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Molecular Cancer
Published in: Molecular Cancer 1/2014

Open Access 01-12-2014 | Research

WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells

Authors: Elin J Ekström, Caroline Bergenfelz, Verena von Bülow, Filiz Serifler, Eric Carlemalm, Göran Jönsson, Tommy Andersson, Karin Leandersson

Published in: Molecular Cancer | Issue 1/2014

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Abstract

Background

Wnt proteins are important for developmental processes and certain diseases. WNT5A is a non-canonical Wnt protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of WNT5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that WNT5A expression in melanoma cell lines increases migration and invasion.

Methods

Malignant melanoma cell lines were treated with rWNT5A or WNT5A siRNA, and mRNA versus protein levels of soluble mediators were measured using RT-PCR, cytokine bead array and ELISA. The induced signaling pathways were analyzed using inhibitors, Rho-GTPase pull down assays and western blot. Ultracentrifugation and electron microscopy was used to analyze microvesicles. Gene expression microarray data obtained from primary malignant melanomas was used to verify our data.

Results

We show that WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins IL-6, VEGF and MMP2 in melanoma cells. The process was independent of the transcriptional machinery and depletion of WNT5A reduced the levels of the exosome-derived proteins. The WNT5A induced exosomal secretion was neither affected by Tetanus toxin nor Brefeldin A, but was blocked by the calcium chelator Bapta, inhibited by a dominant negative version of the small Rho-GTPase Cdc42 and was accompanied by cytoskeletal reorganization. Co-cultures of melanoma/endothelial cells showed that depletion of WNT5A in melanoma cells decreased endothelial cell branching, while stimulation of endothelial cells with isolated rWNT5A-induced melanoma exosomes increased endothelial cell branching in vitro. Finally, gene expression data analysis of primary malignant melanomas revealed a correlation between WNT5A expression and the angiogenesis marker ESAM.

Conclusions

These data indicate that WNT5A has a broader function on tumor progression and metastatic spread than previously known; by inducing exosome-release of immunomodulatory and pro-angiogenic factors that enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize.
Appendix
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Literature
1.
2.
Reischl J, Schwenke S, Beekman JM, Mrowietz U, Sturzebecher S, Heubach JF: Increased expression of Wnt5a in psoriatic plaques. J Invest Dermatol. 2007, 127: 163-169. 10.1038/sj.jid.5700488CrossRefPubMed
3.
Kikuchi A, Yamamoto H, Sato A, Matsumoto S: Wnt5a: its signalling, functions and implication in diseases. Acta Physiol. 2012, 204: 17-33. 10.1111/j.1748-1716.2011.02294.x.CrossRef
4.
Hansen C, Howlin J, Tengholm A, Dyachok O, Vogel WF, Nairn AC, Greengard P, Andersson T: Wnt-5a-induced phosphorylation of DARPP-32 inhibits breast cancer cell migration in a CREB-dependent manner. J Biol Chem. 2009, 284: 27533-27543. 10.1074/jbc.M109.048884PubMedCentralCrossRefPubMed
5.
Dejmek J, Safholm A, Kamp Nielsen C, Andersson T, Leandersson K: Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells. Mol Cell Biol. 2006, 26: 6024-6036. 10.1128/MCB.02354-05PubMedCentralCrossRefPubMed
6.
Houghton AN, Polsky D: Focus on melanoma. Cancer Cell. 2002, 2: 275-278. 10.1016/S1535-6108(02)00161-7CrossRefPubMed
7.
Erhard H, Rietveld FJ, van Altena MC, Brocker EB, Ruiter DJ, de Waal RM: Transition of horizontal to vertical growth phase melanoma is accompanied by induction of vascular endothelial growth factor expression and angiogenesis. Melanoma Res. 1997, 7 (Suppl 2): S19-26.PubMed
8.
Lazar-Molnar E, Hegyesi H, Toth S, Falus A: Autocrine and paracrine regulation by cytokines and growth factors in melanoma. Cytokine. 2000, 12: 547-554. 10.1006/cyto.1999.0614CrossRefPubMed
9.
Wani AA, Jafarnejad SM, Zhou J, Li G: Integrin-linked kinase regulates melanoma angiogenesis by activating NF-kappaB/interleukin-6 signaling pathway. Oncogene. 2011, 30: 2778-2788. 10.1038/onc.2010.644CrossRefPubMed
10.
Ilkovitch D, Lopez DM: Immune modulation by melanoma-derived factors. Exp Dermatol. 2008, 17: 977-985. 10.1111/j.1600-0625.2008.00779.xCrossRefPubMed
11.
Da Forno PD, Pringle JH, Hutchinson P, Osborn J, Huang Q, Potter L, Hancox RA, Fletcher A, Saldanha GS: WNT5A expression increases during melanoma progression and correlates with outcome. Clin Cancer Res. 2008, 14: 5825-5832. 10.1158/1078-0432.CCR-07-5104CrossRefPubMed
12.
Weeraratna AT, Jiang Y, Hostetter G, Rosenblatt K, Duray P, Bittner M, Trent JM: Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell. 2002, 1: 279-288. 10.1016/S1535-6108(02)00045-4CrossRefPubMed
13.
Dissanayake SK, Olkhanud PB, O'Connell MP, Carter A, French AD, Camilli TC, Emeche CD, Hewitt KJ, Rosenthal DT, Leotlela PD, Wade MS, Yang SW, Brant L, Nickoloff BJ, Messina JL, Biragyn A, Hoek KS, Taub DD, Longo DL, Sondak VK, Hewitt SM, Weeraratna AT: Wnt5A regulates expression of tumor-associated antigens in melanoma via changes in signal transducers and activators of transcription 3 phosphorylation. Cancer Res. 2008, 68: 10205-10214. 10.1158/0008-5472.CAN-08-2149PubMedCentralCrossRefPubMed
14.
Stanley AC, Lacy P: Pathways for cytokine secretion. Physiology (Bethesda). 2010, 25: 218-229. 10.1152/physiol.00017.2010CrossRef
15.
Proux-Gillardeaux V, Rudge R, Galli T: The tetanus neurotoxin-sensitive and insensitive routes to and from the plasma membrane: fast and slow pathways?. Traffic. 2005, 6: 366-373. 10.1111/j.1600-0854.2005.00288.xCrossRefPubMed
16.
Hobert ME, Sands KA, Mrsny RJ, Madara JL: Cdc42 and Rac1 regulate late events in Salmonella typhimurium-induced interleukin-8 secretion from polarized epithelial cells. J Biol Chem. 2002, 277: 51025-51032. 10.1074/jbc.M210466200CrossRefPubMed
17.
Sinai P, Nguyen C, Schatzle JD, Wulfing C: Transience in polarization of cytolytic effectors is required for efficient killing and controlled by Cdc42. Proc Natl Acad Sci U S A. 2010, 107: 11912-11917. 10.1073/pnas.0913422107PubMedCentralCrossRefPubMed
18.
Filipazzi P, Burdek M, Villa A, Rivoltini L, Huber V: Recent advances on the role of tumor exosomes in immunosuppression and disease progression. Semin Cancer Biol. 2012, 22: 342-349. 10.1016/j.semcancer.2012.02.005CrossRefPubMed
19.
Bobrie A, Colombo M, Raposo G, Thery C: Exosome secretion: molecular mechanisms and roles in immune responses. Traffic. 2011, 12: 1659-1668. 10.1111/j.1600-0854.2011.01225.xCrossRefPubMed
20.
Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, Garcia-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med. 2012, 18: 883-891. 10.1038/nm.2753PubMedCentralCrossRefPubMed
21.
Shim J, Lee SM, Lee MS, Yoon J, Kweon HS, Kim YJ: Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis. Mol Cell Biol. 2010, 30: 1421-1433. 10.1128/MCB.01463-09PubMedCentralCrossRefPubMed
22.
Ekstrom EJ, Sherwood V, Andersson T: Methylation and loss of Secreted Frizzled-Related Protein 3 enhances melanoma cell migration and invasion. PLoS One. 2011, 6: e18674- 10.1371/journal.pone.0018674PubMedCentralCrossRefPubMed
23.
Schlessinger K, McManus EJ, Hall A: Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity. J Cell Biol. 2007, 178: 355-361. 10.1083/jcb.200701083PubMedCentralCrossRefPubMed
24.
Harbst K, Staaf J, Lauss M, Karlsson A, Masback A, Johansson I, Bendahl PO, Vallon-Christersson J, Torngren T, Ekedahl H, Geisler J, Hoglund M, Ringner M, Lundgren L, Jirstrom K, Olsson H, Ingvar C, Borg A, Tsao H, Jonsson G: Molecular profiling reveals low- and high-grade forms of primary melanoma. Clin Cancer Res. 2012, 18: 4026-4036. 10.1158/1078-0432.CCR-12-0343PubMedCentralCrossRefPubMed
25.
Tellez CS, Shen L, Estecio MR, Jelinek J, Gershenwald JE, Issa JP: CpG island methylation profiling in human melanoma cell lines. Melanoma Res. 2009, 19: 146-155. 10.1097/CMR.0b013e32832b274eCrossRefPubMed
26.
Abi-Habib RJ, Urieto JO, Liu S, Leppla SH, Duesbery NS, Frankel AE: BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin. Mol Cancer Ther. 2005, 4: 1303-1310.CrossRefPubMed
27.
Bergenfelz C, Medrek C, Ekstrom E, Jirstrom K, Janols H, Wullt M, Bredberg A, Leandersson K: Wnt5a induces a tolerogenic phenotype of macrophages in sepsis and breast cancer patients. J Immunol. 2012, 188: 5448-5458. 10.4049/jimmunol.1103378CrossRefPubMed
28.
Bergenfelz C, Janols H, Wullt M, Jirstrom K, Bredberg A, Leandersson K: Wnt5a inhibits human monocyte-derived myeloid dendritic cell generation. Scand J Immunol. 2013, 78: 194-204. 10.1111/sji.12075CrossRefPubMed
29.
Nevins AK, Thurmond DC: A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis. J Biol Chem. 2005, 280: 1944-1952.CrossRefPubMed
30.
Alberts P, Rudge R, Irinopoulou T, Danglot L, Gauthier-Rouviere C, Galli T: Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane. Mol Biol Cell. 2006, 17: 1194-1203.PubMedCentralCrossRefPubMed
31.
32.
Hoang MV, Nagy JA, Senger DR: Cdc42-mediated inhibition of GSK-3beta improves angio-architecture and lumen formation during VEGF-driven pathological angiogenesis. Microvasc Res. 2011, 81: 34-43. 10.1016/j.mvr.2010.09.001PubMedCentralCrossRefPubMed
33.
Kean MJ, Williams KC, Skalski M, Myers D, Burtnik A, Foster D, Coppolino MG: VAMP3, syntaxin-13 and SNAP23 are involved in secretion of matrix metalloproteinases, degradation of the extracellular matrix and cell invasion. J Cell Sci. 2009, 122: 4089-4098. 10.1242/jcs.052761CrossRefPubMed
34.
Miyata T, Ohnishi H, Suzuki J, Yoshikumi Y, Ohno H, Mashima H, Yasuda H, Ishijima T, Osawa H, Satoh K, Sunada K, Kita H, Yamamoto H, Sugano K: Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells. Biochem Biophys Res Commun. 2004, 323: 118-124. 10.1016/j.bbrc.2004.08.064CrossRefPubMed
35.
Schnaeker EM, Ossig R, Ludwig T, Dreier R, Oberleithner H, Wilhelmi M, Schneider SW: Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: prerequisite in human melanoma cell invasion. Cancer Res. 2004, 64: 8924-8931. 10.1158/0008-5472.CAN-04-0324CrossRefPubMed
36.
Li M, Li Z, Sun X: Statins suppress MMP2 secretion via inactivation of RhoA/ROCK pathway in pulmonary vascular smooth muscles cells. Eur J Pharmacol. 2008, 591: 219-223. 10.1016/j.ejphar.2008.06.082CrossRefPubMed
37.
Xiang X, Poliakov A, Liu C, Liu Y, Deng ZB, Wang J, Cheng Z, Shah SV, Wang GJ, Zhang L, Grizzle WE, Mobley J, Zhang HG: Induction of myeloid-derived suppressor cells by tumor exosomes. Int J Cancer. 2009, 124: 2621-2633. 10.1002/ijc.24249PubMedCentralCrossRefPubMed
38.
Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin JP, Boireau W, Rouleau A, Simon B, Lanneau D, De Thonel A, Multhoff G, Hamman A, Martin F, Chauffert B, Solary E, Zitvogel L, Garrido C, Ryffel B, Borg C, Apetoh L, Rebe C, Ghiringhelli F: Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest. 2010, 120: 457-471.PubMedCentralPubMed
39.
Lui WO, Pourmand N, Patterson BK, Fire A: Patterns of known and novel small RNAs in human cervical cancer. Cancer Res. 2007, 67: 6031-6043. 10.1158/0008-5472.CAN-06-0561CrossRefPubMed
40.
Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz LA, Sjoblom T, Barad O, Bentwich Z, Szafranska AE, Labourier E, Raymond CK, Roberts BS, Juhl H, Kinzler KW, Vogelstein B, Velculescu VE: The colorectal microRNAome. Proc Natl Acad Sci U S A. 2006, 103: 3687-3692. 10.1073/pnas.0511155103PubMedCentralCrossRefPubMed
41.
Russell JM, Stephenson GS, Yellowley CE, Benton HP: Adenosine inhibition of lipopolysaccharide-induced interleukin-6 secretion by the osteoblastic cell line MG-63. Calcif Tissue Int. 2007, 81: 316-326. 10.1007/s00223-007-9060-yCrossRefPubMed
42.
Seino S, Shibasaki T: PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis. Physiol Rev. 2005, 85: 1303-1342. 10.1152/physrev.00001.2005CrossRefPubMed
43.
Li Q, Ho CS, Marinescu V, Bhatti H, Bokoch GM, Ernst SA, Holz RW, Stuenkel EL: Facilitation of Ca(2+)-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells. J Physiol. 2003, 550: 431-445. 10.1113/jphysiol.2003.039073PubMedCentralCrossRefPubMed
44.
Jenei V, Sherwood V, Howlin J, Linnskog R, Safholm A, Axelsson L, Andersson T: A t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide functions as a potent antagonist of Wnt5a-dependent melanoma cell invasion. Proc Natl Acad Sci U S A. 2009, 106: 19473-19478. 10.1073/pnas.0909409106PubMedCentralCrossRefPubMed
45.
Hong-Geller E, Cerione RA: Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells. J Cell Biol. 2000, 148: 481-494. 10.1083/jcb.148.3.481PubMedCentralCrossRefPubMed
46.
Chen T, Guo J, Yang M, Zhu X, Cao X: Chemokine-containing exosomes are released from heat-stressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine. J Immunol. 2012, 186: 2219-2228.CrossRef
47.
Taraboletti G, D'Ascenzo S, Giusti I, Marchetti D, Borsotti P, Millimaggi D, Giavazzi R, Pavan A, Dolo V: Bioavailability of VEGF in tumor-shed vesicles depends on vesicle burst induced by acidic pH. Neoplasia. 2006, 8: 96-103. 10.1593/neo.05583PubMedCentralCrossRefPubMed
48.
Yu S, Liu C, Su K, Wang J, Liu Y, Zhang L, Li C, Cong Y, Kimberly R, Grizzle WE, Falkson C, Zhang HG: Tumor exosomes inhibit differentiation of bone marrow dendritic cells. J Immunol. 2007, 178: 6867-6875. 10.4049/jimmunol.178.11.6867CrossRefPubMed
49.
Liu Y, Xiang X, Zhuang X, Zhang S, Liu C, Cheng Z, Michalek S, Grizzle W, Zhang HG: Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells. Am J Pathol. 2010, 176: 2490-2499. 10.2353/ajpath.2010.090777PubMedCentralCrossRefPubMed
50.
Novick P, Guo W: Ras family therapy: Rab, Rho and Ral talk to the exocyst. Trends Cell Biol. 2002, 12: 247-249. 10.1016/S0962-8924(02)02293-6CrossRefPubMed
51.
Logozzi M, De Milito A, Lugini L, Borghi M, Calabro L, Spada M, Perdicchio M, Marino ML, Federici C, Iessi E, Brambilla D, Venturi G, Lozupone F, Santinami M, Huber V, Maio M, Rivoltini L, Fais S: High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients. PLoS One. 2009, 4: e5219- 10.1371/journal.pone.0005219PubMedCentralCrossRefPubMed
52.
Dumitrescu Pene T, Rose SD, Lejen T, Marcu MG, Trifaro JM: Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis. J Neurochem. 2005, 92: 780-789. 10.1111/j.1471-4159.2004.02907.xCrossRefPubMed
53.
Kalwat MA, Wiseman DA, Luo W, Wang Z, Thurmond DC: Gelsolin associates with the N terminus of syntaxin 4 to regulate insulin granule exocytosis. Mol Endocrinol. 2012, 26: 128-141. 10.1210/me.2011-1112PubMedCentralCrossRefPubMed
54.
Ren Y, Yang J, Xie R, Gao L, Yang Y, Fan H, Qian K: Exosomal-like vesicles with immune-modulatory features are present in human plasma and can induce CD4+ T-cell apoptosis in vitro. Transfusion. 2011, 51: 1002-1011. 10.1111/j.1537-2995.2010.02909.xCrossRefPubMed
55.
Gross JC, Chaudhary V, Bartscherer K, Boutros M: Active Wnt proteins are secreted on exosomes. Nat Cell Biol. 2012, 14: 1036-1045. 10.1038/ncb2574CrossRefPubMed
56.
Arbiser JL, Moses MA, Fernandez CA, Ghiso N, Cao Y, Klauber N, Frank D, Brownlee M, Flynn E, Parangi S, Byers HR, Folkman J: Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc Natl Acad Sci U S A. 1997, 94: 861-866. 10.1073/pnas.94.3.861PubMedCentralCrossRefPubMed
57.
Cunha SI, Pardali E, Thorikay M, Anderberg C, Hawinkels L, Goumans MJ, Seehra J, Heldin CH, ten Dijke P, Pietras K: Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs tumor growth and angiogenesis. J Exp Med. 2010, 207: 85-100. 10.1084/jem.20091309PubMedCentralCrossRefPubMed
58.
Pietras A, von Stedingk K, Lindgren D, Pahlman S, Axelson H: JAG2 induction in hypoxic tumor cells alters Notch signaling and enhances endothelial cell tube formation. Mol Cancer Res. 2011, 9: 626-636. 10.1158/1541-7786.MCR-10-0508CrossRefPubMed
59.
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003, 4: 249-264. 10.1093/biostatistics/4.2.249CrossRefPubMed
60.
Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001, 98: 5116-5121. 10.1073/pnas.091062498PubMedCentralCrossRefPubMed
Metadata
Title
WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells
Authors
Elin J Ekström
Caroline Bergenfelz
Verena von Bülow
Filiz Serifler
Eric Carlemalm
Göran Jönsson
Tommy Andersson
Karin Leandersson
Publication date
01-12-2014
Publisher
BioMed Central
Published in
Molecular Cancer / Issue 1/2014
Electronic ISSN: 1476-4598
DOI
https://doi.org/10.1186/1476-4598-13-88

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