MiR-200a Regulates CDK4/6 Inhibitor Effect by Targeting CDK6 in Metastatic Melanoma  Matias A. Bustos, Shigeshi Ono, Diego M. Marzese, Takashi Oyama, Yuuki Iida, Garrett Cheung, Nellie Nelson, Sandy C. Hsu, Qiang Yu, Dave S.B. Hoon  Journal of Investigative Dermatology  Volume 137, Issue 9, Pages 1955-1964 (September 2017) DOI: 10.1016/j.jid.2017.03.039 Copyright © 2017 The Authors Terms and Conditions

Figure 1 MiR-200a is down-regulated in metastatic cutaneous melanoma. (a) Bar chart or (b) boxplot of miR-200a expression from melanoma samples of TCGA database for each group: PRM, LNM, or DOM (Wilcoxon test). (c) Analysis of PEAT tissue from JWCI database by miR-qPCR (Wilcoxon test). (d, e) Melanoma lines (MNC, LNM, and DOM) profiled for miR-200a expression (Wilcoxon test). *P < 0.05, **P < 0.01, ***P < 0.001. DOM, distant organ metastasis; JWCI, John Wayne Cancer Institute; LNM, lymph node metastasis; miR, microRNA; MNC, melanocytes; NEV, benign nevi specimen; PEAT, paraffin-embedded archival tissue; PRM, primary melanoma; RPKM, reads per kilobase of transcript per million mapped reads; qPCR, quantitative PCR; TCGA, The Cancer Genome Atlas. Journal of Investigative Dermatology 2017 137, 1955-1964DOI: (10.1016/j.jid.2017.03.039) Copyright © 2017 The Authors Terms and Conditions

Figure 2 MiR-200a expression is regulated by epigenetic modifications. (a) miR-200b/200a/429 cluster structure (blue) and the CGIs (green) at chromosome 1. (b) DNA methylation levels (beta values) of miR-200b/200a/429 cluster and the two CGIs in melanoma lines (n = 13) depicted as low miR-200a (blue) or high miR-200a (green) expression. (c) Correlation analysis between DNA methylation and miR-200a expression. Each point represents a CpG site throughout the miR-200b/200a/429 cluster and the two CGIs. Orange dashed lines indicated the statistically significant threshold for correlation analysis (P = 0.05). ENCODE data analysis for chromatin accessibility by DHS sequencing. (d) Comparison of chromatin accessibility among primary melanocytes (Melano) and two metastatic melanoma cell lines (Colo 829 and Mel 2183). (e) Chromatin status for the three cell lines is also shown in the merge image. CGI, CpG island; chr, chromosome; DHS, DNase I hypersensitive sites; kb, kilo base pair; MIR/miR, microRNA. Journal of Investigative Dermatology 2017 137, 1955-1964DOI: (10.1016/j.jid.2017.03.039) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Exogenous miR-200a expression targets CDK6 in melanoma lines. (a) Heat map representation for the z-scores values of 390 genes differentially expressed by miR-200a expression. (b) In silico analysis of three different miR databases showed that 5 of 143 genes down-regulated by miR-200a expression were predicted as target of miR-200a. Analysis of (c) miR-200a, (d) CDK6 mRNA, or (e, f) protein levels in metastatic melanoma lines (DP, HM, and WP) transfected with miR-200a or miR-ctrl precursor (t test). (g) Scheme for CDK6 mRNA (blue) depicting the 10Kb 3′-UTR region (light blue) and the seven MREs for miR-200a (black bars). (h) Luciferase activity was measured after the co-transfection of miR-200a or miR-ctrl with a negative control (ctrl-), positive (ctrl+), 3′-UTR-1, or 3′-UTR-2 (t test). Data represented the mean of three experiments ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. ctrl, control; miR, microRNA; MRE, miR-recognition elements; NS, not significant; Rel, relative; UTR, untranslated region. Journal of Investigative Dermatology 2017 137, 1955-1964DOI: (10.1016/j.jid.2017.03.039) Copyright © 2017 The Authors Terms and Conditions

Figure 4 MiR-200a induces cell cycle arrest in melanoma cells. (a) WB analysis of Rb1 or p-Rb1 on Ser780, Ser795, or Ser807/811 after transfection with miR-200a or miR-ctrl (DP, HM, and WP); β-actin was used as loading control. (b) Quantification of p-Rb1 on Ser795, in miR-200a or miR-ctrl transfected cells (t test). Flow cytometry analysis in (c) DP or (d) HM cells transfected with miR-200a or miR-ctrl. Column chart represents the percentage of cells in G0/G1 phases in miR-200a and miR-ctrl groups (t test). WB for (e) E2F1 or (f) its quantification in miR-200a– or miR-ctrl–overexpressing cells; β-actin was used as loading control (t test). (g) Quantification of Ki-67 levels in miR-200a– or miR-ctrl–transfected cells (t test). Data represented the mean of three experiments ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. ctrl, control; miR, microRNA; NS, not significant; p-, phosphorylated; PI, propidium iodide; WB, Western blot. Journal of Investigative Dermatology 2017 137, 1955-1964DOI: (10.1016/j.jid.2017.03.039) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Palbociclib arrests cell cycle by targeting CDK4 and CDK6. (a) WB analysis for CDK6, CDK4, and p-Rb1 Ser780 and Ser807/811 in three melanoma lines overexpressing miR-200a and treated with 100 nmol/L palbociclib. (b) Cells were transfected with miR-200a or miR-ctrl precursors and treated with palbociclib (1, 10, and 100 nmol/L). Then, cells were stained with propidium iodide and analyzed for cell cycle populations by flow cytometry. Bar chart represents the percentage of cells in G0/G1 phases in the miR-200a and miR-ctrl groups after palbociclib treatment (t test). (c) Schematic working model: miR-200a is down-regulated through melanoma progression. In metastatic melanoma, with low miR-200a, CDK6 is up-regulated, making them sensitive to palbociclib. Conversely, overexpression of miR-200a targets CDK6 and limits the effect of palbociclib to CDK4. *P < 0.05. ctrl, control; M, mol/L; miR, microRNA; p-, phosphorylated; PI, propidium iodide; WB, Western blot. Journal of Investigative Dermatology 2017 137, 1955-1964DOI: (10.1016/j.jid.2017.03.039) Copyright © 2017 The Authors Terms and Conditions