,On multivariable analysis, younger age, history of brain metastasis, and higher lactate dehydrogenase at the time of anti-PD-1 discontinuation were associated with recurrence.,Patients with recurrent melanoma were managed with localized treatment, anti-PD-1 therapies, and BRAF-MEK inhibitors.,All patients except one were alive at data cutoff.,In this large real-world, observational cohort study, the majority of patients with metastatic melanoma after 1 year of anti-PD-1 therapy remained without progression on long-term follow-up.,The risk of disease progression even in patients with residual disease on imaging was low.,After prospective validation, elective PD-1 discontinuation at 1 year may reduce financial and immunotherapy-related toxicity without sacrificing outcomes.

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.

Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K+ and Cl− channels now being considered the most active during tumorigenesis.,Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers.,Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily because of the expression of two early regulatory proteins termed small and large tumor antigens (ST and LT, respectively).,Several molecular mechanisms have been attributed to MCPyV-mediated cancer formation but, thus far, no studies have investigated any potential link to cellular ion channels.,Here we demonstrate that Cl− channel modulation can reduce MCPyV ST-induced cell motility and invasiveness.,Proteomic analysis revealed that MCPyV ST up-regulates two Cl− channels, CLIC1 and CLIC4, which when silenced, inhibit MCPyV ST-induced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes.,Consistent with these data, we confirmed that CLIC1 and CLIC4 are up-regulated in primary MCPyV-positive MCC patient samples.,We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation.,Given the intense interest in ion channel modulating drugs for human disease.,This highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor with high mortality rates.,Merkel cell polyomavirus (MCPyV), identified in the majority of MCC, may drive tumorigenesis via viral T antigens.,However, mechanisms underlying pathogenesis in MCPyV-negative MCC remain poorly understood.,To nominate genes contributing to pathogenesis of MCPyV-negative MCC, we performed DNA microarray analysis on 30 MCCs.,MCPyV status of MCCs was determined by PCR for viral DNA and RNA. 1593 probe-sets were differentially expressed between MCPyV-negative and -positive MCC, with significant differential expression defined as at least 2-fold change in either direction and p-value of ≤ 0.05.,MCPyV-negative tumors showed decreased RB1 expression, whereas MCPyV-positive tumors were enriched for immune response genes.,Validation studies included immunohistochemistry demonstration of decreased RB protein expression in MCPyV-negative tumors and increased peritumoral CD8+ T lymphocytes surrounding MCPyV-positive tumors.,In conclusion, our data suggest that loss of RB1 expression may play an important role in tumorigenesis of MCPyV-negative MCC.,Functional and clinical validation studies are needed to determine whether this tumor suppressor pathway represents an avenue for targeted therapy.

Metastatic melanoma is the most aggressive and difficult to treat type of skin cancer, with a survival rate of less than 10%.,Metastatic melanoma has conventionally been considered very difficult to treat; however, recent progress in understanding the cellular and molecular mechanisms involved in the tumorigenesis, metastasis and immune escape have led to the introduction of new therapies.,These include targeted molecular therapy and novel immune-based approaches such as immune checkpoint blockade (ICB), tumor-infiltrating lymphocytes (TILs), and genetically engineered T-lymphocytes such as chimeric antigen receptor (CAR) T cells.,Among these, CAR T cell therapy has recently made promising strides towards the treatment of advanced hematological and solid cancers.,Although CAR T cell therapy might offer new hope for melanoma patients, it is not without its shortcomings, which include off-target toxicity, and the emergence of resistance to therapy (e.g., due to antigen loss), leading to eventual relapse.,The present review will not only describe the basic steps of melanoma metastasis, but also discuss how CAR T cells could treat metastatic melanoma.,We will outline specific strategies including combination approaches that could be used to overcome some limitations of CAR T cell therapy for metastatic melanoma.

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.

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

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

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma.,They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit.,The master regulators driving the expression of resistance-genes remain poorly understood.,Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes.,Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse.,Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists.,We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation.,Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth.,We thus propose AhR-impairment as a strategy to overcome melanoma resistance.,Resistance to BRAF inhibitors limits their clinical benefit in melanoma patients.,Here, the authors show that the Aryl hydrocarbon Receptor (AhR) is a key mediator of resistant genes and use resveratrol, an AhR antagonist, to revert resistance in melanoma bearing mice.

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.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed.,Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature.,Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1.,Mutations and amplification of KIT are also common.,Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common.,Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.,Acral melanoma occurs on the soles of the feet, palms of the hands and in nail beds.,Here, the authors reports the genomic landscape of 87 acral melanomas and find that some tumors harbor a UV signature and that the tumors are diverse at the levels of mutational signatures, structural aberrations and copy number signatures.

Metastatic mucosal melanoma responds poorly to anti-programmed cell death-1 (PD-1) monotherapy.,Vascular endothelial growth factor (VEGF) has been shown to play an important immunosuppressive role in the tumor microenvironment.,The combination of VEGF inhibition and PD-1 blockade provides therapeutic opportunities for patients refractory to either therapy alone.,We conducted a single-center, phase IB trial evaluating the safety and preliminary efficacy of toripalimab, a humanized immunoglobulin G4 monoclonal antibody against PD-1 in combination with the VEGF receptor inhibitor axitinib in patients with advanced melanoma, including patients with chemotherapy-naïve mucosal melanomas (88%).,Patients received toripalimab at 1 or 3 mg/kg via intravenous infusion every 2 weeks, in combination with axitinib 5 mg orally twice a day, in a dose-escalation and cohort-expansion study until confirmed disease progression, unacceptable toxicity, or voluntary withdrawal.,The primary objective was safety.,Secondary objectives included efficacy, pharmacokinetics, pharmacodynamics, immunogenicity, and tumor tissue biomarkers.,Thirty-three patients were enrolled.,No dose-limiting toxicities were observed.,Ninety-seven percent of patients experienced treatment-related adverse events (TRAEs).,The most common TRAEs were mild (grade 1 or 2) and included diarrhea, proteinuria, hand and foot syndrome, fatigue, AST or ALT elevation, hypertension, hypo- or hyperthyroidism, and rash.,Grade 3 or greater TRAEs occurred in 39.4% of patients.,By the cutoff date, among 29 patients with chemotherapy-naïve mucosal melanoma, 14 patients (48.3%; 95% CI, 29.4% to 67.5%) achieved objective response, and the median progression-free survival time was 7.5 months (95% CI, 3.7 months to not reached) per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.,The combination of toripalimab plus axitinib was tolerable and showed promising antitumor activity in patients with treatment-naïve metastatic mucosal melanoma.,Patients enrolled in this study were all Asian, and this combination therapy must be validated in a randomized phase III trial that includes a non-Asian population before it can become a standard of care.

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

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression.,Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors.,However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.,The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically.,There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS.,In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.,Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation.,Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs.,This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor.,These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

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

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

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.

Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity.,In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I).,Although stimulation of melanoma N1 cells (GD3+ and GD3−) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells.,Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells.,When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3− cells or GD3+ cells treated with either one of the stimulants.,Cell growth measured by 5-ethynyl-2‘ deoxyuridine uptake also showed synergistic effects in GD3+ cells.,These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.

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

Ipilimumab is a monoclonal antibody that antagonizes cytotoxic T lymphocyte antigen-4, a negative regulator of the immune system.,The authors report on advanced refractory melanoma patients treated in a compassionate use trial of ipilimumab at the Memorial Sloan-Kettering Cancer Center.,Patients with advanced refractory melanoma were treated in a compassionate use trial with ipilimumab 10 mg/kg every 3 weeks for 4 doses.,Those with evidence of clinical benefit at Week 24 (complete response [CR], partial response [PR], or stable disease [SD]) then received ipilimumab every 12 weeks.,A total of 53 patients were enrolled, with 51 evaluable.,Grade 3/4 immune-related adverse events were noted in 29% of patients, with the most common immune-related adverse events being pruritus (43%), rash (37%), and diarrhea (33%).,On the basis of immune-related response criteria, the response rate (CR + PR) was 12% (95% confidence interval [CI], 5%-25%), whereas 29% had SD (95% CI, 18%-44%).,The median progression-free survival was 2.6 months (95% CI, 2.3-5.2 months), whereas the median overall survival (OS) was 7.2 months (95% CI, 4.0-13.3 months).,Patients with an absolute lymphocyte count (ALC) ≥1000/μL after 2 ipilimumab treatments (Week 7) had a significantly improved clinical benefit rate (51% vs 0%; P = .01) and median OS (11.9 vs 1.4 months; P < .001) compared with those with an ALC <1000/μL.,The results confirm that ipilimumab is clinically active in patients with advanced refractory melanoma.,The ALC after 2 ipilimumab treatments appears to correlate with clinical benefit and OS, and should be prospectively validated.,Cancer 2010.,© 2010 American Cancer Society.,This description of 51 patients with advanced, treatment-refractory melanoma who were enrolled in a compassionate use trial of ipilimumab at Memorial Sloan-Kettering Cancer Center confirms that ipilimumab is active in this disease setting.,In addition, the results suggest that the absolute lymphocyte count after 2 ipilimumab treatments (at Week 7) highly correlates with the rate of clinical benefit at Week 24 and overall survival.

The existence of a dichotomy between immunologically active and quiescent tumor phenotypes has been recently recognized in several types of cancer.,The activation of a Th1 type of immune signature has been shown to confer better prognosis and likelihood to respond to immunotherapy.,However, whether such dichotomy depends on the genetic make‐up of individual cancers is not known yet.,BRAF and NRAS mutations are commonly acquired during melanoma progression.,Here we explored the role of BRAF and NRAS mutations in influencing the immune phenotype based on a classification previously identified by our group.,One‐hundred‐thirteen melanoma metastases underwent microarray analysis and BRAF and NRAS genotyping.,Allele‐specific PCR was also performed in order to exclude low‐frequency mutations.,Comparison between BRAF and NRAS mutant versus wild type samples identified mostly constituents or regulators of MAPK and related pathways.,When testing gene lists discriminative of BRAF, NRAS and MAPK alterations, we found that 112 BRAF‐specific transcripts were able to distinguish the two immune‐related phenotypes already described in melanoma, with the poor phenotype associated mostly with BRAF mutation.,Noteworthy, such association was stronger in samples displaying low BRAF mRNA expression.,However, when testing NRAS mutations, we were not able to find the same association.,This study suggests that BRAF mutation‐related specific transcripts associate with a poor phenotype in melanoma and provide a nest for further investigation.,BRAF and NRAS status was assessed in 113 melanoma metastases by Sanger sequencing and high sensitive allele‐specific PCR.The expression of BRAF‐specific genes categorized the metastases in two divergent groups.The mutant group associated with a poor phenotype.The association between BRAF mutation and the poor phenotype was stronger in samples displaying low BRAF mRNA expression.Functional interpretation of BRAF expression‐discriminative genes revealed pathways related to an unfavorable phenotype.,BRAF and NRAS status was assessed in 113 melanoma metastases by Sanger sequencing and high sensitive allele‐specific PCR.,The expression of BRAF‐specific genes categorized the metastases in two divergent groups.,The mutant group associated with a poor phenotype.,The association between BRAF mutation and the poor phenotype was stronger in samples displaying low BRAF mRNA expression.,Functional interpretation of BRAF expression‐discriminative genes revealed pathways related to an unfavorable phenotype.

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

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

Melanoma is notable for its metastatic propensity, lethality in the advanced setting, and association with ultraviolet (UV) exposure early in life1.,To obtain a comprehensive genomic view of melanoma, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA.,A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-UV exposed hairless skin of the extremities (3 and 14 per Mb genome), intermediate in those originating from hair-bearing skin of the trunk (range = 5 to 55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb).,Analysis of whole-genome sequence data identified PREX2 - a PTEN-interacting protein and negative regulator of PTEN in breast cancer2 - as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas.,PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumor formation of immortalized human melanocytes in vivo.,Thus, whole-genome sequencing of human melanoma tumors revealed genomic evidence of UV pathogenesis and discovered a new recurrently mutated gene in melanoma.

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

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

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

Despite several therapeutic advances, malignant melanoma still remains a fatal disease for which novel and long-term curative treatments are needed.,The successful development of innovative therapies strongly depends on the availability of appropriate pre-clinical models.,For this purpose, several mouse models holding the promise to provide insight into molecular biology and clinical behavior of melanoma have been generated.,The most relevant ones and their contribution for the advancement of therapeutic approaches for the treatment of human melanoma patients will be here summarized.,However, as models, mice do not recapitulate all the features of human melanoma, thus their strengths and weaknesses need to be carefully identified and considered for the translation of the results into the human clinics.,In this panorama, the concept of comparative oncology acquires a priceless value.,The revolutionary importance of spontaneous canine melanoma as a translational model for the pre-clinical investigation of melanoma progression and treatment will be here discussed, with a special consideration to the development of innovative immunotherapeutic approaches.

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

Resistance to immune checkpoint blockade and targeted therapy in melanoma patients is currently one of the major clinical challenges.,With the approval of talimogene laherparepvec (T-VEC), oncolytic viruses are now in clinical practice for locally advanced or non-resectable melanoma.,Here, we describe the usage of T-VEC in stage IVM1b-M1c melanoma patients, who achieved complete remission or stable disease upon systemic treatment but suffered from a loco-regional recurrence.,To our knowledge, there are no case reports so far describing T-VEC as a means to overcome acquired resistance to immune checkpoint blockade or targeted therapy.,All melanoma patients in our department treated with T-VEC in the period of 2016-2018 were evaluated retrospectively.,Data on clinicopathological characteristics, treatment response, and toxicity were analyzed.,Fourteen melanoma patients were treated with T-VEC in our center.,Six patients (43%) received T-VEC first-line.,In eight patients (57%), T-VEC followed a prior systemic therapy.,Three patients with M1b stage and one patient with M1c stage melanoma were treated with T-VEC.,These patients suffered from loco-regional progress, whilst distant metastases had regressed during prior systemic treatment. 64% of patients showed a benefit from therapy with T-VEC.,The durable response rate was 36%.,T-VEC represents an effective and tolerable treatment option.,This is true not only for loco-regionally advanced melanoma patients, but also for patients with stable or regressive systemic metastases who develop loco-regionally acquired resistance upon treatment with immune checkpoint blockade or targeted therapy.,A sensible selection of suitable patients seems to be crucial.

