Mahogunin Ring Finger-1 (MGRN1) is a RING-finger E3 ubiquitin ligase ubiquitously expressed in mammals. MGRN1-null mahoganoid mice display hyperpigmentation and several pathological phenotypes including congenital heart defects and neurodegeneration. Our laboratory has previously shown that MGRN1-KO mouse melanocytes and melanoma cells showed higher dendricity and melanin content, higher adhesion to several supports, reduced motility, and lower colonization potential in an in vivo mouse model. In human melanoma cells, permanent depletion of MGRN1 induced a more dendritic phenotype. Of note, analysis of the melanoma dataset of the TCGA database suggests that low MGRN1 expression is associated with longer patient survival. However, studies on the effects of MGRN1 knockdown on human melanoma cells are scarce.

We selected five human melanoma cell lines with different genetic background to study the effect of MGRN1 silencing in morphology on a collagen I system. We found differences in cells morphology depending on the genetic background. Disruption of MGRN1 expression resulted in an increase in the area of cell aggregates on a collagen matrix only in melanoma cells wildtype for BRAF. In BRAF-mutated cells, transient knockdown of MGRN1 led to increased dendricity. We decided to study the increased intercellular adhesion upon MGRN1 depletion in HBL human melanoma cells (wildtype for NRAS, BRAF, NF1 and MC1R genes) and to investigate whether MGRN1 deletion could affect molecules involved in cytoskeleton dynamics and cell-cell adhesion.

HBL MGRN1-KO human melanoma cells generated by CRISPR-Cas9 technology showed significantly larger cellular aggregates on collagen and more dendricity on plastic than control cells. E-cadherin protein levels were up-regulated in MGRN1-KO HBL cells, likely due to a transcriptional mechanism. Cell membrane-associated E-cadherin also increased after MGRN1 knockdown. E-cadherin negative regulator ZEB1 is decreased in MGRN1-KO cells, which could explain the increase in E-cadherin expression. Furthermore, E-cadherin silencing reduced the area of cellular aggregates in MGRN1-depleted cells. Finally, inhibition of RhoA, Rac1 and Cdc42 GTPases by pharmacological inhibitors mimics the effect of larger cellular aggregates on collagen previously observed in HBL control cells, suggesting a possible role of these Rho-GTPases in the MGRN1-depleted phenotype. Indeed, pulldown assays showed altered activation of RhoA, Rac1 and Cdc42 in the absence of MGRN1.

In summary, permanent knockdown of MGRN1 in human melanoma cells led to higher intercellular adhesion, partly due to an increase in E-cadherin expression at the plasma membrane. ZEB1 repression in MGRN1-KO cells may account for the transcritional regulation of E-cadherin. Moreover, modulation of RhoA, Rac1 and Cdc42 activation, Rho-GTPases involved in regulation of cytoskeletal dynamics, adhesion and migration, might contribute to the MGRN1-depleted phenotype. These findings are consistent with a less aggressive phenotype of MGRN1-KO human melanoma cells.