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In melanoma, the presence of promoter related hypermethylation has previously been reported, however, no methylation-based distinction has been drawn among the diverse melanoma subtypes. Here, we investigated DNA methylation changes associated with melanoma progression and links between methylation patterns and other types of somatic alterations, including the most frequent mutations and DNA copy number changes. Our results revealed that the methylome, presenting in early stage samples and associated with the BRAF(V600E) mutation, gradually decreased in the medium and late stages of the disease. An inverse relationship among the other predefined groups and promoter methylation was also revealed except for histologic subtype, whereas the more aggressive, nodular subtype melanomas exhibited hypermethylation as well. The Breslow thickness, which is a continuous variable, allowed for the most precise insight into how promoter methylation decreases from stage to stage. Integrating our methylation results with a high-throughput copy number alteration dataset, local correlations were detected in the MYB and EYA4 genes. With regard to the effects of DNA hypermethylation on melanoma patients' survival, correcting for clinical cofounders, only the KIT gene was associated with a lower overall survival rate. In this study, we demonstrate the strong influence of promoter localized DNA methylation changes on melanoma initiation and show how hypermethylation decreases in melanomas associated with less favourable clinical outcomes. Furthermore, we establish the methylation pattern as part of an integrated apparatus of somatic DNA alterations.

BackgroundDespite the extensive research approaches applied to characterise malignant melanoma, no specific molecular markers are available that are clearly related to the progression of this disease. In this study, our aims were to define a gene expression signature associated with the clinical outcome of melanoma patients and to provide an integrative interpretation of the gene expression -, copy number alterations -, and promoter methylation patterns that contribute to clinically relevant molecular functional alterations.MethodsGene expression profiles were determined using the Affymetrix U133 Plus2.0 array. The NimbleGen Human CGH Whole-Genome Tiling array was used to define CNAs, and the Illumina GoldenGate Methylation platform was applied to characterise the methylation patterns of overlapping genes.ResultsWE IDENTIFIED TWO SUBCLASSES OF PRIMARY MELANOMA: one representing patients with better prognoses and the other being characteristic of patients with unfavourable outcomes. We assigned 1,080 genes as being significantly correlated with ulceration, 987 genes were downregulated and significantly enriched in the p53, Nf-kappaB, and WNT/beta-catenin pathways. Through integrated genome analysis, we defined 150 downregulated genes whose expression correlated with copy number losses in ulcerated samples. These genes were significantly enriched on chromosome 6q and 10q, which contained a total of 36 genes. Ten of these genes were downregulated and involved in cell-cell and cell-matrix adhesion or apoptosis. The expression and methylation patterns of additional genes exhibited an inverse correlation, suggesting that transcriptional silencing of these genes is driven by epigenetic events.ConclusionUsing an integrative genomic approach, we were able to identify functionally relevant molecular hotspots characterised by copy number losses and promoter hypermethylation in distinct molecular subtypes of melanoma that contribute to specific transcriptomic silencing and might indicate a poor clinical outcome of melanoma.

Abstract H2A.Z-nucleosomes are present in both euchromatin and heterochromatin and it has proven difficult to interpret their disparate roles in the context of their stability features. Using an in situ assay of nucleosome stability and DT40 cells expressing engineered forms of the histone variant we show that native H2A.Z, but not C-terminally truncated H2A.Z (H2A.Z∆C), is released from nucleosomes of peripheral heterochromatin at unusually high salt concentrations. H2A.Z and H3K9me3 landscapes are reorganized in H2A.Z∆C-nuclei and overall sensitivity of chromatin to nucleases is increased. These tail-dependent differences are recapitulated upon treatment of HeLa nuclei with the H2A.Z-tail-peptide (C9), with MNase sensitivity being increased genome-wide. Fluorescence correlation spectroscopy revealed C9 binding to reconstituted nucleosomes. When introduced into live cells, C9 elicited chromatin reorganization, overall nucleosome destabilization and changes in gene expression. Thus, H2A.Z-nucleosomes influence global chromatin architecture in a tail-dependent manner, what can be modulated by introducing the tail-peptide into live cells.

Abstract Osteopontin (OPN) is a secreted integrin‐binding protein that plays a role in inflammation, cellular viability, cell adhesion and migration, cancer development, and diabetes through different mechanisms. The splice variants of OPN can play essential roles in cancer development, progression, and metastasis formation; however, limited data are available about the role of OPN isoforms in human malignant melanoma. Our goal was to define the gene expression patterns of five OPN variants (OPN4, OPN5, OPNa, OPNb, and OPNc), integrin, and CD44 receptor genes in primary and metastatic melanoma‐originated cell lines (n = 19), and to explore the association of the expression patterns with clinicopathological parameters. We evaluated the invasive property of the cell lines and investigated the potential association between the invasion and gene expression of OPN isoforms. We found a significant rise in the expression of OPNc in the invasive cell lines compared to the noninvasive cells and detected significantly higher expression of the OPN splice variants in melanoma cell lines originating from more advanced stages tumors than cell lines originating from early‐stage melanomas. The correlation analysis revealed that all five OPN variants positively correlated with ITGB3 and ITGA9, whereas OPN5 positively correlated with ITGB1, ITGAV, ITGA6, and CD44. OPN can activate extracellular signal‐regulated kinase signaling through binding to α9β1 integrin, promoting melanoma tumor cell migration. It is possible that such associations between OPN splice variants and integrin receptors may play a role in melanoma progression. In conclusion, our findings suggest that high expression of OPNc correlates with the invasive behavior of melanoma cells.

Abstract Due to the widespread applications of metal nanoparticles (NPs), green synthesis strategies have recently advanced, e.g., methods that utilize extracts made from different plant wastes. A particularly innovative approach to reducing large amounts of available household/agricultural green wastes is their application in nanoparticle generation. Regarding this, the aim of our work was to examine the possibility of upgrading green nanoparticle syntheses from an innovative economic and environmental point of view, namely by investigating the multiple recyclabilities of green tea (GT), coffee arabica (CA), and Virginia creeper (Parthenocissus quinquefolia) (VC) waste residues for iron nanoparticle (FeNPs) synthesis. The plant extracts obtained by each extraction round were analyzed individually to determine the amount of main components anticipated to be involved in NPs synthesis. The synthesized FeNPs were characterized by X-ray powder diffraction and transmission electron microscopy. The activity of the generated FeNPs in degrading chlorinated volatile organic compounds (VOC) and thus their future applicability for remediation purposes were also assessed. We have found that VC and especially GT residues could be reutilized in multiple extraction rounds; however, only the first extract of CA was suitable for FeNPs’ generation. All of the obtained FeNPs could degrade VOC with efficiencies GT1-Fe 91.0%, GT2-Fe 83.2%, GT3-Fe 68.5%; CA1-Fe 76.2%; VC1-Fe 88.2%, VC2-Fe 79.7%, respectively, where the number (as in GT3) marked the extraction round. These results indicate that the adequately selected green waste material can be reutilized in multiple rounds for nanoparticle synthesis, thus offering a clean, sustainable, straightforward alternative to chemical methods. Graphical abstract

Recently, liquid biopsy, as a promising approach was introduced for the analysis of different tumor-derived circulating markers including tumor DNA and cell free DNA (ct/cfDNA). Identification of mutations in cfDNA may allow the early detection of tumors, as well as predicting and monitoring treatment responses in a minimally invasive way. In the present study, we used commercially available gene panels to verify the mutation overlap between liquid biopsy and abnormalities detected in colorectal tumor tissue. The two panels (Archer®VariantPlex®Solid Tumor and LIQUIDPlexTM ctDNA) overlap in 23 genes, which enables a comprehensive view of tumor-plasma mutational status by next generation sequencing. We successfully analyzed 16 plasma and 16 tumor samples. We found that 87% of tumor tissues contained 44 mutations in 12 genes and 43.8% of cfDNA harbored 13 mutations in 5 genes. To verify whether the mutation pattern of the tumor DNA could be consistently detected in plasma cfDNA, we compared the alterations between cfDNA and matched tissue DNA in nine patients. Six of the 9 tumor tissues harbored mutations in TP53, KRAS or MET genes, those were not detectable by the ctDNA kit, even eventhough the exons of these genes overlap in both panels. Comparing the mutational patterns of the matched samples, we found that only one cfDNA had the same mutations (KRAS, SMAD4 and TP53) in the paired tissue. The results of the comparison between tumor tissue DNA and matched plasma cfDNA underline the importance of studying the paired solid tumor and plasma samples together.

Osteopontin (OPN) is a multifunctional glycoprotein that physiologically interacts with different types of integrins. It is considered to be a possible prognostic biomarker in certain tumor types; however, various splicing isoforms exist, which have not been investigated in melanoma. We aimed to define the relative expression pattern of five OPN isoforms and clarify the prognostic significance of the splice variants in melanoma. We also aimed to investigate the expression pattern of eight integrins in the same tumors. Gene expression analyses revealed that the relative expression of OPNa, OPNb, and OPNc is significantly higher in metastatic tumors compared to primary lesions (p < 0.01), whereas the expression of OPN4 and OPN5 was low in both. The more aggressive nodular melanomas had higher expression levels compared to the superficial spreading subtype (p ≤ 0.05). The relative expression of the eight tested integrins was low, with only the expression of ITGB3 being detectable in nodular melanoma (Medianlog2 = 1.274). A positive correlation was found between Breslow thickness and the expression of OPNc variant, whereby thicker tumors (>4 mm) had significantly higher expression (p ≤ 0.05). The Breslow thickness was negatively correlated with the expression of OPN4, and similarly with ITGA2. OPNc also exhibited significant positive correlation with the presence of metastasis. Our data show that high expression of OPNa, OPNb, and especially OPNc and low expression of OPN4 and ITGA2 are associated with an advanced stage of tumor progression and poor prognosis in melanoma.

Developing dendritic cells (DCs) from monocytes is a sensitively regulated process. One possible way for cancers to avoid immune recognition and antitumor response is the modulation of DC differentiation. Although several studies are available on the examination of tumor-associated macrophages, a comprehensive analysis focusing on the effects of tumor-formed DCs is not known to date. We provide a comparative analysis of the tumor-edited-monocyte derived DCs differentiated in the presence of adenocarcinomas (MDA, HT29, HeLa)- and primary (WM278, WM983A) or metastatic (WM1617, WM983B) melanomas. The immunomodulatory effect of tumors is mediated at least partly by secreted mediators. We investigated the impact of tumor cell-derived conditioned media on the differentiation of DCs from CD14+ monocytes, sequentially determining the phenotype, cytokine production, phagocytic, and the T cell polarizing capacity of moDCs. We completed our observations by analyzing our data with bioinformatic tools to provide objective correlations between phenotypical and functional properties of different tumor-educated moDCs. The correlation analysis revealed significant differences in the characteristics of adenocarcinomas- or melanomas-edited moDCs. We highlight the functional differences in the properties of moDCs differentiated in the presence of various cancer cell lines. We offer new information and options for the in vitro differentiation protocols of various tumor-conditioned moDCs. Our results confirm that various immunomodulatory properties of different tumor cell lines result in multiple manipulations of DC differentiation.

HA15 is a new anti-melanoma drug that triggers endoplasmic reticulum (ER) stress and causes deleterious effects on melanoma cell viability due to autophagy and apoptosis, regardless of driver mutations or drug resistance. In this study, we investigated the effect of HA15 on the viability/proliferation of BRAFV600E-mutant melanoma cells using different culture conditions. In contrast to the published data, we did not detect significant melanoma cell death under normal culture conditions using HA15 treatment. Indeed, only cells that were cultured under long-term starvation conditions were sensitive to the drug. Quantitative measurements of ER stress and autophagy markers showed that the compound HA15 does not trigger stress alone but synergistically enhances ER stress under starvation conditions. Importantly, we observed that the viability of normal melanocytes decreased significantly with treatment, even at low HA15 concentrations. Finally yet importantly, we were able to generate HA15-resistant cell lines, which failed by Cerezo et al. In summary, HA15 only influences the viability of cells that are starved for several hours before and during treatment. However, this in vitro setting is far from the in vivo conditions. In addition, our data clearly show that melanoma cells can acquire HA15 resistance. Further studies are needed to prove that HA15 is an effective anti-cancer agent.

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).