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Uveal melanoma (UM) is a cancer of eye melanocytes.,Although relatively rare, UM is extremely deadly, as approximately half of all patients develop liver metastases for which there are no approved therapies.,Even therapies that succeed in cutaneous melanoma (CM) treatment have proven ineffectual for UM, highlighting both the distinct nature of these two melanomas and the need to understand the differences between them.,Here, we show that autochthonous UM tumors are rapidly induced by activated YAP and can lack hyperactive ERK, highlighting YAP as a promising therapeutic target.,We further show that MITF functions as a tumor suppressor in UM in contrast to its essential role in CM, establishing that MITF inhibition should not be entertained for UM treatment.,Cutaneous melanoma (CM) and uveal melanoma (UM) both originate from the melanocytic lineage but are primarily driven by distinct oncogenic drivers, BRAF/NRAS or GNAQ/GNA11, respectively.,The melanocytic master transcriptional regulator, MITF, is essential for both CM development and maintenance, but its role in UM is largely unexplored.,Here, we use zebrafish models to dissect the key UM oncogenic signaling events and establish the role of MITF in UM tumors.,Using a melanocytic lineage expression system, we showed that patient-derived mutations of GNAQ (GNAQQ209L) or its upstream CYSLTR2 receptor (CYSLTR2L129Q) both drive UM when combined with a cooperating mutation, tp53M214K/M214K.,The tumor-initiating potential of the major GNAQ/11 effector pathways, YAP, and phospholipase C-β (PLCβ)-ERK was also investigated in this system and thus showed that while activated YAP (YAPAA) induced UM with high potency, the patient-derived PLCβ4 mutation (PLCB4D630Y) very rarely yielded UM tumors in the tp53M214K/M214K context.,Remarkably, mitfa deficiency was profoundly UM promoting, dramatically accelerating the onset and progression of tumors induced by Tg(mitfa:GNAQQ209L);tp53M214K/M214K or Tg(mitfa:CYSLTR2L129Q);tp53M214K/M214K.,Moreover, mitfa loss was sufficient to cooperate with GNAQQ209L to drive tp53-wild type UM development and allowed Tg(mitfa:PLCB4D630Y);tp53M214K/M214K melanocyte lineage cells to readily form tumors.,Notably, all of the mitfa−/− UM tumors, including those arising in Tg(mitfa:PLCB4D630Y);tp53M214K/M214K;mitfa−/− zebrafish, displayed nuclear YAP while lacking hyperactive ERK indicative of PLCβ signaling.,Collectively, these data show that YAP signaling is the major mediator of UM and that MITF acts as a bona fide tumor suppressor in UM in direct opposition to its essential role in CM.

Uveal melanoma (UM) is the most common intraocular malignancy in adults.,Despite improvements in surgical, radiation and chemotherapy treatments, the overall survival of UM and prognosis remain poor.,In the present study, we hypothesized that Sirtuin 1 and 2 (SIRT1/2), class III histone deacetylases (HDACs), were critical in controlling the destiny of bulk tumor cells and cancer stem cells (CSCs) of UM.,We testified this hypothesis in four lines of UM cells (92.1, Mel 270, Omm 1 and Omm 2.3).,Our results showed that inhibition of SIRT1/2 by Tenovin-6 induced apoptosis in UM cells by activating the expression of tumor suppressor genes such as p53 and elevating reactive oxygen species (ROS).,Tenovin-6 inhibited the growth of UM cells.,Tenovin-6 and vinblastine was synergistic in inducing apoptosis of UM cell line 92.1 and Mel 270.,Furthermore, Tenovin-6 eliminated cancer stem cells in 92.1 and Mel 270 cells.,In conclusion, our findings suggest that Tenovin-6 may be a promising agent to kill UM bulk tumor cells and CSCs.

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

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.

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

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

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.

Primary human squamous cell carcinoma (SCCa) are heterogeneous invasive tumors with proliferating outer layers and inner differentiating cell masses.,To determine if tumor initiating cells (TIC) are present in SCCa, we utilized newly developed reliable in vitro and in vivo xenograft assays that propagate human SCCa, and demonstrated that a small subset of SCCa cells (~1%) expressing Prominin-1 (CD133) in the outer layers of SCCa were highly enriched for TIC (~1/400) compared to unsorted SCCa cells (TIC ~1/106).,Xenografts of CD133+ SCCa recreated the original SCCa tumor histology and organizational hierarchy, while CD133- cells did not, and only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies.,We present a model of human SCCa in which tumor projections expand with outer leading edges that contain CD133+ TIC.,Successful cancer treatment will likely require that the TIC identified in cancers be targeted therapeutically.,The demonstration that TIC are present in SCCa and are enriched in a CD133-expressing subpopulation to our knowledge has not previously been reported.

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

Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy.,Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules.,LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored.,In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1.,Collectively, our results present MIRAT’s direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer.

Sun exposure is a major environmental risk factor for skin cancers and is also an important source of vitamin D.,However, while experimental evidence suggests that vitamin D may have a protective effect on skin cancer risk, epidemiologic studies investigating the influence of 25-hydroxyvitamin D (25(OH)D) level and/or vitamin D intake on skin cancer risk are conflicting.,A systematic review and dose-response meta-analyses of prospective studies was conducted to clarify these associations.,Relevant studies were identified by searching the PubMed database up to 30th August 2019.,Random effects dose-response meta-analyses were used to estimate summary relative risks (SRRs) and 95% confidence intervals (CIs).,Overall, thirteen prospective studies were included.,Circulating level of 25(OH)D was associated with higher risks of melanoma (SRR (95% CI) per 30 nmol = 1.42 (1.17-1.72)) and keratinocyte cancer (KC) (SRR (95% CI) per 30 nmol/L = 1.30 (1.13-1.49)).,The SRR (95% CI) per 30 nmol/L increase in 25(OH) D level was 1.41 (1.19-1.67), and 1.57 (0.64-3.86), for basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs), respectively.,However, while we found that vitamin D intake (from diet, supplemental and total) was not associated with risks of melanoma and SCC, vitamin D intake was associated with slightly increased BCC risk, albeit with no heterogeneity across skin cancer type.,This meta-analysis suggests positive associations between circulating 25(OH)D level and risk of melanoma and KC, however, this finding is most likely confounded by sun exposure.,We found no associations between vitamin D intake skin cancers, except positive associations with BCC risk.

Basal cell carcinoma (BCC) is the most common human cancer, characterized by aberrant activation of the hedgehog (HH) signaling pathway resulting from mutations in the patched 1 (PTCH1) or smoothened (SMO) genes.,In the present study, to uncover the expression profile of HH signaling-related molecules, we thoroughly examined the mRNA and protein expression levels of six molecules including GLI1, GLI2, PTCH1, PTCH2, SHH, and SMO in BCC and various other cutaneous tumors.,Real-time PCR analysis demonstrated that BCC showed remarkably enhanced mRNA expression of all HH molecules, except SMO compared to other skin tumors.,However, immunohistochemical analysis revealed that only GLI1 protein was specifically upregulated in BCC, while the other HH-related proteins did not show any significant differences between the tumors.,Notably, other skin malignancies such as squamous cell carcinoma, sebaceous carcinoma, and malignant melanoma showed no GLI1 expression and there was no difference in GLI1 expression between the BCC subtypes.,In addition, GLI1 and GLI2 expression were strongly associated with the hair follicle stem cell markers, LGR4 and LGR5, which are known target genes of the Wnt pathway.,Our results suggest that GLI1 has the potential to be a diagnostically useful marker for differentiating BCC from other skin malignancies and an interaction between the HH and Wnt signaling pathways may be involved in the development of BCCs.

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

Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ∼50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized.,Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAFV600E protein.,To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAFV600E expression.,To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAFV600E-mutant human melanomas.,BRAFV600E regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts.,These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription.,Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs.,BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration.,BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11.,Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.

Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment.,Genomic alterations in these cells remain a point of contention.,We report that CAFs from skin squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous NOTCH1 gene amplification and overexpression that also occur, to a lesser extent, in dermal fibroblasts of apparently unaffected skin.,The fraction of the latter cells harboring NOTCH1 amplification is expanded by chronic UVA exposure, to which CAFs are resistant.,The advantage conferred by NOTCH1 amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade.,In an orthotopic model of skin SCC, genetic or pharmacological inhibition of NOTCH1 activity suppresses cancer/stromal cells expansion.,Here we show that NOTCH1 gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-cancer intervention.,The presence of genomic alterations in cancer associated fibroblasts (CAFs) is largely unexplored.,The authors show that frequent NOTCH1 gene amplification and overexpression render CAFs resistant to the UVA-induced DNA damage response (DDR) and promote cancer/stromal cells expansion, which can be reversed by NOTCH inhibition.

We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS.,We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling.,This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS.,Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice.,Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression.,They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.,► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice

An important component of research using animal models is ensuring rigor and reproducibility.,This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy.,Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC).,Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied.,The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation.,We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank.,When tumors reached 190-230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine).,Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia.,Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors.,SC tumors reached the target tumor volume significantly faster compared with ID tumors.,Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not.,Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth.,These findings were then extended to additional immunotherapy regimens in four separate tumor models.,These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment.,Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

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

Long noncoding RNAs (LncRNAs), including MALAT1, are critical regulators of tumor development.,However, the roles and molecular mechanisms of LncRNAs in cutaneous squamous cell carcinoma (cSCC) remain underexplored.,In this study, functional studies using in vitro cellular and in vivo xenograft models confirmed the pro-carcinogenic roles of MALAT1 in cSCC.,Further, MALAT1 was identified to regulate epidermal growth factor receptor (EGFR) protein expression but did not affect EGFR mRNA expression.,Transcriptomic sequencing identified kinectin 1 (KTN1) as the key mediator for MALAT1 regulation of EGFR.,Mechanistic study revealed that MALAT1 interacts with c-MYC to form a complex and directly binds to the promoter region of KTN1 gene and enhances its transactivation to positively regulate EGFR protein expression.,Our findings, therefore, establish a novel c-MYC-assisted MALAT1-KTN1-EGFR axis, which contributes to cSCC development and may serve as novel target for therapeutic intervention.

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

Anti-PD-1 therapy has shown significant clinical activity in advanced melanoma.,We developed and validated a clinical prediction scale for response to anti- PD-1 monotherapy.,A total of 315 patients with advanced melanoma treated with pembrolizumab (2 or 10 mg kg−1 Q2W or Q3W) or nivolumab (3 mg kg−1 Q2W) at four cancer centres between 2011 to 2013 served as the setting for the present cohort study.,Variables with significant association to response on a univariate analysis were entered into a forward stepwise logistic regression model and were given a score based on ORs to calculate a clinical prediction scale.,The developed clinical prediction scale included elevated LDH (1 point), age <65 years (1 point), female sex (1 point), history of ipilimumab treatment (2 points) and the presence of liver metastasis (2 points).,The scale had an area under the receiver-operating curve (AUC) of 0.73 (95% CI 0.67, 0.80) in predicting response to therapy.,The predictive performance of the score was maintained in the validation cohort (AUC 0.70 (95% CI 0.58, 0.81)) and the goodness-to-fit model demonstrated good calibration.,Based on a large cohort of patients, we developed and validated a simple five-factor prediction scale for the clinical activity of PD-1 antibodies in advanced melanoma patients.,This scale can be used to stratify patients participating in clinical trials.

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.

Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated.,Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease.,Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively.,Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures.,Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs.,Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.

MicroRNAs (miRNAs) are small non-coding RNAs (18-24 nucleotides) that have recently been shown to regulate gene expression during cancer progression.,Dicer, a central enzyme in the multi-component miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms.,Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.,Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12).,Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001).,The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001).,In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009).,Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro.,A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.,Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma.,Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.

Vitamin D is a precursor for secosteroidal hormones, which demonstrate pleiotropic biological activities, including the regulation of growth and the differentiation of normal and malignant cells.,Our previous studies have indicated that the inhibition of melanoma proliferation by a short side-chain, low calcemic analog of vitamin D-21(OH)pD is not fully dependent on the expression of vitamin D receptor (VDR).,We have examined the effects of classic vitamin D metabolites, 1,25(OH)2D3 and 25(OH)D3, and two low calcemic vitamin D analogs, (21(OH)pD and calcipotriol), on proliferation, mRNA expression and vitamin D receptor (VDR) translocation in three human melanoma cell lines: WM98, A375 and SK-MEL-188b (subline b of SK-MEL-188, which lost responsiveness to 1,25(OH)2D3 and became VDR−/−CYP27B1−/−).,All tested compounds efficiently inhibited the proliferation of WM98 and A375 melanoma cells except SK-MEL-188b, in which only the short side-chain vitamin D analog-21(OH)pD was effective.,Overall, 21(OH)pD was the most potent compound in all three melanoma cell lines in the study.,The lack of responsiveness of SK-MEL-188b to 1,25(OH)2D3, 25(OH)D3 and calcipotriol is explained by a lack of characteristic transcripts for the VDR, its splicing variants as well as for vitamin D-activating enzyme CYP27B1.,On the other hand, the expression of VDR and its splicing variants and other vitamin D related genes (RXR, PDIA3, CYP3A4, CYP2R1, CYP27B1, CYP24A1 and CYP11A1) was detected in WM98 and A375 melanomas with the transcript levels being modulated by vitamin D analogs.,The expression of VDR isoforms in WM98 cells was stimulated strongly by calcipotriol.,The antiproliferative activities of 21(OH)pD appear not to require VDR translocation to the nucleus, which explains the high efficacy of this noncalcemic pregnacalciferol analog in SK-MEL-188b melanoma, that is, VDR−/−.,Therefore, we propose that 21(OH)pD is a good candidate for melanoma therapy, although the mechanism of its action remains to be defined.

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas.,SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear.,Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss.,Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3’ss-sequence context.,SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants.,Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage.,Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.,Mutations in the splicing factor SF3B1 are found in uveal melanoma.,Here, Alsafadi et al. use RNA-sequencing data from these cancers and experimental models, and show that mutant SF3B1 promotes alternative branchpoints in a specific gene subset and that the mutant protein gains a new function.

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas.,We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients.,This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi).,We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients.,Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi.,In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells.,Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes.,Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi.,Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.

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

Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these patients develop liver metastases.,Exosomes are small RNA containing nano-vesicles released by most cells, including malignant melanoma cells.,This clinical translational study included patients undergoing isolated hepatic perfusion (IHP) for metastatic uveal melanoma, from whom exosomes were isolated directly from liver perfusates.,The objective was to determine whether exosomes are present in the liver circulation, and to ascertain whether these may originate from melanoma cells.,Exosomes were isolated from the liver perfusate of twelve patients with liver metastases from uveal melanoma undergoing IHP.,Exosomes were visualised by electron microscopy, and characterised by flow cytometry, Western blot and real-time PCR.,Furthermore, the concentration of peripheral blood exosomes were measured and compared to healthy controls.,The liver perfusate contained Melan-A positive and RNA containing exosomes, with similar miRNA profiles among patients, but dissimilar miRNA compared to exosomes isolated from tumor cell cultures.,Patients with metastatic uveal melanoma had a higher concentration of exosomes in their peripheral venous blood compared to healthy controls.,Melanoma exosomes are released into the liver circulation in metastatic uveal melanoma, and is associated with higher concentrations of exosomes in the systemic circulation.,The exosomes isolated directly from liver circulation contain miRNA clusters that are different from exosomes from other cellular sources.,The online version of this article (doi:10.1186/1471-2407-14-962) contains supplementary material, which is available to authorized users.

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.

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

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.

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.

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

Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies.,While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive.,This study used low-attachment based generation of spheroids composed of up to three cell types.,Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses.,Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student’s t-test.,The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages.,Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside.,Regularly, some melanoma cells were also found to invade the fibroblast core.,In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells.,Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells.,Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells.,Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity.,In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described.,This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.,The online version of this article (10.1186/s12885-019-5606-4) contains supplementary material, which is available to authorized users.

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

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

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

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.

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.

Talimogene laherparepvec (T-VEC) is a licensed therapy for use in melanoma patients of stage IIIB-IVM1a with injectable, unresectable metastatic lesions in Europe.,Approval was based on the Oncovex Pivotal Trial in Melanoma study, which also included patients with distant metastases and demonstrated an overall response rate (ORR) of 40.5% and a complete response (CR) rate of 16.6%.,The aim of this study was to assess the outcome of melanoma patients treated with T-VEC in a real-life clinical setting.,Based on data from 10 melanoma centers in Austria, Switzerland and southern Germany, we conducted a retrospective chart review, which included 88 patients (44 male, 44 female) with a median age of 72 years (range 36-95 years) treated with T-VEC during the period from May 2016 to January 2020.,88 patients fulfilled the inclusion criteria for analysis.,The ORR was 63.7%. 38 patients (43.2%) showed a CR, 18 (20.5%) had a partial response, 8 (9.1%) had stable disease and 24 (27.3%) patients had a progressive disease.,The median treatment period was 19 weeks (range: 1-65), an average of 11 doses (range: 1-36) were applied.,39 (45.3%) patients developed adverse events, mostly mild, grade I (64.1%).,This real-life cohort treatment with T-VEC showed a high ORR and a large number of durable CRs.

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy.,Delineating mechanisms of secondary resistance to this strategy is increasingly important.,Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb).,Sustained IFNβ transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC).,Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation.,Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature.,Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy.

RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61.,Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch.,Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma.,When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis.,RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors.,These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.,•RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes•RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK•RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis•RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes,RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK,RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis,RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S is a common mutation in human cutaneous melanoma.,Lionarons et al. show that RAC1P29S induces a melanocytic to mesenchymal switch via an SRF/MRTF-mediated gene expression program, cooperates with BRAF in melanomagenesis, and drives BRAF inhibitor resistance, which is reversed by SRF/MRTF inhibition.

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.

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.

The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity.,Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties.,Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells.,We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium.,Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers.,They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts.,Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells.,In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities.,In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts.,Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity.,The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures.,While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells.,Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

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

The acquisition of invasive properties in melanoma is associated with a high proclivity for metastasis, but the underlying pathways are poorly characterized.,The Hippo pathway plays an important role in organ size control and is dysregulated in some types of tumors.,The present study, “Pro-invasive activity of the Hippo pathway effectors YAP and TAZ in cutaneous melanoma” by Nallet-Staub et al., provides the first in-depth analysis of expression of the Hippo pathway effectors YAP (yes-associated protein) and TAZ (Tafazzin) in human melanocytic lesions.,Importantly, results from this study demonstrate a causal relationship between YAP/TAZ levels and melanoma cell tumorigenicity and invasiveness.

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis.,We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs).,Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration.,Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos.,The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells.,A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation.,This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos.,We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.,The online version of this article (10.1007/s00018-020-03707-4) contains supplementary material, which is available to authorized users.

Targeted inhibitors elicit heterogeneous clinical responses in genetically stratified groups of patients.,While most studies focus on tumor intrinsic properties, factors in the tumor microenvironment were recently found to modulate the response to inhibitors.,Here, we show that in cutaneous BRAF V600E melanoma, the cytokine TNFα blocks RAF-inhibitor-induced apoptosis via activation of nuclear factor κB (NFκB).,Several NFκB-dependent factors are up-regulated following TNFα and RAF inhibitor treatment.,Of these factors, we show that death receptor inhibitor cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) is required for TNFα-induced protection against RAF inhibitor.,Overexpression of c-FLIP_S or c-FLIP_L isoform decreased RAF inhibitor-induced apoptosis in the absence of TNFα.,Importantly, targeting NFκB enhances response to RAF inhibitor in vitro and in vivo.,Together, our results show mechanistic evidence for cytokine-mediated resistance to RAF inhibitor and provide a preclinical rationale for the strategy of co-targeting the RAF-MEK-ERK1/2 pathway and the TNFα/NFκB axis to treat mutant BRAF melanomas.

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis.,Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival.,PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo.,Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells.,We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo.,This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior.,In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed.,Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.

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

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.

Cutaneous melanoma is a malignant tumor of skin melanocytes that are pigment-producing cells located in the basal layer (stratum basale) of epidermis.,Accumulation of genetic mutations within their oncogenes or tumor-suppressor genes compels melanocytes to aberrant proliferation and spread to distant organs of the body, thereby resulting in severe and/or lethal malignancy.,Metastatic melanoma’s heavy mutational load, molecular heterogeneity and resistance to therapy necessitate the development of novel biomarkers and drug-based protocols that target key proteins involved in perpetuation of the disease.,To this direction, we have herein employed a nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) proteomics technology to profile the deep-proteome landscape of WM-266-4 human metastatic melanoma cells.,Our advanced melanoma-specific catalogue proved to contain 6,681 unique proteins, which likely constitute the hitherto largest single cell-line-derived proteomic collection of the disease.,Through engagement of UNIPROT, DAVID, KEGG, PANTHER, INTACT, CYTOSCAPE, dbEMT and GAD bioinformatics resources, WM-266-4 melanoma proteins were categorized according to their sub-cellular compartmentalization, function and tumorigenicity, and successfully reassembled in molecular networks and interactomes.,The obtained data dictate the presence of plastically inter-converted sub-populations of non-cancer and cancer stem cells, and also indicate the oncoproteomic resemblance of melanoma to glioma and lung cancer.,Intriguingly, WM-266-4 cells seem to be subjected to both epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) programs, with 1433G and ADT3 proteins being identified in the EMT/MET molecular interface.,Oncogenic addiction of WM-266-4 cells to autocrine/paracrine signaling of IL17-, DLL3-, FGF(2/13)- and OSTP-dependent sub-routines suggests their critical contribution to the metastatic melanoma chemotherapeutic refractoriness.,Interestingly, the 1433G family member that is shared between the BRAF- and EMT/MET-specific interactomes likely emerges as a novel and promising druggable target for the malignancy.,Derailed proliferation and metastatic capacity of WM-266-4 cells could also derive from their metabolic addiction to pathways associated with glutamate/ammonia, propanoate and sulfur homeostasis, whose successful targeting may prove beneficial for advanced melanoma-affected patients.

Circular RNAs (circRNAs) are reported to be aberrantly expressed and perform different functions in numerous types of tumor; however, their expression levels in cutaneous squamous cell carcinoma (CSCC) remain largely unclear.,Thus, the purpose of the present study was to investigate the function of circRNA_001937 in CSCC.,Differential circRNA expression profiles of CSCC were analyzed using the Arraystar Human circRNAs chip and reverse transcription-quantitative PCR (RT-qPCR); and the effects of circRNA_001937 on cell behavior, in particular its regulation over the microRNA (miRNA)-597-3p/Fos-related antigen 2 (FOSL2) pathway, was investigated using a dual-luciferase reporter assay, and verified using RT-qPCR and western blotting. circRNA_001937 expression levels were significantly increased in CSCC tissues and cell lines compared with the corresponding adjacent tissues and control cells (P<0.05).,The genetic silencing of circRNA_001937 with small interfering RNA significantly inhibited cell proliferation, and induced cell apoptosis (P<0.05). circRNA_001937 was observed to directly bind to miRNA-597-3p and serve as a sponge, which indirectly increased the expression levels of FOSL2, a miRNA-597-3p target gene.,In conclusion, circRNA_001937 expression was increased in CSCC and silencing circRNA_001937 gene expression may inhibit CSCC progression by sponging the miRNA-597-3p/FOSL2 pathway.

We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS.,We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling.,This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS.,Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice.,Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression.,They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.,► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice

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

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

Ipilimumab (IPI) and BRAF inhibitors (BRAFi) improve survival in melanoma, but not all patients will benefit and toxicity can be significant.,Pretreatment neutrophil to lymphocyte ratio (NLR) has been associated with outcome in IPI-treated patients, but has not been studied during treatment or in BRAFi-treated patients.,Using a prospectively maintained database, patients with unresectable stage III or IV melanoma treated with IPI or a BRAFi (vemurafenib or dabrafenib as monotherapy) from 2006 to 2011 were identified.,NLR was calculated before treatment and at 3-week intervals after treatment initiation until 9 weeks.,Baseline NLR was tested for association with overall survival (OS), progression free survival (PFS), and clinical response to treatment.,On-treatment NLRs were tested for association with the same outcomes using landmark survival analyses and time-dependent Cox regression models.,The association of relative change of NLR from baseline with outcomes was also examined.,A multivariate model tested the association of NLR and OS/PFS with additional clinical factors.,There were 197 IPI patients and 65 BRAFi patients.,In multivariable analysis adjusting for M stage, and disease type (in OS)/gender (in PFS), an NLR value of 5 or above at every timepoint was associated with worse OS (HR 2.03-3.37, p < 0.001), PFS (HR 1.81-2.51, p < 0.001), and response to therapy (OR 3.92-9.18, p < 0.007), in the IPI cohort.,In addition, a > 30% increase in NLR above baseline at any timepoint was associated with a worse OS and PFS (HR 1.81 and 1.66, p < 0.004).,In BRAFi patients, NLR was not consistently associated with outcomes.,A high NLR, whether measured prior to or during treatment with IPI, is associated with worse OS, PFS, and clinical response in patients with advanced melanoma.,An increasing NLR from baseline during treatment was correlated with worse OS and PFS in IPI-treated patients.,In comparison, as NLR was not associated with outcomes in BRAFi patients, NLR may have a uniquely predictive value in patients treated with immunotherapy.,•Neutrophil to lymphocyte ratio is associated with important clinical outcomes in melanoma patients treated with ipilimumab.,•Changes in neutrophil to lymphocyte ratio from baseline during treatment with ipilimumab correlate with clinical outcomes•Neutrophil to lymphocyte ratio is not associated with outcomes in those treated with BRAF inhibitors,Neutrophil to lymphocyte ratio is associated with important clinical outcomes in melanoma patients treated with ipilimumab.,Changes in neutrophil to lymphocyte ratio from baseline during treatment with ipilimumab correlate with clinical outcomes,Neutrophil to lymphocyte ratio is not associated with outcomes in those treated with BRAF inhibitors,Baseline neutrophil to lymphocyte ratio (NLR) and changes in NLR during treatment associate with important clinical outcomes, including overall survival, progression-free survival, and clinical response in advanced melanoma patients treated with immunotherapy, and therefore may have a valuable role in selecting patients most likely or least likely to benefit from treatment, or for monitoring response to treatment over time.,This marker is not useful in patients treated with BRAF inhibitors, perhaps reflecting its unique value in immunotherapy.

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

Risk of melanoma is in part determined by genetic factors.,Currently the only established high penetrance familial melanoma genes are CDKN2A and CDK4.,Recent studies reported germline variants in POT1 in melanoma families.,In the present study, we sequenced the entire POT1 gene in 694 patients from the M3-study.,Patients with multiple primary melanomas (n = 163) or with a positive family history (n = 133) were classified as high-risk melanoma patients.,Additionally, 200 single primary melanoma patients and 198 non-melanoma controls were sequenced.,For prediction analysis 10 different tools were used.,In total 53 different variants were found, of which 8 were detected in high-risk melanoma patients, only.,Two out of these 8 variants were located in exons and were non-synonymous: g.124510982 G>A (p.R80C) and g.124491977 T>G (p.N300H).,While g.124491977 T>G was predicted to be neutral, 80% of the prediction tools classified g.124510982 G>A as deleterious.,The variant, g.124467236 T>C, which possibly causes a change in the splice site was identified in a case with a positive family history in the present study.,Another variant in the 5-UTR, g.124537261 A>G, was found in 2 high-risk patients.,So, in conclusion, melanoma associated POT1 germline variants seem to be rare.,Further studies are required to evaluate the role of POT1 for genetic counseling.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown.,Using whole exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy.,Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance.,Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of CDKN2A mutations in this population.,These variants were not found in public databases or in 2,038 genotyped Italian controls.,We also identified two rare recurrent POT1 variants in American and French familial melanoma cases.,Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

This study aims to investigate the difference of gene expression and its prognostic significance in younger women with melanoma.,Significantly upregulated genes in tumors compared to normal skin tissues were extracted.,Among these genes, genes that significantly affected survival according to expression level were selected, and pathway annotation was performed.,The patient proportion with high/low expression of the most significant pathways was analyzed in each age (< 50, 50-59, ≥ 60) and gender group.,Survival was analyzed according to age, gender, and pathways.,The most significant pathways that were upregulated in tumor tissues and also had impacts on survival were programmed cell death protein [PD]-1, interferon-γ, and interferon-α/β pathways.,In women, the immune signaling rate in patients was higher than men and decreased with age (63.5%, 53.8%, and 47.6%).,In men, the decreasing tendency was minimal (47.6%, 50.0%, and 41.6%).,In patients aged < 60 years, women had a favorable survival rate than men (p = 0.055).,Except for patients with high immune signaling, no survival difference was observed between genders (p = 0.6).,In conclusion, younger female melanoma patients had high immune signaling than older women and men.,This immune signaling improved survival of the younger female patients.

Mitotic rate is no longer considered a staging criterion for thin melanoma in the 8th edition of the American Joint Committee on Cancer Staging Manual.,The aim of this observational study was to identify prognostic factors for thin melanoma and predictors and prognostic significance of sentinel lymph node (SLN) involvement in a large multicenter cohort of patients with melanoma from nine tertiary care hospitals.,A total of 4249 consecutive patients with thin melanoma diagnosed from January 1, 1998 to December 31, 2016 were included.,The main outcomes were disease‐free interval and melanoma‐specific survival for the overall population and predictors of SLN metastasis (n = 1083).,Associations between survival and SLN status and different clinical and pathologic variables (sex, age, tumor location, mitosis, ulceration, regression, lymphovascular invasion, histologic subtype, Clark level, and Breslow thickness) were analyzed by Cox proportional hazards regression and logistic regression.,SLN status was the most important prognostic factor for melanoma‐specific survival (hazard ratio, 13.8; 95% CI, 6.1‐31.2; P < 0.001), followed by sex, ulceration, and Clark level for patients who underwent SLNB.,A mitotic rate of >2 mitoses/mm2 was the only factor associated with a positive SLN biopsy (odds ratio, 2.9; 95% CI, 1.22‐7; P = 0.01.,SLN status is the most important prognostic factor in thin melanoma.,A high mitotic rate is associated with metastatic SLN involvement.,SLN biopsy should be discussed and recommended in patients with thin melanoma and a high mitotic rate.

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

Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma.,However, many patients do not display therapeutic response or eventually relapse.,Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies.,Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown.,Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma.,Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers.,Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression.,TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens.,This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.,Most melanoma patients do not respond to anti-PD1 therapy.,Here, the authors show that TNFα blockade synergizes with anti-PD-1 by preventing anti-PD-1-induced CD8+ T cell death and TIM-3 expression on such cells.

Predicting metastasis in melanoma patients is important for disease management and could help to identify those who might benefit from adjuvant treatment.,The aim of this study was to investigate whether the tumor microenvironment-derived protein S100A8/A9 qualifies as prognostic marker for melanoma patients, also in the setting of immunotherapy.,S100A8/A9 gene and protein expression were analyzed on melanocytic nevi, primary melanomas and metastases using a cDNA library and three independent tissue-microarrays (TMA).,Serum levels of S100A8/A9 were measured using a specific ELISA in two independent cohorts of 354 stage III and stage IV melanoma patients as well as in two independent cohorts of patients treated with the PD-1 antibody pembrolizumab.,cDNA analysis revealed an upregulation of S100A8 and S100A9 gene expression in melanoma metastases compared to primary melanomas.,Significantly higher numbers of infiltrating S100A8/A9 positive cells were found in tissue samples of metastasizing primary melanomas compared to non-metastasizing melanomas (P < .0001) and in melanomas of short-term survivors compared to long-term survivors (P < .0001).,Serum S100A8/A9 levels > 5.5 mg/l were associated with impaired overall survival in two independent cohorts (both P < .0001).,Importantly, patients with serum elevated S100A8/A9 treated with pembrolizumab showed significantly impaired survival compared to patients with lower S100A8/A9 levels (cohort 1: P = .0051; cohort 2: P < .0001).,The tumor microenvironment-associated protein S100A8/A9 serves as a novel prognostic marker for metastasis and survival of metastatic melanoma patients and predicts response to immunotherapy with pembrolizumab.,These data underscore the significance of tumor microenvironment-derived factors as suitable biomarkers for melanoma.

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.

Dysregulation of receptor tyrosine kinases (RTKs) contributes to several aspects of oncogenesis including drug resistance.,In melanoma, distinct RTKs have been involved in BRAF inhibitors (BRAFi) resistance, yet the utility of RTKs expression pattern to identify intrinsically resistant tumors has not been assessed.,Transcriptional profiling of RTKs and integration with a previous classification, reveals three robust subtypes in two independent datasets of melanoma cell lines and one cohort of melanoma samples.,This classification was validated by Western blot in a panel of patient-derived melanoma cell lines.,One of the subtypes identified here for the first time displayed the highest and lowest expression of EGFR and ERBB3, respectively, and included BRAF-mutant tumors all intrinsically resistant to BRAFi PLX4720, as assessed by analysis of the Cancer Cell Line Encyclopedia pharmacogenomic study and by in vitro growth inhibition assays.,High levels of EGFR were detected, even before therapy, in tumor cells of one of three melanoma patients unresponsive to BRAFi.,Use of different pharmacological inhibitors highlighted the relevance of PI3K/mTOR signaling for growth of this PLX4720-resistant subtype.,Our results identify a specific molecular profile of melanomas intrinsically resistant to BRAFi and suggest the PI3K/mTOR pathway as a potential therapeutic target for these tumors.

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.

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

Despite remarkable advances in the genomic characterization of adult melanoma, the molecular pathogenesis of pediatric melanoma remains largely unknown.,We analyzed 15 conventional melanomas (CMs), 3 melanomas arising in congenital nevi (CNMs), and 5 spitzoid melanomas (SMs), using various platforms, including whole genome or exome sequencing, the molecular inversion probe assay, and/or targeted sequencing.,CMs demonstrated a high burden of somatic single-nucleotide variations (SNVs), with each case containing a TERT promoter (TERT-p) mutation, 13/15 containing an activating BRAF V600 mutation, and >80% of the identified SNVs consistent with UV damage.,In contrast, the three CNMs contained an activating NRAS Q61 mutation and no TERT-p mutations.,SMs were characterized by chromosomal rearrangements resulting in activated kinase signaling in 40%, and an absence of TERT-p mutations, except for the one SM that succumbed to hematogenous metastasis.,We conclude that pediatric CM has a very similar UV-induced mutational spectrum to that found in the adult counterpart, emphasizing the need to promote sun protection practices in early life and to improve access to therapeutic agents being explored in adults in young patients.,In contrast, the pathogenesis of CNM appears to be distinct.,TERT-p mutations may identify the rare subset of spitzoid melanocytic lesions prone to disseminate.

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

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

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

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

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

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

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.

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

Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab).,Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG.,While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB.,All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB+ cells.,This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance.,As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.,Agonistic 4-1BB antibodies developed for cancer immunotherapy have suffered from either hepatotoxicity or insufficient anti-cancer activity.,Here the authors determine the contribution of FcγR binding and agonistic strength to these outcomes, and engineer a 4-1BB antibody with potent anti-tumor effect and no liver toxicity in mice.

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.

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

Clinical outcomes for advanced malignant melanoma (MM) are often poor due to tumor invasiveness, metastasis, recurrence, and multidrug resistance.,We investigated whether apoptosis, cell cycle regulation, oxidative status, and redox balance were altered by changes in the expression of the long noncoding RNA, growth arrest-specific transcript 5 (GAS5), in MM cells.,Analysis of clinical samples from MM patients showed that the rate of reduced GAS5 expression, relative to that in adjacent noncancerous tissues, was significantly lower for tumors from patients with advanced disease (76.6%, P < 0.001), as evidenced by larger tumor size, higher TNM stage, and higher incidences of ulceration and metastasis (P < 0.001 for all).,Cell culture experiments showed that siRNA-mediated knockdown of GAS5 increased the viability of A375-GAS5si cells.,Flow cytometry and western blotting showed that GAS5 knockdown increased MM cell proliferation by inducing G1/S cell cycle progression through increases in Cyclin D1, CDK4, and p27 expression (P < 0.05 for all) and by inhibiting apoptosis through an increase in Bcl-2 expression (P < 0.001).,Knockdown of GAS5 also increased levels of superoxide anion (P < 0.01), NADP+(P < 0.001), and oxidized glutathiones (P < 0.01) through increases in NOX4 expression (P < 0.001), G6PD expression (P < 0.01), and NOX activity (P < 0.05), and RNA co-immunoprecipitation showed that GAS5 induced these changes through a physical interaction between GAS5 and the G6PD protein.,Our findings show GAS5 contributes to regulation of the apoptosis, cell cycle, homeostasis of reactive oxygen species, and redox balance in MM cells, and suggest that reduced GAS5 expression contributes to disease progression in MM patients.,The online version of this article (10.1007/s00432-018-2820-4) contains supplementary material, which is available to authorized users.

Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer.,The costs and morbidity associated with skin cancer are increasing, and therefore identifying molecules that can help prevent skin carcinogenesis is important.,In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK).,Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar simulated light (SSL)-induced skin carcinogenesis in mouse models.,In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues.,We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling.,Importantly, topical application of either rocuronium bromide or betamethasone decreased SSL-induced epidermal hyperplasia, neovascularization and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers, COX-2, cyclin D1 and MMP-9.,This study is the first to demonstrate that targeting PRPK could be useful against sUV-induced cSCC development.

Wrightia tinctoria is a constituent of several ayurvedic preparations against skin disorders including psoriasis and herpes, though not yet has been explored for anticancer potential.,Herein, for the first time, we report the significant anticancer properties of a semi-purified fraction, DW-F5, from the dichloromethane extract of W. tinctoria leaves against malignant melanoma.,DW-F5 exhibited anti-melanoma activities, preventing metastasis and angiogenesis in NOD-SCID mice, while being non-toxic in vivo.,The major pathways in melanoma signaling mediated through BRAF, WNT/β-catenin and Akt-NF-κB converging in MITF-M, the master regulator of melanomagenesis, were inhibited by DW-F5, leading to complete abolition of MITF-M.,Purification of DW-F5 led to the isolation of two cytotoxic components, one being tryptanthrin and the other being an unidentified aliphatic fraction.,The overall study predicts Wrightia tinctoria as a candidate plant to be further explored for anticancer properties and DW-F5 as a forthcoming drug formulation to be evaluated as a chemotherapeutic agent against malignant melanoma.

Over the past several years the incidence of cutaneous melanoma has rapidly increased.,This tumor develops often in-transit metastases that significantly reduce patient survival at 5 years.,To improve prognosis and quality of life in patients with melanoma metastases, a mini invasive procedure like electrochemotherapy (ECT) is adopted to remove superficial tissue lesions.,To detect the melanoma metastases, high frequency (HF) ultrasound (US) is used.,This technique, though, can be time-consuming and it needs an expert operator and a high performing machine.,Therefore, we asked whether the US could be replaced or integrated with other less time-consuming techniques such as 18-FDG positron emission tomography/computed tomography (PET-CT) and telethermography (TT).,Fifteen patients (4 males and 11 females - age range: 63-91) affected whit advanced stage melanoma were enrolled.,They presented 52 in-transit metastases as detected by the three techniques used, HF-US, PET/CT and TT within 30 days before ECT.,All the 52 lesions were detected by HF-US (100%), 24/52 were detected by PET-CT (42,6%) and 15/52 were detected by TT (27,7%).,PET-CT reported 3.7% false positives, while no false positive were reported by TT.,As US detected 100% lesions, compared to the other two techniques used, US, along with clinical examination, has still to be considered as gold standard in the diagnosis of metastatic lesions.,US, associated with an exhaustive anamnesis and accurate clinical examination, cannot be replaced by either PET-CT or TT.,When US performing devices and experienced operators are not available, though, it is highly recommended to integrate US with at least one of the other techniques.,Under certain circumstances, as in the case of obese and non-collaborating patients or in patients with lymphatic stasis, these techniques should be integrated to obtain exact in-transit metastases evaluation.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1.,To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3.,Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential.,New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories.,Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested.,Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations.,Details about these techniques and comparisons are available in the literature 4.,Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature.,There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6.,Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease.,However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable.,This situation changed dramatically in the late 1990s-2000s.,Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.,In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13.,In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging.,We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results.,We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

Recent evidence suggests that immunotherapy efficacy in melanoma is modulated by gut microbiota.,Few studies have examined this phenomenon in humans, and none have incorporated metatranscriptomics, important for determining expression of metagenomic functions in the microbial community.,In melanoma patients undergoing immunotherapy, gut microbiome was characterized in pre-treatment stool using 16S rRNA gene and shotgun metagenome sequencing (n = 27).,Transcriptional expression of metagenomic pathways was confirmed with metatranscriptome sequencing in a subset of 17.,We examined associations of taxa and metagenomic pathways with progression-free survival (PFS) using 500 × 10-fold cross-validated elastic-net penalized Cox regression.,Higher microbial community richness was associated with longer PFS in 16S and shotgun data (p < 0.05).,Clustering based on overall microbiome composition divided patients into three groups with differing PFS; the low-risk group had 99% lower risk of progression than the high-risk group at any time during follow-up (p = 0.002).,Among the species selected in regression, abundance of Bacteroides ovatus, Bacteroides dorei, Bacteroides massiliensis, Ruminococcus gnavus, and Blautia producta were related to shorter PFS, and Faecalibacterium prausnitzii, Coprococcus eutactus, Prevotella stercorea, Streptococcus sanguinis, Streptococcus anginosus, and Lachnospiraceae bacterium 3 1 46FAA to longer PFS.,Metagenomic functions related to PFS that had correlated metatranscriptomic expression included risk-associated pathways of l-rhamnose degradation, guanosine nucleotide biosynthesis, and B vitamin biosynthesis.,This work adds to the growing evidence that gut microbiota are related to immunotherapy outcomes, and identifies, for the first time, transcriptionally expressed metagenomic pathways related to PFS.,Further research is warranted on microbial therapeutic targets to improve immunotherapy outcomes.

This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT) response in metastatic melanoma patients.,Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT-particularly with BRAF-wild-type disease.,In preclinical studies, specific gut microbiota promotes regression of melanoma in mice.,We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN), or pembrolizumab (P).,IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease.,ICT responders for all types of therapies were enriched for Bacteroides caccae.,Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis.,Among P responders, the microbiome was enriched for Dorea formicogenerans.,Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders.,Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.

Women diagnosed with cutaneous melanoma have a survival advantage compared to men, which has been hypothesized to be due to difference in behavior and/or biology (sex hormones).,It remains controversial whether this advantage is dependent on age or stage of disease.,We sought to compare melanoma‐specific survival between females in pre, peri, and postmenopausal age groups to males in the same age group, adjusting for stage of disease.,This is a retrospective population‐based cohort study using the Surveillance, Epidemiology, and End Results (SEER) database.,Patients diagnosed from 1 January 1992 through 31 January 2011 with primary invasive cutaneous melanoma were included in our cohort.,Melanoma‐specific survival was the main outcome studied.,Of the 106,511 subjects that were included, 45% were female.,Females in all age groups (18-45, 46-54, and ≥55) with localized and regional disease, were less likely to die from melanoma compared to males in the same age group.,Among patients with localized and regional disease, the relative risk of death due to melanoma increased with advancing age at diagnosis; this increase was more pronounced among females than males.,In contrast, we observed no female survival advantage among patients with distant disease and no effect of age on relative risk of death from melanoma.,Females with localized and regional melanoma have a decreased risk of death compared to males within all age groups.,Our data show no differences in survival between men and women with metastatic melanoma, indicating that the influence of sex on survival is limited to early stage disease but not confined to pre or perimenopausal age groups.

Age has been reported as an independent prognostic factor for melanoma-specific survival (MSS).,We tested the hypothesis that age impacts the host anti-tumor immune response, accounting for age-specific survival outcomes in three unique melanoma patient cohorts.,We queried the U.S. population-based Surveillance, Epidemiology, and End Results Program (SEER), the prospective tertiary care hospital-based Interdisciplinary Melanoma Cooperative Group (IMCG) biorepository, and the Cancer Genome Atlas (TCGA) biospecimen database to test the association of patient age at time of melanoma diagnosis with clinicopathologic features and survival outcomes.,Age groups were defined as ≤45 (young), 46-65 (intermediate), and >65 (older).,Each age group in the IMCG and TCGA cohorts was stratified by tumor infiltrating lymphocyte (TIL) measurements and tested for association with MSS.,Differential expression of 594 immunoregulatory genes was assessed in a subset of primary melanomas in the IMCG and TCGA cohorts using an integrative pathway analysis.,We analyzed 304, 476 (SEER), 1241 (IMCG), and 292 (TCGA) patients.,Increasing age at melanoma diagnosis in both the SEER and IMCG cohorts demonstrated a positive correlation with tumor thickness, ulceration, stage, and mortality, however age in the TCGA cohort did not correlate with mortality.,Older age was associated with shorter MSS in all three cohorts.,When the young age group in both the IMCG and TCGA cohorts was stratified by TIL status, there were no differences in MSS.,However, older IMCG patients with brisk TILs and intermediate aged TCGA patients with high lymphocyte scores (3-6) had improved MSS.,Gene expression analysis revealed top pathways (T cell trafficking, communication, and differentiation) and top upstream regulators (CD3, CD28, IFNG, and STAT3) that significantly changed with age in 84 IMCG and 43 TCGA primary melanomas.,Older age at time of melanoma diagnosis is associated with shorter MSS, however age’s association with clinicopathologic features is dependent upon specific characteristics of the study population.,TIL as a read-out of the host immune response may have greater prognostic impact in patients older than age 45.,Recognition of age-related factors negatively impacting host immune responses may provide new insights into therapeutic strategies for the elderly.,The online version of this article (doi:10.1186/s12967-016-1026-2) contains supplementary material, which is available to authorized users.

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663).,At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised.,In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire.,Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor.,Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules.,Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver.,Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma.,Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes.,Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function.,Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol.,By CDR3-T-cell receptor β (TCRβ) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization.,A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood.,In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization.,These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood.,This is the first report of the modulation of the TCRβ repertoire from a melanoma patient immunized with the CSF-470 vaccine.,After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

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

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

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.

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

Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma.,However, many patients do not display therapeutic response or eventually relapse.,Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies.,Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown.,Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma.,Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers.,Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression.,TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens.,This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.,Most melanoma patients do not respond to anti-PD1 therapy.,Here, the authors show that TNFα blockade synergizes with anti-PD-1 by preventing anti-PD-1-induced CD8+ T cell death and TIM-3 expression on such cells.

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.

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.

BRAF mutations are frequent in cutaneous melanomas and BRAF inhibitors(BRAFi) have shown remarkable clinical efficacy in BRAF mutant melanoma patients.,However, acquired drug resistance can occur rapidly and tumor(s) often progress thereafter.,Various mechanisms of BRAFi resistance have recently been described; however, the mechanism of resistance remains controversial.,In this study we developed BRAFi resistant melanoma cell lines and found that metastasis related EMT properties of BRAFi resistant cells were enhanced significantly.,Upregulation of EGFR was observed in BRAFi resistant cell lines and patient tumors due to demethylation of EGFR regulatory DNA elements.,EGFR induced PI3K/AKT pathway activation in BRAFi resistant cells through epigenetic regulation.,Treatment of EGFR inhibitor was effective in BRAFi resistant melanoma cell lines.,The study demonstrates that EGFR epigenetic activation has important implications in BRAFi resistance in melanoma.

The weight that gene copy number plays in transcription remains controversial; although in specific cases gene expression correlates with copy number, the relationship cannot be inferred at the global level.,We hypothesized that genes steadily expressed by 15 melanoma cell lines (CMs) and their parental tissues (TMs) should be critical for oncogenesis and their expression most frequently influenced by their respective copy number.,Functional interpretation of 3,030 transcripts concordantly expressed (Pearson's correlation coefficient p-value < 0.05) by CMs and TMs confirmed an enrichment of functions crucial to oncogenesis.,Among them, 968 were expressed according to the transcriptional efficiency predicted by copy number analysis (Pearson's correlation coefficient p-value < 0.05).,We named these genes, "genomic delegates" as they represent at the transcriptional level the genetic footprint of individual cancers.,We then tested whether the genes could categorize 112 melanoma metastases.,Two divergent phenotypes were observed: one with prevalent expression of cancer testis antigens, enhanced cyclin activity, WNT signaling, and a Th17 immune phenotype (Class A).,This phenotype expressed, therefore, transcripts previously associated to more aggressive cancer.,The second class (B) prevalently expressed genes associated with melanoma signaling including MITF, melanoma differentiation antigens, and displayed a Th1 immune phenotype associated with better prognosis and likelihood to respond to immunotherapy.,An intermediate third class (C) was further identified.,The three phenotypes were confirmed by unsupervised principal component analysis.,This study suggests that clinically relevant phenotypes of melanoma can be retraced to stable oncogenic properties of cancer cells linked to their genetic back bone, and offers a roadmap for uncovering novel targets for tailored anti-cancer therapy.

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.

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

The 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.

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

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

Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these patients develop liver metastases.,Exosomes are small RNA containing nano-vesicles released by most cells, including malignant melanoma cells.,This clinical translational study included patients undergoing isolated hepatic perfusion (IHP) for metastatic uveal melanoma, from whom exosomes were isolated directly from liver perfusates.,The objective was to determine whether exosomes are present in the liver circulation, and to ascertain whether these may originate from melanoma cells.,Exosomes were isolated from the liver perfusate of twelve patients with liver metastases from uveal melanoma undergoing IHP.,Exosomes were visualised by electron microscopy, and characterised by flow cytometry, Western blot and real-time PCR.,Furthermore, the concentration of peripheral blood exosomes were measured and compared to healthy controls.,The liver perfusate contained Melan-A positive and RNA containing exosomes, with similar miRNA profiles among patients, but dissimilar miRNA compared to exosomes isolated from tumor cell cultures.,Patients with metastatic uveal melanoma had a higher concentration of exosomes in their peripheral venous blood compared to healthy controls.,Melanoma exosomes are released into the liver circulation in metastatic uveal melanoma, and is associated with higher concentrations of exosomes in the systemic circulation.,The exosomes isolated directly from liver circulation contain miRNA clusters that are different from exosomes from other cellular sources.,The online version of this article (doi:10.1186/1471-2407-14-962) contains supplementary material, which is available to authorized users.

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

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

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.

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 skin field cancerization is a cutaneous area with subclinical changes resultant from chronic sun exposure, with a higher predisposition to development of pre-neoplastic and neoplastic lesions.,So far, there are no well-defined objective parameters that can indicate their degree of activity.,To describe and compare morphometric aspects and expression of factors related to apoptosis and cell proliferation in actinic keratosis (AK), in both photoexposed and photoprotected epidermis.,A cross-sectional study of patients with actinic keratosis in the forearms, biopsied at two points: the actinic keratosis and the axillary region.,The biopsies of the actinic keratosis, perilesional area, and axilla were evaluated through keratinocyte intraepithelial neoplasia (KIN), and immunohistochemistry of p53, survivin, and Ki67.,Nuclear morphometry of basal layer cells was performed through digital image analysis: entropy, area, perimeter, Ra, fractal dimension, circularity, color intensity, and largest diameter.,There were 13 patients included and 38 actinic keratosis biopsied.,In morphometry, 1039 nuclei were analyzed, of which 228 represented axillary skin, 396 demonstrated actinic keratosis, and 415 represented the perilesional area to the actinic keratosis.,There was a significant difference (p < 0.05) in all variables tested for the topographies evaluated.,A significant correlation was identified between nucellar morphometric elements, KIN, proliferation markers, and apoptosis.,Joint patterns of p53, Ki67, and KIN discriminated the topographies sampled.,This was a cross-sectional study with a small number of patients.,There are patterns of proliferation, resistance to apoptosis, and different cellular morphometrics between photoprotected skin and photoexposed skin.,The joint expression of p53, Ki67, and KIN can characterize skin field cancerization activity.

Targeted immunotherapy using dendritic cell vaccine has been employed for the treatment of solid tumors.,Topical 5-aminolevulinic acid-mediated photodynamic therapy, an established approach for topical cancers, can induce an effective antitumor immune response.,We have previously shown that 5-aminolevulinic acid-mediated photodynamic therapy-induced tumor lysates could considerably enhance antigen-presenting capacity of ex vivo-generated dendritic cells.,The current study further demonstrates that 5-aminolevulinic acid-mediated photodynamic therapy dendritic cell vaccine can induce immune responses against cancers.,Dendritic cells pulsed by photodynamic therapy-treated skin squamous cell carcinoma cells inhibited squamous cell carcinoma to a greater extent than tumor lysates treated by photodynamic therapy alone or dendritic cells pulsed by freeze-thawed treated tumor cells.,Immunohistochemistry showed that photodynamic therapy dendritic cell vaccine could increase the activity of CD4+ and CD8+ T cells in the tumor implantation sites.,Flow cytometry assays showed that CD4+ and CD8+ T cells in the spleens of photodynamic therapy dendritic cell vaccine immunized mice increased significantly.,Furthermore, we observed increased amounts of interleukin 12 and Interferon gamma (IFN-γ) and decreased amounts of interleukin 10 in the splenocytes and peripheral blood of photodynamic therapy dendritic cell vaccine immunized mice by enzyme linked immunosorbent assay (ELISA).,Taken together, our findings suggest that photodynamic therapy dendritic cell vaccination is an effective prophylactic therapy for squamous cell carcinoma.

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

Melanoma is an aggressive tumor with increasing incidence.,To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood.,In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis.,Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis.,This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.,Here, we analyzed Xmrk-induced gene expression using a microarray approach.,Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors.,The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA.,Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.,Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6).,Interestingly, most genes were blocked in presence of a SRC kinase inhibitor.,Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes.,Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration.,Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines.,The identified molecules constitute new possible molecular players in melanoma development.,Specifically, a role of FOSL1 in melanomagenic processes is demonstrated.,These data are the basis for future detailed analyses of the investigated target genes.

PD-L1 expression in tumour tissues is widely used to select patients to receive anti-PD-1/PD-L1 antibodies, but data are lacking on the correlation of plasma PD-L1 levels with the effect of treatments.,To investigate the association between PD-L1 mRNA in plasma-derived exosomes and response to nivolumab and pembrolizumab in patients with melanoma (n=18) and NSCLC (n=8), blood was obtained at time point 0 and after 2 months.,Exosomal PD-L1 mRNA was measured by digital droplet PCR.,The mean±s.e.m.,PD-L1 levels in patients with complete and partial responses were 830.4±231.3 and 242.5±82.5 copies per ml at time 0 vs 2 months, respectively (P=0.016).,In patients with stable disease the mean±s.e.m. values were 298.8±97.2 vs 247.5±29.8 copies per ml (P=0.586), while in progressive disease, PD-L1 mRNA levels were 204.0±68.8 vs 416.0±87.8 copies per ml at time 0 vs 2 months, respectively (P=0.001).,This study demonstrates that exosomal PD-L1 is significantly associated with response to treatment.

Ultraviolet light (UV) is an important risk factor for cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma of the skin.,These cancers most commonly affect persons with fair skin and blue eyes who sunburn rather than suntan.,However, each of these cancers appears to be associated with a different pattern of UV exposure and to be mediated by different intracellular molecular pathways.,Some melanocortin 1 receptor (MC1R) gene variants play a direct role in the pathogenesis of cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma apart from their role in determining a cancer-prone pigmentory phenotype (fair skin, red hair, blue eyes) through their interactions with other genes regulating immuno-inflammatory responses, DNA repair or apoptosis.,In this short review we focus on the aetiological role of UV in cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma and cutaneous melanoma of the skin, and on some associated biopathological events.

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

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.

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.

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.

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations.,In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin.,Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line.,High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively.,Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM.,Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation.,According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success.,Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes.,Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a−/−Pten−/− YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1).,We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy.,Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma.,The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

Checkpoint inhibitors (CPIs) are thought to be effective against cutaneous melanoma in part because of the large burden of somatic mutations (neoantigens) generated from exposure to ultraviolet radiation.,However, rare melanoma subtypes arising from acral skin, mucosal surfaces, and the uveal tract are largely sun-shielded.,Genomic studies show these sun-shielded melanomas have a paucity of neoantigens and unique biology; they are thought to be largely resistant to immunotherapy.,It has not been definitively shown that CPI improves survival in metastatic sun-shielded melanoma.,We reviewed a single institutional experience using antibodies against CTLA-4, PD-1 and/or PD-L1 to treat patients with metastatic melanoma.,Primary tumor histology was categorized as cutaneous, unknown, acral, mucosal, or uveal.,We studied demographic data, treatment characteristics, and overall survival (OS) after CPI.,We treated 428 patients with metastatic melanoma from 2007 to 2019.,Primary tumors were cutaneous in 283 (66%), unknown in 55 (13%), acral in 22 (5%), mucosal in 38 (9%), and uveal in 30 (7%).,Patients with metastatic disease from cutaneous primary tumors had median OS after CPI of 45 months compared with 17 months for acral (p=0.047), 18 months for mucosal (p=0.003), and 12 months for uveal (p<0.001).,For all patients with sun-shielded melanoma (n=90), first treatment with anti-PD-1 or anti-PD-L1 was followed by a median OS of 9 months compared with 18 months after anti-CTLA-4 (p=0.010) and 20 months after combination therapy (p=0.003).,There were 21 patients who achieved actual 3-year survival; 20 received both anti-CTLA-4 and anti-PD-1, either sequentially or in combination.,Over 80% of 3-year survivors with progressive disease were treated with local therapy after CPI.,Long survival in patients with metastatic melanoma from acral, mucosal, and uveal primary tumors was associated with receipt of both anti-CTLA-4 and anti-PD-1 antibodies.,Complete responses were rare, and local therapy was frequently employed to control disease progression.,While sun-shielded melanomas exhibit worse outcomes after CPI than cutaneous melanomas, with an aggressive multidisciplinary approach, 5-year survival is still possible for 25%-32% of these patients.

Uveal melanoma (UM) represents the most common intraocular malignancy in adults and accounts for about 5% of all melanomas.,Primary disease can be effectively controlled by several local therapy options, but UM has a high potential for metastatic spread, especially to the liver.,Despite its clinical and genetic heterogeneity, therapy of metastatic UM has largely been adopted from cutaneous melanoma (CM) with discouraging results until now.,The introduction of antibodies targeting CTLA-4 and PD-1 for immune checkpoint blockade (ICB) has revolutionized the field of cancer therapy and has achieved pioneering results in metastatic CM.,Thus, expectations were high that patients with metastatic UM would also benefit from these new therapy options.,This review provides a comprehensive and up-to-date overview on the role of ICB in UM.,We give a summary of UM biology, its clinical features, and how it differs from CM.,The results of several studies that have been investigating ICB in metastatic UM are presented.,We discuss possible reasons for the lack of efficacy of ICB in UM compared to CM, highlight the pitfalls of ICB in this cancer entity, and explain why other immune-modulating therapies could still be an option for future UM therapies.

Fibroblasts become cancer-associated fibroblasts (CAFs) in the tumor microenvironment after activation by transforming growth factor-β (TGF-β) and are critically involved in cancer progression.,However, it is unknown whether the TGF superfamily member Nodal, which is expressed in various tumors but not expressed in normal adult tissue, influences the fibroblast to CAF conversion.,Here, we report that Nodal has a positive correlation with α-smooth muscle actin (α-SMA) in clinical melanoma and colorectal cancer (CRC) tissues.,We show the Nodal converts normal fibroblasts to CAFs, together with Snail and TGF-β signaling pathway activation in fibroblasts.,Activated CAFs promote cancer growth in vitro and tumor-bearing mouse models in vivo.,These results demonstrate that intercellular crosstalk between cancer cells and fibroblasts is mediated by Nodal, which controls tumor growth, providing potential targets for the prevention and treatment of tumors.

The incidence of malignant melanoma is rapidly increasing and current medicine is offering only limited options for treatment of the advanced disease.,For B-Raf mutated melanomas, treatment with mutation-specific drug inhibitors may be used.,Unfortunately, tumors frequently acquire resistance to the treatment.,Tumor microenvironment, namely cancer-associated fibroblasts, largely influence this acquired resistance.,In the present study, fibroblasts were isolated from a patient suffering from acrolentiginous melanoma (Breslow, 4.0 mm; Clark, IV; B-Raf V600E mutated).,The present study focused on the expression of structural and functional markers of fibroblast activation in melanoma-associated fibroblasts (MAFs; isolated prior to therapy initiation) as well as in autologous control fibroblasts (ACFs) of the same patient isolated during B-Raf inhibitor therapy, yet before clinical progression of the disease.,Analysis of gene transcription was also performed, as well as DNA methylation status analysis at the genomic scale of both isolates.,MAFs were positive for smooth muscle actin (SMA), which is a marker of myofibroblasts and the hallmark of cancer stoma.,Surprisingly, ACF isolated from the distant uninvolved skin of the same patient also exhibited strong SMA expression.,A similar phenotype was also observed in control dermal fibroblasts (CDFs; from different donors) exclusively following stimulation by transforming growth factor (TGF)-β1.,Immunohistochemistry confirmed that melanoma cells potently produce TGF-β1.,Significant differences were also identified in gene transcription and in DNA methylation status at the genomic scale.,Upregulation of SMA was observed in ACF cells at the protein and transcriptional levels.,The present results support recent experimental findings that tumor microenvironment is driving resistance to B-Raf inhibition in patients with melanoma.,Such an activated microenvironment may be viable for the growth of circulating melanoma cells.

The immune system fights cancer and sometimes temporarily eliminates it or reaches an equilibrium stage of tumor growth.,However, continuous immunological pressure also selects poorly immunogenic tumor variants that eventually escape the immune control system.,Here, we focus on metastatic melanoma, a highly immunogenic tumor, and on anti-melanoma immunotherapies, which recently, especially following the FDA approval of Ipilimumab, gained interest from drug development companies.,We describe new immunomodulatory approaches currently in the development pipeline, focus on the novel CEACAM1 immune checkpoint, and compare its potential to the extensively described targets, CTLA4 and PD1.,This paper combines multi-disciplinary approaches and describes anti-melanoma immunotherapies from molecular, medical, and business angles.

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

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

Molecular signatures specific to particular tumor types are required to design treatments for resistant tumors.,However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures.,We developed similarity core analysis (SCA), a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines.,We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases.,The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion.,About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7) and miRNAs (211-5p, 221-3p, and 10a-5p).,The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.,Cancer cell lines are at the forefront of drug discovery but are often limited in representing the tumor of origin due to the artificial culture conditions.,Rambow et al. develop a computational approach for identifying tumor cell lineage expression cores.,These core genes reveal relevant molecular dependencies linking aggressiveness, patient survival, and drug sensitivity.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs).,In some respects, HFs can be defined as ‘ordered’ skin appendage growths, while BCCs can be regarded as ‘disordered’ skin appendage growths.,The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage.,Human nodular BCCs, along with HFs and non-follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry.,Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1).,Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively.,Specific molecular mechanisms were found to be involved in the process of cell self-renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways.,However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs.,Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation.,The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs.,Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.

