Regulation of Melanoma Progression through the TCF4/miR-125b/NEDD9 Cascade  Florian Rambow, Audrey Bechadergue, Flavie Luciani, Gwendoline Gros, Melanie Domingues, Jacky Bonaventure, Guillaume Meurice, Jean-Christophe Marine, Lionel Larue  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1229-1237 (June 2016) DOI: 10.1016/j.jid.2016.02.803 Copyright © 2016 The Authors Terms and Conditions

Figure 1 miRNA expression profile of phenotypically different melanoma cells. (a) Migratory (wound scratch assay) and invasive (Matrigel assay, Corning) capacities of nine human melanoma cell lines. Class 1 melanoma cell lines (WM793, WM1366, Lu1205, WM852, and A375M) are more aggressive than class 2 melanoma cell lines (501mel, T1, G1, and MNT-1). (b) miRNA expression profiling of the nine melanoma cell lines showed an underexpression of miR-513a-5p, miR-185, and miR-211 and an overexpression of miR-125b, miR-21, miR-100, and miR-199-5p in aggressive melanoma cells (FC > 2, adj. P < 0.01). (c) Poorly aggressive miRNAs were assayed in in vivo melanoma samples (TCGA SKCM cohort). Melanoma samples were classified into invasive (Inv), immune (Imm), and proliferative (Pro) molecular phenotypes based on global mRNA expression according to (Verfaillie et al., 2015). miR-513a, miR-185, and miR-211 were overexpressed in the proliferative category of melanomas. Mann-Whitney test, error bars = 95% confidence interval, ∗P < 0.05, ∗∗P < 10–2, ∗∗∗P < 10–3, and ∗∗∗∗P < 10–4. (d) Aggressive miRNAs were quantified in melanoma samples (TCGA SKCM cohort). miR-125b-1, miR-21, miR-100, and miR-199b were overexpressed in the invasive category of melanomas. adj.P, adjusted P-value; AU, arbitrary unit; FC, fold change; Imm, immune; Inv, invasive; miR, microRNA; miRNA, microRNA; mRNA, messenger RNA; ns, not significant; Pro, proliferative; SKCM, skin cutaneous melanoma; TCGA, The Cancer Genome Atlas. Journal of Investigative Dermatology 2016 136, 1229-1237DOI: (10.1016/j.jid.2016.02.803) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Elevated miR-125b levels are associated with shorter overall survival and aggressiveness in vitro. (a) miR-125b expression levels (Mann Whitney test, P = 3 × 10–4) are 75% higher in metastatic than primary melanoma cells (TCGA SKCM). Error bars represent 95% confidence interval. Mann-Whitney test, ∗∗∗P < 10–3. (b) Survival analysis of metastatic melanoma patients based on high and low miR-125b levels (TCGA SKCM) using 91 patients in both cases. The Kaplan Meier curves for high and low miR-125b levels are significantly different (P < 0.05) as assessed by the Mantel-Cox test. (c) miR-125b is heterogeneously expressed in human melanoma cell lines and primary melanocyte cultures. dp, darkly pigmented; Met, metastatic; miR, microRNA; NHEM, normal human epidermal melanocyte; Prim, primary; RPMMM, reads per million miRNA mapped; SKCM, skin cutaneous melanoma; TCGA, The Cancer Genome Atlas. Journal of Investigative Dermatology 2016 136, 1229-1237DOI: (10.1016/j.jid.2016.02.803) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Inhibition of miR-125b reduces melanoma cell migration and invasion. (a) miR-125b levels in Lu1205 melanoma cells are significantly reduced after antagomiR treatment (quantitative real-time reverse transcription PCR). The control inhibitor was cel-miR-239b. (b) The proliferative capacity of Lu1205 melanoma cells, assessed using FACS-based BrdU incorporation assay, was unaltered after miR-125b inhibition. (c) Migratory capacity, assessed using wound healing assay (wound closure after 15 hours), of Lu1205 melanoma cells was significantly reduced upon miR-125b inhibition. (d) The Matrigel (Corning) invasion capacity of Lu1205 melanoma cells was significantly reduced upon miR-125b inhibition (Mann Whitney test, P = 0.033). ∗P < 0.05, ∗∗P < 10−2. Ctrl, control; ns, not significant; r.u., relative unit. Journal of Investigative Dermatology 2016 136, 1229-1237DOI: (10.1016/j.jid.2016.02.803) Copyright © 2016 The Authors Terms and Conditions

Figure 4 NEDD9 is a miR-125b target involved in melanoma cell migration. (a) Luciferase activity assay validates predicted miR-125b binding site in the NEDD9 3′ untranslated region. The predicted binding site (WT) and a mutated version (MUT) were inserted into the pmiRGLO system. The mutated nucleotides are in red. Upon miR-125b overexpression, luciferase activity was significantly reduced by the presence of the WT sequence. (b) NEDD9 mRNA and protein levels were reduced upon miR-125b overexpression in 501mel melanoma cells, expressing miR-125b at a low level (miR-125blow). (c) NEDD9 mRNA and protein levels were increased upon miR-125b inhibition in Lu1205 melanoma cells, expressing miR-125b at a high level (miR-125bhigh). (d) Single-cell migration speed of miR-125blow 501mel cells was increased and NEDD9 mRNA levels decreased upon miR-125b overexpression. Single-cell migration speed of miR-125bhigh Lu1205 cells was increased and NEDD9 mRNA levels decreased upon siRNA mediated knockdown of NEDD9. Mann Whitney test, error bars = 95% confidence interval; ∗P < 0.05, ∗∗P < 10–2, and ∗∗∗P < 10–3. h, hours; luc, luciferase; miR, microRNA; mRNA, messenger RNA; MUT, mutated form; ns, not significant; NEDD9, neural precursor cell expressed developmentally down-regulated protein 9; RQ, relative quantity; ru, relative unit; siRNA, small interfering RNA; UTR, untranslated region; WT, wild type. Journal of Investigative Dermatology 2016 136, 1229-1237DOI: (10.1016/j.jid.2016.02.803) Copyright © 2016 The Authors Terms and Conditions

Figure 5 TCF4 induces mir-125b-1 expression. MM034 melanoma cells were stably infected with a lentivirus for TCF4 (MM034_TCF). (a) Semi-quantitative reverse transcription-PCR analysis shows up-regulation of TCF4 and mir-125b-1 and down-regulation of NEDD9 in cells stably overexpressing TCF4 relative to MM034 wild-type cells (MM034_WT). (b) Western blot analysis showing the overexpression of TCF4 after viral infection and the reduction of the amount of NEDD9 protein. Actin was used as loading control. (c) Schematic model for the TCF4/miR-125b/NEDD9 cascade. TCF4 drives miR-125b-1 expression, which upon maturation targets the 3′ UTR of NEDD9, leading to a reduction of NEDD9 transcript and protein levels and an increase in melanoma cell migration and invasion. FC, fold change; miR, microRNA; NEDD9, neural precursor cell expressed developmentally down-regulated protein 9; TCF4, transcription factor 4; UTR, untranslated region; WT, wild type. Journal of Investigative Dermatology 2016 136, 1229-1237DOI: (10.1016/j.jid.2016.02.803) Copyright © 2016 The Authors Terms and Conditions