Semaphorin-5A downregulation is associated with enhanced migration and invasion of BRAF-positive melanoma cells under vemurafenib treatment in melanomas with heterogeneous BRAF status.

Tumor heterogeneity affects the efficacy of anticancer treatment as tumor subclones with distinct molecular patterns may be present within one tumor, leading to differing sensitivities to chemotherapeutic agents. In the present study, six melanoma tissue fragments were obtained from different parts of tumor of four patients and then the effect of vemurafenib treatment on biological characteristics and molecular processes of cell cultures was estimated by using MTT-test, apoptosis, migration and invasion assays, PCR real time. There was different BRAF status determined between cells derived from the central and peripheral regions of primary melanoma tumors. BRAF-positive melanoma cells showed an increased apoptotic rate under vemurafenib treatment, as well as increased migration and invasion rates, whereas BRAF-negative melanoma cells did not exhibit such tendency. Furthermore, semaphorin-5A levels were diminished in BRAF-positive cells, but not in BRAF-negative ones, which could be related to increased migration and invasion. Melanoma cells derived from different regions of the same tumor may differ by mutations status, molecular processes and biological response to target therapy. The downregulation of semaphorin-5A may be involved in divergent effects of anticancer agents on tumor cell biology.

Differences in microRNA expression between melanoma and healthy adjacent skin.

BACKGROUND: The tumor microenvironment is composed of cancer-associated fibroblasts, tumor-associated macrophages, endothelial cells, immune cells, signaling molecules and extracellular matrix structures, which closelycommunicate with the tumor via multiple mechanisms. MicroRNAs are paracrine regulators that provide a direct interaction between the microenvironment and cancer cells. In the presentstudy, we aimed to identify the microRNA expression profile in melanoma compared with thatin healthy adjacent skin, with a further assessment of altered microRNA signaling pathways and target genes. METHODS: Formalin-fixed paraffin-embedded (FFPE) melanoma tissue samples were separated by dissection into tumor and surrounding health tissue fragments. MicroRNA expression profiles were obtained by microarray using Gene Atlas Microarray System (Affymetrix, California, USA). To confirm microarray results real-time PCR was carried out. Bioinformatic analysis was performed using the DIANA-miRPath v.3.0 database. Target genes for miR-146a-5p were determined using three algorithms: TargetScan 7.0, miRWalk 2.0 and miRTarBase v.4.5. RESULTS: A microarray profiling revealed 143 microRNAs asdifferent in tumor versus adjacent tissues. Expression level of hsa-miR-146a-5p showedto be higher in melanoma cells as compared to thehealthy adjacent skin. The bioinformatic study has determined several signaling cascades associated with miR-146a-5p:Toll-like receptor pathway, NF-κB pathway, ErB pathway, and measles signaling pathway. The 38 target genes have been shown for miR-146a-5p of which NRAS gene is known asone of the most frequent mutated in melanoma. CONCLUSIONS: Elucidation of the role of miR-146-a-5p in complex interactions between the tumor and the cells of healthy adjacent skin is necessary for our understanding of the mechanisms oftumor progression. Significant differences found between cancer cells and adjacent tissues in microRNA expression profile corresponding to divergent mRNA/protein levels in these structures should be taken into account when tumor samples characterization estimatedby high-throughput methods.

miR-204-5p and miR-3065-5p exert antitumor effects on melanoma cells.

MicroRNA (miR)-204-5p was previously identified to be downregulated in melanoma compared with melanocytic nevi. This observation prompted a functional study on miR-204-5p and the newly-identified miR-3065-5p, two miRNAs suggested to be tumor-suppressive oncomiRs. Application of miR-204-5p mimics or inhibitors resulted in a decrease or increase, respectively, in melanoma cell proliferation and colony formation. miR-204-5p mimics hindered invasion, whereas miR-204-5p inhibitors stimulated cancer cell migration. Modulation of miR-3065-5p led to a decrease in melanoma cell proliferation, altered cell cycle distribution and increased expression levels of its target genes HIPK1 and ITGA1, possibly due to functional modifications identified in these cells. miR-204-5p and miR-3065-5p demonstrated antitumor capacities that may need to be taken into account in the development of melanoma treatment approaches.

Genetic analysis of melanocortin 1 receptor red hair color variants in a Russian population of Eastern Siberia.

The melanocortin 1 receptor is a Gs protein-coupled receptor implicated in melanogenesis regulation. The receptor gene is highly polymorphic, which accounts for the association of several of its single-nucleotide polymorphisms (SNPs) with an increased risk of melanoma. The present study aimed to evaluate the distribution of melanocortin 1 receptor gene variants R151C, R160W, and D294H within the Russian population of Eastern Siberia and its association with melanoma development. Melanoma patients (n=95) admitted to Krasnoyarsk Territorial Oncological Center and healthy controls (n=334) were enrolled in the study. A clinical examination of patients was performed to evaluate the phenotypic features of melanoma patients. SNPs were analyzed by real-time PCR. Clinical examination indicated a more frequent occurrence of fair skin type, blue eyes, blonde and red hair, and more frequent localization of freckles on the neck, trunk, and extremities in the melanoma group of patients. The R151C melanocortin 1 receptor gene variant was found in 18% of melanoma patients and associated with an increased likelihood of melanoma development (odds ratio=6.4; 95% confidence interval: 2.8-14.3; P=0.0001). The two remaining variant alleles of the melanocortin 1 receptor gene occurred with low frequency both in controls and in the melanoma group. The R160W SNP was identified neither in controls nor in melanoma patients. The D294H heterozygous variant was observed in 0.3% of individuals in the control group and in 1.1% of the patients in the melanoma group. Such an asymmetric distribution of the melanocortin 1 receptor within red hair color genotypes in the population under study compared with other populations may be because of Russian genetic homogeneity. Carriers of the mutant R151C allele should exercise caution in terms of exposure to the sun to avoid the risk of melanoma development.

MicroRNA in skin diseases.

MicroRNAs are essential regulators of various cellular processes such as cell growth, differentiation, apoptosis, and the immune response, acting as factors for translational repression and/or degradation of target messenger RNA. Currently, microRNAs are considered as promising biomarkers and therapeutic targets for different pathological conditions. Skin may serve as a convenient model for microRNA modulation studies due to the comparatively easy access to targets cells. Cutaneous diseases are characterized by multiple intercellular communication pathways, triggered by diverse stimuli and mediated by heterogenous regulators, including microRNAs. The goal of this article is to summarize the state of research in dermatology concerning the action of microRNAs as epigenetic modulators.

Antiproliferative and Pro-Apoptotic Effects of MiR-4286 Inhibition in Melanoma Cells.

INTRODUCTION: MicroRNAs are essential regulators of gene expression at the post-transcriptional level. Their expression is altered in cancer tissues, and evaluation of these alterations is considered a promising tool used to diagnose and identify prognostic markers. MATERIALS AND METHODS: The microRNA expression profiles of formalin-fixed, paraffin-embedded melanoma and melanocytic nevi samples were estimated with a microarray and subsequently validated by real-time PCR. Melanoma cells were transfected with miR-4286 inhibitor to evaluate the influence of this microRNA on the viability, proliferation, apoptosis, migration, and invasion of melanoma cells. RESULTS: The microarray revealed that the expression of 1,171 microRNAs was altered in melanoma samples compared to melanocytic nevi. Real-time PCR validation experiments found the microRNA expression levels to correspond to the melanoma/melanocytic nevi microarray results. The pathway analysis identified 52 modulated pathways in melanoma. Moreover, the application of miR-4286 inhibitor to BRO melanoma cells resulted in a 2.6-fold increase in the apoptosis rate and a 1.7-fold decrease in the cell proliferation/viability but did not affect the invasiveness and migration of these cells. Furthermore, the use of miR-4286 inhibitor altered the mRNA expression of several miR-4286 gene targets: folylpolyglutamate synthase, RNA polymerase I-specific transcription initiation factor, apelin, G-protein-coupled receptor 55, and high-mobility group A1 protein, which have been implicated in cell proliferation/apoptosis regulation. Lastly, the transiently transfected SK-MEL-1 cells with miR-4286 inhibitor decreased proliferation rate and modulated folylpolyglutamate synthase rates of these cells. CONCLUSION: Our results demonstrate that miR-4286 mediates proliferation and apoptosis in melanoma cells, these findings may represent a novel mechanism underlying these processes.