Ultraviolet B (UVB) radiation from the sun may lead to photocarcinogenesis of the skin.,Sunscreens were used to protect the skin by reducing UVB irradiance, but sunscreen use did not reduce sunburn episodes.,It was shown that UVB-induced erythema depends on surface exposure but not irradiance of UVB.,We previously showed that irradiance plays a critical role in UVB-induced cell differentiation.,This study investigated the impact of irradiance on UVB-induced photocarcinogenesis.,For hairless mice receiving equivalent exposure of UVB radiation, the low irradiance (LI) UVB treated mice showed more rapid tumor development, larger tumor burden, and more keratinocytes harboring mutant p53 in the epidermis as compared to their high irradiance (HI) UVB treated counterpart.,Mechanistically, using cell models, we demonstrated that LI UVB radiation allowed more keratinocytes harboring DNA damages to enter cell cycle via ERK-related signaling as compared to its HI UVB counterpart.,These results indicated that at equivalent exposure, UVB radiation at LI has higher photocarcinogenic potential as compared to its HI counterpart.,Since erythema is the observed sunburn at moderate doses and use of sunscreen was not found to associate with reduced sunburn episodes, the biological significance of sunburn with or without sunscreen use warrants further investigation.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, whose incidence continues to rise.,Epidemiological studies reveal that the major etiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2.,We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor (HGF/SF) transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and etiologic criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4.,However, mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown.,Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation.,We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion.,UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons.,IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2.,Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival.,IFN-γ-producing macrophages were also identified in 70% of human melanomas examined.,Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, and suggest that IFN-γ-R signaling represents a novel therapeutic melanoma target.

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

The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts.,Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively.,The strong antimetastatic activity of MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10 melanoma lung metastases in drug-treated mice.,Our data also show that MC3181 affects the lactate production in the high glycolytic WM266.4 cell line.,To unveil the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the degree of activation of different MAPK pathways, as well as the expression/activity levels of several proteins involved in angiogenesis, invasion, and survival (i.e.,AP2, MCAM/MUC18, N-cadherin, VEGF and MMP-2).,Our data disclosed both a decrease of the phospho-active form of JNK and an increased expression of the transcription factor AP2, events that occur in the very early phase of drug treatment and may be responsible of the antimetastatic effects of MC3181.

A melanocyte malignant transformation model was developed in our laboratory, in which different melanoma cell lines were obtained after submitting the non-tumorigenic melanocyte lineage melan-a to sequential cycles of anchorage impediment.,Our group has already showed that increased superoxide level leads to global DNA hypermemethylation as well increased Dnmt1 expression few hours after melanocyte anchorage blockade.,Here, we showed that Ras/Rac1/ERK signaling pathway is activated in melanocytes submitted to anchorage impediment, regulating superoxide levels, global DNA methylation, and Dnmt1 expression.,Interestingly, Ras and Rac1 activation is not related to codon mutations, but instead regulated by superoxide.,Moreover, the malignant transformation was drastically compromised when melan-a melanocytes were submitted to sequential cycles of anchorage blockage in the presence of a superoxide scavenger.,This aberrant signaling pathway associated with a sustained stressful condition, which might be similar to conditions such as UV radiation and inflammation, seems to be an early step in malignant transformation and to contribute to an epigenetic reprogramming and the melanoma development.

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.

Malignant melanoma is an aggressive skin tumor with a good prognosis when treated in an early tumor stage, but has a poor prognosis with distant metastases.,The incidence of malignant melanoma has increased continuously over the last decades, with little change in mortality.,One explanation for this is that melanomas are increasingly detected in early stages, especially after the establishment of statutory skin cancer screening in 2008, which allows a free skin examination every 2 years for people older than 35 years.,In this study incidence and mortality of malignant melanoma were correlated with the dermatologist density in Bavarian administrative regions.,In addition, the incidence data were compared before and after the introduction of statutory skin cancer screening.,There was a significant correlation between the incidence of malignant melanoma and dermatologist density (r = 0.258, p = 0.044), but no correlation between mortality and dermatologist density (r = 0.201, p = 0.121).,Similarly, the increase of malignant melanoma incidence following the introduction of statutory skin cancer screening in 2008 was independent of dermatologist density (r = 0.021, p = 0.873).,The dermatologist density in Bavaria correlates positively with the incidence of malignant melanoma.,Despite an increased incidence, mortality was not elevated in the respective administrative regions.

The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response.,This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell.,Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists.,However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g.,MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g.,HIF, MITF).,In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol.,The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria.,Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression.,Detection of cytosolic NADH by Peredox-mCherry Assay.,Detection of OCR, and ECAR using Seahorse XF96.,Measurement of produced O2•− with MitoTracker Red CMXRos.,1H NMR analysis of metabolites present in cell suspension, and medium.,By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS.,Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis.,Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.

Recent experimental studies have demonstrated an essential role for the Hippo-Yes-associated protein (YAP) pathway in GNAQ/GNA11-induced tumorigenesis in uveal melanoma (UM).,However, the association between YAP activity and clinical outcomes remains elusive.,We investigated possible associations between YAP activity and clinicopathological features including survival outcomes in patients with UM using The Cancer Genome Atlas (TCGA) cohort and our local cohort.,We estimated YAP activity by mRNA expression levels, Gene Set Variation Analysis (GSVA) for the TCGA cohort, and immunohistochemical YAP staining for the local cohort.,In the TCGA cohort, most clinicopathological features including tumor stage, mitotic counts, mutation of genes, and tumor sizes did not significantly differ between low and high YAP activity groups.,In the local cohort, YAP nuclear-positive staining was observed in 30 (42%) of 72 patients with primary UM.,UM-specific survival was not significantly different between tumors with low and high YAP activities.,Unlike mesothelioma cells harboring a mutation of negative regulators of YAP, the survival of multiple UM cell lines was not significantly reduced by YAP/TAZ depletion.,Our results suggest that the effect of YAP on development, growth, and invasion of UM in actual patients is less than previously demonstrated in experimental studies.

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.

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.

Thousands of putative enhancers are characterized in the human genome, yet few have been shown to have a functional role in cancer progression.,Inhibiting oncokinases, such as EGFR, ALK, ERBB2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by up-regulation of the HGF receptor, MET.,The mechanisms mediating such genomic responses to targeted therapy are unknown.,Here, we identify lineage-specific enhancers at the MET locus for multiple common tumor types, including a melanoma lineage-specific enhancer 63 kb downstream from the MET TSS.,This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition.,Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function.,Targeted genomic deletion (<7 bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation.,Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of a dominant transcription factor and block innate resistance to oncokinase therapy.

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

The presence of immune cells in the tumor microenvironment has been associated with response to immunotherapies across several cancer types, including melanoma.,Despite its therapeutic relevance, characterization of the melanoma immune microenvironments remains insufficiently explored.,To distinguish the immune microenvironment in a cohort of 180 metastatic melanoma clinical specimens, we developed a method using promoter CpG methylation of immune cell type‐specific genes extracted from genome‐wide methylation arrays.,Unsupervised clustering identified three immune methylation clusters with varying levels of immune CpG methylation that are related to patient survival.,Matching protein and gene expression data further corroborated the identified epigenetic characterization.,Exploration of the possible immune exclusion mechanisms at play revealed likely dependency on MITF protein level and PTEN loss‐of‐function events for melanomas unresponsive to immunotherapies (immune‐low).,To understand whether melanoma tumors resemble other solid tumors in terms of immune methylation characteristics, we explored 15 different solid tumor cohorts from TCGA.,Low‐dimensional projection based on immune cell type‐specific methylation revealed grouping of the solid tumors in line with melanoma immune methylation clusters rather than tumor types.,Association of survival outcome with immune cell type‐specific methylation differed across tumor and cell types.,However, in melanomas immune cell type‐specific methylation was associated with inferior patient survival.,Exploration of the immune methylation patterns in a pan‐cancer context suggested that specific immune microenvironments might occur across the cancer spectrum.,Together, our findings underscore the existence of diverse immune microenvironments, which may be informative for future immunotherapeutic applications.,The importance of the tumor immune microenvironment has been highlighted in multiple cancers, but information regarding the contribution of individual immune cell types is lacking for melanoma.,In this study, we have sought to fill the gap by identifying distinct DNA methylation patterns for specifc immune cells.,These were corroborated by other molecular correlates and found to be shared across other types of solid tomors.

Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood.,Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression.,Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis.,Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10.,Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation.,To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression.,Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program.,Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression.,Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.

Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis.,By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma.,We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes.,We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation.,For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level.,Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.

MicroRNAs (miRNAs) are attractive therapeutic targets for various therapy-resistant tumors.,However, the association between miRNA and BRAF inhibitor resistance in melanoma remains to be elucidated.,We used microarray analysis to comprehensively study the miRNA expression profiling of vemurafenib resistant (VemR) A375 melanoma cells in relation to parental A375 melanoma cells.,MicroRNA-7 (miR-7) was identified to be the most significantly down-regulated miRNA in VemR A375 melanoma cells.,We also found that miR-7 was down-regulated in Mel-CVR cells (vemurafenib resistant Mel-CV melanoma cells).,Reestablishment of miR-7 expression could reverse the resistance of both cells to vemurafenib.,We showed that epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R) and CRAF were over-expressed in VemR A375 melanoma cells.,Introduction of miR-7 mimics could markedly decrease the expressions of EGFR, IGF-1R and CRAF and further suppressed the activation of MAPK and PI3K/AKT pathway in VemR A375 melanoma cells.,Furthermore, tumor growth was inhibited in an in vivo murine VemR A375 melanoma tumor model transfected with miR-7 mimics.,Collectively, our study demonstrated that miR-7 could reverse the resistance to BRAF inhibitors in certain vemurafenib resistant melanoma cell lines.,It could advance the field and provide the basis for further studies in BRAF inhibitor resistance in melanoma.

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

Intratumoral immunotherapies aim to trigger local and systemic immunologic responses via direct injection of immunostimulatory agents with the goal of tumor cell lysis, followed by release of tumor‐derived antigens and subsequent activation of tumor‐specific effector T cells.,In 2019, a multitude of intratumoral immunotherapies with varied mechanisms of action, including nononcolytic viral therapies such as PV‐10 and toll‐like receptor 9 agonists and oncolytic viral therapies such as CAVATAK, Pexa‐Vec, and HF10, have been extensively evaluated in clinical trials and demonstrated promising antitumor activity with tolerable toxicities in melanoma and other solid tumor types.,Talimogene laherparepvec (T‐VEC), a genetically modified herpes simplex virus type 1-based oncolytic immunotherapy, is the first oncolytic virus approved by the U.S.,Food and Drug Administration for the treatment of unresectable melanoma recurrent after initial surgery.,In patients with unresectable metastatic melanoma, T‐VEC demonstrated a superior durable response rate (continuous complete response or partial response lasting ≥6 months) over subcutaneous GM‐CSF (16.3% vs.,2.1%; p < .001).,Responses were seen in both injected and uninjected lesions including visceral lesions, suggesting a systemic antitumor response.,When combined with immune checkpoint inhibitors, T‐VEC significantly improved response rates compared with single agent; similar results were seen with combinations of checkpoint inhibitors and other intratumoral therapies such as CAVATAK, HF10, and TLR9 agonists.,In this review, we highlight recent results from clinical trials of key intratumoral immunotherapies that are being evaluated in the clinic, with a focus on T‐VEC in the treatment of advanced melanoma as a model for future solid tumor indications.,This review provides oncologists with the latest information on the development of key intratumoral immunotherapies, particularly oncolytic viruses.,Currently, T‐VEC is the only U.S.,Food and Drug Administration (FDA)‐approved oncolytic immunotherapy.,This article highlights the efficacy and safety data from clinical trials of T‐VEC both as monotherapy and in combination with immune checkpoint inhibitors.,This review summarizes current knowledge on intratumoral therapies, a novel modality with increased utility in cancer treatment, and T‐VEC, the only U.S.,FDA‐approved oncolytic viral therapy, for medical oncologists.,This review evaluates approaches to incorporate T‐VEC into daily practice to offer the possibility of response in selected melanoma patients with manageable adverse events as compared with other available immunotherapies.,This review highlights recent results from clinical trials of key intratumoral immunotherapies that are being evaluated in the clinic, with a focus on talimogene laherparepvec in the treatment of advanced melanoma as a model for future solid tumor indications.

Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment.,In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints.,As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed.,We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation.,Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation.,Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable.,Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity.,These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target.,By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

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.

In a relatively short period of time, treatment strategies for metastatic melanoma have radically changed leading to an unprecedented improvement in patient survival.,In this period, immunotherapy options have evolved from cytokine-based approaches to antibody-mediated inhibition of immune checkpoints, cancer vaccines and pharmacological modulation of the melanoma microenvironment.,Combination of immunotherapy strategies and the association of immune checkpoint inhibitors (ICIs) with BRAF V600 targeted therapy show encouraging results.,The future of drug development in this field is promising.,The comprehension of primary and acquired resistance mechanisms to ICIs and the dissection of melanoma immunobiology will be instrumental for the development of new treatment strategies and to improve clinical trial design.,Moreover, biomarker discovery will help patient stratification and management during immunotherapy treatment.,In this review, we summarize landmark clinical trials of immune checkpoint inhibitors in advanced melanoma and discuss the rational for immunotherapy combinations.,Immunotherapy approaches at early stage of clinical development and recent advances in melanoma immunotherapy biomarker development are also discussed.

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses.,NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance.,We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival.,Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy.,Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis.,Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types.

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

Cellular plasticity of cancer cells is often associated with phenotypic heterogeneity and drug resistance and thus remains a major challenge for the treatment of melanoma and other types of cancer.,Melanoma cells have the capacity to switch their phenotype during tumor progression, from a proliferative and differentiated phenotype to a more invasive and dedifferentiated phenotype.,However, the molecular mechanisms driving this phenotype switch are not yet fully understood.,Considering that cellular heterogeneity within the tumor contributes to the high plasticity typically observed in melanoma, it is crucial to generate suitable models to investigate this phenomenon in detail.,Here, we discuss the use of complete and partial reprogramming into induced pluripotent cancer (iPC) cells as a tool to obtain new insights into melanoma cellular plasticity.,We consider this a relevant topic due to the high plasticity of melanoma cells and its association with a strong resistance to standard anticancer treatments.

