miR-138-5p induces aggressive traits by targeting Trp53 expression in murine melanoma cells, and correlates with poor prognosis of melanoma patients.

Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.

Gene expression and promoter methylation of angiogenic and lymphangiogenic factors as prognostic markers in melanoma.

The high mortality rate of melanoma is broadly associated with its metastatic potential. Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis. Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients. Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay. Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P-value = 0.044) and the lymphangiogenic receptor VEGFR-3 (P-value = 0.002) were independent prognostic factors of overall survival in melanoma patients. Enhanced reduced representation bisulfite sequencing-based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells. In patients, VEGFC (P-value = 0.031), ANGPT2 (P-value < 0.001), and SIX1 (P-value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival. Hence, our data suggest that these angio- and lymphangiogenesis factors are potential biomarkers of melanoma prognosis. Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.

SIRT1 regulates Mxd1 during malignant melanoma progression.

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.