Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.

Cutaneous melanoma is one of the most malignant types of skin cancer, with an extremely poor prognosis. Immune cells infiltrated in the tumor microenvironment (TME) affects melanoma initiation, progression, prognosis and immunotherapy strategies in melanoma. The potential utility of TME-related genes as a prognostic model for melanoma and as a predictor of immunotherapeutic response merits further exploration. In this study, we determined that an immune-related gene, protein tyrosine phosphatase receptor type C (PTPRC), was positively correlated with the positive prognosis of melanoma patients. Integration of this gene with TNM classification created a predictive model that showed better performance in determining overall survival than others. PTPRC expression was positively correlated with the levels of immune checkpoint molecules, and PTPRC knockdown significantly enhanced the migration, invasion, and proliferation of melanoma cells. Finally, immunohistochemical results from HPA and Real-time quantitative PCR of clinical tissues confirmed that PTPRC expression was higher in melanoma than in normal skin. In conclusion, PTPRC served as a potential predictor of survival and response to immunotherapy in melanoma patients. The risk model combining the PTPRC and TNM classifications holds the potential to be a promising tool for prognostic prediction of cutaneous melanoma. This will help in the effective clinical management of melanoma patients.

Apelin, a ligand of the APJ receptor, is overexpressed in several human cancers and plays an important role in tumor angiogenesis and growth in various experimental systems. We investigated the role of apelin signaling in the malignant behavior of cutaneous melanoma. Murine B16 and human A375 melanoma cell lines were stably transfected with apelin encoding or control vectors. Apelin overexpression significantly increased melanoma cell migration and invasion in vitro, but it had no impact on its proliferation. In our in vivo experiments, apelin significantly increased the number and size of lung metastases of murine melanoma cells. Melanoma cell proliferation rates and lymph and blood microvessel densities were significantly higher in the apelin-overexpressing pulmonary metastases. APJ inhibition by the competitive APJ antagonist MM54 significantly attenuated the in vivo pro-tumorigenic effects of apelin. Additionally, we detected significantly elevated circulating apelin and VEGF levels in patients with melanoma compared to healthy controls. Our results show that apelin promotes blood and lymphatic vascularization and the growth of pulmonary metastases of skin melanoma. Further studies are warranted to validate apelin signaling as a new potential therapeutic target in this malignancy.

Lymphocyte cytosolic protein 2 (LCP2) is one of the SLP-76 family of adapters, which are critical intermediates in signal cascades downstream of several receptors. LCP2 regulates immunoreceptor signaling (such as T-cell receptors) and is also required for integrin signaling in neutrophils and platelets. However, the role of LCP2 in the tumor microenvironment is still unknown. In this study, we found a significant increase of mRNA and protein expression of LCP2 in metastatic skin cutaneous melanoma compared to normal skin. The upregulation of LCP2 was associated with good overall survival of patients with metastatic skin cutaneous melanoma, who received pharmacotherapy and radiation. GSEA signaling pathways analysis showed that LCP2 was involved in multiple pathways of immune response and correlation analysis revealed LCP2 was positively correlated with molecules in TCR signaling and 11 immune checkpoints, while LCP2 negatively correlated with 2 immune checkpoints in the metastatic skin cutaneous melanoma. According to the different expressions of LCP2, high LCP2 expression was positively correlated with more tumor-infiltrating CD8+ T cells. Furthermore, Kaplan-Meier plot indicated that LCP2 acted as a prognostic biomarker for progression-free survival of patients with metastatic skin cutaneous melanoma receiving anti-PD1 immunotherapy. In conclusion, our results integrated both the expression and function of LCP2 in melanoma using multiple tools, shedding light on the potential role of LCP2 in melanoma, and suggesting LCP2 serves as a prognostic biomarker and therapeutic target in anti-tumor immunity.

Dysregulation of signaling networks controlling self-renewal and migration of developmental cell lineages is closely linked to the proliferative and invasive properties of tumors. Identification of such signaling pathways and their critical regulators is vital for successful design of effective targeted therapies against neoplastic tissue growth. The neurotrophin receptor (CD271/NGFR/p75NTR) is a key regulator of the melanocytic cell lineage through its ability to mediate cell growth, survival, and differentiation. Using clinical melanoma samples, normal melanocytes and global gene expression profiling we have investigated the role of CD271 in rewiring signal transduction networks of melanoma cells during neoplastic transformation. Our analysis demonstrates that depending on the cell fate of tumor initiation vs normal development, elevated levels of CD271 can serve as a switch between proliferation/survival and differentiation/cell death. Two divergent arms of neurotrophin signaling hold the balance between positive regulators of tumor growth controlled by E2F, MYC, SREBP1 and AKT3 pathways on the one hand, and differentiation, senescence, and apoptosis controlled by TRAF6/IRAK-dependent activation of AP1 and TP53 mediated processes on the other hand. A molecular network map revealed in this study uncovers CD271 as a context-specific molecular switch between normal development and malignant transformation.

Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of 1H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines.

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

Peripheral CD4+CD8+ double positive (DP) T cells are a phenotypically and functionally heterogeneous population depending on their origin and pathologic context. We previously identified among tumour infiltrating lymphocytes in melanoma, a tumour-reactive MHC class-I restricted CD4lowCD8high DP αβ T-cell subpopulation with CD4-like function. In this study, we used an in-depth comparative transriptomic analysis of intra-melanoma DP T cells and CD4 and CD8 single positive (SP) T cells, to better comprehend the origin of this DP phenotype, and define the transcriptomic signature of activated DP T cells. We observed that intra-melanoma DP T cells were transcriptome-wise closer to their CD8 SP T-cell counterparts in terms of number of genes differentially expressed (97 in common with CD8 SP T cells and 15 with CD4 SP T cells) but presented hallmarks of a transition to a CD4-like functional profile (CD40LG) with a decreased cytotoxic signature (KLRC1) in favour of an increased cytokine-receptor interaction signature (IL4, IL24, IL17A…). This unleashed CD4-like program could be the results of the observed unbalanced expression of the THPOK/Runx3 transcription factors in DP T cells. Overall, this study allow us to speculate that intra-melanoma DP T cells arise from CD8 SP T cells being reprogrammed to a helper function.

Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities.

Melanoma is the most aggressive form of skin cancer, with metastatic melanoma being refractory to currently available conventional therapies. In this study, we evaluated the inhibitory effect of coronatine (COR) on the proliferation of metastatic melanoma cells. COR inhibited the proliferation of melanoma cells but negligibly affected the proliferation of normal melanocytes. Comparative metabolic and lipidomic profiling using gas chromatography-mass spectrometry and direct infusion-mass spectrometry was performed to investigate COR-induced metabolic changes. These analyses identified 33 metabolites and 82 lipids. Of these, the levels of lactic acid and glutamic acid, which are involved in energy metabolism, significantly decreased in COR-treated melanoma cells. Lipidomic profiling indicated that ceramide levels increased in COR-treated melanoma cells, suggesting that ceramides could function as a suppressor of cancer cell proliferation. In contrast, the levels of phosphatidylinositol (PI) species, including PI 16:0/18:0, 16:0/18:1, 18:0/18:0, and 18:0/18:1, which were found to be potential biomarkers of melanoma metastasis in our previous study, were lower in the COR-treated cells than in control cells. The findings of metabolomic and lipidomic profiling performed in the present study provide new insights on the anticancer mechanisms of COR and can be used to apply COR in cancer treatment.

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

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

Cutaneous melanoma is a highly aggressive cancer with a propensity for distant metastasis to various organs. In contrast, melanoma arising in pigmented uveal layers of the eye metastasizes mostly in the liver. The mechanisms of these metastases, which are ultimately resistant to therapy, are still unclear. Metastasis via intravascular dissemination of tumour cells is widely accepted as a central paradigm. However, we have previously described an alternative mode of tumour dissemination, extravascular migratory metastasis, based on clinical and experimental data. This mechanism is characterised by the interaction of cancer cells with the abluminal vascular surface, which defines angiotropism. Here, we employed our 3D co-culture approach to monitor cutaneous and uveal human melanoma cells dynamics in presence of vascular tubules. Using time-lapse microscopy, we evaluated angiotropism, the migration of tumour cells along vascular tubules and the morphological changes occurring during these processes. Cutaneous and uveal melanoma cells were injected in zebrafish embryos in order to develop xenografts. Employing in vivo imaging coupled with 3D reconstruction, we monitored the interactions between cancer cells and the external surface of zebrafish vessels. Overall, our results indicate that cutaneous and uveal melanoma cells spread similarly along the abluminal vascular surfaces, in vitro and in vivo.

Melanoma is a type of cancer with abnormal proliferation of melanocytes and is one of the most diagnosed cancer types. In traditional Chinese medicine, pangolin scales have been used to treat various diseases, including human cancers. However, its efficacy has not been scientifically proven. Here we studied the anticancer effect and mechanism of pangolin scale extract (PSE) on melanoma cell lines using scientific approaches. Our cell viability assay shows that PSE exhibits up to approximately 50-80% inhibition on SK-MEL-103 and A375 melanoma cell lines. Mechanically, PSE inhibits melanoma cell proliferation, migration, and causes changes in cell morphology. The apoptosis assay showed a significant chromosomal condensation inside the PSE-treated melanoma cells. The sequencing and analysis of A375 melanoma cell transcriptomes revealed 3077 differentially expressed genes in the 6 h treatment group and 8027 differentially expressed genes in the 72 h treatment group. Transcriptome analysis suggests that PSE may cause cell cycle arrest in melanoma cells and promote apoptosis mainly by up-regulating the p53 signaling pathway and down-regulating the PI3K-Akt signaling pathway. In this study, the anticancer effect of PSE was demonstrated by molecular biological means. PSE shows a significant inhibition effect on melanoma cell proliferation and cell migration in vitro, causes cell cycle arrest and promotes apoptosis through p53 and PI3K-AKT pathways. This study provides better insights into the anti-cancer efficacy and underlying mechanism of PSE and a theoretical basis for mining anticancer compounds or the development of new treatments for melanoma in the future. It is worth noting that this study does not advocate the use of the pangolin scale for disease treatment, but only to confirm its usefulness from a scientific research perspective and to encourage subsequent research around the development of active compounds to replace pangolin scales to achieve the conservation of this endangered species.

Melanoma is the most invasive type of skin cancer, in which the immune system plays a vital role. In this study, we aimed to establish a prognostic prediction nomogram for melanoma patients that incorporates immune-related genes (IRGs). Ninety-seven differentially expressed IRGs between melanoma and normal skin were screened using gene expression omnibus database (GEO). Among these IRGs, a two-gene signature consisting of CCL8 and DEFB1 was found to be closely associated with patient prognosis using the cancer genome atlas (TCGA) database. Survival analysis verified that the IRGs score based on the signature gene expressions efficiently distinguished between high- and low-risk patients, and was identified to be an independent prognostic factor. A nomogram integrating the IRGs score, age and TNM stage was established to predict individual prognosis for melanoma. The prognostic performance was validated by the TCGA/GEO-based concordance indices and calibration plots. The area under the curve demonstrated that the nomogram was superior than the conventional staging system, which was confirmed by the decision curve analysis. Overall, we developed and validated a nomogram for prognosis prediction in melanoma based on IRGs signatures and clinical parameters, which could be valuable for decision making in the clinic.

The aim of the present study was to investigate combined effects of cold atmospheric plasma (CAP) and the chemotherapeutic drug doxorubicin (DOX) on murine and human melanoma cells, and normal cells. In addition to free drug, the combination of CAP with a liposomal drug (DOX-LIP) was also studied for the first time. Thiazolyl blue tetrazolium bromide (MTT) and Trypan Blue exclusion assays were used to evaluate cell viability; the mechanism of cell death was evaluated by flow cytometry. Combined treatment effects on the clonogenic capability of melanoma cells, was also tested with soft agar colony formation assay. Furthermore the effect of CAP on the cellular uptake of DOX or DOX-LIP was examined. Results showed a strong synergistic effect of CAP and DOX or DOX-LIP on selectively decreasing cell viability of melanoma cells. CAP accelerated the apoptotic effect of DOX (or DOX-LIP) and dramatically reduced the aggressiveness of melanoma cells, as the combination treatment significantly decreased their anchorage independent growth. Moreover, CAP did not result in increased cellular uptake of DOX under the present experimental conditions. In conclusion, CAP facilitates DOX cytotoxic effects on melanoma cells, and affects their metastatic potential by reducing their clonogenicity, as shown for the first time.

Melanomas frequently metastasize to distant organs and especially intracranial metastases still represent a major clinical challenge. Epigenetic reprogramming of intracranial metastases is thought to be involved in therapy failure, but so far only little is known about patient-specific DNA-methylation differences between intra- and extracranial melanoma metastases. Hierarchical clustering of the methylomes of 24 patient-matched intra- and extracranial melanoma metastases pairs revealed that intra- and extracranial metastases of individual patients were more similar to each other than to metastases in the same tissue from other patients. Therefore, a personalized analysis of each metastases pair was done by a Hidden Markov Model to classify methylation levels of individual CpGs as decreased, unchanged or increased in the intra- compared to the extracranial metastasis. The predicted DNA-methylation alterations were highly patient-specific differing in the number and methylation states of altered CpGs. Nevertheless, four important general observations were made: (i) intracranial metastases of most patients mainly showed a reduction of DNA-methylation, (ii) cytokine signaling was most frequently affected by differential methylation in individual metastases pairs, but also MAPK, PI3K/Akt and ECM signaling were often altered, (iii) frequently affected genes were mainly involved in signaling, growth, adhesion or apoptosis, and (iv) an enrichment of functional terms related to channel and transporter activities supports previous findings for a brain-like phenotype. In addition, the derived set of 17 signaling pathway genes that distinguished intra- from extracranial metastases in more than 50% of patients included well-known oncogenes (e.g. PRKCA, DUSP6, BMP4) and several other genes known from neuronal disorders (e.g. EIF4B, SGK1, CACNG8). Moreover, associations of gene body methylation alterations with corresponding gene expression changes revealed that especially the three signaling pathway genes JAK3, MECOM, and TNXB differ strongly in their expression between patient-matched intra- and extracranial metastases. Our analysis contributes to an in-depth characterization of DNA-methylation differences between patient-matched intra- and extracranial melanoma metastases and may provide a basis for future experimental studies to identify targets for new therapeutic approaches.

Melanoma is the deadliest form of skin cancer, and little is known about the impact of deregulated expression of long noncoding RNAs (lncRNAs) in the progression of this cancer. In this study, we explored RNA-Seq data to search for lncRNAs associated with melanoma progression. We found distinct lncRNA gene expression patterns across melanocytes, primary and metastatic melanoma cells. Also, we observed upregulation of the lncRNA ZEB1-AS1 (ZEB1 antisense RNA 1) in melanoma cell lines. Data analysis from The Cancer Genome Atlas (TCGA) confirmed higher ZEB1-AS1 expression in metastatic melanoma and its association with hotspot mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) gene and RAS family genes. In addition, a positive correlation between ZEB1-AS1 and ZEB1 (zinc finger E-box binding homeobox 1) gene expression was verified in primary and metastatic melanomas. Using gene expression signatures indicative of invasive or proliferative phenotypes, we found an association between ZEB1-AS1 upregulation and a transcriptional profile for invasiveness. Enrichment analysis of correlated genes demonstrated cancer genes and pathways associated with ZEB1-AS1. We suggest that the lncRNA ZEB1-AS1 could function by activating ZEB1 gene expression, thereby influencing invasiveness and phenotype switching in melanoma, an epithelial-to-mesenchymal transition (EMT)-like process, which the ZEB1 gene has an essential role.

Skin cutaneous melanoma (SKCM) is the most lethal form of skin cancers owing to high invasiveness and high metastatic potential. Tumor microenvironment (TME) provides powerful evidences for discerning SKCM, raising the prospect to identify biomarkers of SKCM. Based on the transcriptome profiles of patients with SKCM and the corresponding clinical information from The Cancer Genome Atlas (TCGA), we used ESTIMATE algorithm to calculate ImmuneScore and StromalScore and identified the TME-Related differentially expressed genes (DEGs), than the intersected TME-Related DEGs were used for subsequent functional enrichment analysis. Protein-protein interaction (PPI) analysis was used to identify the functionality-related DEGs and univariate Cox regression analysis was used to identify the survival-related DEGs. Furthermore, SKCM-related DEGs were identified based on two Gene Expression Omnibus (GEO) datasets. Finally, we intersected functionality-related DEGs, survival-related DEGs, and SKCM-related DEGs, ascertaining that six DEGs (CCL4, CXCL10, CCL5, GZMB, C1QA, and C1QB) function as core TME-related genes (CTRGs). Significant differences of GZMB, C1QA, and C1QB expressions were found in gender and clinicopathologic staging of SKCM. High levels of GZMB, C1QA, and C1QB expressions were associated with favorable prognosis. Gene set enrichment analysis (GSEA) showed that cell-cell interaction, cell behavior, and intracellular signaling transduction may be mainly involved in both C1QA, C1QB and GZMB expressions and metabolism of phospholipid and amino acid, transcription, and translation may be implicated in low GZMB expressions. C1QA, C1QB, and GZMB are novel SKCM-relating CTRGs, providing promising immune-related prognostic biomarkers for SKCM.

Understanding the biological factors that are characteristic of metastasis in melanoma remains a key approach to improving treatment. In this study, we seek to identify a gene signature of metastatic melanoma. We configured a new network-based computational pipeline, combined with a machine learning method, to mine publicly available transcriptomic data from melanoma patient samples. Our method is unbiased and scans a genome-wide protein-protein interaction network using a novel formulation for network scoring. Using this, we identify the most influential, differentially expressed nodes in metastatic as compared to primary melanoma. We evaluated the shortlisted genes by a machine learning method to rank them by their discriminatory capacities. From this, we identified a panel of 6 genes, ALDH1A1, HSP90AB1, KIT, KRT16, SPRR3 and TMEM45B whose expression values discriminated metastatic from primary melanoma (87% classification accuracy). In an independent transcriptomic data set derived from 703 primary melanomas, we showed that all six genes were significant in predicting melanoma specific survival (MSS) in a univariate analysis, which was also consistent with AJCC staging. Further, 3 of these genes, HSP90AB1, SPRR3 and KRT16 remained significant predictors of MSS in a joint analysis (HR = 2.3, P = 0.03) although, HSP90AB1 (HR = 1.9, P = 2 × 10-4) alone remained predictive after adjusting for clinical predictors.