BAP1 regulates developmental switch in lineages commonly affected by BAP1-mutant cancers.,The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome.,It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression.,Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development.,Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma.,The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4.,Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion.,These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

Nivolumab combined with ipilimumab have shown activity in melanoma brain metastasis (MBM).,However, in most of the clinical trials investigating immunotherapy in this subgroup, patients with symptomatic MBM and/or prior local brain radiotherapy were excluded.,We studied the efficacy of nivolumab plus ipilimumab alone or in combination with local therapies regardless of treatment line in patients with asymptomatic and symptomatic MBM.,Patients with MBM treated with nivolumab plus ipilimumab in 23 German Skin Cancer Centers between April 2015 and October 2018 were investigated.,Overall survival (OS) was evaluated by Kaplan-Meier estimator and univariate and multivariate Cox proportional hazard analyses were performed to determine prognostic factors associated with OS.,Three hundred and eighty patients were included in this study and 31% had symptomatic MBM (60/193 with data available) at the time of start nivolumab plus ipilimumab.,The median follow-up was 18 months and the 2 years and 3 years OS rates were 41% and 30%, respectively.,We identified the following independently significant prognostic factors for OS: elevated serum lactate dehydrogenase and protein S100B levels, number of MBM and Eastern Cooperative Oncology Group performance status.,In these patients treated with checkpoint inhibition first-line or later, in the subgroup of patients with BRAFV600-mutated melanoma we found no differences in terms of OS when receiving first-line either BRAF and MEK inhibitors or nivolumab plus ipilimumab (p=0.085).,In BRAF wild-type patients treated with nivolumab plus ipilimumab in first-line or later there was also no difference in OS (p=0.996).,Local therapy with stereotactic radiosurgery or surgery led to an improvement in OS compared with not receiving local therapy (p=0.009), regardless of the timepoint of the local therapy.,Receiving combined immunotherapy for MBM in first-line or at a later time point made no difference in terms of OS in this study population (p=0.119).,Immunotherapy with nivolumab plus ipilimumab, particularly in combination with stereotactic radiosurgery or surgery improves OS in asymptomatic and symptomatic MBM.

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

High C reactive protein (CRP) levels have been reported to be associated with a poor clinical outcome in a number of malignancies and with programmed cell death protein 1 immune checkpoint blockade in patients with advanced cancer.,Little is known about the direct effects of CRP on adaptive immunity in cancer.,Therefore, we investigated how CRP impacted the function of T cells and dendritic cells (DCs) from patients with melanoma.,The effects of CRP on proliferation, function, gene expression and phenotype of patient T cells and DCs, and expansion of MART-1 antigen-specific T cells were analyzed by multicolor flow cytometry and RNA-seq.,Additionally, serum CRP levels at baseline from patients with metastatic melanoma treated on the Checkmate-064 clinical trial were assessed by a Luminex assay.,In vitro, CRP inhibited proliferation, activation-associated phenotypes and the effector function of activated CD4+ and CD8+ T cells from patients with melanoma.,CRP-treated T cells expressed high levels of interleukin-1β, which is known to enhance CRP production from the liver.,CRP also suppressed formation of the immune synapse and inhibited early events in T-cell receptor engagement.,In addition, CRP downregulated the expression of costimulatory molecules on mature DCs and suppressed expansion of MART-1-specific CD8+ T cells in a dose-dependent manner by impacting on both T cells and antigen-presenting cells.,High-serum CRP levels at baseline were significantly associated with a shorter survival in both nivolumab-treated and ipilimumab-treated patients.,These findings suggest that high levels of CRP induce an immunosuppressive milieu in melanoma and support the blockade of CRP as a therapeutic strategy to enhance immune checkpoint therapies in cancer.,NCT01783938 and NCT02983006.

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

Skin lesions are a severe disease globally.,Early detection of melanoma in dermoscopy images significantly increases the survival rate.,However, the accurate recognition of melanoma is extremely challenging due to the following reasons: low contrast between lesions and skin, visual similarity between melanoma and non-melanoma lesions, etc.,Hence, reliable automatic detection of skin tumors is very useful to increase the accuracy and efficiency of pathologists.,In this paper, we proposed two deep learning methods to address three main tasks emerging in the area of skin lesion image processing, i.e., lesion segmentation (task 1), lesion dermoscopic feature extraction (task 2) and lesion classification (task 3).,A deep learning framework consisting of two fully convolutional residual networks (FCRN) is proposed to simultaneously produce the segmentation result and the coarse classification result.,A lesion index calculation unit (LICU) is developed to refine the coarse classification results by calculating the distance heat-map.,A straight-forward CNN is proposed for the dermoscopic feature extraction task.,The proposed deep learning frameworks were evaluated on the ISIC 2017 dataset.,Experimental results show the promising accuracies of our frameworks, i.e., 0.753 for task 1, 0.848 for task 2 and 0.912 for task 3 were achieved.

Background.,One of the fatal disorders causing death is malignant melanoma, the deadliest form of skin cancer.,The aim of the modern dermatology is the early detection of skin cancer, which usually results in reducing the mortality rate and less extensive treatment.,This paper presents a study on classification of melanoma in the early stage of development using SVMs as a useful technique for data classification.,Method.,In this paper an automatic algorithm for the classification of melanomas in their early stage, with a diameter under 5 mm, has been presented.,The system contains the following steps: image enhancement, lesion segmentation, feature calculation and selection, and classification stage using SVMs.,Results.,The algorithm has been tested on 200 images including 70 melanomas and 130 benign lesions.,The SVM classifier achieved sensitivity of 90% and specificity of 96%.,The results indicate that the proposed approach captured most of the malignant cases and could provide reliable information for effective skin mole examination.,Conclusions.,Micro-melanomas due to the small size and low advancement of development create enormous difficulties during the diagnosis even for experts.,The use of advanced equipment and sophisticated computer systems can help in the early diagnosis of skin lesions.

Apigenin, an aromatic compound, exhibits antioxidant, anti-inflammatory and anti-viral effects.,The present study aimed to investigate the effects of apigenin on cell proliferation and apoptosis of human melanoma cells A375P and A375SM.,Therefore, melanoma cells were treated with apigenin to determine its anti-proliferative and survival effects, using wound healing and MTT assays.,The results revealed that melanoma cell viability was decreased in a dose-dependent manner.,Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose-dependent manner, as demonstrated by DAPI staining.,In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V-propidium iodide staining.,The changes in the expression levels of apoptosis-related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis.,The results demonstrated that the protein expression levels of Bcl-2 were decreased, whereas those of Bax, cleaved poly ADP-ribose polymerase, cleaved caspase-9 and p53 were upregulated in a dose-dependent manner in apigenin-treated cells compared with those noted in untreated cells.,In addition, in apigenin-treated A375P cells, phosphorylated (p)-p38 was upregulated and p-extracellular signal-regulated kinase (ERK), p-c-Jun N-terminal kinase (JNK) and p-protein kinase B (Akt) were downregulated.,However, in A375SM cells, apigenin treatment increased p-ERK and p-JNK and decreased p-p38 and p-Akt protein expression levels.,Subsequently, the inhibitory effect of apigenin on tumor growth was investigated in vivo.,Tumor volume was significantly reduced in the 25 and 50 mg/kg apigenin-treated groups compared with the control group.,Additionally, a TUNEL assay was performed to detect apoptotic cells.,Immunohistochemical staining also revealed elevated p-ERK expression in the apigenin-treated group compared with the control group.,Overall, the findings of the present study indicated that apigenin attenuated the growth of A375SM melanoma cells by inducing apoptosis via regulating the Akt and mitogen-activated protein kinase signaling pathways.

Apigenin (4′,5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley.,This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound.,In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release.,Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 μM and 60 μM.,Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining.,The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production.,The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api.,Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%.,Api elicited antiangiogenic properties in a dose-dependent manner.,Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.

Despite recent genetic advances and numerous ongoing therapeutic trials, malignant melanoma remains fatal, and prognostic factors as well as more efficient treatments are needed.,The development of such research strongly depends on the availability of appropriate models recapitulating all the features of human melanoma.,The concept of comparative oncology, with the use of spontaneous canine models has recently acquired a unique value as a translational model.,Canine malignant melanomas are naturally occurring cancers presenting striking homologies with human melanomas.,As for many other cancers, dogs present surprising breed predispositions and higher frequency of certain subtypes per breed.,Oral melanomas, which are much more frequent and highly severe in dogs and cutaneous melanomas with severe digital forms or uveal subtypes are subtypes presenting relevant homologies with their human counterparts, thus constituting close models for these human melanoma subtypes.,This review addresses how canine and human melanoma subtypes compare based on their epidemiological, clinical, histological, and genetic characteristics, and how comparative oncology approaches can provide insights into rare and poorly characterized melanoma subtypes in humans that are frequent and breed-specific in dogs.,We propose canine malignant melanomas as models for rare non-UV-induced human melanomas, especially mucosal melanomas.,Naturally affected dogs offer the opportunity to decipher the genetics at both germline and somatic levels and to explore therapeutic options, with the dog entering preclinical trials as human patients, benefiting both dogs and humans.

Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development.,Different mechanisms underlie this activation in different tumour settings.,The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown.,Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses.,Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts.,The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing.,The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting.,Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting.,We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway.,The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases.,Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses.,These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis.,These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.,The online version of this article (doi:10.1186/1471-2407-14-857) contains supplementary material, which is available to authorized users.

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

Targeted therapies as BRAF and MEK inhibitor combination have been approved as first-line treatment for BRAF-mutant melanoma.,However, disease progression occurs in most of the patients within few months of therapy.,Metabolic adaptations have been described in the context of acquired resistance to BRAF inhibitors (BRAFi).,BRAFi-resistant melanomas are characterized by an increase of mitochondrial oxidative phosphorylation and are more prone to cell death induced by mitochondrial-targeting drugs.,BRAFi-resistant melanomas also exhibit an enhancement of oxidative stress due to mitochondrial oxygen consumption increase.,To understand the mechanisms responsible for survival of BRAFi-resistant melanoma cells in the context of oxidative stress, we have established a preclinical murine model that accurately recapitulates in vivo the acquisition of resistance to MAPK inhibitors including several BRAF or MEK inhibitors alone and in combination.,Using mice model and melanoma cell lines generated from mice tumors, we have confirmed that the acquisition of resistance is associated with an increase in mitochondrial oxidative phosphorylation as well as the importance of glutamine metabolism.,Moreover, we have demonstrated that BRAFi-resistant melanoma can adapt mitochondrial metabolism to support glucose-derived glutamate synthesis leading to increase in glutathione content.,Besides, BRAFi-resistant melanoma exhibits a strong activation of NRF-2 pathway leading to increase in the pentose phosphate pathway, which is involved in the regeneration of reduced glutathione, and to increase in xCT expression, a component of the xc-amino acid transporter essential for the uptake of cystine required for intracellular glutathione synthesis.,All these metabolic modifications sustain glutathione level and contribute to the intracellular redox balance to allow survival of BRAFi-resistant melanoma cells.

This study aimed to estimate the latest magnitudes and temporal trends of melanoma burden at the national, regional, and global levels.,The data on melanoma incidence, deaths, and disability-adjusted life-years (DALYs) in 204 countries and territories between 1990 and 2019 came from the Global Burden of Disease 2019 Study.,Estimated annual percentage change (EAPC) was calculated to depict the temporal trends and Spearman rank correlation was used to analyze the influential factors of EAPC.,From 1990 to 2019, the incident cases of melanoma increased by 170% to 289,950, death increased by 90% to 62,840, and DALYs increased by 67% to 1,707,800 globally.,The age-standardized incidence rate (ASIR) of melanoma increased globally by an average of 1.13 [95% confidence interval (CI): 0.93-1.32], while the age-standardized rates of death and DALYs both declined with the EAPC of −0.27 (95% CI: −0.36 to −0.19) and −0.49 (95% CI: −0.57 to −0.41).,In 2019, the highest burden of melanoma was observed in Australasia, followed by high-income North America and Europe regions, which all presented an incremental growth in ASIR.,The positive association between the EAPC in ASIR and socio-demographic index (SDI) in 2019 (ρ = 0.600, P < 0.001) suggested that countries with higher SDI have experienced a more rapid increase in ASIR of melanoma.,In conclusion, the burden of melanoma is increasing globally but differed greatly across the world.,Notably, the high burden areas are facing a continuing increase in incidence, which implies more targeted strategies should be taken for reducing the increasing melanoma burden.

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.

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.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma.,We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells.,HIF-1α protein accumulation in normoxia was inhibited by rapamycin.,As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX.,Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels.,In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels.,Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia.,We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt.,Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity.,In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling.,Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

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

Simultaneous chemotherapy with vascular endothelial growth factor (VEGF) inhibition has not shown additional benefit over chemotherapy alone in advanced melanoma.,We tested administration of the potent VEGF inhibitor axitinib followed by paclitaxel/carboplatin to determine whether enhanced tumour proliferation during axitinib withdrawal leads to sustained chemosensitivity.,We conducted a prospective phase II trial in metastatic melanoma patients with ECOG performance status 0-1 and normal organ function.,Axitinib 5 mg PO b.i.d. was taken on days 1-14 of each 21-day treatment cycle, and carboplatin (AUC=5) with paclitaxel (175 mg m−2) was administered on day 1 starting with cycle 2.,3′-Deoxy-3′-18F-fluorothymidine (18F-FLT)-PET scans were performed in five patients to assess tumour proliferation on days 1, 14, 17, and 20 of cycle 1.,Molecular profiling for BRAF was performed for all patients with cutaneous, acral, or mucosal melanoma.,The treatment was well tolerated.,The most common grade 3 AEs were hypertension, neutropenia, and anaemia.,Grade 4 non-haematologic AEs were not observed.,Four of five patients completing 18F-FLT-PET scans showed increases (23-92%) in SUV values during the axitinib holiday.,Of 36 evaluable patients, there were 8 confirmed PRs by Response Evaluation Criteria in Solid Tumors.,Overall, 20 patients had SD and 8 had PD as the best response.,The median PFS was 8.7 months and the median overall survival was 14.0 months.,Five BRAFV600E/K patients had significantly worse PFS than patients without these mutations.,Axitinib followed by carboplatin and paclitaxel was well tolerated and effective in BRAF wild-type metastatic melanoma.,3′-Deoxy-3′-18F-fluorothymidine-PET scans showed increased proliferation during axitinib withdrawal.

The study objective was to measure the rates of inclusion of populations at risk of advanced melanoma in a pilot targeted screening project involving general practitioners.,This cross-sectional database study compared the inclusion rates of patients who signed inclusion in a targeted screening project with those of patients who did not, during a period in which both groups of patients consulted investigators.,Data were extracted from the national healthcare insurance records in western France from 11 April to 30 October 2011.,Patients, older than 18, considered for the data extraction had consulted one of the 78 participating GPs during the study period, and were affiliated with the national healthcare insurance.,Inclusion in the screening was the main outcome measure.,Patients at risk of advanced melanoma were characterized by male gender, age over 50, low income, rural residence, farmer, and presence of chronic disease.,A total of 57,279 patients consulted GPs during the inclusion period and 2711 (4.73%) were included in the targeted screening.,Populations at risk of advanced melanoma were less included: men (OR = 0.67; 95%CI [0.61-0.73]; p < 0.001), older than 50 (OR = 0.67; 95%CI [0.60-0.74]; p < 0.001), low income (OR = 0.65; 95%CI [0.55-0.77]; p < 0.001), farmer (OR = 0.23; 95%CI [0.17-0.30]; p < 0.001) and presence of a chronic disease (OR = 0.87; 95%CI [0.77-0.98]; p < 0.028).,This study demonstrated inequalities in the inclusion of patients in a melanoma screening.,Patients at risk of advanced cancer were screened less often.,Further studies should focus on GPs ability to identify and screen these patients.,Advanced melanoma is more frequently diagnosed in men, older patients and socioeconomically disadvantaged populations, which leads to survival inequalities.,• Despite the involvement of general practitioners, the implementation of targeted melanoma screening did not avoid inclusion inequalities.,• Men, older patients, patients suffering from chronic diseases, and low-income patients were less likely to benefit from screening.,• The display of a conventional or an alarmist poster in the waiting room did not statistically reduce these inclusion inequalities.,Advanced melanoma is more frequently diagnosed in men, older patients and socioeconomically disadvantaged populations, which leads to survival inequalities.,• Despite the involvement of general practitioners, the implementation of targeted melanoma screening did not avoid inclusion inequalities.,• Men, older patients, patients suffering from chronic diseases, and low-income patients were less likely to benefit from screening.,• The display of a conventional or an alarmist poster in the waiting room did not statistically reduce these inclusion inequalities.

Melanoma incidence in the UK has doubled over two decades, yet there is conflicting evidence about factors which prompt or delay patients seeking advice.,Aim: To explore patient understanding of pigmented skin lesions (moles) and skin cancer, and factors which influence seeking help in primary care.,Semi-structured interviews with forty MoleMate Trial participants, analysed using the theoretical framework of the Safer-Andersen model of Total Patient Delay.,Patient understanding and awareness was influenced by personal, family and friends' experiences of moles, skin cancer and other cancers, knowledge of risk factors, and the lay media.,The route to consulting was complex and often iterative.,For lesions that people could see, detecting and appraising change was influenced by comparisons with a normal mole on themselves, a family member, friend or image.,Inferring illness came about with recognition of changes (particularly size) as serious, and associated 'internal' symptoms such as pain.,For lesions that people could not see, family, friends and health professionals detected and appraised changes.,Deciding to seek help was often prompted by another person or triggered by rapid or multiple changes in a mole.,Three of four people subsequently diagnosed with melanoma did not seek help; instead, their GP opportunistically noticed the lesion.,Changing moles are often perceived as trivial and not signifying possible skin cancer.,This study contributes to current national strategies to improve patient awareness and earlier diagnosis of cancer by highlighting factors that can trigger or act as barriers to seeking help.,(ISRCTN79932379)

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

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

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.

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

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

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma.,We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells.,HIF-1α protein accumulation in normoxia was inhibited by rapamycin.,As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX.,Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels.,In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels.,Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia.,We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt.,Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity.,In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling.,Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

Melanoma is an aggressive disease that accounts for approximately 75% of skin cancer-related deaths.,Historically, treatment options for patients with advanced stage melanoma have been limited by modest response rates and failure to improve overall survival.,The treatment landscape for advanced stage melanoma was revolutionized in 2011 with the approval of ipilimumab and vemurafenib, both of which improved overall survival in phase III clinical trials.,More recently, the targeted inhibitors dabrafenib and trametinib have demonstrated similar therapeutic profiles.,To (a) discuss emerging treatment options for advanced melanoma, specifically ilpilimumab, vemurafenib, dabrafenib, and trametinib, in the context of their mechanisms of action and their potential for long-term improvement in patient outcome, and (b) to consider the impact of these agents on the current treatment landscape.,A literature search was conducted to collect data from clinical trials involving ipilimumab, vemurafenib, dabrafenib, and trametinib.,Emphasis was placed on outcome measures related to long-term clinical benefit.,Ipilimumab, a fully human monoclonal antibody, exploits the natural ability of the immune system to eradicate primary cancer cells.,It inhibits the binding of cytotoxic T-lymphocyte antigen-4 to its ligands, thereby potentiating T-cell response and antitumor immunity.,In a phase III clinical trial, ipilimumab at 3 mg/kg improved overall survival in previously treated patients with metastatic melanoma.,Components of the mitogen-activated protein kinase (MAPK) pathway are particularly relevant in melanoma and have been targeted by small molecular inhibitors.,Vemurafenib and dabrafenib inhibit the BRAF V600 mutation, which prevents oncogenic activities such as uncheck proliferation and evasion of immune response.,Data from phase III clinical trials suggest that both vemurafenib and dabrafenib improve patient outcomes, with vemurafenib showing an overall survival benefit and dabrafenib showing improved median progression-free survival.,The targeted-therapy approach in melanoma continued to gain momentum with the development of trametinib, which inhibits the MEK protein, the only known substrate of the BRAF V600 protein.,Inhibition of MEK leads to decreased cell signaling and proliferation in cancer cells.,In phase III trials, trametinib demonstrated significant improvement in median progression-free survival and median overall survival compared with chemotherapy treatment, making this treatment a valuable addition to the current armamentarium.,The adverse events associated with these new treatments are generally tolerable and mild to moderate in severity; however, care should be taken when selecting a therapy, since the specific adverse events associated with these treatments are unique, and serious events have been reported.,The immunotherapy ipilimumab and the MAPK-targeted inhibitors vemurafenib, dabrafenib, and trametinib have forever changed the treatment landscape for melanoma.,Indeed, these new therapies have demonstrated long-term improvement in patient outcome, a benefit not afforded by traditional therapeutics.,Important research continues on the molecular basis of melanoma, and new targets are likely to emerge.,Other areas of work include optimization of sequencing and/or combination of current treatments, which may increase the number of patients who experience clinical benefit.

The common mutation BRAFV600 in primary melanomas activates the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway and the introduction of proto-oncogene B-Raf (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors (BRAFi and MEKi) was a breakthrough in the treatment of these cancers.,However, 15-20% of tumors harbor primary resistance to this therapy, and moreover, patients develop acquired resistance to treatment.,Understanding the molecular phenomena behind resistance to BRAFi/MEKis is indispensable in order to develop novel targeted therapies.,Most often, resistance develops due to either the reactivation of the MAPK/ERK pathway or the activation of alternative kinase signaling pathways including phosphatase and tensin homolog (PTEN), neurofibromin 1 (NF-1) or RAS signaling.,The hyperactivation of tyrosine kinase receptors, such as the receptor of the platelet-derived growth factor β (PDFRβ), insulin-like growth factor 1 receptor (IGF-1R) and the receptor for hepatocyte growth factor (HGF), lead to the induction of the AKT/3-phosphoinositol kinase (PI3K) pathway.,Another pathway resulting in BRAFi/MEKi resistance is the hyperactivation of epidermal growth factor receptor (EGFR) signaling or the deregulation of microphthalmia-associated transcription factor (MITF).

Background: Melanoma is a cancer of the skin with potential to spread to other organs and is responsible for most deaths due to skin cancer.,It is imperative to identify immune biomarkers for early melanoma diagnosis and treatment.,Results: 63 immune-related genes of the total 1039 unique IRGs retrieved were associated with overall survival of melanoma.,A multi-IRGs classifier constructed using eight IRGs showed a powerful predictive ability.,The classifier had better predictive power compared with the current clinical data.,GSEA analysis showed multiple signaling differences between high and low risk score group.,Furthermore, biomarker was associated with multiple immune cells and immune infiltration in tumor microenvironment.,Conclusions: The immune-related genes prognosis biomarker is an effective potential prognostic classifier in the immunotherapies and surveillance of melanoma.,Methods: Melanoma samples of genes were retrieved from TCGA and GEO databases while the immune-related genes (IRGs) were retrieved from the ImmPort database.,WGCNA, Cox regression analysis and LASSO analysis were used to classify melanoma prognosis.,ESTIMATE and CIBERSORT algorithms were used to explore the relationship between risk score and tumor immune microenvironment.,GSEA analysis was performed to explore the biological signaling pathway.

Skin cutaneous melanoma (SKCM) is one of most aggressive type of cancers worldwide.,Serglycin (SRGN) is an intracellular proteoglycan that playing an important role in various tumors.,However, its effect on immune infiltrates and whether it associates with survival of SKCM and SKCM-metastasis patients has not been explored.,We evaluated SRGN expression via the databases of Oncomine, Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA).,The influence of SRGN expression on survival of SKCM and SKCM-metastasis patients was analyzed using TIMER database.,Furthermore, the correlations between SRGN expression and immune infiltrates or gene marker sets of immune infiltrates were also analyzed via TIMER database.,We found that the expression of SRGN in SKCM and SKCM-metastasis tissues was significantly increased compared to the normal skin tissues (P < 0.001).,Interestingly, it was showed that lower level of SRGN expression and lower immune infiltrates of B cell, CD8+ T cell, Neutrophil, and Dendritic cell were correlated with poor survival rate of SKCM and SKCM-metastasis patients (P < 0.001) but not SKCM primary patients.,We also demonstrated that SRGN expression was positively associated with the immune infiltrates and diverse immune marker sets in SKCM and SKCM-metastasis.,Our findings indicated that SRGN was associated with the survival of SKCM and SKCM-metastasis patients.,SRGN may be a new immune therapy target for treating SKCM and SKCM-metastasis.

The tumor milieu consists of numerous cell types each existing in a different environment.,However, a characterization of metabolic heterogeneity at single-cell resolution is not established.,Here, we develop a computational pipeline to study metabolic programs in single cells.,In two representative human cancers, melanoma and head and neck, we apply this algorithm to define the intratumor metabolic landscape.,We report an overall discordance between analyses of single cells and those of bulk tumors with higher metabolic activity in malignant cells than previously appreciated.,Variation in mitochondrial programs is found to be the major contributor to metabolic heterogeneity.,Surprisingly, the expression of both glycolytic and mitochondrial programs strongly correlates with hypoxia in all cell types.,Immune and stromal cells could also be distinguished by their metabolic features.,Taken together this analysis establishes a computational framework for characterizing metabolism using single cell expression data and defines principles of the tumor microenvironment.,Each cell type in the tumour microenvironment has unique metabolic demands enabling specific functions.,Here the authors use published single-cell RNA-seq data and develop a computational framework to better understand the heterogeneity of tumour metabolism, highlighting the discordance between results obtained from single cells and bulk tumours.

A comparative analysis of genome-scale transcriptomic data of two types of skin cancers, melanoma and basal cell carcinoma in comparison with other cancer types, was conducted with the aim of identifying key regulatory factors that either cause or contribute to the aggressiveness of melanoma, while basal cell carcinoma generally remains a mild disease.,Multiple cancer-related pathways such as cell proliferation, apoptosis, angiogenesis, cell invasion and metastasis, are considered, but our focus is on energy metabolism, cell invasion and metastasis pathways.,Our findings include the following. (a) Both types of skin cancers use both glycolysis and increased oxidative phosphorylation (electron transfer chain) for their energy supply. (b) Advanced melanoma shows substantial up-regulation of key genes involved in fatty acid metabolism (β-oxidation) and oxidative phosphorylation, with aerobic metabolism being far more efficient than anaerobic glycolysis, providing a source of the energetics necessary to support the rapid growth of this cancer. (c) While advanced melanoma is similar to pancreatic cancer in terms of the activity level of genes involved in promoting cell invasion and metastasis, the main metastatic form of basal cell carcinoma is substantially reduced in this activity, partially explaining why this cancer type has been considered as far less aggressive.,Our method of using comparative analyses of transcriptomic data of multiple cancer types focused on specific pathways provides a novel and highly effective approach to cancer studies in general.

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

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

In 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 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.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed.,Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature.,Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1.,Mutations and amplification of KIT are also common.,Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common.,Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.,Acral melanoma occurs on the soles of the feet, palms of the hands and in nail beds.,Here, the authors reports the genomic landscape of 87 acral melanomas and find that some tumors harbor a UV signature and that the tumors are diverse at the levels of mutational signatures, structural aberrations and copy number signatures.

Treating advanced or recurrent melanoma remains a challenge.,Cancer cells can evade the immune system by blocking T‐cell activation through overexpression of the inhibitory receptor programmed death 1 (PD‐1) ligands.,The PD‐1 inhibitor nivolumab blocks the inhibitory signal in T cells, thus overcoming the immune resistance of cancer cells.,Nivolumab has shown promising anticancer activity in various cancers.,We carried out a single‐arm, open‐label, multicenter, phase II study to investigate the efficacy and safety of nivolumab in previously untreated Japanese patients with advanced melanoma.,Twenty‐four patients with stage III/IV or recurrent melanoma were enrolled and received i.v. nivolumab 3 mg/kg every 2 weeks until disease progression or unacceptable toxicity.,The primary endpoint was overall response rate evaluated by an independent radiology review committee.,The independent radiology review committee‐assessed overall response rate was 34.8% (90% confidence interval, 20.8-51.9), and the overall survival rate at 18 months was 56.5% (90% confidence interval, 38.0-71.4).,Treatment‐related adverse events (AEs) of grade 3 or 4 only occurred in three patients (12.5%).,Two patients discontinued nivolumab because of AEs, but all AEs were considered manageable by early diagnosis and appropriate treatment.,Subgroup analyses showed that nivolumab was clinically beneficial and tolerable regardless of BRAF genotype, and that patients with treatment‐related select AEs and with vitiligo showed tendency for better survival.,In conclusion, nivolumab showed favorable efficacy and safety profiles in Japanese patients with advanced or recurrent melanoma, with or without BRAF mutations.,(Trial registration no.,JapicCTI‐142533.)

The response of BRAF-mutant melanoma patients to BRAF inhibitors is dramatically impaired by secondary resistances and rapid relapse.,So far, the molecular mechanisms driving these resistances are not completely understood.,Here, we show that, in BRAF-mutant melanoma cells, inhibition of BRAF or its target MEK induces RHOB expression by a mechanism that depends on the transcription factor c-Jun.,In those cells, RHOB deficiency causes hypersensitivity to BRAF and MEK inhibitors-induced apoptosis.,Supporting these results, loss of RHOB expression in metastatic melanoma tissues is associated with an increased progression-free survival of BRAF-mutant patients treated with vemurafenib.,Following BRAF inhibition, RHOB activates AKT whose inhibition causes hypersensitivity of BRAF-mutant melanoma cells to BRAF inhibitors.,In mice, AKT inhibition synergizes with vemurafenib to block tumor growth of BRAF-mutant metastatic melanoma.,Our findings reveal that BRAF inhibition activates a c-Jun/RHOB/AKT pathway that promotes tumor cell survival and further support a role of this pathway in the resistance of melanoma to vemurafenib.,Our data also highlight the importance of using RHOB tumor levels as a biomarker to predict vemurafenib patient's response and to select those that would benefit of the combination with AKT inhibitors.

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.

Melanoma is a cancer of the pigment-producing cells of the body and its incidence is rising.,Targeted inhibitors that act against kinases in the MAPK pathway are approved for BRAF-mutant metastatic cutaneous melanoma and increase patients’ survival.,Response to these therapies is limited by drug resistance and is less durable than with immune checkpoint inhibition.,Conversely, rare melanoma subtypes have few therapeutic options for advanced disease and MAPK pathway targeting agents show minimal anti-tumor effects.,Nevertheless, there is a future for targeted kinase inhibitors in melanoma: in new applications such as adjuvant or neoadjuvant therapy and in novel combinations with immunotherapies or other targeted therapies.,Pre-clinical studies continue to identify tumor dependencies and their corresponding actionable drug targets, paving the way for rational targeted kinase inhibitor combinations as a personalized medicine approach for melanoma.

This study shows that melanoma-associated fibroblasts (MAFs) suppress cytotoxic T lymphocyte (CTL) activity and reveals a pivotal role played by arginase in this phenomenon.,MAFs and normal dermal fibroblasts (DFs) were isolated from surgically resected melanomas and identified as Melan-A-/gp100-/FAP+ cells.,CTLs of healthy blood donors were activated in the presence of MAF- and DF-conditioned media (CM).,Markers of successful CTL activation, cytotoxic degranulation, killing activity and immune checkpoint regulation were evaluated by flow cytometry, ELISPOT, and redirected killing assays.,Soluble mediators responsible for MAF-mediated effects were identified by ELISA, flow cytometry, inhibitor assays, and knock-in experiments.,In the presence of MAF-CM, activated/non-naïve CTLs displayed dysregulated ERK1/2 and NF-κB signaling, impeded CD69 and granzyme B production, impaired killing activity, and upregulated expression of the negative immune checkpoint receptors TIGIT and BTLA.,Compared to DFs, MAFs displayed increased amounts of VISTA and HVEM, a known ligand of BTLA on T cells, increased l-arginase activity and CXCL12 release.,Transgenic arginase over-expression further increased, while selective arginase inhibition neutralized MAF-induced TIGIT and BTLA expression on CTLs.,Our data indicate that MAF interfere with intracellular CTL signaling via soluble mediators leading to CTL anergy and modify immune checkpoint receptor availability via l-arginine depletion.,The online version of this article (10.1007/s00018-020-03517-8) contains supplementary material, which is available to authorized users.

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

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5).,Mouse models have been broadly utilized to study tissue morphogenesis in vivo.,However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans.,Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis.,The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types.,The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis.,Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction.,The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin.,As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes.,Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo.,Recently, dermal stem cells have been identified in the human dermis (6).,These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

Melanoma is the deadliest cutaneous neoplasm.,To prevent metastasis, early diagnosis and surgical treatment is vital.,Long non-coding RNAs (lncRNAs) may serve as biomarkers and therapeutic targets in tumors.,We investigated the molecular mechanisms of lncRNA KCNQ1OT1 in melanoma.,Real time PCR demonstrated that KCNQ1OT1 expression is up-regulated in melanoma tissues and cells.,KCNQ1OT1 promoted cell proliferation and metastasis in melanoma.,By directly bindin to miR-153, KCNQ1OT1 acted as a competing endogenous RNA (ceRNA) to de-repress MET expression.,Our results may provide the basis for a novel strategy for early detection and/or treatment of melanoma.

Aberrant HGF-MET signaling activation via interactions with surrounding stromal cells in tumor microenvironment plays significant roles in malignant tumor progression.,However, extracellular proteolytic regulation of HGF activation which is influenced by the tumor microenvironment and its consequential effects on melanoma malignancy remain uncharacterized.,In this study we identified SPINT2: a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which plays a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression.,SPINT2 expression is significantly lower in metastatic melanoma tissues compared to those in early stage primary melanomas which also corresponded with DNA methylation levels isolated from tissue samples.,Treatment with the DNA hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas.,Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits HGF induced MET-AKT signaling pathway and decreases malignant phenotype potential such as cell motility, and invasive growth of melanoma cells.,These results suggest that SPINT2 is associated with tumor suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF which is typically activated by tumor-stromal interactions.,These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression.

The presence of immune cells in the tumor microenvironment has been associated with response to immunotherapies across several cancer types, including melanoma.,Despite its therapeutic relevance, characterization of the melanoma immune microenvironments remains insufficiently explored.,To distinguish the immune microenvironment in a cohort of 180 metastatic melanoma clinical specimens, we developed a method using promoter CpG methylation of immune cell type‐specific genes extracted from genome‐wide methylation arrays.,Unsupervised clustering identified three immune methylation clusters with varying levels of immune CpG methylation that are related to patient survival.,Matching protein and gene expression data further corroborated the identified epigenetic characterization.,Exploration of the possible immune exclusion mechanisms at play revealed likely dependency on MITF protein level and PTEN loss‐of‐function events for melanomas unresponsive to immunotherapies (immune‐low).,To understand whether melanoma tumors resemble other solid tumors in terms of immune methylation characteristics, we explored 15 different solid tumor cohorts from TCGA.,Low‐dimensional projection based on immune cell type‐specific methylation revealed grouping of the solid tumors in line with melanoma immune methylation clusters rather than tumor types.,Association of survival outcome with immune cell type‐specific methylation differed across tumor and cell types.,However, in melanomas immune cell type‐specific methylation was associated with inferior patient survival.,Exploration of the immune methylation patterns in a pan‐cancer context suggested that specific immune microenvironments might occur across the cancer spectrum.,Together, our findings underscore the existence of diverse immune microenvironments, which may be informative for future immunotherapeutic applications.,The importance of the tumor immune microenvironment has been highlighted in multiple cancers, but information regarding the contribution of individual immune cell types is lacking for melanoma.,In this study, we have sought to fill the gap by identifying distinct DNA methylation patterns for specifc immune cells.,These were corroborated by other molecular correlates and found to be shared across other types of solid tomors.

In the recent past years, many discoveries in the tumor microenvironment have led to changes in the management of melanoma and it is rising up hopes, specially, to those in advanced stages.,FDA approved seven new drugs from 2011 to 2014.,They are: Vemurafenib, Dabrafenib and Trametinib, kinases inhibitors used for patients that have BRAFV600E mutation; Ipilimumab (anti-CTLA4), Pembrolizumab (anti-PD-1) and Nivolumab (anti-PD-1), monoclonal antibodies that stimulate the immune system; and Peginterferon alfa-2b, an anti-proliferative cytokine used as adjuvant therapy.,In this article, we will review the molecular bases for these new metastatic melanoma therapeutic agents cited above and also analyze new molecular discoveries in melanoma study, as Cancer-Testis antigens (CT).,They are capable of induce humoral and cellular immune responses in cancer patients and because of this immunogenicity and their restrict expression in normal tissues, they are considered an ideal candidate for vaccine development against cancer.,Among CT antigens, NY-ESO-1 is the best characterized in terms of expression patterns and immunogenicity.,It is expressed in 20-40% of all melanomas, more in metastatic lesions than in primary ones, and it is very heterogeneous inter and intratumoral.,Breslow index is associate with NY-ESO-1 expression in primary cutaneous melanomas, but its relation to patient survival remains controversial.

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.

Nivolumab 1 mg/kg plus ipilimumab 3 mg/kg (NIVO1+IPI3) is approved for first-line treatment of patients with advanced melanoma in several countries.,We conducted a phase IIIb/IV study (CheckMate 511) to determine if nivolumab 3 mg/kg plus ipilimumab 1 mg/kg (NIVO3+IPI1) improves the safety profile of the combination.,Patients (N = 360) age 18 years or older with previously untreated, unresectable stage III or IV melanoma were randomly assigned 1:1 to NIVO3+IPI1 or NIVO1+IPI3 once every 3 weeks for four doses.,After 6 weeks, all patients received NIVO 480 mg once every 4 weeks until disease progression or unacceptable toxicity.,The primary end point was a comparison of the incidence of treatment-related grade 3 to 5 adverse events (AEs) between groups.,Secondary end points included descriptive analyses of objective response rate, progression-free survival, and overall survival.,The study was not designed to formally demonstrate noninferiority of NIVO3+IPI1 to NIVO1+IPI3 for efficacy end points.,At a minimum follow-up of 12 months, incidence of treatment-related grade 3 to 5 AEs was 34% with NIVO3+IPI1 versus 48% with NIVO1+IPI3 (P = .006).,In descriptive analyses, objective response rate was 45.6% in the NIVO3+IPI1 group and 50.6% in the NIVO1+IPI3 group, with complete responses in 15.0% and 13.5% of patients, respectively.,Median progression-free survival was 9.9 months in the NIVO3+IPI1 group and 8.9 months in the NIVO1+IPI3 group.,Median overall survival was not reached in either group.,The CheckMate 511 study met its primary end point, demonstrating a significantly lower incidence of treatment-related grade 3-5 AEs with NIVO3+IPI1 versus NIVO1+IPI3.,Descriptive analyses showed that there were no meaningful differences between the groups for any efficacy end point, although longer follow up may help to better characterize efficacy outcomes.

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

A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression.,The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis.,Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others.,We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration.,Individual tumor cells can invade in either elongated, “mesenchymal-type” or rounded, “amoeboid-like” modes and these two modes of invasion are inter-convertible [1].,In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion.,MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion.,Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions.,In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology.,Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited.,Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms.,We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients.,Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures.,Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations.,In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients.,We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells.,These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.

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.

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.

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

SPRED1 is a negative regulator of the MAPK pathway frequently deleted in human melanoma.,Through in vivo modeling in zebrafish and mechanistic analyses in human cell lines, Ablain et al. demonstrate that SPRED1 inactivation confers resistance to targeted inhibition of V600 mutant BRAF, the most common driver of melanoma.,Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies.,We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma.,Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF.,We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo.,Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity.,Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy.,These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma.

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

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status.,We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting.,Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR).,Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques.,CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas.,HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting.,CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

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.

Metastatic melanoma is the most aggressive and difficult to treat type of skin cancer, with a survival rate of less than 10%.,Metastatic melanoma has conventionally been considered very difficult to treat; however, recent progress in understanding the cellular and molecular mechanisms involved in the tumorigenesis, metastasis and immune escape have led to the introduction of new therapies.,These include targeted molecular therapy and novel immune-based approaches such as immune checkpoint blockade (ICB), tumor-infiltrating lymphocytes (TILs), and genetically engineered T-lymphocytes such as chimeric antigen receptor (CAR) T cells.,Among these, CAR T cell therapy has recently made promising strides towards the treatment of advanced hematological and solid cancers.,Although CAR T cell therapy might offer new hope for melanoma patients, it is not without its shortcomings, which include off-target toxicity, and the emergence of resistance to therapy (e.g., due to antigen loss), leading to eventual relapse.,The present review will not only describe the basic steps of melanoma metastasis, but also discuss how CAR T cells could treat metastatic melanoma.,We will outline specific strategies including combination approaches that could be used to overcome some limitations of CAR T cell therapy for metastatic melanoma.

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

Recent experimental studies have demonstrated an essential role for the Hippo-Yes-associated protein (YAP) pathway in GNAQ/GNA11-induced tumorigenesis in uveal melanoma (UM).,However, the association between YAP activity and clinical outcomes remains elusive.,We investigated possible associations between YAP activity and clinicopathological features including survival outcomes in patients with UM using The Cancer Genome Atlas (TCGA) cohort and our local cohort.,We estimated YAP activity by mRNA expression levels, Gene Set Variation Analysis (GSVA) for the TCGA cohort, and immunohistochemical YAP staining for the local cohort.,In the TCGA cohort, most clinicopathological features including tumor stage, mitotic counts, mutation of genes, and tumor sizes did not significantly differ between low and high YAP activity groups.,In the local cohort, YAP nuclear-positive staining was observed in 30 (42%) of 72 patients with primary UM.,UM-specific survival was not significantly different between tumors with low and high YAP activities.,Unlike mesothelioma cells harboring a mutation of negative regulators of YAP, the survival of multiple UM cell lines was not significantly reduced by YAP/TAZ depletion.,Our results suggest that the effect of YAP on development, growth, and invasion of UM in actual patients is less than previously demonstrated in experimental studies.

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

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

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

The p.E318K variant of the Melanocyte Inducing Transcription Factor (MITF) has been implicated in genetic predisposition to melanoma as an intermediate penetrance allele.,However, the impact of this variant on clinico-phenotypic, as well as on dermoscopic patterns features of affected patients is not entirely defined.,The purpose of our study was to assess the association between the p.E318K germline variant and clinic-phenotypical features of MITF+ compared to non-carriers (MITF−), including dermoscopic findings of melanomas and dysplastic nevi.,we retrospectively analyzed a consecutive series of 1386 patients recruited between 2000 and 2017 who underwent genetic testing for CDKN2A, CDK4, MC1R and MITF germline variants in our laboratory for diagnostic/research purposes.,The patients were probands of melanoma-prone families and apparently sporadic single or multiple primary melanoma patients.,For all, we collected clinical, pathological information and dermoscopic images of the histopathologically diagnosed melanomas and dysplastic nevi, when available.,After excluding patients positive for CDKN2A/CDK4 pathogenic variants and those affected by non-cutaneous melanomas, our study cohort comprised 984 cutaneous melanoma patients, 22 MITF+ and 962 MITF−.,MITF+ were more likely to develop dysplastic nevi and multiple primary melanomas.,Nodular melanoma was more common in MITF+ patients (32% compared to 19% in MITF−).,MITF+ patients showed more frequently dysplastic nevi and melanomas with uncommon dermoscopic patterns (unspecific), as opposed to MITF− patients, whose most prevalent pattern was the multicomponent.,MITF+ patients tend to develop melanomas and dysplastic nevi with histopathological features, frequency and dermoscopic patterns often different from those prevalent in MITF− patients.,Our results emphasize the importance of melanoma prevention programs for MITF+ patients, including dermatologic surveillance with digital follow-up.

Previous studies have investigated the protective effect of vitamin D serum levels, at diagnosis and during the follow-up period after treatment, on melanoma outcome.,In the present study we assess whether vitamin D supplementation, in the follow-up period after diagnosis and surgical resection of the primary tumor, has a protective effect on relapse of cutaneous malignant melanoma and whether this protective effect correlates with vitamin D levels in serum and Vitamin D Receptor immunoreactivity in the primary tumor.,This study is a multicenter randomized double blind placebo- controlled phase III trial.,Patients between the age of 18 and 80 years diagnosed and treated surgically for a melanoma stage IB-III are eligible for randomization in a 1:1 ratio to active treatment or placebo.,The study drug is taken each month and consists of either 100,000 International Unit cholecalciferol or arachidis oleum raffinatum used as a placebo.,The primary endpoint is relapse free survival.,The secondary endpoints are 25 hydroxyvitamin D3 serum levels at diagnosis and at 6 month intervals, melanoma subtype, melanoma site and stage of melanoma at diagnosis according to the 2009 American Joint Committee on Cancer melanoma staging and classification.,At randomization a bloodsample is taken for DNA analysis.,The study is approved by the local Ethics Committees.,If we can confirm our hypothesis that vitamin D supplementation after removal of the tumor has a protective effect on relapse of cutaneous malignant melanoma we may reduce the burden of CMM at several levels.,Patients, diagnosed with melanoma may have a better clinical outcome and improved quality of life.,There will be a decrease in health care costs related to treatment of metastatic disease and there will be a decrease in loss of professional years, which will markedly reduce the economic burden of the disease.,Clinical Trial.gov, NCT01748448, 05/12/2012

Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic capacity.,According to the “phenotype switching” model, the aggressive nature of melanoma cells results from their intrinsic potential to dynamically switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state.,Here we identify the low affinity neurotrophin receptor CD271 as a key effector of phenotype switching in melanoma.,CD271 plays a dual role in this process by decreasing proliferation, while simultaneously promoting invasiveness.,Dynamic modification of CD271 expression allows tumor cells to grow at low levels of CD271, to reduce growth and invade when CD271 expression is high, and to re-expand at a distant site upon decrease of CD271 expression.,Mechanistically, the cleaved intracellular domain of CD271 controls proliferation, while the interaction of CD271 with the neurotrophin receptor Trk-A modulates cell adhesiveness through dynamic regulation of a set of cholesterol synthesis genes relevant for patient survival.,The aggressive nature of melanoma cells relies on their ability to switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state; however, the mechanisms governing this switch are unclear.,Here, using in vivo models of human melanoma, the authors show that CD271 is a key regulator of phenotype switching and metastasis formation.

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.

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.

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

Early detection of melanoma can be lifesaving but this remains a challenge.,Recent diagnostic studies have revealed the superiority of artificial intelligence (AI) in classifying dermoscopic images of melanoma and nevi, concluding that these algorithms should assist a dermatologist’s diagnoses.,The aim of this study was to investigate whether AI support improves the accuracy and overall diagnostic performance of dermatologists in the dichotomous image-based discrimination between melanoma and nevus.,Twelve board-certified dermatologists were presented disjoint sets of 100 unique dermoscopic images of melanomas and nevi (total of 1200 unique images), and they had to classify the images based on personal experience alone (part I) and with the support of a trained convolutional neural network (CNN, part II).,Additionally, dermatologists were asked to rate their confidence in their final decision for each image.,While the mean specificity of the dermatologists based on personal experience alone remained almost unchanged (70.6% vs 72.4%; P=.54) with AI support, the mean sensitivity and mean accuracy increased significantly (59.4% vs 74.6%; P=.003 and 65.0% vs 73.6%; P=.002, respectively) with AI support.,Out of the 10% (10/94; 95% CI 8.4%-11.8%) of cases where dermatologists were correct and AI was incorrect, dermatologists on average changed to the incorrect answer for 39% (4/10; 95% CI 23.2%-55.6%) of cases.,When dermatologists were incorrect and AI was correct (25/94, 27%; 95% CI 24.0%-30.1%), dermatologists changed their answers to the correct answer for 46% (11/25; 95% CI 33.1%-58.4%) of cases.,Additionally, the dermatologists’ average confidence in their decisions increased when the CNN confirmed their decision and decreased when the CNN disagreed, even when the dermatologists were correct.,Reported values are based on the mean of all participants.,Whenever absolute values are shown, the denominator and numerator are approximations as every dermatologist ended up rating a varying number of images due to a quality control step.,The findings of our study show that AI support can improve the overall accuracy of the dermatologists in the dichotomous image-based discrimination between melanoma and nevus.,This supports the argument for AI-based tools to aid clinicians in skin lesion classification and provides a rationale for studies of such classifiers in real-life settings, wherein clinicians can integrate additional information such as patient age and medical history into their decisions.

Overlapping histological features between benign and malignant lesions and a lack of firm diagnostic criteria for malignancy result in high rates of inter-observer variation in the diagnosis of melanocytic lesions.,We aimed to investigate the differential expression of five miRNAs (21, 200c, 204, 205, and 211) in benign naevi (n = 42), dysplastic naevi (n = 41), melanoma in situ (n = 42), and melanoma (n = 42) and evaluate their potential as diagnostic biomarkers of melanocytic lesions.,Real-time PCR showed differential miRNA expression profiles between benign naevi; dysplastic naevi and melanoma in situ; and invasive melanoma.,We applied a random forest machine learning algorithm to classify cases based on their miRNA expression profiles, which resulted in a ROC curve analysis of 0.99 for malignant melanoma and greater than 0.9 for all other groups.,This indicates an overall very high accuracy of our panel of miRNAs as a diagnostic biomarker of benign, dysplastic, and malignant melanocytic lesions.,However, the impact of variable lesion percentage and spatial expression patterns of miRNAs on these real-time PCR results was also considered.,In situ hybridisation confirmed the expression of miRNA 21 and 211 in melanocytes, while demonstrating expression of miRNA 205 only in keratinocytes, thus calling into question its value as a biomarker of melanocytic lesions.,In conclusion, we have validated some miRNAs, including miRNA 21 and 211, as potential diagnostic biomarkers of benign, dysplastic, and malignant melanocytic lesions.,However, we also highlight the crucial importance of considering tissue morphology and spatial expression patterns when using molecular techniques for the discovery and validation of new biomarkers.,The online version of this article (10.1007/s00428-020-02817-5) contains supplementary material, which is available to authorized users.

BAP1 regulates developmental switch in lineages commonly affected by BAP1-mutant cancers.,The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome.,It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression.,Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development.,Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma.,The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4.,Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion.,These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

High-dose interleukin-2 (HD IL-2) was approved for treatment of metastatic renal cell carcinoma (mRCC) in 1992 and for metastatic melanoma (mM) in 1998, in an era predating targeted therapies and immune checkpoint inhibitors.,The PROCLAIMSM registry was established to collect and analyze data for patients treated with HD IL-2 in the current era.,This analysis includes 170 patients with mM and 192 patients with mRCC treated between 2005 and 2012 with survival data current as of July 27, 2015.,For patients with mM, complete response (CR) was observed in 5 %, partial response (PR) in 10 %, stable disease (SD) in 22 %, and 63 % had progressive disease (PD).,The median overall survival (mOS) for these patients was 19.6 months, with a median follow-up of 43.1 months.,The mOS was not reached for patients achieving CR or PR, and was 33.4 months for patients with SD.,For patients with mRCC, 6 % achieved CR, 9 % had PR, 22 % had SD, and 62 % had PD.,The mOS was 41 months, with a median follow-up of 46.6 months.,The mOS for patients who had CR and PR was not reached and was 49.6 months for patients with SD.,There were no treatment-related deaths among 362 patients.,The duration of mOS for patients with mM and mRCC is longer than historically reported.,These data support a continued role for IL-2 in the treatment of eligible patients with mM or mRCC and warrant further evaluation of HD IL-2 in combination or sequence with other therapeutic agents.,The online version of this article (doi:10.1007/s00262-016-1910-x) contains supplementary material, which is available to authorized users.

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.

Matrix metalloproteinase-2 (MMP-2) is a key regulator in the migration of tumor cells. αvβ3 integrin has been reported to play a critical role in cell adhesion and regulate the migration of tumor cells by promoting MMP-2 activation.,However, little is known about the effects of MMP-2 on αvβ3 integrin activity and αvβ3 integrin-mediated adhesion and migration of tumor cells.,Human melanoma cells were seeded using an agarose drop model and/or subjected to in vitro analysis using immunofluorescence, adhesion, migration and invasion assays to investigate the relationship between active MMP-2 and αvβ3 integrin during the adhesion and migration of the tumor cells.,We found that MMP-2 was localized at the leading edge of spreading cells before αvβ3 integrin. αvβ3 integrin-mediated adhesion and migration of the tumor cells were inhibited by a MMP-2 inhibitor.,MMP-2 cleaved fibronectin into small fragments, which promoted the adhesion and migration of the tumor cells.,MMP-2 cleaves fibronectin into small fragments to enhance the adhesion and migration of human melanoma cells mediated by αvβ3 integrin.,These results indicate that MMP-2 may guide the direction of the tumor cell migration.

The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma.,Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response.,Therefore, the precise identification of the underlying somatic mutations is essential.,Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.,Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples.,DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction.,BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC).,All mutations were independently reassessed by Sanger sequencing.,Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).,There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%).,All mutations down to 6.6% allele frequency could be detected with 100% specificity.,In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity.,The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib.,NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity.,IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.,Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

The cobas 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to select patients with metastatic melanoma for treatment with the selective BRAF inhibitor vemurafenib.,We describe the pre-approval validation of this test in two external laboratories.,Melanoma specimens were tested for BRAF V600 mutations at two laboratories with the: cobas BRAF Mutation Test; ABI BRAF test; and bidirectional direct sequencing.,Positive (PPA) and negative (NPA) percent agreements were determined between the cobas test and the other assays.,Specimens with discordant results were tested with massively parallel pyrosequencing (454).,DNA blends with 5% mutant alleles were tested to assess detection rates.,Invalid results were observed in 8/116 specimens (6·9%) with Sanger, 10/116 (8·6%) with ABI BRAF, and 0/232 (0%) with the cobas BRAF test.,PPA was 97·7% for V600E mutation for the cobas BRAF test and Sanger, and NPA was 95·3%.,For the cobas BRAF test and ABI BRAF, PPA was 71·9% and NPA 83·7%.,For 16 cobas BRAF test-negative/ABI BRAF-positive specimens, 454 sequencing detected no codon 600 mutations in 12 and variant codon 600 mutations in four.,For eight cobas BRAF test-positive/ABI BRAF-negative specimens, four were V600E and four V600K by 454 sequencing.,Detection rates for 5% mutation blends were 100% for the cobas BRAF test, 33% for Sanger, and 21% for the ABI BRAF.,Reproducibility of the cobas BRAF test was 111/116 (96%) between the two sites.,It is feasible to evaluate potential companion diagnostic tests in external laboratories simultaneously to the pivotal clinical trial validation.,The health authority approved assay had substantially better performance characteristics than the two other methods.,The overall success of the cobas BRAF test is a proof of concept for future biomarker development.

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.

The aim of this study was to investigate the feasibility of using serum miR-221 as a noninvasive prognostic biomarker for cutaneous malignant melanoma (CMM).,We measured the expression levels of miR-221 in serum samples from 72 CMM patients and 54 healthy controls by real-time quantitative polymerase chain reaction (RT-PCR).,The overall survival (OS) and disease-free survival (DFS) were calculated using the Kaplan-Meier method.,The differences between the survival curves were tested by using the log-rank test.,The COX proportional hazards regression model was used to determine the joint effects of several variables on survival.,The serum miR-221 levels were significantly higher in patients with CMM than in healthy controls (p<0.0001).,Patients with high serum miR-221 levels had a significantly lower 5-year OS rate (22.1% vs.,54.6%; P=0.018) and RFS rate (12.5% vs.,45.2%; P=0.008) than those with low serum miR-221 level.,In a multivariate Cox model, we found that miR-221 expression was an independent predictor of poor 5-year OS (hazards ratio [HR]=3.189, 95% confidence interval [CI]=1.782-6.777, P=0.007) and 5-year DFS (HR=2.119, CI=1.962-8.552, P=0.01) in CMM patients.,Our data indicate that serum miR-221expression level has prognostic value in patients with CMM.

Familial history of melanoma is a well-known risk factor for the disease, and 7% melanoma patients were reported to have a family history of melanoma.,Data relating to the frequency and clinical and pathological characteristics of both familial and non-familial melanoma in Spain have been published, but these only include patients from specific areas of Spain and do not represent the data for the whole of Spain.,An observational study conducted by the Spanish Group of Melanoma (GEM) analyzed the family history of patients diagnosed with melanoma between 2011 and 2013 in the dermatology and oncology departments.,In all, 1047 patients were analyzed, and 69 (6.6%) fulfilled criteria for classical familial melanoma (two or more first-degree relatives diagnosed with melanoma).,Taking into account other risk factors for familial melanoma, such as multiple melanoma, pancreatic cancer in the family or second-degree relatives with melanoma, the number of patients fulfilling the criteria increased to 165 (15.8%).,Using a univariate analysis, we determined that a Breslow index of less than 1 mm, negative mitosis, multiple melanoma, and a history of sunburns in childhood were more frequent in familial melanoma patients, but a multivariate analysis revealed no differences in any pathological or clinical factor between the two groups.,Similar to that observed in other countries, familial melanoma accounts for 6.6% of melanoma diagnoses in Spain.,Although no differences in the multivariate analysis were found, some better prognosis factors, such as Breslow index, seem more frequent in familial melanoma, which reflect a better early detection marker and/or a different biological behavior.

Atypical Mole Syndrome is the most important phenotypic risk factor for developing cutaneous melanoma, a malignancy that accounts for about 80% of deaths from skin cancer.,Because the diagnosis of melanoma at an early stage is of great prognostic relevance, the identification of Atypical Mole Syndrome carriers is essential, as well as the creation of recommended preventative measures that must be taken by these patients.

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

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

Emerging data support the rationale of combined therapies in advanced melanoma.,Specifically, the combined use of drugs with different mechanisms of action can reduce the probability of selecting resistant clones.,To identify agents active against melanoma cells, we screened a library of 349 anti‐cancer compounds, currently in clinical use or trials, and selected PIK‐75, an inhibitor of the phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) pathway, as the ‘top active’ drug.,PIK‐75 was then used alone or in combination with vemurafenib, the first BRAF inhibitor approved for patients with melanoma harboring BRAF mutations.,We identified a combined dose of PIK‐75 and vemurafenib that inhibited both the PI3K/AKT and mitogen‐activated protein kinase pathways, thereby overcoming any compensatory activation.,In view of the important tumor suppressor function induced by restoring expression of microRNA (miR)‐126 in metastatic melanoma cells, we examined whether miR‐126 has a synergistic role when included in a triple combination alongside PIK‐75 and vemurafenib.,We found that enforced expression of miR‐126 (which alone can reduce tumorigenicity) significantly increased PIK‐75 activity when used as either a single agent or in combination with vemurafenib.,Interestingly, PIK‐75 proved to be effective against early passage cell lines derived from patients’ biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, thus suggesting that it potentially has the capability to overcome drug resistance.,Finally, the synergistic role played by miR‐126 in combination with vemurafenib and/or PIK‐75 was demonstrated in vivo in mouse xenograft models, in which tumor growth inhibition was associated with increased apoptosis.,These results not only show the efficacy of PIK‐75 and vemurafenib co‐treatment but also indicate that restoration of miR‐126 expression in advanced melanoma can enhance their antitumor activity, which may possibly allow dose reduction to decrease adverse events without reducing the therapeutic benefits.

Ferroptosis is a regulated form of cell death driven by small molecules or conditions that induce lipid-based reactive oxygen species (ROS) accumulation.,This form of iron-dependent cell death is morphologically and genetically distinct from apoptosis, necroptosis, and autophagy. miRNAs are known to play crucial roles in diverse fundamental biological processes.,However, to date no study has reported miRNA-mediated regulation of ferroptosis.,Here we show that miR-137 negatively regulates ferroptosis by directly targeting glutamine transporter SLC1A5 in melanoma cells.,Ectopic expression of miR-137 suppressed SLC1A5, resulting in decreased glutamine uptake and malondialdehyde (MDA) accumulation.,Meanwhile, antagomir-mediated inactivation of endogenous miR-137 increased the sensitivity of melanoma cells to erastin- and RSL3-induced ferroptosis.,Importantly, knockdown of miR-137 increased the antitumor activity of erastin by enhancing ferroptosis both in vitro and in vivo.,Collectively, these data indicate that miR-137 plays a novel and indispensable role in ferroptosis by inhibiting glutaminolysis and suggest a potential therapeutic approach for melanoma.

The critical long non-coding RNAs (lncRNAs) involved in the carcinogenesis and progression of malignant melanoma (MM) have not been fully investigated.,In the present study, it was identified that lncRNA activated by transforming growth factor-β (lncRNA-ATB) was upregulated in MM tissues and cells compared with benign nevus cells and human melanocytes, via comparative lncRNA screening from Gene Expression Omnibus datasets and reverse transcription-quantitative polymerase chain reaction analysis.,Furthermore, lncRNA-ATB promoted the cell proliferation, cell migration, and cell invasion of MM cells in vitro, and tumor growth in vivo.,It was additionally identified that lncRNA-ATB attenuated cell cycle arrest and inhibited cellular apoptosis in MM cells.,Finally, it was demonstrated that lncRNA-ATB functions as a competing endogenous RNA (ceRNA) to enhance Yes associated protein 1 expression by competitively sponging microRNA miR-590-5p in MM cells.,In conclusion, the present study revealed the expression and roles of lncRNA-ATB in MM, and indicated that lncRNA-ATB functions as a ceRNA to promote MM proliferation and invasion by sponging miR-590-5p.

The present study aimed to investigate the potential role of the long non-coding RNA (lncRNA) Pvt1 oncogene (non-protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms.,The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A-375 and sk-mel-5 cell lines, was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analyses.,The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively.,The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay.,The effect of PVT1 on microRNA-200c (miR-200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay.,PVT1 was highly expressed in the melanoma tissues and cells.,Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage.,Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells.,The expression of E-cadherin was significantly increased and the expression of N-cadherin and vimentin was significantly decreased in the PVT1-silenced group.,The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control.,The ChIP assay revealed that the expression of miR-200c was decreased significantly in the PVT1-silenced group compared with the controls.,Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR-200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.

Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic capacity.,According to the “phenotype switching” model, the aggressive nature of melanoma cells results from their intrinsic potential to dynamically switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state.,Here we identify the low affinity neurotrophin receptor CD271 as a key effector of phenotype switching in melanoma.,CD271 plays a dual role in this process by decreasing proliferation, while simultaneously promoting invasiveness.,Dynamic modification of CD271 expression allows tumor cells to grow at low levels of CD271, to reduce growth and invade when CD271 expression is high, and to re-expand at a distant site upon decrease of CD271 expression.,Mechanistically, the cleaved intracellular domain of CD271 controls proliferation, while the interaction of CD271 with the neurotrophin receptor Trk-A modulates cell adhesiveness through dynamic regulation of a set of cholesterol synthesis genes relevant for patient survival.,The aggressive nature of melanoma cells relies on their ability to switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state; however, the mechanisms governing this switch are unclear.,Here, using in vivo models of human melanoma, the authors show that CD271 is a key regulator of phenotype switching and metastasis formation.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations.,Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes.,Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS.,In this study we show that β-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi.,We further demonstrate that β-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo.,Unexpectedly, β-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of β-catenin in melanoma progression and therapy resistance.,We further demonstrate that β-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.,Scheme of the change of interacting proteins with β-catenin during the acquisition of resistance to BRAFi from TCF/LEF family members to novel interactants like Stat3.,Binding of ligands to different receptor tyrosine kinases like c-met and Egfr family members activate the MAPK signaling pathway and PI3K signaling pathway independently of mutated BRAF.,Additional MAPK signaling reactivating mechanisms like the acquisition of novel mutations (asterisk), the formation of Raf heterodimers to the BRAFi.,In this environment a switch in the interaction partners of β-catenin to Stat3 occurs further enhancing resistance and contributing to the relapse of the tumor.,Dotted lines indicated weak activating potential or indirect mechanisms.Image 1

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

Bacteria within the skin microbiome of some individuals produce an antimetabolite that inhibits tumor growth.,We report the discovery that strains of Staphylococcus epidermidis produce 6-N-hydroxyaminopurine (6-HAP), a molecule that inhibits DNA polymerase activity.,In culture, 6-HAP selectively inhibited proliferation of tumor lines but did not inhibit primary keratinocytes.,Resistance to 6-HAP was associated with the expression of mitochondrial amidoxime reducing components, enzymes that were not observed in cells sensitive to this compound.,Intravenous injection of 6-HAP in mice suppressed the growth of B16F10 melanoma without evidence of systemic toxicity.,Colonization of mice with an S. epidermidis strain producing 6-HAP reduced the incidence of ultraviolet-induced tumors compared to mice colonized by a control strain that did not produce 6-HAP.,S. epidermidis strains producing 6-HAP were found in the metagenome from multiple healthy human subjects, suggesting that the microbiome of some individuals may confer protection against skin cancer.,These findings show a new role for skin commensal bacteria in host defense.

The critical long non-coding RNAs (lncRNAs) involved in the carcinogenesis and progression of malignant melanoma (MM) have not been fully investigated.,In the present study, it was identified that lncRNA activated by transforming growth factor-β (lncRNA-ATB) was upregulated in MM tissues and cells compared with benign nevus cells and human melanocytes, via comparative lncRNA screening from Gene Expression Omnibus datasets and reverse transcription-quantitative polymerase chain reaction analysis.,Furthermore, lncRNA-ATB promoted the cell proliferation, cell migration, and cell invasion of MM cells in vitro, and tumor growth in vivo.,It was additionally identified that lncRNA-ATB attenuated cell cycle arrest and inhibited cellular apoptosis in MM cells.,Finally, it was demonstrated that lncRNA-ATB functions as a competing endogenous RNA (ceRNA) to enhance Yes associated protein 1 expression by competitively sponging microRNA miR-590-5p in MM cells.,In conclusion, the present study revealed the expression and roles of lncRNA-ATB in MM, and indicated that lncRNA-ATB functions as a ceRNA to promote MM proliferation and invasion by sponging miR-590-5p.

Melanoma is the deadliest cutaneous neoplasm.,To prevent metastasis, early diagnosis and surgical treatment is vital.,Long non-coding RNAs (lncRNAs) may serve as biomarkers and therapeutic targets in tumors.,We investigated the molecular mechanisms of lncRNA KCNQ1OT1 in melanoma.,Real time PCR demonstrated that KCNQ1OT1 expression is up-regulated in melanoma tissues and cells.,KCNQ1OT1 promoted cell proliferation and metastasis in melanoma.,By directly bindin to miR-153, KCNQ1OT1 acted as a competing endogenous RNA (ceRNA) to de-repress MET expression.,Our results may provide the basis for a novel strategy for early detection and/or treatment of melanoma.

Allium vegetables such as garlic (Allium sativum L.) are rich in organosulfur compounds that prevent human chronic diseases, including cancer.,Of these, diallyl trisulfide (DATS) exhibits anticancer effects against a variety of tumors, including malignant melanoma.,Although previous studies have shown that DATS increases intracellular calcium (Ca2+) in different cancer cell types, the role of Ca2+ in the anticancer effect is obscure.,In the present study, we investigated the Ca2+ pathways involved in the anti-melanoma effect.,We used melittin, the bee venom that can activate a store-operated Ca2+ entry (SOCE) and apoptosis, as a reference.,DATS increased apoptosis in human melanoma cell lines in a Ca2+-dependent manner.,It also induced mitochondrial Ca2+ (Ca2+mit) overload through intracellular and extracellular Ca2+ fluxes independently of SOCE.,Strikingly, acidification augmented Ca2+mit overload, and Ca2+ channel blockers reduced the effect more significantly under acidic pH conditions.,On the contrary, acidification mitigated SOCE and Ca2+mit overload caused by melittin.,Finally, Ca2+ channel blockers entirely inhibited the anti-melanoma effect of DATS.,Our findings suggest that DATS explicitly evokes Ca2+mit overload via a non-SOCE, thereby displaying the anti-melanoma effect.

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.

Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance.,In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133.,Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells.,Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity.,A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down.,In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest.,To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375.,We found CD271+ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases.,Strikingly, only 2/12 cell strains harbored a CD133+ sub-set that in addition comprised a fraction of cells of a CD271+/CD133+ phenotype.,Those cells were found in the label-retaining fraction and in vitro deduced from CD271+ but not CD271 knock-down cells.,Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes.,Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.

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

Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies.,Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib.,Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition.,In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo.,We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines.,The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201.,This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance.,Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA.,Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested.,E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro.,In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1.,Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background.,Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling.,In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways.,MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations.,In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055.,While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only.,In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model.,We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells.,For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis.,Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis.,These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Several biomarkers such as tumor mutation burden (TMB), neoantigen load (NAL), programmed cell‐death receptor 1 ligand (PD‐L1) expression, and lactate dehydrogenase (LDH) have been developed for predicting response to immune checkpoint inhibitors (ICIs) in melanoma.,However, some limitations including the undefined cut‐off value, poor uniformity of test platform, and weak reliability of prediction have restricted the broad application in clinical practice.,In order to identify a clinically actionable biomarker and explore an effective strategy for prediction, we developed a genetic mutation model named as immunotherapy score (ITS) for predicting response to ICIs therapy in melanoma, based on whole‐exome sequencing data from previous studies.,We observed that patients with high ITS had better durable clinical benefit and survival outcomes than patients with low ITS in three independent cohorts, as well as in the meta‐cohort.,Notably, the prediction capability of ITS was more robust than that of TMB.,Remarkably, ITS was not only an independent predictor of ICIs therapy, but also combined with TMB or LDH to better predict response to ICIs than any single biomarker.,Moreover, patients with high ITS harbored the immunotherapy‐sensitive characteristics including high TMB and NAL, ultraviolet light damage, impaired DNA damage repair pathway, arrested cell cycle signaling, and frequent mutations in NF1 and SERPINB3/4.,Overall, these findings deserve prospective investigation in the future and may help guide clinical decisions on ICIs therapy for patients with melanoma.,This study provided evidences that the genetic mutation model (named as ITS) identified a melanoma population with multiple genetic patterns of sensitivity to ICIs, who might potentially benefit from ICIs therapy.,Preliminary data from three independent cohorts strongly suggested better treatment outcomes from ICIs therapy in melanoma patients with high ITS.,Remarkably, the combination strategy of ITS and TMB or LDH showed better prediction efficacy compared with any single biomarker.

