Dissecting the complex crosstalk network between endothelin-1 axis and vascular endothelial growth factor system in melanoma cells

Francesca Spinella, Valentina Caprara, Laura Rosanò, Valeriana Di Castro, Roberta Cianfrocca, Pier Giorgio Natali, Anna Bagnato. Regina Elena National Cancer Institute, Molecular Pathology Laboratory, Italy.

Phenotypic and genotypic analyses of cutaneous melanoma have identified endothelin B receptor (ETB) as tumor progression marker, thus representing a potential therapeutic target. We previously reported that the binding of ET-1 to ETB stimulates angiogenesis and lymphangiogenesis directly on blood and lymphatic endothelial cells and by stimulating vascular endothelial growth factor (VEGF)-A and VEGF-C production. In this study we investigated as to whether in melanoma cells ET-1 axis may interact with VEGF-A and VEGF-C signaling pathways to heighten cellular responsiveness. We found that primary and metastatic melanoma cell lines expressed besides VEGF-A, also VEGF-C and its selective receptor VEGFR-3, at mRNA and protein level. Following ET-1 stimulation, VEGF-A and VEGF-C mRNA were upregulated, showing a 4.5 and 2.5 fold increase compared to the control, respectively, and VEGFR-3 and downstream signaling intermediates, such as p42/44 MAPK and Akt, were rapidly phosphorylated. The use of selective ETB antagonist, BQ788, completely blocked the VEGFR-3 phosphorylation and down-stream signaling pathways, demonstrating that ET-1-induced VEGFR-3 activation progress through ETB. Inhibition of c-Src activity by PP2 reduced ET-1-induced VEGFR-3 phosphorylation, demonstrating that ET-1 transactivates VEGFR-3 through an intracellular mechanism mediated by c-Src to expand the signaling network. Stimulation with ET-1 in combination with VEGF-A or VEGF-C increased p42/44 MAPK and AKT phosphorylation, and resulted in a greater degree, 4 fold increase, of migrated melanoma cell compared to a single factor. Furthermore, this combination significantly (p< 0.005) enhanced (3.5 fold increase compared to ET-1 alone) the ET-1-induced vasculogenic differentiation of melanoma cell in tube-like structures, a phenomenon defined as vasculogenic mimicry and associated with high aggressive phenotype, indicating that crosstalk between ET-1/ETB axis with VEGFR-3/VEGF-C system may enhance cancer cell motility and invasiveness and contributes to the promotion of cancer metastasis. Finally, in melanoma xenografts, ETB antagonist induced a 60% inhibition of tumor growth compared to control mice, and a reduction in neovascularization-related effectors, indicating that targeting ETB related signalling cascade may represent a novel treatment of melanoma by impairing the crosstalk between ET axis and VEGF in melanoma. Supported by AIRC.