Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.

Cecilia Herraiz,Dorothy C Bennett,María Castejón-Griñán,Idoya Martínez-Vicente,Celia Jiménez-Cervantes,Julia Sirés-Campos,Marta Abrisqueta,Conchi Olivares,Jose Carlos García-Borrón

doi:10.3390/cancers12102840

Abstract

Simple SummaryMelanoma, the most aggressive skin cancer, accounts for the majority of deaths due to this disease. Therefore, identification of genes/proteins involved in melanoma genesis and/or progression is urgent. Mutations abrogating expression of Mahogunin Ring Finger 1 (MGRN1) in mice cause complex phenotypes with hyperpigmentation, and known MGRN1 interactors are important regulators of cell shape and movement. This suggests that MGRN1 may modulate the malignant phenotype of melanoma cells. Analysis of MGRN1-KO mouse melanocytes and melanoma cells showed that lack of MGRN1 leads to cell cycle defects and to a more differentiated, less aggressive phenotype, with increased adhesion to various matrices, decreased motility and high genomic instability. The higher aggressivity of MGRN1-expressing melanoma cells was confirmed in an in vivo mouse melanoma model and is consistent with higher survival of human melanoma patients expressing low levels of MGRN1. Therefore, MGRN1 appears an important determinant of the malignant phenotype of melanoma.The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

Highlights

Mahogunin Ring Finger-1 (MGRN1) is a C3HC4 RING-Finger motif-containing nuclear-cytoplasmic E3 ubiquitin ligase well conserved throughout the phylogenetic scale [1,2] and ubiquitously expressed in mammals [3]
Clones were selected based on lack of MGRN1 expression (Figure S1B) and confirmed by Mgrn1 gene amplification and sequencing (Figure S1C)
We showed that Mgrn1 knockdown in normal melanocytes and melanoma cells induced a phenotype characterized by: (i) cell morphology changes with increased size, higher number of dendrites, longer protrusions and higher melanin content, (ii) impaired cell motility and higher adhesion to collagen matrices, (iii) abnormal cell cycle progression with increased percentage of cells in S phase, and (iv) decreased genome stability with accumulation of DNA damage

Summary

Introduction

Mahogunin Ring Finger-1 (MGRN1) is a C3HC4 RING-Finger motif-containing nuclear-cytoplasmic E3 ubiquitin ligase well conserved throughout the phylogenetic scale [1,2] and ubiquitously expressed in mammals [3] In mice, it is encoded by Mgrn, site of the coat color mutant allele mahoganoid (Mgrn1md ) [3] on chromosome 16. Adult homozygous animals develop progressive spongiform neurodegeneration with central nervous system (CNS) vacuolation and features of prion diseases, but without accumulation of prion proteins [2,6] These mice show mitochondrial dysfunction, with reduced expression and activity of electron transport chain proteins and increased oxidative stress in the CNS [7], aberrant patterning of the left-right body axis, congenital heart defects [8], abnormal cranial shape [9] and high embryonic lethality [8]. To date no phenotype like mahoganoid has been described in humans and MGRN1 point mutations are rare (cancer.sanger.ac.uk/cosmic) [11,12]

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