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. 2019 May;38(22):4384-4396.
doi: 10.1038/s41388-019-0729-2. Epub 2019 Feb 1.

An actionable axis linking NFATc2 to EZH2 controls the EMT-like program of melanoma cells

Valentina Perotti  1 , Paola Baldassari  1 , Alessandra Molla  1 , Gabriella Nicolini  1 , Ilaria Bersani  1 , Giulia Grazia  1 , Fabio Benigni  2 , Andrea Maurichi  3 , Mario Santinami  3 , Andrea Anichini  1 , Roberta Mortarini  4
Affiliations

An actionable axis linking NFATc2 to EZH2 controls the EMT-like program of melanoma cells

Valentina Perotti et al. Oncogene. 2019 May.

Abstract

Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2+ EZH2+ melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
NFATc2 expression correlates with EMT-related markers in melanoma. a Western blot analysis for NFATc2, ZEB1, SNAIL, N-cadherin, α-catulin, AXL, MITF, E-cadherin, ZEB2, and TWIST, in melanoma cell lines with the indicated genotype for BRAFV600E or NRASQ61R mutations. b Hierarchical clustering of log2-transformed and median-centered qPCR mRNA expression levels (2−ΔCt) for MITF, NFATc2, AXL, SNAI1, CDH2, ZEB1 genes in 30 melanoma cell lines with the indicated genotype for BRAFV600E or NRASQ61R mutations. c Spearman correlation analysis of mRNA expression values obtained by qPCR for the indicated genes in the panel of 30 melanoma cells lines shown in b. Positive and negative r-values are indicated in light blue and yellow, respectively.*p < 0.05; **p < 0.01; ***p < 0.001. d Spearman correlation analysis of the indicated genes in the TCGA melanoma dataset; r-values indicated as in c
Fig. 2
Fig. 2
Targeting of NFATc2 in melanoma cells downregulates EMT-related markers and AXL. a Expression by western blotting of NFATc2, ZEB1, N-cadherin, α-catulin, SNAIL, and E-cadherin in three melanoma cell lines (Me71, Me79, Me98) at 72 h after transfection with two different NFATc2-specific Stealth siRNA (NFATc2 siRNA-1 and siRNA-2) or with control siRNA (ctrl siRNA). b, c Expression of NFATc2, ZEB1, N-cadherin, α-catulin, SNAIL, and E-cadherin in b NFATc2 shRNA stable transfectants (NFATc2 shRNA_86a and NFATc2 shRNA_87a) or in control cells (ctrl shRNA), or c in melanoma cell lines Me71, Me79, and Me98 treated or not for 144 h with the NFATc2 inhibitor AM404 (at 20 μM). d Expression of NFATc2 and AXL in melanoma cell lines Me51 (top panels) and Me67 (bottom panels) after NFATc2 targeting by siRNA as in a, or by AM404 as in c
Fig. 3
Fig. 3
Targeting of NFATc2 in melanoma cells downregulates c-Myc, FOXM1, and EZH2. a Expression by western blotting of NFATc2, c-Myc, FOXM1, EZH2, and H3K27me3 in three melanoma cell lines (Me71, Me79, Me98) at 72 h after transfection with two different NFATc2-specific Stealth siRNA (NFATc2 siRNA-1 and siRNA-2) or with control siRNA (ctrl siRNA). b, c Expression of NFATc2, c-Myc, FOXM1, EZH2, and H3K27me3 in b NFATc2 shRNA stable transfectants (NFATc2 shRNA_86a, and NFATc2 shRNA_87a) or in control cells (ctrl shRNA), or c in melanoma cell lines Me71, Me79, and Me98 treated or not for 144 h with the NFATc2 inhibitor AM404 (at 20 μM)
Fig. 4
Fig. 4
Regulation of FOXM1 and EZH2 by c-Myc, of EZH2 by FOXM1 and downregulation of EMT-related markers by targeting of c-Myc, FOXM1 and EZH2. a Expression by western blotting of c-Myc, FOXM1, EZH2, H3K27me3 and of different EMT-related markers in melanoma cell line Me79 at 72 h after transfection with two different c-Myc-specific Stealth siRNA (c-Myc siRNA-1 and siRNA-2) or with control siRNA (ctrl siRNA), or at 48 h after treatment with c-Myc inhibitor 10058-F4 at 20 μM. b, c expression by western blotting of the indicated proteins after transfection of Me79 at 72 h with two different FOXM1-specific siRNA b or with two different EZH2-specific siRNA c or with control siRNA, or at 48 h after treatment with FOXM1 inhibitor Siomycin A at 2 μM b or with EZH2 inhibitor GSK126 at 5 μM c
Fig. 5
Fig. 5
Targeting of NFATc2, Myc, FOXM1, and EZH2 inhibits melanoma migratory and invasive activity. a Top panels: reduced melanoma cell migration in NFATc2 shRNA transfectants (NFATc2 shRNA_86a and NFATc2 shRNA_87a) compared to control transfectants (Ctrl_shRNA), evaluated by the wound closure assay. Bottom panels: representative images of the wound closure assay performed with NFATc2 shRNA transfectants and control transfectants. b Inhibition of Me71 melanoma migration by the wound closure assay by treatment for 48 h with inhibitors of NFATc2 (AM404), c-Myc (10058-F4), FOXM1 (Siomycin A), or EZH2 (GSK126). Results expressed as % closed wound area. c Inhibition of melanoma cell invasive activity, evaluated at 8 h by the CultrexCoat BME Cell Invasion Assay. Top panels: NFATc2 shRNA transfectants compared to control transfectants. Middle and bottom panels: melanoma cell line Me71 pre-treated for 48 h with the indicated inhibitors as in a. Statistical analysis in a, b, c by Student's t-test. ****p < 0.001. Error bars indicate mean ± SD. Data from four-independent experiments
Fig. 6
Fig. 6
Promotion of melanoma apoptosis and upregulation of ATF3 by combinatorial treatment with NFATc2 and EZH2 inhibitors. a, b Evaluation of apoptosis by PI/Annexin-V assay on BRAFV600E-mutant and BRAF-inhibitor-resistant melanoma cell lines Me99, Me78, and Me27 (PLX4720 IC50 values: 5.638 μM, 1.593 μM, 0.582 μM, respectively, as reported in ref. 35), at 48 h after treatment with NFATc2 inhibitor AM404 (15 μM), or EZH2 inhibitor GSK126 (10 μM), or their combination, or the BRAFV600E-specific inhibitor PLX4720 (at 1 μM on Me99 and Me78; at 0.25 μM on Me27). Data of a representative experiment shown in a, data from three-independent experiments shown in b. c Expression of ATF3, by western blot analysis, in 7 BRAF-mutant melanoma cell lines, upon treatment o/n with PLX4720 or with the combination of AM404 and GSK126. d Promotion of apoptosis, by Annexin-V/PI assay at 48 h, in BRAF/NRAS wild type (top panels) or NRASQ61R-mutant (bottom panels) melanoma cell lines after treatment with NFATc2 inhibitor AM404 (15 μM), or with EZH2 inhibitor GSK126 (10 μM), or with the combination of AM404 and GSK126. Data from three-independent experiments. b, d results expressed as % live cells (PI− Annexin-V− cells). Statistical analysis in b, d by one-way ANOVA followed by SNK test; *p < 0.05; **p < 0.01; ***p < 0.001. Error bars indicate mean ± SD
Fig. 7
Fig. 7
Modulation of subcellular localization of NFATc2 by Zoledronic acid and anti-tumor effect of the combination of Zoledronic acid with GSK126 and of a GSK-3β inhibitor with GSK126. a Expression of NFATc2 by western blot analysis in cytoplasmic (C) or nuclear (N) fractions of melanoma cells treated or not with zoledronic acid (indicated as Zol, 150 μM, 72 h). b Anti-proliferative effect by MTT assay of Zoledronic acid (at three different doses in each cell line), GSK126 or their combinations on melanoma cell lines belonging to distinct mutational subset. c Apoptosis by annexin-V/PI assay at 72 h in melanoma cells treated with Zoledronic acid, GSK126 or their combinations. d Apoptosis by annexin-V/PI assay at 72 h in melanoma cells treated for 48 h with GSK-3β inhibitor AR-014418 (at 7.5 μM) and GSK126 (10 μM) or their combinations. Data from three-independent experiments. Statistical analysis in b, c, d by ANOVA and SNK post test. *p < 0.05; **p < 0.001, ***p < 0.001. Error bars indicate mean ± SD
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