MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.This research was funded by grant SAF2018_RTI2018-094929-B-I00 financed by FEDER/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación (Spain) (to C.J.-C. and J.C.G.-B.), and by grant UPV/EHU GIU20/035 (to S.A and M.D.B.)

Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters

A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA

Common variants of the MC1R gene coding the α-melanocyte stimulating hormone receptor are associated with light skin, poor tanning, blond or red hair, and increased melanoma risk, due to pigment-dependent and -independent effects. This complex phenotype is usually attributed to impaired activation of cAMP signaling. However, several MC1R variants show significant residual coupling to cAMP and efficiently activate mitogenic extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Yet, residual signaling and the key actions of wildtype and variant MC1R have never been assessed under strictly comparable conditions in melanocytic cells of identical genetic background. We devised a strategy based on CRISPR-Cas9 knockout of endogenous MC1R in a human melanoma cell line wildtype for BRAF, NRAS and NF1, followed by reconstitution with epitope-labeled MC1R constructs, and functional analysis of clones expressing comparable levels of wildtype, R151C or D294H MC1R. The proliferation rate, shape, adhesion, motility and sensitivity to oxidative DNA damage were compared. The R151C and D294H RHC variants displayed impaired cAMP signaling, intracellular stability similar to the wildtype, triggered ERK1/2 activation as effectively as the wildtype, and afforded partial protection against oxidative DNA damage, although less efficiently than the wildtype. Therefore, common melanoma-associated MC1R variants display biased signaling and significant genoprotective activity

Heterodimerization of MC1R and MC1R-TUBB3 chimeric isoforms.

<p>(A) Co-immunoprecipitation of MC1R-001 and the MC1R-TUBB3 chimeric isoforms. HEK293T cells expressing the indicated constructs were lysed and immunoprecipitated for Flag-labelled MC1R-001, Iso1 or Iso2 using an anti-Flag monoclonal antibody. The pellets were electrophoresed and blotted for HA-labelled MC1R-001 (with a specific anti-HA monoclonal antibody) or for Flag-labelled MC1R-001, Iso1 or Iso2 (with anti-Flag monoclonal antibody) as a control for efficient immunoprecipitation. Total lysates were also electrophoresed and blotted as expression controls (n = 3, representative blots are shown). (B) Co-immunoprecipitation of V60L or R151C variant MC1R-001 and WT MC1R-TUBB3 intergenic splice isoform Iso1. The indicated constructs were expressed in HEK293T cells and immunoprecipitated for Flag-labelled MC1R-001, V60L or R151C using an anti-Flag monoclonal antibody. Immunoblots for Flag-tagged constructs and TUBB3 are shown for immunoprecipitated and total lysates. (C) Representative confocal images of MC1R-001 (green) and Iso1 or Iso2 (red) immunostaining in HBL cells transiently transfected with HA-labelled MC1R-001 and Flag-labelled MC1R-TUBB3 chimeric isoforms. Scale bar, 10 μm. Representative line scan (right panel) from multiple experimental repeats across the cell (location indicated in merged image) shows co-localization of canonical MC1R-001 and chimeric proteins. Line scan, 20 μm for MC1R-001+Iso1, and 31 μm for MC1R-001+Iso2. (D) Effect of heterodimerization on functional coupling to cAMP. Intracellular cAMP levels in HEK293T cells expressing WT, V60L or R151C MC1R-001 alone or in combination with Iso1 and Iso2 upon stimulation with 10⁻⁷ M NDP-MSH for 30 min. Results are presented as residual cAMP production relative to WT MC1R-001 (for which cAMP levels were 0.096±0.043 and 0.889±0.071 pmol/μg protein in resting and stimulated conditions respectively) (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values *p< 0.05, **p< 0.01, ***p< 0.001). (E) Specific binding of [¹²⁵I]-NDP-MSH (5x10^-11 M and 5x10⁴ cpm) to HEK293T cells expressing MC1R-001 or the MC1R-TUBB3 isoforms Iso1 and Iso2, alone or in combination (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, ***p< 0.001). (F) Agonist internalization index in HEK293T cells co-transfected with MC1R-001 and Iso1 or Iso2 upon incubation with [¹²⁵I]-labelled NDP-MSH (5x10^-11 M and 5x10⁴ cpm) for 90 min (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, ***p< 0.001).</p

MC1R transcripts and intergenic splice isoforms of MC1R and TUBB3.

<p>(A) Schematic panel showing the exon organization of MC1R splice variants (MC1R-001 and MC1R-002) and MC1R-TUBB3 chimeric transcripts, Iso1 and Iso2. Exons of all MC1R derived transcripts are represented in colored boxes and the number of nucleotides in the ORF is shown below. (B) Diagram representing the structural domains of MC1R-001, MC1R-002, β-tubulin III (TUBB3) and chimeric proteins Iso1 and Iso2. Structural and functional domains are depicted in colored boxes and the number of key residues in the proteins is shown. TM indicates transmembrane regions of MC1R. Dashed lines indicate residues of MC1R and TUBB3 linked in fused proteins Iso1 and Iso2. (C) MC1R-001, Iso1 and Iso2 expression in human melanoma cell lines. Data are shown as relative expression of each isoform (as indicated in each bar graph) as compared with the levels of the isoform in HBL cells. (D) Expression of Iso1 and Iso2 mRNA as a function of the levels of the canonical MC1R-001 transcript in a panel of human melanoma cell lines. Data are represented as mRNA expression of the two intergenic splicing forms relative to MC1R-001 in each cell line.</p

Effects of intergenic splicing on the functional coupling of MC1R to the cAMP and ERK1/2 pathways.

<p>(A) Agonist-induced cAMP production in HEK293T cells expressing MC1R-001, Iso1, Iso2, or the natural MC1R-001 variant allelesV60L, V92M, R151C and D294H. Cells were incubated with 10⁻⁷ M NDP-MSH for 30 min and cAMP levels were determined by an immunoassay (n = 6, error bars are ±SEM, two-sided Student´s t test was used to generate p values, *p< 0.05, *** p< 0.001). (B-C) Representative immunoblots (B) and quantification (C) of ERK1 and ERK2 phosphorylation in PC12 cells transfected to express MC1R-001, Iso1 or Iso2 and stimulated with NDP-MSH (10⁻⁷ M) for the times indicated (n = 5, error bars are ±SEM, two-sided Student´s t test was used to generate p values, *p< 0.05, ** p< 0.01).</p

Electrophoretic analysis and intracellular stability of MC1R-TUBB3 isoforms.

<p>(A) Expression of canonical and chimeric MC1R proteins in heterologous HEK293T cells. HEK293T cells were transiently transfected to express Flag-labelled WT MC1R-001, Iso1 and Iso2. Cells were detergent-solubilized, electrophoresed and blotted. For MC1R detection, cell lysates were probed with an anti-Flag monoclonal antibody (upper blot). Membranes were also probed for TUBB3 (middle blot) and ERK2 (lower blot), as loading control (n = 5, representative blots are shown). (B) Electrophoretic pattern of MC1R-TUBB3 transcripts expressed in HBL human melanoma cells. Representative immunoblots for MC1R, TUBB3 and ERK2 are shown as in panel A (n = 5, representative blots are shown). (C) Intracellular stability of MC1R-TUBB3 chimeric fusion proteins in HEK293T cells. Flag-labelled MC1R-001, Iso1 and Iso2 were expressed in HEK293T cells. Cells were incubated with the protein synthesis inhibitor cycloheximide (Chx, 0.1 mM) for the times indicated, lysed and the levels of residual proteins in cell extracts were detected by Western blot. Representative immunoblots probed for MC1R-001, Iso1 or Iso2 with anti-Flag are shown. (D) Semi-log graph for calculation of half-lives. The intensity of receptor bands in the blots as in panel C was quantitated with ImageJ and the semi-log of residual signals was plotted against time. Half-life (t½) values correspond to the slope of the resulting lines.</p

Radioligand binding and intracellular trafficking properties of MC1R-TUBB3 isoforms.

<p>(A-B) Competition binding assay of HEK293T cells transfected with MC1R-001, Iso1 and Iso2. Cells were incubated with ¹²⁵I-labelled NDP-MSH (5x10^-11 M) and increasing concentrations of non-labelled competing NDP-MSH, from 10⁻¹² to 10⁻⁷ M, extensively washed and counted for radioactivity. Non-specific binding was determined with non-transfected cells or with transfected cells incubated with the radioactive tracer in the presence of excess (10⁻⁶ M) non-labelled peptide, with the same results. Values are represented as specifically bound [¹²⁵I]-NDP-MSH (A) and as percentage of residual binding (B) at the different ligand concentrations (n = 3, data are given as mean ±SEM). (C) Flow cytometric analysis of HEK293T cells expressing MC1R-001 and MC1R-TUBB3 chimeric isoforms. Non-permeabilized cells expressing the indicated proteins were incubated with an anti-Flag antibody labelled with phycoerythrin. Since the Flag epitope was fused in-frame to the extracellular N-terminus of the MC1R sequence, only cells expressing the constructs of the plasma membrane should be detected. Histograms represent cell number (counts) as a function of Flag surface staining, on a logarithmic scale. The gray filled curve refers to cells transfected with an empty pcDNA3 (n = 3, representative histograms are shown). (D) Left panel: Representative confocal images of MC1R-001 or the chimeric isoforms (red) and calnexin (green) immunostaining in HEK293T cells transiently transfected with Flag-labelled MC1R-001 and MC1R-TUBB3 constructs. Scale bar, 10 μm. Representative line scan (right panel) from multiple experimental repeats across the cell (location indicated in merged image) shows co-localization of MC1R-TUBB3 transcripts and calnexin. Line scan, 19 μm for MC1R-001, 17 μm for Iso1 and 18 μm for Iso2. (E) Radioligand internalization assay performed on HEK293T cells expressing MC1R-001, Iso1 or Iso2 incubated with ¹²⁵I-labelled NDP-MSH. The radioactive tracer was isotopically diluted to achieve a final concentration of 5x10^-11 M and 5x10⁴ counts/well. Externally bound agonist was separated by an acid wash procedure. Both the externally bound ligand present in the acid washes and the internalized ligand associated with the cell pellets were counted. The internalization index represents the percentage of ligand internalized referred to total radioligand bound (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, *p<0.05, **p<0.01).</p