Wnt1 Is Anti-Lymphangiogenic in a Melanoma Mouse Model Heide Niederleithner, Magdalena Heinz, Stefanie Tauber, Martin Bilban, Hubert Pehamberger, Stefan Sonderegger, Martin Knöfler, Andreas Bracher, Walter Berger, Robert Loewe, Peter Petzelbauer  Journal of Investigative Dermatology  Volume 132, Issue 9, Pages 2235-2244 (September 2012) DOI: 10.1038/jid.2012.138 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Wnt1 suppresses melanoma-derived vascular endothelial growth factor C (VEGF-C) expression. (a) Wnt1 or VEGF-C protein and mRNA expression from cell extracts or supernatants of A375 cells overexpressing Wnt1, VEGF-C, or control (Co) vector. (b) Lymphatic endothelial spheroids incubated with supernatants (SNs) from A375 melanoma overexpressing a control vector, Wnt1, VEGF-C, or indicated combinations, or with recombinant (Rec.) Wnt3a (100ngml−1) or medium alone for 6hours. Numbers of sprouts were counted (mean±SD of three independent experiments). *P<0.05. (c) Melanoma lines transiently or stably transfected with Wnt1 or stimulated with SNs of Wnt1-expressing A375 cells or with recombinant Wnt3a (100ngml−1). VEGF-C mRNA was determined by real-time PCR. FCS, fetal calf serum. Journal of Investigative Dermatology 2012 132, 2235-2244DOI: (10.1038/jid.2012.138) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Wnt1 reduces vascular endothelial growth factor C (VEGF-C) expression independent of β-catenin and glycogen synthase kinase-3β (GSK3β). (a, b) VEGF-C mRNA expression in A375 cells (a) stably or (b) transiently overexpressing control (Co; pLNCX) or indicated plasmids. The inserted western blot shows VEGF-C protein expression from supernatants of control and Wnt1+ A375 cells. (c) A375 cells treated with GSK3β inhibitors for 16hours. (d) A375 cells stimulated with ionomycin or transiently transfected with Wnt1 or WNT5a plus ionomycin (8μM, 16hours). (e) A375 cells stably overexpressing Wnt1 treated with cyclosporine A (CsA). (f) A375 cells transiently expressing control vector, Wnt1, or WNT5a and co-transfected with empty vector or dominant-negative nuclear factor of activated T cells (dnNFAT). The right graph confirms functionality of dnNFAT: cells transfected with control vector or dnNFAT plus luciferase reporter controlled by NFAT-responsive elements were stimulated with DMSO, ionomycin (4μM), or ionomycin/phorbol 12-myristate 13-acetate (PMA; 100ngml−1) for 16hours. Mean±SD of three independent experiments, *P<0.05. DKK-1, dickkopf 1; Tcf, T-cell factor; ΔNTcf-4, dominant-negative Tcf-4 lacking β-catenin-binding site. Journal of Investigative Dermatology 2012 132, 2235-2244DOI: (10.1038/jid.2012.138) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Wnt1 overexpression delays melanoma metastasis in severe combined immunodeficient (SCID) mice. (a) Immunohistochemistry with indicated antibodies of control or Wnt1-transduced melanoma. (b) In vitro proliferation of control and Wnt1-expressing A375 cells. (c) Primary A375 tumor growth times until excision. (d) Time until lymph node metastasis. Numbers denote positive sentinel lymph nodes. Wnt1 A375 tumors did not metastasize during an observation period of 60 days (P<0.001 compared with controls). (e) vascular endothelial growth factor (VEGF)-C and VEGF-A mRNA expression by real-time PCR from A375-melanoma expressing control or Wnt1 vectors treated with or without cyclosporine A (CsA). Mean±SD, *P<0.05; n=5 per group. (f) Heat map for lymphangiogenic genes showing scaled ratios of mRNA of each gene to the median of this gene over all arrays from A375 or M24met melanoma (“C” for control; “W” for Wnt1 transfected) and A375 cells treated with CsA (n=5 per group; red color denotes high expression, and blue color indicates low expression). Journal of Investigative Dermatology 2012 132, 2235-2244DOI: (10.1038/jid.2012.138) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Wnt1 overexpression reduces lymphangiogenesis in vivo. (a) Example for hematoxylin and eosin (H&E) staining and immunohistochemistry (anti-Lyve-1 and CD34 stain in red) of M24met melanoma (control vs. Wnt1 overexpression). Bar graphs show quantification of lymph (Lyve-1 stains) or blood vessel (CD34 stains) areas; *P<0.05; n=30 sections per group; bar=100μm. NS, not significant. Right images show double immunofluorescence for human Vimentin (identifies human melanoma cells) and Lyve-1 (identifies lymphatics). (b, c) Real-time PCR for mouse Lyve-1 and Prox-1 mRNA from (b) M24met and (c) A375 tumors overexpressing the indicated plasmids or treated with cyclosporine A (CsA); mean values±SD, *P<0.05; n=5 severe combined immunodeficient (SCID) mice per group. Journal of Investigative Dermatology 2012 132, 2235-2244DOI: (10.1038/jid.2012.138) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions