NFATc2 Is a Potential Therapeutic Target in Human Melanoma

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NFATc2 Is a Potential Therapeutic Target in Human Melanoma Valentina Perotti, Paola Baldassari, Ilaria Bersani, Alessandra Molla, Claudia Vegetti, Elena Tassi, Jessica Dal Col, Riccardo Dolcetti, Andrea Anichini, Roberta Mortarini Journal of Investigative Dermatology Volume 132, Issue 11, Pages 2652-2660 (November 2012) DOI: 10.1038/jid.2012.179 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Antiproliferative and proapoptotic effects of CsA on human melanoma cells. (a) IC50s (μM) of CsA, by 3-(4,5) dimethylthiazol-2,5-diphenyltetrazolium bromide assay, on 37 melanoma cells lines with mutant or wild-type (WT) BRAF and NRAS. Black symbols, tumors with mutant p53; empty symbols, tumors with p53wt. (b) Cell cycle analysis, at 48hours, of melanoma cells (Me#21158) cultured with/without CsA. Numbers in each panel indicate % of cells in (left to right) sub-G1, G1, S, and G2M phases of the cell cycle. (c) Cell cycle analysis of CsA-treated Me#21158 melanoma cells at 24, 48, and 72hours. (d) Annexin-V/propidium iodide stainings of CsA-treated melanoma cells. CsA, cyclosporin A; FCS, fetal calf serum. Journal of Investigative Dermatology 2012 132, 2652-2660DOI: (10.1038/jid.2012.179) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 NFATc1 and NFATc2 expression in melanoma cells and NFATc2 inhibition by CsA treatment. (a) NFATc2 and NFATc1 expression by flow cytometry in melanoma cell lines (n=37 for NFATc2 and n=18 for NFATc1). (b) NFATc1 and NFATc2 expression by immunohistochemistry in a lymph node metastasis from a melanoma patient. Right panels, two fields showing NFATc2 expression in nuclei and cytoplasm of neoplastic cells (upper panel) and in tumor-associated lymphocytes (lower panel). Bar=50μm (c) Expression of NFATc1 and NFATc2 in nuclear (N) and cytoplasmic (C) fractions of melanoma cell lines cultured O/N with (+) or without (-) FCS (4%). (d) NFATc2 expression by western blot in melanoma cell lines treated with or without CsA (10μM) for 48hours. (e) NFATc2 and HLA class I antigen expression (after staining with w6/32mAb, Parham et al., 1979) by flow cytometry in melanoma cells treated with or without CsA (10μM) for 48hours. Gray histogram, staining with secondary antibody only. CsA, cyclosporin A; Ctrl, control; FCS, fetal calf serum; MFI, mean fluorescence intensity; n.d., not done; NFATc2, nuclear factor of activated T cells c2; siRNA, small interfering RNA; WT, wild type. Journal of Investigative Dermatology 2012 132, 2652-2660DOI: (10.1038/jid.2012.179) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Silencing of NFATc2 in melanoma cells and promotion of apoptosis. (a) NFATc2 expression by flow cytometry after transfection with control or NFATc2-specific small interfering RNA (siRNA). Gray histograms, staining with secondary antibody only. (b) NFATc2 expression by western blot analysis at 48hours after transfection with control (lane #1 and #3) or NFATc2-specific (lane #2 and #4) siRNAs. (c) NFATc2 expression by flow cytometry at 48hours after transfection as in panel a. (d) Release of soluble factors in a BRAFV600E p53wt melanoma cell line (Me#21158) at 72hours after transfection with control (black histogram) or NFATc2-specific siRNA (empty histogram). (e) Cell cycle analysis after transfection of a BRAFV600E p53wt melanoma cell line (Me#2934) with control or NFATc2-specific siRNA. Numbers in each panel indicate % of cells in (left to right) sub-G1, G1, S, and G2M phases of the cell cycle. (f) Annexin-V/propidium iodide stainings of a BRAFV600E p53wt melanoma cell line (Me#2934) after transfection with control or NFATc2-specific siRNA. CsA, cyclosporin A; Ctrl, control; n.d., not done; NFATc2, nuclear factor of activated T cells c2; siRNA, small interfering RNA; WT, wild type. ***P<0.001. Journal of Investigative Dermatology 2012 132, 2652-2660DOI: (10.1038/jid.2012.179) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CsA treatment or NFATc2 silencing activates caspase-dependent melanoma apoptosis. (a, b) Caspase-2 and -3 activity at different time points after melanoma (Me#2934) treatment with 4μM CsA (a), or (b) after transfection with control (-)- or NFATc2-specific siRNA (+) in the presence or absence of the pan-caspase inhibitor z-VAD-fmk or of the negative control z-FA-fmk. Statistical analysis in panels a and b by analysis of variance followed by Student-Newman–Keuls post-test. (c, d) Melanoma apoptosis (in Me#2934 cells) by CsA (4μM) at 72hours (c), or NFATc2 silencing (96hours, d), was inhibited by the pancaspase inhibitor z-VAD-fmk, but not by the negative control z-FA-fmk. Apoptosis assay by annexin-V/propidium iodide flow cytometry. Numbers in dot plots indicate the % of cells in each quadrant. CsA, cyclosporin A; Ctrl, control; NFATc2, nuclear factor of activated T cells c2; RFU, relative fluorescence units; siRNA, small interfering RNA. ***P<0.001. Journal of Investigative Dermatology 2012 132, 2652-2660DOI: (10.1038/jid.2012.179) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Calcineurin inhibition or NFATc2 silencing downregulates the expression of the antiapoptotic protein Apollon. (a, b) Apollon expression by western blot (a) or flow cytometry (b) analyses in melanoma cell lines treated with or without CsA (10μM, 48hours). (c) Apollon and p53 expression by western blot analysis in Me#2934 cells treated with or without CsA (10μM, 48hours) or transfected with control- or NFATc2-specific siRNA (72hours). (d) Apollon expression in Me# 2934 cells (left and middle panels) or in p53-mutant Me#14362 cells (right panel), by flow cytometry, after CsA treatment or NFATc2 silencing. (e) Apollon expression by flow cytometry in Me#21158 melanoma cells treated with or without the NFATc2 inhibitor AM404 (10μM, 72hours). Gray histograms in b, d, and e indicate cells stained with secondary antibody only. CsA, cyclosporin A; Ctrl, control; n.d., not done; NFATc2, nuclear factor of activated T cells c2; siRNA, small interfering RNA; WT, wild type. Journal of Investigative Dermatology 2012 132, 2652-2660DOI: (10.1038/jid.2012.179) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions