Inhibition of CRM1-Mediated Nucleocytoplasmic Transport: Triggering Human Melanoma Cell Apoptosis by Perturbing Multiple Cellular Pathways  Gaurav Pathria, Christine Wagner, Stephan N. Wagner  Journal of Investigative Dermatology  Volume 132, Issue 12, Pages 2780-2790 (December 2012) DOI: 10.1038/jid.2012.233 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Chromosome region maintenance 1 (CRM1) expression is significantly upregulated in human metastatic melanoma. Relative mRNA expression levels (y-axis) of CRM1, RAN (RAN-GTPase), RANBP1, and RANGAP1 in melanocytic nevi (n=9), primary human melanoma (n=31), human melanoma metastases (n=52), and human melanoma cell lines (n=15). ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05. Error bars indicate ±SD. Journal of Investigative Dermatology 2012 132, 2780-2790DOI: (10.1038/jid.2012.233) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Chromosome region maintenance 1 (CRM1) inhibition induces cytoplasmic extracellular signal–regulated kinase (Erk) depletion. (a, left) Immunofluorescent staining for Erk1/2 in the indicated cell lines upon 6 hours of treatment with leptomycin B (LMB; left panel). Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; middle panel). Overlay of Erk1/2 and nuclear stain (right panel). (a, right) Quantification of Erk1/2 cellular distribution. Cells were scored for predominant cytoplasmic (C>N), equal cytoplasmic and nuclear (C=N) or predominant nuclear (C<N) distribution of Erk1/2. More than 80 cells were scored and the distribution was plotted (y-axis) as the percentage of all the cells scored. Error bars indicate ±SD from the representative experiment performed in duplicate. (b) Immunofluorescence-based time-course analysis of Erk1/2 localization (left panel) in UACC62 cells. Cells were stained at 6, 18, or 48 hours after leptomycin B (LMB) treatment. Nuclei stained with DAPI (middle panel). Right panel shows the overlay. (SK28—SK-MEL-28). Journal of Investigative Dermatology 2012 132, 2780-2790DOI: (10.1038/jid.2012.233) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Chromosome region maintenance 1 (CRM1) inhibition mediates extracellular signal–regulated kinase (Erk)-signaling hyperactivation. (a, left) Immunofluorescence staining for phospho-Erk1/2 (p-Erk1/2) in the indicated cell lines upon 6 hours of leptomycin B (LMB) treatment. (a, right) Quantification was performed as discussed in Figure 2a. (b) Western blotting for p-Erk1/2, Erk1/2, phospho-p90RSK1 (p-p90RSK1), and p90RSK1 in the indicated cell lines treated with LMB for 6 or 18 hours. (c) Western blotting for CRM1, p-Erk1/2, and Erk1/2 in the indicated cell lines upon 72 hours of treatment with control small interfering RNA (Ctrl-siRNA) and siRNA–CRM1 (si-CRM1#1 or si-CRM1#2). (d, upper panel) UACC62 cells were treated with an increasing dose of LMB (as indicated) for 6 hours, followed by western blotting for p-Erk1/2 and Erk1/2. (d, lower panel) Densitometric units of p-Erk1/2 normalized with Erk1/2. (e) UACC62 cells were treated for 6 hours as indicated, followed by assessment of phospho-p90RSK1 and p90RSK1 levels by western blotting. (f) Amino-acid sequence alignment of a potential nuclear export sequence (NES) in p90RSK1 (aa 174–183) with established NES from the indicated proteins. (g, top) Upper panel, immunofluorescence-based analysis of p90RSK1 distribution in UACC62 cells upon 6 hours of LMB treatment. Middle panel, nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Lower panel, overlay of p90RSK1 and the nuclear stain. (g, bottom) Quantification of p90RSK1 cellular distribution. (h) Indicated cell lines were treated with LMB for 18 hours, followed by western blotting for c-Fos. (SK5—SK-MEL-5). Journal of Investigative Dermatology 2012 132, 2780-2790DOI: (10.1038/jid.2012.233) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Chromosome region maintenance 1 (CRM1) inhibition suppresses melanoma cell viability through apoptotic induction. (a) Indicated cell lines were left untreated or treated as shown (x-axis). Cell viability estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is expressed as absorbance at 492nm (y-axis). Error bars indicate ±SD from the representative experiment performed in triplicate. (b, left) Western blotting for CRM1 in the indicated cell lines 72 hours after control small interfering RNA (Ctrl-siRNA) or si-CRM1#1 treatment. (b, right) Increase in absolute cell numbers (y-axis) was determined by subtracting cell number at the time of seeding from the cell number at 96 hours post si-CRM1#1 transfection. Error bars indicate ±SD from the representative experiment performed in triplicate. (c) Cell-cycle analysis of UACC62 and M14 cells 24 hours post leptomycin B (LMB) treatment. (d) UACC62 and M14 cells were treated with LMB for 48 hours, followed by AnnexinV/propidium iodide (PI) staining. Numbers on the top right quadrant indicate the proportion of AnnexinV-positive (early apoptosis) and AnnexinV+PI-positive (late apoptosis) cells. (e) UACC62 and M14 cells were treated for 18 hours with LMB followed by western blotting for cleaved caspase-3. (f) UACC62 and M14 cells were treated with indicated drug combinations for 48 hours followed by AnnexinV/PI staining. (g) p′Mel-BRAFV600E and IMR90 cells were treated with LMB for 48 hours followed by AnnexinV/PI staining. (h) UACC62 and M14 cells were treated with LMB for 18 hours, followed by detection of cleaved caspase-9 by western blotting. (i) Indicated cell lines were treated with LMB for 18 hours; caspase-8 (uncleaved and cleaved fragments) and uncleaved-Bid were detected by western blotting. (j) UACC62 and M14 cells were treated with LMB for 32 hours followed by western blotting for Bcl-2. (k) AnnexinV/PI staining in UACC62, SK28, and M14 cells upon the indicated set of treatments for 48 hours. Z-IETD-FMK (20μM), caspase-8 inhibitor; Z-VAD-FMK (40μM), pan-caspase inhibitor; DMSO, solvent for caspase inhibitors. Journal of Investigative Dermatology 2012 132, 2780-2790DOI: (10.1038/jid.2012.233) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Chromosome region maintenance 1 (CRM1) inhibition induces Survivin nuclear entrapment and downregulation. (a) Relative Survivin mRNA levels (y-axis) in melanocytic nevi, primary melanoma, metastatic melanoma, and melanoma cell lines as determined by complementary DNA microarray analysis. ****P≤0.0001, ***P≤0.001. Error bars indicate ±SD. (b) Immunofluorescence staining for Survivin in MeWo and UACC62 cells upon 6 hours of leptomycin B (LMB) treatment. (c) Western blot analysis of Survivin levels in MeWo and UACC62 cells after 18 hours of LMB or (d) 72 hours of small interfering RNA–CRM1 (si-CRM1#1 or si-CRM1#2) treatment. Ctrl, control. Journal of Investigative Dermatology 2012 132, 2780-2790DOI: (10.1038/jid.2012.233) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Chromosome region maintenance 1 (CRM1) inhibition initiates multiple molecular alterations to induce G1 arrest. (a, left) Cell-cycle analysis of M14, MeWo, and SK5 cells treated with leptomycin B (LMB) for 18 hours. (a, right) Western blotting for phospho(Ser780)-Rb in the same set of cell lines upon 18 hours of LMB treatment. (b, upper panel) Western blotting for p53 and p21 in the indicated cell lines upon 18 hours of LMB treatment; (b, middle panel) wild type (wt) and mutant (Mut) correspond to wt p53 and Mut p53, respectively (please refer to Supplementary Table S1 online for details); (b, lower panel) western blotting for Mdm2 with the same set of lysates as in the upper panel. (c) Immunofluorescence-based detection of p53 in UACC62 and M14 cells upon 4 hours of LMB treatment. (d) UACC62 cells were treated as indicated for 18 hours. Mdm2 and p53 levels were assessed by western blotting. (e, left) Western blot analysis of p53 in UACC62 and M14 cells upon the indicated treatments. In the small interfering RNA (siRNA)-p53+LMB group, siRNA-p53 treatment was initiated 6 hours before LMB treatment. Cell lysates were prepared 48 hours after the addition of LMB. (e, right) AnnexinV/propidium iodide (PI) staining of UACC62 and M14 cells, 48 hours after the indicated treatments. (f) AnnexinV/PI staining of UACC62 and M14 cells after the indicated treatments for 48 hours. (g) Summary of the changes in the protein levels of multiple cell-cycle regulatory proteins in the indicated cell lines. Figure summarizes the western blotting results from Figure 6b and Supplementary Figures S3d and S9a–e online. Journal of Investigative Dermatology 2012 132, 2780-2790DOI: (10.1038/jid.2012.233) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions