Sonia Cerdido Ochoa¹, Marta Abrisqueta González¹, Ana Lambertos Escudero¹, CELIA JIMENEZ-CERVANTES FRIGOLS¹, JOSE CARLOS GARCIA-BORRON MARTINEZ¹, Cecilia María Herraiz Serrano¹

(1) CONTROL MOLECULAR DE LA PROLIFERACIÓN Y DIFERENCIACIÓN, IMIB-Arrixaca, España

Mahogunin Ring Finger-1 (MGRN1) is a RING-finger nucleo-cytoplasmic E3 ubiquitin ligase ubiquitously expressed in mammals. In mahoganoid mutant mice, loss of MGRN1 gene expression causes fur darkening, mitochondrial dysfunction, congenital heart defects and spongiform neurodegeneration. Mgrn1-null mahoganoid melanocytes exhibit a senescent-like differentiated phenotype and accumulate DNA damage. Abrogation of Mgrn1 expression in mouse melanoma cells decreases their motility and invasive potential (Martínez-Vicente et al, 2020). The effects of MGRN1 knockdown on human melanoma cells are unknown, but Kaplan-Meier analysis of data in the TCGA database suggest that low MGRN1 gene expression is associated with longer patient survival. Accordingly, we investigated whether MGRN1-null human melanoma cells display a low motility, low invasion phenotype comparable to Mgrn1-KO mouse melanoma cells and the molecular basis of this effect.

We used HBL human melanoma cells (a gift from Prof G Ghanem, Free University of Brussels) wildtype for the following genes frequently mutated in melanoma: NRAS, BRAF, MC1R and TP53. The MGRN1 gene was stably knocked down by CRISPR-Cas9. MGRN-KO clones were selected and validated by sequencing the MGRN1 gene and lack of MGRN1 protein expression by Western blot. Three independent clones were analyzed, each one obtained with a different sgRNA during the CRISPR-Cas9 procedure to minimize the likelihood of artefacts

Knockdown of MGRN1 led to a more differentiated phenotype of HBL human melanoma cells grown on 2D cultures in plastic plates. MGRN1-KO cells also showed reduced migration in 2D assays. Cells seeded on a collagen I system better mimicking the dermal extracellular matrix tended to grow as cellular aggregates that were significantly bigger for MGRN1-KO cells compared with wildtype MGRN1-expressing cells. Moreover, in short-term adhesion assays MGRN1-KO cells showed higher adhesion to a collagen I matrix than control cells. This suggested that MGRN1 might be involved in the regulation of genes/proteins involved in cell-cell adhesion. Consistent with this possibility, Western blot analysis showed that MGRN1-KO cells expressed higher levels of E-cadherin, the main component of adherent junctions that occur at cell–cell and cell–matrix junctions in epithelial tissues. This was confirmed by immunostaining control and MGRN-KO HBL cells and quantification of the staining intensity in confocal microscopy images. Induction of E-cadherin in MGRN1-null cells was not due to an increased intracellular stability of the protein, since its half-life in cycloheximide-treated cells did not increase upon ablation of the MGRN1 gene. On the other hand, the levels of the mRNA corresponding to CDH1 gene encoding for E-cadherin were higher in MGRN1-KO cells compared with controls, suggesting that MGRN1 could regulate E-cadherin expression by a transcriptional mechanism. In MGRN1-KO cells, we found a higher co-localization of β-catenin and E-cadherin and a lower percentage of nuclear β-catenin as estimated by confocal microscopy

Permanent MGRN1 knockdown in human melanoma cells led to a high adhesion-low motility phenotype, with higher expression of E-cadherin and partial sequestration of β-catenin outside nuclei. These findings are consistent with the less aggressive phenotype of MGRN1-KO cells and the longer survival of patients carrying melanomas expressing low levels of MGRN1, and support the use of MGRN1 as a useful prognostic marker