Basal cell carcinoma (BCC) is the most common malignancy.,Exposure to sunlight is the most important risk factor.,Most, if not all, cases of BCC demonstrate overactive Hedgehog signaling.,A variety of treatment modalities exist and are selected based on recurrence risk, importance of tissue preservation, patient preference, and extent of disease.,The pathogenesis, epidemiology, clinical features, diagnosis, histopathology, and management of BCC will be discussed in this review.

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.

Cell-type specific signalling determines cell fate under physiological conditions, but it is increasingly apparent that also in cancer development the impact of any given oncogenic pathway on the individual cancer pathology is dependent on cell-lineage specific molecular traits.,For instance in colon and liver cancer canonical Wnt signalling produces increased cytoplasmic and nuclear localised beta-catenin, which correlates with invasion and poor prognosis.,In contrast, in melanoma increased cytoplasmic and nuclear beta-catenin is currently emerging as a marker for good prognosis and thus appears to have a different function compared to other cancer types; however this function is unknown.,We discovered that in contrast to its function in other cancers, in melanoma, beta-catenin blocks invasion.,We demonstrate that this opposing role of nuclear beta-catenin in melanoma is mediated through MITF, a melanoma-specific protein that defines the lineage background of this cancer type.,Downstream of beta-catenin MITF not only suppresses the Rho-GTPase regulated cell-morphology of invading melanoma cells, but also interferes with beta-catenin induced expression of the essential collagenase MT1-MMP, thus affecting all aspects of an invasive phenotype.,Importantly, overexpression of MITF in invasive colon cancer cells modifies beta-catenin directed signalling and induces a ‘melanoma-phenotype’.,In summary, the cell type specific presence of MITF in melanoma affects beta-catenin’s pro-invasive properties otherwise active in colon or liver cancer.,Thus our study reveals the general importance of considering cell-type specific signalling for the accurate interpretation of tumour markers and ultimately for the design of rational therapies.

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

Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility.,How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood.,Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development.,Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion.,Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature.,We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces.,Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis.,CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients.,Its expression is also enriched in the invasive fronts of lesions.,Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth.,We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.,•TGF-β-SMAD promotes amoeboid migration in melanoma•Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility•Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients•TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,TGF-β-SMAD promotes amoeboid migration in melanoma,Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility,Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients,TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,Cantelli et al. find that, in melanoma, TGF-β-SMAD-CITED1 controls amoeboid behavior through activation of a transcriptional program.,As a result, melanoma cells detach from keratinocytes, increase their invasive potential, and efficiently colonize the lung.,This is a new function of TGF-β, independent of epithelial-to-mesenchymal transition.

Genes in the S100 family are abnormally expressed in a variety of tumor cells and are associated with clinical pathology, but their prognostic value in melanoma patients has not yet been fully elucidated.,In this study, we extracted and profiled S100 family mRNA expression data and corresponding clinical data from the Gene Expression Omnibus database to analyze how expression of these genes correlates with clinical pathology.,Compared with normal skin, S100A1, S100A13, and S100B were expressed at significantly higher levels in melanoma samples.,S100A2, S100A7, S100A8, S100A9, S100A10, S100A11, and S100P were all highly expressed in primary melanoma samples but were expressed at low levels in metastatic melanoma, and all of these genes were strongly correlated with each other (P<0.001).,We found the expression of these S100 family genes to be significantly correlated with both lymphatic and distant melanoma metastasis, as well as with American Joint Committee on Cancer grade but not with Clark’s grade, age, or sex.,This suggests that expression of these genes may be related to the degree of tumor invasion.,Although further validation through basic and clinical trials is needed, our results suggest that the S100 family genes have the potential to play an important role in the diagnosis of melanoma.,S100 expression may be related to tumor invasion and may facilitate the early diagnosis of melanoma, allowing for a more accurate prognosis.,Targeted S100 therapies are also potentially viable strategies in the context of melanoma.

While melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising.,Interferon-γ (IFN-γ) produced by immune cells plays a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic.,In the current study, we found that IFN-γ enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival.,IFN-γ increased phosphorylation of AKT Ser473 and upregulated total and cell surface expression of CD74 in human melanoma cell lines tested.,CD74 was highly expressed in melanoma tissues.,Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-γ levels in melanoma patient samples.,In our analysis of melanoma cell lines, all produced MIF constitutively.,Blockade of CD74-MIF interaction reduced AKT phosphorylation and expression of pro-tumorigenic molecules, including interleukin-6, interleukin-8 and BCL-2.,Inhibition of CD74-MIF interaction significantly suppressed tumor growth in the presence of IFN-γ in our xenograft mouse model.,Thus, we conclude that IFN-γ promotes melanoma cell survival by regulating CD74-MIF signaling, suggesting that targeting the CD74-MIF interaction under IFN-γ-stimulatory conditions would be an effective therapeutic approach for melanoma.

Uveal melanoma is the most common primary intraocular malignancy in adults.,Despite successful control of the primary tumor, metastatic disease will ultimately develop in approximately 50% of patients, with the liver being the most common site for metastases.,The median survival for patients with liver metastases is between 6 and 12 months, and no treatment has in randomized trials ever been shown to prolong survival.,A previous phase II trial using isolated hepatic perfusion (IHP) has suggested a 14-month increase in overall survival compared with a historic control group consisting of the longest surviving patients in Sweden during the same time period (26 versus 12 months).,This is the protocol for a multicenter phase III trial randomizing patients with isolated liver metastases of uveal melanoma to IHP or best alternative care (BAC).,Inclusion criteria include liver metastases (verified by biopsy) and no evidence of extra-hepatic tumor manifestations by positron emission tomography-computed tomography (PET-CT).,The primary endpoint is overall survival at 24 months, with secondary endpoints including response rate, progression-free survival, and quality of life.,The planned sample size is 78 patients throughout five years.,Patients with isolated liver metastases of uveal melanoma origin have a short expected survival and no standard treatment option exists.,This is the first randomized clinical trial to evaluate IHP as a treatment option with overall survival being the primary endpoint.,ClinicalTrials.gov registration number: NCT01785316 (registered 1 February 2013).,EudraCT registration number: 2013-000564-29.

When mutations in two different genes produce the same mutant phenotype, it suggests that the encoded proteins either interact with each other, or act in parallel to fulfill a similar purpose.,Haploinsufficiency of Neurofibromin and over-expression of Endothelin 3 both cause increased numbers of melanocytes to populate the dermis during mouse development, and thus we are interested in how these two signaling pathways might intersect.,Neurofibromin is mutated in the human genetic disease, neurofibromatosis type 1, which is characterized by the development of Schwann cell based tumors and skin hyper-pigmentation.,Neurofibromin is a GTPase activating protein, while the Endothelin 3 ligand activates Endothelin receptor B, a G protein coupled receptor.,In order to study the genetic interactions between endothelin and neurofibromin, we defined the deletion breakpoints of the classical Ednrb piebald lethal allele (Ednrbs-l) and crossed these mice to mice with a loss-of-function mutation in neurofibromin, Dark skin 9 (Dsk9).,We found that Neurofibromin haploinsufficiency requires Endothelin receptor B to darken the tail dermis.,In contrast, Neurofibromin haploinsufficiency increases the area of the coat that is pigmented in Endothelin receptor B null mice.,We also found an oncogenic mutation in the G protein alpha subunit, GNAQ, which couples to Endothelin receptor B, in a uveal melanoma from a patient with neurofibromatosis type 1.,Thus, this data suggests that there is a complex relationship between Neurofibromin and Endothelin receptor B.

Significant tumor regressions have been observed in up to 70% of patients receiving adoptively transferred autologous melanoma-reactive tumor infiltrating lymphocytes (TIL) 1,2, and in pilot trials, 40% of treated patients experienced complete regressions of all measurable lesions for at least five years following treatment 3.,To evaluate the potential association between the ability of TIL to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, a novel screening approach was developed that involved mining whole exome sequence data to identify the mutated proteins that were expressed in patient tumors.,Candidate mutated T cell epitopes that were identified using an MHC binding algorithm 4 were then synthesized and evaluated for recognition by TIL.,Using this approach, mutated antigens expressed on autologous tumor cells were identified as targets of three TIL that were associated with objective tumor regressions following adoptive transfer.,This simplified approach, which avoids the need to generate and laboriously screen cDNA libraries from tumors, may represent a generally applicable method for identifying mutated T cell antigens expressed in melanoma as well as other tumor types.

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.

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.

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

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

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

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

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

Circulating tumors cells (CTCs) can be detected in the blood of metastatic melanoma patients (MMPs) both as isolated circulating tumor cells (iCTCs) and circulating tumor microemboli (CTMs), but their clinical significance remains unknown.,The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell (ISET) method.,We characterized the phenotype of CTCs using anti‐PS100, anti‐SOX10, anti‐CD10, and anti‐TRF2 antibodies.,128 MMPs and 37 control healthy individuals with benign nevi were included in this study.,Results were compared to the follow‐up of patients.,109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs.,PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs.,SOX10, CD10, and TRF2 were mainly expressed in CTMs.,None of the control subjects demonstrated circulating malignant tumor cells.,Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies.,In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs.

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

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

Wnt5a signaling has been implicated in the progression of cancer by regulating multiple cellular processes, largely migration and invasion, epithelial-mesenchymal transition (EMT), and metastasis.,Since Wnt5a signaling has also been involved in inflammatory processes in infectious and inflammatory diseases, we addressed the role of Wnt5a in regulating NF-κB, a pivotal mediator of inflammatory responses, in the context of cancer.,The treatment of melanoma cells with Wnt5a induced phosphorylation of the NF-κB subunit p65 as well as IKK phosphorylation and IκB degradation.,By using cDNA overexpression, RNA interference, and dominant negative mutants we determined that ROR1, Dvl2, and Akt (from the Wnt5a pathway) and TRAF2 and RIP (from the NF-κB pathway) are required for the Wnt5a/NF-κB crosstalk.,Wnt5a also induced p65 nuclear translocation and increased NF-κB activity as evidenced by reporter assays and a NF-κB-specific upregulation of RelB, Bcl-2, and Cyclin D1.,Further, stimulation of melanoma cells with Wnt5a increased the secretion of cytokines and chemokines, including IL-6, IL-8, IL-11, and IL-6 soluble receptor, MCP-1, and TNF soluble receptor I.,The inhibition of endogenous Wnt5a demonstrated that an autocrine Wnt5a loop is a major regulator of the NF-κB pathway in melanoma.,Taken together, these results indicate that Wnt5a activates the NF-κB pathway and has an immunomodulatory effect on melanoma through the secretion of cytokines and chemokines.

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.

B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression.,B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity.,B7-H3 expression and prognostic utility was shown by RT-qPCR and IHC analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double blind study.,B7-H3 mRNA expression was shown to be significantly increased with stage of melanoma(P<0.0001) and significantly associated with melanoma-specific survival(MSS) in both stage III(P<0.0001) and stage IV(P<0.012) melanoma patients.,B7-H3 expression was related to migration and invasion; overexpression B7-H3 increased migration and invasion while knockdown of B7-H3 reduced cell migration and invasion.,MiR-29c expression was shown to inversely regulate B7-H3 expression.,Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to p-STAT3 activity level in melanoma tissues and cell lines.,These studies demonstrate that B7-H3 is a significant factor in melanoma progression, and events of metastasis.

Despite genetics being accepted as the primary cause of familial aggregation for most diseases, it is still unclear whether afflicted families are likely to share a single highly penetrant rare variant, many minimally penetrant common variants, or a combination of the two types of variants.,We therefore use recent estimates of SNP heritability and the liability threshold model to estimate the proportion of afflicted families likely to carry a rare, causal variant.,We then show that Polygenic Risk Scores (PRS) may be useful for identifying families likely to carry such a rare variant and therefore for prioritizing families to include in sequencing studies with that aim.,Specifically, we introduce a new statistic that estimates the proportion of individuals carrying causal rare variants based on the family structure, disease pattern, and PRS of genotyped individuals.,Finally, we consider data from the MelaNostrum consortium and show that, despite an estimated PRS heritability of only 0.05 for melanoma, families carrying putative causal variants had a statistically significantly lower PRS, supporting the idea that PRS prioritization may be a useful future tool.,However, it will be important to evaluate whether the presence of rare mendelian variants are generally associated with the proposed test statistic or lower PRS in future and larger studies.

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

This study aimed to evaluate the efficacy of carbon‐ion radiotherapy in combination with chemotherapy using dacarbazine, nimustine, and vincristine (DAV therapy) in mucosal melanoma.,Twenty‐one patients with clinically localized mucosal melanoma of the head and neck were enrolled.,The primary endpoint was 3‐year overall survival (OS).,Secondary endpoints included local control, progression‐free survival (PFS), and adverse event occurrence.,Carbon‐ion radiotherapy with a dose of 57.6‐64.0 Gy (relative biological effectiveness) in 16 fractions was delivered concurrently with DAV therapy, and 2 cycles of adjuvant DAV therapy were administered every 6 weeks.,The median follow‐up periods were 15.5 months for all patients, and 31.2 months for 12 surviving patients.,All patients had locally advanced T4a or T4b disease in the rhino‐sinus area.,In 16 patients (76.2%), 3 cycles of planned DAV therapy were completed.,The 3‐year OS and PFS rates were 49.2% and 37.0% respectively.,The 3‐year local control rate was 92.3%.,Eleven patients (52%) developed distant metastasis, which was the most frequent pattern of the first failure.,Commonly presenting acute grade 2‐3 toxicities associated with radiotherapy and chemotherapy were mucositis (11 patients [53%]) and leukopenia (9 patients [43%]), which improved with conservative therapy.,None of the patients developed grade 3 or greater late toxicities.,Carbon‐ion radiotherapy in combination with DAV therapy led to excellent local control for advanced mucosal melanoma within acceptable toxicities.,The efficacy of additional DAV therapy in improving survival was weaker than expected as distant metastases still occurred frequently.,Trial registration no.,UMIN000007939.,It is the first prospective trial to evaluate the clinical outcomes of carbon‐ion radiotherapy combined with chemotherapy.

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

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

Melanoma 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.

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

Uveal melanoma is the most common primary intraocular malignancy in adults.,Despite successful control of the primary tumor, metastatic disease will ultimately develop in approximately 50% of patients, with the liver being the most common site for metastases.,The median survival for patients with liver metastases is between 6 and 12 months, and no treatment has in randomized trials ever been shown to prolong survival.,A previous phase II trial using isolated hepatic perfusion (IHP) has suggested a 14-month increase in overall survival compared with a historic control group consisting of the longest surviving patients in Sweden during the same time period (26 versus 12 months).,This is the protocol for a multicenter phase III trial randomizing patients with isolated liver metastases of uveal melanoma to IHP or best alternative care (BAC).,Inclusion criteria include liver metastases (verified by biopsy) and no evidence of extra-hepatic tumor manifestations by positron emission tomography-computed tomography (PET-CT).,The primary endpoint is overall survival at 24 months, with secondary endpoints including response rate, progression-free survival, and quality of life.,The planned sample size is 78 patients throughout five years.,Patients with isolated liver metastases of uveal melanoma origin have a short expected survival and no standard treatment option exists.,This is the first randomized clinical trial to evaluate IHP as a treatment option with overall survival being the primary endpoint.,ClinicalTrials.gov registration number: NCT01785316 (registered 1 February 2013).,EudraCT registration number: 2013-000564-29.

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

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.

Human skin cutaneous melanoma is the most common and dangerous skin tumour, but its pathogenesis is still unclear.,Although some progress has been made in genetic research, no molecular indicators related to the treatment and prognosis of melanoma have been found.,In various diseases, dysregulation of lncRNA is common, but its role has not been fully elucidated.,In recent years, the birth of the “competitive endogenous RNA” theory has promoted our understanding of lncRNAs.,To identify the key lncRNAs in melanoma, we reconstructed a global triple network based on the “competitive endogenous RNA” theory.,Gene Ontology and KEGG pathway analysis were performed using DAVID (Database for Annotation, Visualization, and Integration Discovery).,Our findings were validated through qRT-PCR assays.,Moreover, to determine whether the identified hub gene signature is capable of predicting the survival of cutaneous melanoma patients, a multivariate Cox regression model was performed.,According to the “competitive endogenous RNA” theory, 898 differentially expressed mRNAs, 53 differentially expressed lncRNAs and 16 differentially expressed miRNAs were selected to reconstruct the competitive endogenous RNA network.,MALAT1, LINC00943, and LINC00261 were selected as hub genes and are responsible for the tumorigenesis and prognosis of cutaneous melanoma.,MALAT1, LINC00943, and LINC00261 may be closely related to tumorigenesis in cutaneous melanoma.,In addition, MALAT1 and LINC00943 may be independent risk factors for the prognosis of patients with this condition and might become predictive molecules for the long-term treatment of melanoma and potential therapeutic targets.

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

Randomized phase III trial (JCOG1309) has been started to confirm the superiority of adjuvant therapy with locoregional interferon beta over surgery alone in overall survival for patients with stage II/III cutaneous melanoma.,The Dermatologic Oncology Group of Japan Clinical Oncology Group has started a randomized phase III trial to confirm the superiority of adjuvant therapy with locoregional interferon beta in overall survival over surgery alone for patients with pathological stage II/III cutaneous melanoma (JCOG1309).,Patients in the interferon beta arm receive intra- or subcutaneous injections of interferon beta directly into the surgical site at a flat dose of 3 million units once per day.,Treatment is repeated for 10 consecutive days every 8 weeks for a total of 3 courses during the induction phase, then 1-day injection every 4 weeks for 2.5 years.,A total of 240 patients will be accrued from 17 Japanese institutions within 6.5 years.,Primary endpoint is overall survival.,Secondary endpoints are relapse-free survival, distant metastasis-free survival, pattern of recurrence, and adverse events.,This trial has been registered at the UMIN Clinical Trials Registry as UMIN000017494 [http://www.umin.ac.jp/ctr/index.htm].

Ultraviolet radiation is a risk factor for BRAF V600 mutations frequently found in melanomas that cause constitutive BRAF activation.,Primary sites of melanoma and the frequency of BRAF mutations might differ between races.,Melanoma is rare in Japan (1500-2000 cases/year compared with 132 000/year worldwide) and the frequency and distribution of BRAF V600 mutations are unknown.,We aimed to investigate the frequency of BRAF V600 mutations in a cohort of Japanese patients with melanoma and determine the relationship between mutations and clinical/pathologic features.,DNA was extracted from 80 formalin-fixed, paraffin-embedded tumours from individuals diagnosed with melanoma.,BRAF V600 mutations were detected using the Cobas 4800 System with z480 Analyzer and Cobas 4800 BRAF V600 Mutation Test reagents.,BRAF V600 mutations were detected in 41.8% of tested tumours, with an invalid rate of 1.3%.,The mutation rate was more than 60% in patients aged less than 60 years and more than 36% in patients with stage III/IV disease.,No sex difference in the mutation rate was observed.,BRAF V600 mutations were detected in 18.8% of acral lentiginous melanomas (ALMs), 64.7% of superficial spreading melanomas, 50.0% of lentigo maligna melanomas and 20.0% of nodular melanomas.,Although the mutation rate was low in ALMs, 36.4% were mutation positive at stage III/IV compared with 9.5% at stage I/II.,This study confirmed associations among BRAF V600 mutations, pathological features and subtypes of melanoma.,BRAF V600 mutations were more frequent in late-stage ALMs than in early-stage ALMs.,Superficial spreading melanomas had similar mutation rates at all stages.,These insights suggest improved treatment predictions for stage III/IV melanoma patients.

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

Genes, proteins, or cells influence each other and consequently create patterns, which can be increasingly better observed by experimental biology and medicine.,Thereby, descriptive methods of statistics and bioinformatics sharpen and structure our perception.,However, additionally considering the interconnectivity between biological elements promises a deeper and more coherent understanding of melanoma.,For instance, integrative network-based tools and well-grounded inductive in silico research reveal disease mechanisms, stratify patients, and support treatment individualization.,This review gives an overview of different modeling techniques beyond statistics, shows how different strategies align with the respective medical biology, and identifies possible areas of new computational melanoma research.

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

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading.,Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event.,This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively.,Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors.,Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma.,Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.,The key regulators that allow transition from proliferative to invasive phenotype in melanoma cells have not been identified yet.,The authors perform chromatin and transcriptome profiling followed by comprehensive bioinformatics analysis identifying new candidate regulators for two distinct cell states of melanoma.

Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes‐associated protein (YAP).,Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance.,Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood.,Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils.,We also confirm the accumulation of amyloid‐like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies.,Mechanistically, beta‐secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity.,We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling.,Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta‐secretase inhibitors as potential therapeutic approach for metastatic melanoma.,The accumulation of PMEL fibrils in the metastatic melanoma microenvironment modulates YAP activity.,Preventing aggregate formation reduces metastatic cell proliferation and chemosensitivity, suggesting beta‐secretase inhibition as treatment for metastatic melanoma.

Heat-shock factor 1 (HSF1) orchestrates the heat-shock response in eukaryotes.,Although this pathway has been evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy.,However, the mechanisms that regulate HSF1 and thus cellular stress response are poorly understood.,Here we show that the ubiquitin ligase FBXW7 α interacts with HSF1 through a conserved motif phosphorylated by GSK3β and ERK1.,FBXW7α ubiquitylates HSF1 and loss of FBXW7α results in impaired degradation of nuclear HSF1 and defective heat-shock response attenuation.,FBXW7α is either mutated or transcriptionally downregulated in melanoma and HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression.,FBXW7α deficiency and subsequent HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells.,These findings identify a post-translational mechanism of regulation of the HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

Clonal selection and transcriptional reprogramming (e.g., epithelial-mesenchymal transition or phenotype switching) are the predominant theories thought to underlie tumor progression.,However, a “division of labor” leading to cooperation among tumor-cell subpopulations could be an additional catalyst of progression.,Using a zebrafish-melanoma xenograft model, we found that in a heterogeneous setting, inherently invasive cells, which possess protease activity and deposit extracellular matrix (ECM), co-invade with subpopulations of poorly invasive cells, a phenomenon we term “cooperative invasion”.,Whereas the poorly invasive cells benefit from heterogeneity, the invasive cells switch from protease-independent to an MT1-MMP-dependent mode of invasion.,We did not observe changes in expression of the melanoma phenotype determinant MITF during cooperative invasion, thus ruling out the necessity for phenotype switching for invasion.,Altogether, our data suggest that cooperation can drive melanoma progression without the need for clonal selection or phenotype switching and can account for the preservation of heterogeneity seen throughout tumor progression.,•Inherently invasive and poorly invasive melanoma subpopulations co-invade•Leader cells are inherently invasive and provide MT1-MMP and deposit ECM•Follower cells affect the mode of invasion and thereby elicit protease dependency•Cooperative invasion bypasses phenotype switching and maintains tumor heterogeneity,Inherently invasive and poorly invasive melanoma subpopulations co-invade,Leader cells are inherently invasive and provide MT1-MMP and deposit ECM,Follower cells affect the mode of invasion and thereby elicit protease dependency,Cooperative invasion bypasses phenotype switching and maintains tumor heterogeneity,Tumor heterogeneity has been viewed as the outcome of adaptive competition or differential transcriptional reprogramming (phenotype switching).,Alternatively, heterogeneity may reflect a division of labor between tumor cell subpopulations with complementary acquired characteristics.,Chapman et al. now show reciprocal interactions between heterogeneous melanoma cell subpopulations that lead to cooperative invasion without competition and without phenotype switching.,Thus, cooperative behavior emerges as a property of heterogeneity relevant for tumor biology and worth targeting therapeutically.

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas and targeting MEK as central kinase of this pathway is currently tested in clinical trials.,However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response.,Apart from dose-limitations, a reason for the low response to MEK targeting drugs is thought to be the up-regulation of counteracting signalling cascades as a direct response to MEK inhibition.,Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of drug being used.,We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352 while efficiently suppressing proliferation stimulates increased invasiveness.,Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion.,Most importantly, and surprisingly MEK inhibition results in a significant increase in MMP-2 and MT1-MMP expression.,All together MEK inhibition in melanoma cells induces a ‘mesenchymal’ phenotype that is characterised by protease driven invasion.,This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are main regulators of this process, the SRC kinase inhibitor saracatinib (AZD0530) completely abolished the MEK inhibitor induced invasion.,Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in 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.

Understanding the molecular and cellular processes underlying melanoma plasticity and heterogeneity is of paramount importance to improve the efficiency of current treatment and to overcome resistance to chemotherapy drugs.,The notion of plasticity and heterogeneity implies the existence of melanoma cell populations with different phenotypic and tumorigenic properties.,Using melanoma cell lines and melanoma cells freshly isolated from patient biopsies, we investigated the relationship between ABCB5+, CD271+ and low-MITF, expressing populations that were reported to display melanoma initiating cell properties.,Here, we showed that ABCB5+ and CD271+ populations poorly overlap.,However, we found that the CD271+ population is enriched in low-MITF cells and expresses a higher level of stemness genes, such as OCT4, NANOG and NES.,These features could explain the increased tumorigenicity of the CD271+ cells.,The rapid conversion of CD271+ to CD271− cells in vitro demonstrates the plasticity ability of melanoma cells.,Finally, we observed that the transient slow-growing population contains only CD271+ cells that are highly tumorigenic.,However, the fast growing/CD271+ population exhibits a poor tumorigenic ability.,Taking together, our data show that CD271 is an imperfect marker for melanoma initiating cells, but may be useful to identify melanoma cells with an increased stemness and tumorigenic potential.

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.

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

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.

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.

Melanoma is the leading cause of skin-cancer related deaths in North America.,Metastatic melanoma is difficult to treat and chemotherapies have limited success.,Furthermore, chemotherapies lead to toxic side effects due to nonselective targeting of normal cells.,Curcumin is a natural product of Curcuma longa (turmeric) and has been shown to possess anti-cancer activity.,However, due to its poor bioavailability and stability, natural curcumin is not an effective cancer treatment.,We tested synthetic analogs of curcumin that are more stable.,One of these derivatives, Compound A, has shown significant anti-cancer efficacy in colon, leukemia, and triple-negative inflammatory breast cancer cells.,However, the effects of Compound A against melanoma cells have not been studied before.,In this study, for the first time, we demonstrated the efficacy of Compound A for the selective induction of apoptosis in melanoma cells and its interaction with tamoxifen, taxol, and cisplatin.,We found that Compound A induced apoptosis selectively in human melanoma cells by increasing oxidative stress.,The anti-cancer activity of Compound A was enhanced when combined with tamoxifen and the combination treatment did not result in significant toxicity to noncancerous cells.,Additionally, Compound A did not interact negatively with the anti-cancer activity of taxol and cisplatin.,These results indicate that Compound A could be developed as a selective and effective melanoma treatment either alone or in combination with other non-toxic agents like tamoxifen.

Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study.,In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed.,Thirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously.,Tumor BRAFV600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR).,Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples.,The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation.,One patient had a RECIST partial response (PR) lasting 175 days.,Three patients experienced stable disease (SD) with a mean duration of 37 weeks.,Routine BRAFV600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors.,No correlation was seen between BRAFV600E mutational status and clinical activity.,No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples.,Sorafenib monotherapy has limited activity in advanced melanoma patients.,BRAFV600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib.,Clinical Trials.gov NCT00119249

The melanocortin-1 receptor (MC1R), a G protein-coupled receptor, plays a crucial role in human and mouse pigmentation1-8.,Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signaling and melanin production and enhances DNA repair after UV irradiation (UVR)10-16.,Individuals carrying MC1R variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-variants), are associated with higher risk of melanoma5,17,18,19,20.,However, how MC1R activity might be modulated by UV irradiation, why redheads are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit remain unresolved questions.,Here we demonstrate a potential MC1R-targeted intervention strategy to rescue loss-of-function MC1R in MC1R RHC-variants for therapeutic benefit based on activating MC1R protein palmitoylation.,Specifically, MC1R palmitoylation, primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling that triggers increased pigmentation, UVB-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo.,Using C57BL/6J-MC1Re/eJ mice expressing MC1R RHC-variants we show that pharmacological activation of palmitoylation rescues the defects of MC1R RHC-variants and prevents melanomagenesis.,The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

The major genetic determinants of cutaneous melanoma risk in the general population are disruptive variants (R alleles) in the melanocortin 1 receptor (MC1R) gene.,These alleles are also linked to red hair, freckling, and sun sensitivity, all of which are known melanoma phenotypic risk factors.,Here we report that in melanomas and for somatic C>T mutations, a signature linked to sun exposure, the expected single-nucleotide variant count associated with the presence of an R allele is estimated to be 42% (95% CI, 15-76%) higher than that among persons without an R allele.,This figure is comparable to the expected mutational burden associated with an additional 21 years of age.,We also find significant and similar enrichment of non-C>T mutation classes supporting a role for additional mutagenic processes in melanoma development in individuals carrying R alleles.,Deleterious germline variants in the MC1R gene are associated with red hair and freckles, and with an increased risk of developing melanoma.,Here, the authors investigate melanoma samples from patients with and without these variants and find that their presence is predictive of a higher overall mutation prevalence.

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.

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.

While the role of genetic risk factors in the etiology of uveal melanoma (UM) has been strongly suggested, the genetic susceptibility to UM is currently vastly unexplored.,Due to shared epidemiological risk factors between cutaneous melanoma (CM) and UM, in this study we have selected 28 SNPs identified as risk variants in previous genome-wide association studies on CM or CM-related host phenotypes (such as pigmentation and eye color) and tested them for association with UM risk.,By logistic regression analysis of 272 UM cases and 1782 controls using an additive model, we identified five variants significantly associated with UM risk, all passing adjustment for multiple testing.,The three most significantly associated variants rs12913832 (OR = 0.529, 95% CI 0.415-0.673; p = 8.47E-08), rs1129038 (OR = 0.533, 95% CI 0.419-0.678; p = 1.19E-07) and rs916977 (OR = 0.465, 95% CI 0.339-0.637; p = 3.04E-07) are correlated (r2 > 0.5) and map at 15q12 in the region of HERC2/OCA2, which determines eye-color in the human population.,Our data provides first evidence that the genetic factors associated with pigmentation traits are risk loci of UM susceptibility.

MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs.,Recent studies have demonstrated that miRNAs are implicated in the development of cancer.,However, the role of miR-144 in uveal melanoma metastasis remains largely unknown.,MiR-144 was downregulated in both uveal melanoma cells and tissues.,Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion.,After identification of two putative miR-144 binding sites within the 3' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites.,In addition, the expression of c-Met protein was inhibited by miR-144.,Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells.,In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.

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.

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

Bone metastases occur rarely in patients suffering from malignant melanoma, although their onset severely worsens both prognosis and quality of life.,Extracellular vesicles (EVs) including exosomes (Exos) are active players in melanoma progression involved in the formation of the pre-metastatic niche.,Trans-well assays explored the basal migratory and invasive potential of four melanoma cell lines and investigated their different propensity to be attracted toward the bone.,Exosomes were purified from cell supernatants by ultracentrifugation and explored in their ability to influence the bone tropism of melanoma cells.,The molecular machinery activated during this process was investigated by RT-PCR, droplet digital-PCR, flow-cytometry and Western blot, while loss of function studies with dedicated siRNAs defined the single contribute of CXCR4 and CXCR7 molecules.,Melanoma cells revealed a variable propensity to be attracted toward bone fragments.,Gene profiling of both osteotropic and not-osteotropic cells did not show a different expression of those genes notoriously correlated to chemotaxis and bone metastasis.,However, bone conditioned medium significantly increased CXCR4, CXCR7 and PTHrP expression solely to osteotropic cells, while their Exos were able to revert the original poor bone tropism of not-osteotropic cells through CXCR7 up-regulation.,Silencing experiments also demonstrated that membrane expression of CXCR7 is required by melanoma cells to promote their chemotaxis toward SDF-1 gradients.,Our data correlated the osteotropism of melanoma cells to the activation of the SDF-1/CXCR4/CXCR7 axis following the exposition of tumor cells to bone-derived soluble factors.,Also, we demonstrated in vitro that tumor-derived Exos can reprogram the innate osteotropism of melanoma cells by up-regulating membrane CXCR7.,These results may have a potential translation to future identification of druggable targets for the treatment of skeletal metastases from malignant melanoma.,The online version of this article (10.1186/s12967-019-1982-4) contains supplementary material, which is available to authorized users.

Patients with locally advanced basal cell carcinoma (laBCC) or metastatic BCC (mBCC), two difficult‐to‐treat populations, have had limited treatment options.,Sonidegib, a hedgehog pathway inhibitor (HPI), was approved in laBCC based on results from the BOLT trial.,To evaluate long‐term efficacy and safety of sonidegib in laBCC and mBCC in the BOLT 18‐ and 30‐month analyses.,BOLT (NCT01327053, ClinicalTrials.gov), a double‐blind phase 2 study, enrolled patients from July 2011 until January 2013.,Eligible HPI‐treatment-naïve patients with laBCC not amenable to curative surgery/radiotherapy or mBCC were randomized 1 : 2 to sonidegib 200 mg (laBCC, n = 66; mBCC, n = 13) or 800 mg (laBCC, n = 128; mBCC, n = 23).,Tumour response was assessed per central and investigator review.,With 30 months of follow‐up, among patients treated with sonidegib 200 mg (approved dose), objective response rates were 56.1% (central) and 71.2% (investigator) in laBCC and 7.7% (central) and 23.1% (investigator) in mBCC.,Tumour responses were durable as follows: median duration of response was 26.1 months (central) and 15.7 months (investigator) in laBCC and 24.0 months (central) and 18.1 months (investigator) in mBCC.,Five patients with laBCC and three with mBCC in the 200‐mg arm died.,Median overall survival was not reached in either population; 2‐year overall survival rates were 93.2% (laBCC) and 69.3% (mBCC).,In laBCC, efficacy was similar regardless of aggressive or non‐aggressive histology.,Sonidegib 200 mg continued to have a better safety profile than 800 mg, with lower rates of grade 3/4 adverse events (43.0% vs.,64.0%) and adverse events leading to discontinuation (30.4% vs.,40.0%).,Sonidegib continued to demonstrate long‐term efficacy and safety in these populations.,These data support the use of sonidegib 200 mg per local treatment guidelines.

Stromal fibroblasts are an integral part of the tumor stroma and constantly interact with cancer cells to promote their initiation and progression.,However, the role and function of dermal fibroblasts during the early stage of melanoma development remain poorly understood.,We, therefore, designed a novel genetic approach to deactivate stromal fibroblasts at the onset of melanoma formation by targeted ablation of β‐catenin.,To our surprise, melanoma tumors formed from β‐catenin‐deficient group (B16F10 mixed with β‐catenin‐deficient fibroblasts) appeared earlier than tumors formed from control group (B16F10 mixed with normal dermal fibroblasts).,At the end point when tumors were collected, mutant tumors were bigger and heavier than control tumors.,Further analysis showed that there were fewer amounts of stromal fibroblasts and myofibroblasts inside mutant tumor stroma.,Melanoma tumors from control group showed reduced proliferation, down‐regulated expression of cyclin D1 and increased expression of cyclin‐dependent kinase inhibitor p16, suggesting dermal fibroblasts blocked the onset of melanoma tumor formation by inducing a cell cycle arrest in B16F10 melanoma cells.,Furthermore, we discovered that dermal fibroblasts prevented epithelial‐mesenchymal transition in melanoma cells.,Overall, our findings demonstrated that dermal fibroblasts crosstalk with melanoma cells to regulate in vivo tumor development via multiple mechanisms, and the outcomes of their reciprocal interactions depend on activation states of stromal fibroblasts and stages of melanoma 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.

Melanoma is a fatal skin malignant tumor with a poor prognosis.,We found that long noncoding RNA BASP1 ‐AS1 is essential for the development and prognosis of melanoma.,The methylation, RNA sequencing, copy number variation, mutation data, and sample follow‐up information of melanoma from The Cancer Genome Atlas (TCGA) were analyzed using weighted gene co‐expression network analysis and 366 samples common to the three omics were selected for multigroup clustering analysis.,A four‐gene prognostic model (BASP1‐AS1, LOC100506098, ARHGAP27P1, and LINC01532) was constructed in the TCGA cohort and validated using the GSE65904 series.,The expression of BASP1‐AS1 was upregulated in melanoma tissues and various melanoma cell lines.,Functionally, the ectopic expression of BASP1‐AS1 promoted cell proliferation, migration, and invasion in both A375 and SK‐MEL‐2 cells.,Mechanically, BASP1‐AS1 interacted with YBX1 and recruited it to the promoter of NOTCH3, initiating its transcription process.,The activation of the Notch signaling then resulted in the transcription of multiple oncogenes, including c‐MYC, PCNA, and CDK4, which contributed to melanoma progression.,Thus, BASP1‐AS1 could act as a potential biomarker for cutaneous malignant melanoma.,We identified a new type of long noncoding RNA (LncRNA) BASP1 ‐AS1, which can promote melanoma development both in vivo and in vitro.,Detailed studies on the molecular mechanism showed that BASP1‐AS1 promoted the proliferation, invasion, and migration of melanoma cells by regulating YBX1.,In addition, LncRNA BASP1‐AS1 is a poor prognostic indicator and a potential therapeutic target for melanoma.

Melanoma of the ocular region (ocular melanoma) comprises about 5% of all patients with melanoma and covers posterior uveal melanoma, iris melanoma, and conjunctival melanoma.,The risk of metastasis is much higher in patients with ocular melanoma compared to a primary melanoma of the skin.,The subtypes of ocular melanoma have distinct genetic features, which should be taken into consideration when making clinical decisions.,Most relevant for current practice is the absence of BRAF mutations in posterior uveal melanoma, although present in some iris melanomas and conjunctival melanomas.,In this review, we discuss the genetic biomarkers of the subtypes of ocular melanoma and their impacts on the clinical care of these patients.