MGRN1 como determinante de la integridad genómica y del fenotipo maligno de las células de melanoma

Abstract

Mahogunin (Mgrn1) is an E3-ubiquitin ligase encoded by a gene whose loss-of-function mutations cause the mahoganoid mouse phenotype, mainly characterized by coat color darkening, spongiform neurodegeneration and congenital heart defects. One of the best-known functions of MGRN1 is the modulation of the melanocortin 1 receptor (MC1R) signaling. The MC1R gene, the major determinant of skin pigmentation and sensitivity to ultraviolet radiation, is highly polymorphic and some of its allelic variants are associated with melanoma. Furthermore, MC1R produces different alternative splicing transcripts, including a minority spliceoform, MC1R-203, with unknown function. The influence of Mgrn1 on MC1R signaling and its functions in endolysosomal trafficking, mitochondrial homeostasis, oxidative stress and mitotic spindle orientation, could contribute to the development of melanoma and/or to the modulation of the phenotype of malignant melanocytes. Within this context, we proposed the following objectives: i) to functionally characterize the MC1R-203 isoform, ii) to study the role of Mgrn1 as a modulator of the malignant phenotype of mouse melanoma cells, iii) to analyze the role of MGRN1 as a determinant of human melanoma aggressiveness and iv) to study the molecular basis of the phenotypic actions of MGRN1 in human melanoma cells. We found that MC1R-203 expression in HBL cells was much lower than that of the canonical form (MC1R). However, MC1R and MC1R-203 showed similar intracellular stability when transiently expressed in heterologous HEK293T cells. Functional experiments indicated that MC1R-203 did not efficiently activate the cAMP pathway, but stimulated normally the ERK1/2 cascade. Unlike MC1R, MC1R-203 was incapable of promoting β-arrestin-2 (ARRB2) ubiquitylation. Since this post-translational ARRB2 modification dependens on the formation of an MGRN1-MC1R-ARRB2 ternary complex, this suggested that the MC1R-203 spliceoform was unable to scaffold ARRB2 and MGRN1. The study of Mgrn1 as a modulator of the phenotype of normal mouse melanocytes (melan-a6) and melanoma cells (B16F10) indicated that Mgrn1 deficiency (decreased expression upon by transient silencing with siRNA or knockout by CRISPR/Cas9) promoted a differentiated phenotype with increased pigmentation, dendricity and adhesion, decreased motility, and low rates of lung colonization. In addition, we observed by flow cytometry that Mgrn1-deficient cells showed an increased percentage of cells in the S phase of the cell cycle and an impaired genome stability, with accumulation of DNA damage (mainly evaluated by comet assays). These results suggested that Mgrn1 could regulate the malignant phenotype of mouse melanoma cells. To determine whether MGRN1 expression could act as a determinant of prognosis in melanoma patients, we analyzed public data (TCGA) and patient samples along with their clinical data. The results indicated that i) MGRN1 expression is higher in human melanoma than in normal skin or nevi, ii) melanomas display a significant rate of mutations in MGRN1 comparable with other cancer-associated genes, and iii) low MGRN1 expression is significantly associated with higher patient survival. These results indicated that MGRN1 could play an important role as determinant of human melanoma aggressiveness. The molecular basis of the phenotypic effects of MGRN1 was studied in HBL human melanoma cells. Lack of MGRN1 in human melanoma cells led to aberrant cell cycle progression and increased DNA damage. DNA fiber assays showed that the absence of MGRN1 increased cellular replication stress, with accumulation of stalled replication forks and impaired activation of the ATR-CHK1 signaling axis. Immunoprecipitation assays and proteomic approaches showed the interaction of MGRN1 with CDK2 and RPA1. Downregulation of MGRN1 decreased the levels of CDK2, and experiments with a selective inhibitor of the kinase suggested that the MGRN1-CDK2 interaction contributed to the normal response to replicative stress by participating in the efficient activations of the ATR-CHK1 signaling axis