The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors

Abstract

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

Fig. 1. miR-579-3p and MITF are co-regulated in BRAF-mutant melanomas.

A A bubble plot illustrating a selection of KEGG-enriched pathways obtained from ShinyGO with a False Discovery Rate (FDR) below 20% (bubble sizes represent Fold Enrichment). The gene set used for the pathway analysis was derived from miR-579-3p putative targets with a binding score higher than 0.8, as predicted by miRWalk. B Schematic illustration of the cell lines tested for miR-579-3p expression levels (BRAF-mutant; n = 12) (BRAF wild type; n = 8). C Box plot representing miR-579-3p expression levels by qRT-PCR expressed in Log of relative expression in the 20 different melanoma cell lines. U6 was evaluated to normalize the results. D Spearman correlation calculated using qRT-PCR data of MITF and miR-579-3p in BRAF-mutant melanoma cells. E Western blot analyses have been performed on total protein lysates coming from five different melanoma cell lines (WM115, A375, M14, LOX IMVI and WM266) for the indicated antibodies. GAPDH protein has been used as housekeeping for the equal loading. F Heat maps representing the expression levels of p-ERK and MITF proteins (calculated by Image J) and miR-579-3p (Log of relative expression) in the five above indicated BRAF-mutant melanoma cell lines. G Spearman correlation of MITF/miR-579-3p vs p-ERK activation levels in the same cell lines. qRT-PCR data are represented as the mean of at least three independent experiments ± SD. The results are expressed in terms of relative expression of the indicated markers on the appropriate internal controls (GAPDH for MITF and p-ERK; U6 for miR-579-3p). Student’s t test was performed to determine statistical significance (p value < 0.05).

Fig. 2. MITF transcription factor controls miR-579-3p expression.

A Schematic illustration of the promoter region of ZFR/miR-579 gene showing the two MITF binding sites located −1182 bp and −361 bp upstream of the transcription start site (TSS). B Spearman’s correlation coefficient was calculated on 74 matched samples from the GSE54467 dataset, using miRNA/mRNA expression levels. C Quantification of miR-579-3p, MITF and TYR by using qRT–PCR in LOX IMVI, WM266 and M14 cell lines following 48 hours of transient transfection with scrambled (SCR) sequences or MITF siRNA. U6 and GAPDH were evaluated to normalize the results through ΔΔCt method. D Chromatin immunoprecipitation (ChIP) was performed on DNA extracted from LOX IMVI cells incubated with an anti-MITF antibody or with anti-mouse IgG, used as control. PCR analyses were used to evaluate MITF binding on ZFR/miR-579 or TYR promoter regions, using specific primers. Luciferase reporter assays of the constructs containing a region of 1000 bp with the two MITF binding sites in miR-579 promoter (E) (each point represent a biological replicate) or with the deletion of MITF binding sites (F) (Del1, Del2 or Double del) were used to test the capability of MITF to bind these regions. pGL3 plasmid (Basic) was used as control. Transient transfections of the above mentioned plasmids (500 ng each) have been performed in the presence or not of MITF siRNA or SCR for 48 hours. pLX313-Renilla plasmid (50 ng) has been used to normalize results. Student’s t test was performed to determine statistical significance *p < 0.05; **p < 0.01; and ***p < 0.001. qRT-PCR data are represented as mean (n = 3) ± SD; luciferase results are expressed as the mean of at least three independent experiments ±SEM.

Fig. 3. miR-579-3p is able to stabilize MITF protein and to induce its own transcription.

A Western blot analyses have been performed on total protein lysates extracted from M14 (upper panel) or WM266 (lower panel) cell lines treated or not with Dabrafenib (500 nM) for 4, 16, 24 and 48 hours. GAPDH protein has been used as housekeeping for the equal loading. B qRT-PCR analyses have been performed to detect miR-579-3p expression levels in the same experimental conditions. U6 was evaluated to normalize the results through ΔΔCt method. C Western blot analyses for the indicated antibodies have been performed on M14 (upper panel) or WM266 (lower panel) cell lines following 72 hours of transient transfection with SCR sequences or miR-579-3p mimic sequences. α−Tubulin protein has been used as housekeeping for the equal loading. D qRT-PCR analyses have been performed to detect pri-miR-579 expression levels in the same experimental conditions. GAPDH was evaluated to normalize the results through ΔΔCt method. Student’s t test was performed to determine statistical significance *p < 0.05; **p < 0.01; and ***p < 0.001. qRT-PCR data are represented as mean (n = 3) ± SD.

Fig. 4. miR-579-3p overexpression induces senescence features in BRAF-mutant melanoma cells.

A M14 and WM266 cells left untreated, treated with Dabrafenib, transfected with miR-579-3p mimic or with SCR sequences have been stained after 72 hours with crystal violet to measure proliferation. B M14 and WM266 cells treated as reported above were fixed and stained for b-galactosidase after 72 hours of the relative treatments. Results have been then quantified counting b-Gal positive cells over the total cells present in ten different fields. C Representative images are reported at original magnification= ×20, the arrows indicate b-Gal positive cells. Student’s t test was performed to determine statistical significance ****p < 0.0001.

Fig. 5. miR-579-3p and MITF levels are lost in melanoma cells and tumors from patients relapsing after targeted therapies.

A LOX IMVI melanoma cells have been subjected to increasing concentrations of Dabrafenib for two months (from 50 nM to 1 μM) and at each step of drug increase total RNAs was collected to test the levels of mir-579-3p, MITF, TYR, and AXL by qRT-PCR. Results were expressed in Log of relative expression over untreated cells; U6 and GAPDH were used to normalize the results. Representative pictures of the cellular pellets are showed in the same experimental conditions. B Western blot analyses (left panel) have been performed on total protein lysates extracted from LOX IMVI sensitive melanoma cells or rendered resistant to Dabrafenib. GAPDH protein has been used as housekeeping for the equal loading. qRT-PCR analyses for mir-579-3p expression levels (right panel) have been performed in the same cell lines and U6 was evaluated to normalize the results through ΔΔCt method. C Spearman’s correlation coefficient was calculated on 74 matched samples from the GSE54467 dataset, using miRNA/mRNA expression levels. D LOX IMVI BRAFi-resistant melanoma cells have been transiently transfected with an expression vector coding for MITF (500 ng) and relative empty control (500 ng) for 48 hours. Cells have been then subjected to Western blot (left panel) and qRT-PCR (right panel) analyses for the indicated markers. GAPDH protein and U6 have been used to normalize results. E The same experimental conditions (LOX IMVI res cells transfected with empty or MITF vectors) have been treated with Dabrafenib (as BRAFi, 500 nM) alone or in combo with Trametinib (as MEKi, 10 nM) and after 72 hours cells have been stained with crystal violet. The relative adsorbance (595 nm) was read using a microplate ELISA reader after dissolving the dye trapped in the adherent cells using a methanol/SDS solution. F miR-579-3p and MITF expression levels have been evaluated following RNA extraction from matched formalin-fixed paraffin-embedded (FFPE) melanoma samples before initiation of targeted therapy (Pre) and after disease progression (PD) (n = 14). GAPDH and U6 have been used to normalize results. Student’s t test was performed to determine statistical significance *p < 0.05; **p < 0.01. qRT-PCR data are represented as mean (n = 3) ± SD; cell viability results are expressed as the mean of at least three independent experiments ±SEM.

Fig. 6. The model depicting the reciprocal interplay between miR-579-3p and MITF in BRAF-mutant melanoma cells through the modulation of MAPK signaling pathway.

This image was created with BioRender (https://biorender.com/).