Learning from Embryogenesis—A Comparative Expression Analysis in Melanoblast Differentiation and Tumorigenesis Reveals miRNAs Driving Melanoma Development

Abstract

Malignant melanoma is one of the most dangerous tumor types due to its high metastasis rates and a steadily increasing incidence. During tumorigenesis, the molecular processes of embryonic development, exemplified by epithelial–mesenchymal transition (EMT), are often reactivated. For melanoma development, the exact molecular differences between melanoblasts, melanocytes, and melanoma cells are not completely understood. In this study, we aimed to identify microRNAs (miRNAs) that promote melanoma tumorigenesis and progression, based on an in vitro model of normal human epidermal melanocyte (NHEM) de-differentiation into melanoblast-like cells (MBrCs). Using miRNA-sequencing and differential expression analysis, we demonstrated in this study that a majority of miRNAs have an almost equal expression level in NHEMs and MBrCs but are significantly differentially regulated in primary tumor- and metastasis-derived melanoma cell lines. Further, a target gene analysis of strongly regulated but functionally unknown miRNAs yielded the implication of those miRNAs in many important cellular pathways driving malignancy. We hypothesize that many of the miRNAs discovered in our study are key drivers of melanoma development as they account for the tumorigenic potential that differentiates melanoma cells from proliferating or migrating embryonic cells.

Alpha-Synuclein and Its Role in Melanocytes

“…We compared these two groups via the web tool GEO2R and found a higher expression of aSyn in melanocytes compared to IPs (Figure 2A) and that aSyn is regulated depending on the differentiation status. To confirm our hypothesis, we cultivated de-differentiated melanoblast-related cells (MBrcs) generated by growth factor treatment of differentiated melanocytes [42,43]. Analysis of MBrcs on mRNA (Figure 2B, Supplementary Table S1) and protein level (Figure 2C,D) revealed a strong reduction of aSyn expression in MBrc compared to untreated NHEMs.…”

“…Human primary epidermal melanocytes derived from normal skin (NHEM, dark pigmented donor; PromoCell, Heidelberg, Germany) were cultivated in M2 melanocyte growth medium (M2 with Supplementary Mix containing growth factors and hormones (e.g., pituitary gland extracts with alpha-MSH), PromoCell, Heidelberg, Germany) under a humidified atmosphere of 5% CO 2 at 37 • C. They were used between Passages 4 and 14 and split once per week at a ratio of 1:4. Melanocytes were de-differentiated into melanoblast-related cells (MBrcs) and cultured as previously described [42,43]. Co-culture experiments were based on previously described methods with modifications [44][45][46].…”

Sox9 regulates melanocytic fate decision of adult hair follicle stem cells

“… 29 In contrast to our findings in the HF, we did not detect Sox9 expression in epidermal melanocytes in vivo ( Figure 3 A). In silico analysis from our group 30 and in vitro analysis revealed low mRNA and protein expression of Sox9 in epidermal melanocytes and an upregulation in melanoblasts, the embryonic progenitor cells of melanocytes ( Figures 3 B–3D and Table S2 ). These findings indicate once more that Sox9 is expressed in early melanocytic cells.…”

NF1-Dependent Transcriptome Regulation in the Melanocyte Lineage and in Melanoma

“…Last, we wanted to focus on the 263 genes which were regulated in both NF1-mutated melanoblasts and in NF1-low expressing metastatic melanoma ( Figure 2 C) because genes involved in melanocyte development are likely to be reactivated during melanoma progression [ 23 , 61 ]. The genes likely regulated by NF1 were involved in common signalings.…”

“…The cell plasticity of lineage-committed melanoblasts is also observed in certain clones of melanoma tumors which have undergone dedifferentiation and which are usually refractory to treatments [ 21 , 22 ]. Recently, miRNAs identified in a human model of melanoblasts were described as potential drivers of melanoma development [ 23 ]. Malignant melanoma has been classified in four subgroups according to its main genetic drivers by The Cancer Genome Atlas: mutant BRAF , mutant NRAS , mutant NF1 , or triple wild-type tumors [ 24 ].…”