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Intercellular communication is a cell-type and stimulus-dependent event driven not only by soluble factors but also by extracellular vesicles (EVs). EVs include vesicles of different size and origin that contain a myriad of molecules. Among them, small EVs (sEV; <200 nm) have been shown to modulate not just regional cell responses but also distant organ behavior. In cancer, distant organ modulation by sEVs has been associated to disease dissemination, which is one of the main concerns in melanoma. Description of broadly conserved alterations in sEV-contained molecules represents a strategy to identify key modifications in cellular communication as well as new disease biomarkers. Here, we characterize proteomes of cutaneous melanocyte and melanoma-derived sEVs to deepen on the landscape of normal and disease-related cell communication. Results reveal the presence of unique protein signatures for melanocytes and melanoma cells that reflect cellular transformation-related profound modifications. Melanocyte-derived sEVs are enriched in oxidative metabolism (e.g., aconitase 2, ACO2) or pigmentation (e.g., tyrosinase, TYR) related proteins while melanoma-derived sEVs reflect a generalized decrease in mature melanocytic markers (e.g., melanoma antigen recognized by T-cells 1, MART-1, also known as MLANA) and an increase in epithelial to mesenchymal transition (EMT)-related adhesion molecules such as tenascin C (TNC)., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the "malignancy lipid signature" features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.

We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype.

Background: The observed correlation between ultraviolet light incidence and skin color, together with the geographical apportionment of skin reflectance among human populations, suggests an adaptive value for the pigmentation of the human skin. We have used Affymetrix U133a v2.0 gene expression microarrays to investigate the expression profiles of a total of 9 melanocyte cell lines (5 from lightly pigmented donors and 4 from darkly pigmented donors) plus their respective unirradiated controls. In order to reveal signatures of selection in loci with a bearing on skin pigmentation in humans, we have resequenced between 4 to 5 kb of the proximal regulatory regions of three of the most differently expressed genes, in the expectation that variation at regulatory regions might account for intraespecific morphological diversity, as suggested elsewhere., Results: Contrary to our expectations, expression profiles did not cluster the cells into unirradiated versus irradiated melanocytes, or into lightly pigmented versus darkly pigmented melanocytes. Instead, expression profiles correlated with the presence of Bovine Pituitary Extract (known to contain alpha-MSH) in the media. This allowed us to differentiate between melanocytes that are synthesizing melanin and those that are not. TYR, TYRP1 and DCT were among the five most differently expressed genes between these two groups. Population genetic analyses of sequence haplotypes of the proximal regulatory flanking-regions included Tajima's D, HEW and DHEW neutrality tests analysis. These were complemented with EHH tests (among others) in which the significance was obtained by a novel approach using extensive simulations under the coalescent model with recombination. We observe strong evidence for positive selection for TYRP1 alleles in Africans and for DCT and TYRP1 in Asians. However, the overall picture reflects a complex pattern of selection, which might include overdominance for DCT in Europeans., Conclusion: Diversity patterns clearly evidence adaptive selection in pigmentation genes in Africans and Asians. In Europeans, the evidence is more complex, and both directional and balancing selection may be involved in light skin. As a result, different non-African populations may have acquired light skin by alternative ways, and so light skin, and perhaps dark skin too, may be the result of convergent evolution.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker. [ABSTRACT FROM AUTHOR]

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12–1.48; p-value = 4×10−4). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocytes. [ABSTRACT FROM AUTHOR]