Melanoma is increasing rapidly in incidence and prevalence, especially in younger females and older males.,Treatment options have expanded beyond high-dose interleukin 2 and adoptive T-cell therapy to include inhibitors of immune checkpoints programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and small molecular inhibitors of pathways activated in melanoma, in particular the mitogen-activated protein kinase (MAPK) pathway.,PD-1/CTLA-4 inhibitors and inhibitors of MAPK such as BRAF/MEK inhibitors have significantly improved survival in both the metastatic and, more recently, adjuvant settings.,In this review, we discuss the preclinical data, clinical development, and potential use of novel MEK inhibitor binemetinib, particularly in the setting of NRAS mutant melanoma.

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

Malignant melanoma is an exceptionally aggressive, drug-resistant and heterogeneous cancer.,Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common, but markers distinguishing such cells from cells lacking these abilities have not been identified.,There is therefore no definite evidence that an exclusive cell subpopulation, i.e. cancer stem cells (CSC), exists in malignant melanoma.,Rather, it is suggested that multiple cell populations are implicated in initiation and progression of the disease, making it of importance to identify subpopulations with elevated aggressive properties.,In several other cancer forms, Aldehyde Dehydrogenase (ALDH), which plays a role in stem cell biology and resistance, is a valuable functional marker for identification of cells that show enhanced aggressiveness and drug-resistance.,Furthermore, the presence of ALDH+ cells is linked to poor clinical prognosis in these cancers.,By analyzing cell cultures, xenografts and patient biopsies, we showed that aggressive melanoma harboured a large, distinguishable ALDH+ subpopulation.,In vivo, ALDH+ cells gave rise to ALDH− cells, while the opposite conversion was rare, indicating a higher abilities of ALDH+ cells to reestablish tumour heterogeneity with respect to the ALDH phenotype.,However, both ALDH+ and ALDH− cells demonstrated similarly high abilities for clone formation in vitro and tumour initiation in vivo.,Furthermore, both subpopulations showed similar sensitivity to the anti-melanoma drugs, dacarbazine and lexatumumab.,These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells, implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma, and arguing against ALDH as a “universal” marker.,Besides, it was shown that the ability to reestablish tumour heterogeneity is not necessarily linked to the more aggressive phenotype.

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

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

The common mutation BRAFV600 in primary melanomas activates the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway and the introduction of proto-oncogene B-Raf (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors (BRAFi and MEKi) was a breakthrough in the treatment of these cancers.,However, 15-20% of tumors harbor primary resistance to this therapy, and moreover, patients develop acquired resistance to treatment.,Understanding the molecular phenomena behind resistance to BRAFi/MEKis is indispensable in order to develop novel targeted therapies.,Most often, resistance develops due to either the reactivation of the MAPK/ERK pathway or the activation of alternative kinase signaling pathways including phosphatase and tensin homolog (PTEN), neurofibromin 1 (NF-1) or RAS signaling.,The hyperactivation of tyrosine kinase receptors, such as the receptor of the platelet-derived growth factor β (PDFRβ), insulin-like growth factor 1 receptor (IGF-1R) and the receptor for hepatocyte growth factor (HGF), lead to the induction of the AKT/3-phosphoinositol kinase (PI3K) pathway.,Another pathway resulting in BRAFi/MEKi resistance is the hyperactivation of epidermal growth factor receptor (EGFR) signaling or the deregulation of microphthalmia-associated transcription factor (MITF).

Treatment with programmed death receptor-1 (PD-1) antibodies is associated with high response rates in patients with advanced melanoma.,Reliable markers for early response and outcome are still sparse.,We evaluated 66 consecutive patients with advanced/metastatic melanoma treated with nivolumab or pembrolizumab between 2013 and 2014.,The main objectives of this study were to investigate whether, first, serum lactate dehydrogenase (LDH) at baseline (normal vs above the upper limit of normal) correlates with overall survival (OS), and, second, whether the change of LDH during treatment predicts response before the first scan and OS in patients with an elevated baseline LDH.,After a median follow-up of 9 months, patients with an elevated baseline LDH (N=34) had a significantly shorter OS compared with patients with normal LDH (N=32; 6-month OS: 60.8% vs 81.6% and 12-month OS: 44.2% vs 71.5% (log-rank P=0.0292).,In those 34 patients with elevated baseline LDH, the relative change during treatment was significantly associated with an objective response on the first scan: the 11 (32%) patients with partial remission had a mean reduction of −27.3% from elevated baseline LDH.,In contrast, patients with progressive disease (N=15) had a mean increase of +39%.,Patients with a relative increase over 10% from elevated baseline LDH had a significantly shorter OS compared with patients with ⩽10% change (4.3 vs 15.7 months, log-rank P<0.00623).,LDH could be a useful marker at baseline and during treatment to predict early response or progression in patients with advanced melanoma who receive anti-PD-1 therapy.

Even though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues.,In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo.,Human (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations.,Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment.,NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses.,Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM.,Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay.,Cytokine/chemokine plasmatic levels were measured by xMAP technology.,Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses.,NAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro.,NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001).,About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice.,The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response.,The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival.,Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples.,NAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.

Nicotinamide (NAM) is an amide form of vitamin B3 and the precursor of nicotinamide adenine dinucleotide (NAD+), an essential co-enzyme of redox reactions for adenosine triphosphate (ATP) production and for other metabolic processes.,As NAD+ status is critical in maintaining cellular energy, vitamin B3 deficiency mainly affects tissues that need high cellular energy causing pellagra and skin sun sensitivity.,In animal models, NAD+ deficiency leads to UV sensitivity of the skin, impairs DNA damage response, and increases genomic instability and cancer incidence.,Furthermore, NAD+ depletion is associated with human skin aging and cancer.,NAM prevents the UV-induced ATP depletion boosting cellular energy and enhances DNA repair activity in vitro and in vivo.,Moreover, NAM reduces skin cancer incidence and prevents the immune-suppressive effects of UV in mice.,Thus, NAM is involved in the maintenance of genomic stability and may have beneficial effects against skin aging changes and tumor development.,Clinical studies showed that topical use of NAM reduces cutaneous aging.,Furthermore, oral NAM administration reduces the level of UV-mediated immunosuppression and lowers the rate of non-melanoma skin cancers in high-risk patients.,Therefore, NAM replenishment strategy may be a promising approach for skin cancer chemoprevention.

Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies.,Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments.,Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2.,Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics.,In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines.,Four well characterized UM PDXs were used for in vivo experiments.,S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent.,Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC).,S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect.,However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models.,In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts.,Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration.,The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine.,These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

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

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.

In the adjuvant setting for malignant melanoma, interferon (IFN)‐α‐2b and pegylated (PEG) IFN‐α‐2b were approved in several countries including the USA before these were approved in Japan.,To resolve the “drug‐lag” issue, this phase I study was designed to evaluate the safety and tolerability in Japanese patients with stage II or III malignant melanoma who had undergone surgery, by treating with PEG IFN‐α‐2b.,As with a previously reported phase III study, patients were to receive PEG IFN‐α‐2b 6 μg/kg per week s.c. during an 8‐week induction phase, followed by a maintenance phase at a dose of 3 μg/kg per week up to 5 years.,Dose‐limiting toxicity and pharmacokinetics were assessed during the initial 8 weeks.,Of the nine patients enrolled, two patients had dose‐limiting toxicities that resolved after discontinuation of treatment.,The most frequently reported drug‐related adverse events (DRAE) included pyrexia, decreased neutrophil and white blood cell counts, and arthralgia.,Grade 3 DRAE included decreased neutrophil count.,No deaths, serious adverse events and grade 4 adverse events were reported.,Distant metastasis occurred in one patient.,No apparent differences in area under the concentration-time curve and maximum observed serum concentration were observed between Japanese and historical non‐Japanese pharmacokinetic data, suggesting no marked racial differences.,No neutralizing antibody was detected in these patient samples.,PEG IFN‐α‐2b was tolerated in Japanese patients, and eventually approved in Japan in May 2015 for adjuvant therapy in patients with stage III malignant melanoma.,Because the number of patients was limited, further investigation would be crucial.

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.

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

Metastases to the spleen are rare and are generally part of a multi-visceral metastatic disease.,The most common sources of splenic metastases include breast, lung and colorectal malignancies as well as melanoma and ovarian carcinoma.,Solitary splenic metastasis is very uncommon.,We present a case of a 44-year-old man who presented at our department for gallstones symptoms.,He had a past medical history of neck cutaneous melanoma (T3bN0M0-Stage IIb).,He had not attended follow-up schedule for personal reasons.,However, abdominal ultrasound revealed the presence of a solitary solid lesion in the spleen.,Preoperative workup was completed with CT scan that confirmed the presence of a large splenic lesion with subcapsular fluid collection, also compatible with a post-traumatic lesion.,Preoperative findings could not exclude malignancy and patient was therefore submitted to surgery.,At laparoscopy, a condition of peritoneal melanosis was present.,Splenectomy was carried out.,Histological report confirmed the peritoneal melanosis and the diagnosis of metastatic spleen lesion from melanoma.,Patient was observed, but died of metastatic disease 14 months after surgery.,Splenic metastases are uncommon.,Isolated metastases from melanoma are rare and could be found several months after primary diagnosis of melanoma.,Surgery remains the most effective treatment, especially for metachronous disease, offering the best chance of long-term survival.,Prognosis remains poor, as metachronous disease is indicative of aggressive widespread of the disease.

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

The tumor microenvironment (TME) involves infiltration of multiple immune cell subsets, which could influence the prognosis and clinical characteristics.,The increasing evidence on the role of tumor-infiltrating lymphocytes (TILs) in primary and metastatic melanomas supports that the immune system is involved in the progression and outcomes of melanoma.,However, the immune infiltration landscape in melanoma has not been systematically elucidated.,In this study, we used CIBERSORT and ESTIMATE algorithms to analyze immune infiltration pattern of 993 melanoma samples.,Then we screened differential expression genes (DEGs) related to immune subtypes and survival.,The immune cell infiltration (ICI) score was constructed by using principal-component analysis (PCA) based on immune signature genes from DGEs.,Gene set enrichment analysis (GSEA) was applied to explore high and low ICI score related pathways.,Finally, the predictive ability of ICI score was evaluated in survival prognosis and immunotherapy benefit.,We identified three ICI clusters and three gene clusters associated with different immune subtypes and survival outcomes.,Then the ICI score was constructed, and we found that high ICI score exhibited activated immune characteristics and better prognosis.,High ICI score was significantly enriched in immune pathways and highly expressed immune signature genes.,More importantly, we confirmed that melanoma patients with high ICI score had longer overall survival and rate of response to immunotherapy.,We presented a comprehensive immune infiltration landscape in melanoma.,Our results will facilitate understanding of the melanoma tumor microenvironment and provide a new immune therapy strategy.

Skin cutaneous melanoma (SKCM) is one of most aggressive type of cancers worldwide.,Serglycin (SRGN) is an intracellular proteoglycan that playing an important role in various tumors.,However, its effect on immune infiltrates and whether it associates with survival of SKCM and SKCM-metastasis patients has not been explored.,We evaluated SRGN expression via the databases of Oncomine, Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA).,The influence of SRGN expression on survival of SKCM and SKCM-metastasis patients was analyzed using TIMER database.,Furthermore, the correlations between SRGN expression and immune infiltrates or gene marker sets of immune infiltrates were also analyzed via TIMER database.,We found that the expression of SRGN in SKCM and SKCM-metastasis tissues was significantly increased compared to the normal skin tissues (P < 0.001).,Interestingly, it was showed that lower level of SRGN expression and lower immune infiltrates of B cell, CD8+ T cell, Neutrophil, and Dendritic cell were correlated with poor survival rate of SKCM and SKCM-metastasis patients (P < 0.001) but not SKCM primary patients.,We also demonstrated that SRGN expression was positively associated with the immune infiltrates and diverse immune marker sets in SKCM and SKCM-metastasis.,Our findings indicated that SRGN was associated with the survival of SKCM and SKCM-metastasis patients.,SRGN may be a new immune therapy target for treating SKCM and SKCM-metastasis.

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

A phase II randomised discontinuation trial assessed cabozantinib (XL184), an orally bioavailable inhibitor of tyrosine kinases including VEGF receptors, MET, and AXL, in a cohort of patients with metastatic melanoma.,Patients received cabozantinib 100 mg daily during a 12-week lead-in.,Patients with stable disease (SD) per Response Evaluation Criteria in Solid Tumours (RECIST) at week 12 were randomised to cabozantinib or placebo.,Primary endpoints were objective response rate (ORR) at week 12 and postrandomisation progression-free survival (PFS).,Seventy-seven patients were enroled (62% cutaneous, 30% uveal, and 8% mucosal).,At week 12, the ORR was 5% 39% of patients had SD.,During the lead-in phase, reduction in target lesions from baseline was seen in 55% of evaluable patients overall and in 59% of evaluable patients with uveal melanoma.,Median PFS after randomisation was 4.1 months with cabozantinib and 2.8 months with placebo (hazard ratio of 0.59; P=0.284).,Median PFS from study day 1 was 3.8 months, 6-month PFS was 33%, and median overall survival was 9.4 months.,The most common grade 3/4 adverse events were fatigue (14%), hypertension (10%), and abdominal pain (8%).,One treatment-related death was reported from peritonitis due to diverticular perforation.,Cabozantinib has clinical activity in patients with metastatic melanoma, including uveal melanoma.,Further clinical investigation is warranted.