Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: Role of epithelial cell kinase (Eck/EphA2)

During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endot helial cells, which form a primitive, patterned vasculogenic network This i s followed by angiogenesis, the sprouting of nea vessels from preexisting v asculature, to yield a more refined microcirculation. However, we and our c ollaborators have recently described a process termed "vasculogenic mimicry ," which consists of the formation of patterned, tubular networks by aggres sive melanoma tumor cells (in three-dimensional cultures in vitro), that mi mics endothelial-formed vasculogenic networks and correlates dth poor clini cal prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cell s revealed a significant increased expression of tyrosine kinases associate d with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction pro teins that are critical for many cellular processes (e.g., cell adhesion, m igration, and invasion), we examined whether protein tyrosine kinases are i nvolved in melanoma vasculogenic mimicry. Immunofluorescence analysis of ag gressive melanoma cells forming tubular networks in vitro showed that tyros ine phosphorylation activity colocalized specifically within areas of tubul ar network formation, A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks),,Most notably, we identified epith elial cell kinase (EphA2) as being one receptor tyrosine kinase expressed a nd phosphorylated exclusively in the aggressive metastatic melanoma cells, Furthermore, general inhibitors of protein tyrosine kinases hindered tube f ormation, and transient knockout of EphA2 abrogated the ability of tumor ce lls to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular netw orks by aggressive melanoma tumor cells in vitro, which may represent a nov el therapeutic target for further clinical investigation.