WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells.
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.
Elin J
EJ
Ekström
Caroline
C
Bergenfelz
Verena
V
von Bülow
Filiz
F
Serifler
Eric
E
Carlemalm
Göran
G
Jönsson
Tommy
T
Andersson
Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö SE-20502, Sweden. karin.leandersson@med.lu.se.
Karin
K
Leandersson
D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000098:blood supply / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000098:blood supply / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D013744:Tetanus Toxin / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism; D000071818:Wnt-5a Protein; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D015815:Cell Adhesion Molecules; C568779:ESAM protein, human; C508600:IL6 protein, human; D015850:Interleukin-6; D011518:Proto-Oncogene Proteins; D013744:Tetanus Toxin; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D020126:Brefeldin A; D004533:Egtazic Acid; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020764:cdc42 GTP-Binding Protein; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium