EphA2 kinase regulates cell shape, adhesion, and motility and is frequently overexpressed in several cancers, including melanoma, prostate, breast, and colon cancers and lung carcinoma. Although a function in both tumor onset and metastasis has been proposed, the role played by EphA2 in tumor progression is still debated. In melanoma, EphA2 has been reported to affect cell migration and invasiveness allowing cells to move by a proteolysis-independent strategy, commonly referred as amoeboid motility. With the aim to understand the role of EphA2 in prostate cancer metastatic spreading, we stably silenced EphA2 expression in a model of aggressive metastatic prostate carcinoma. Our results show that EphA2 drives the metastatic program of prostate carcinoma, although its involvement greatly differs among metastatic steps. Indeed, EphA2 expression (i) greatly affects prostate carcinoma cell motility style, guiding an amoeboid movement based on Rho-mediated cell rounding and independent from metalloprotases, (ii) is ineffective on transendothelial migration, adhesion onto extracellular matrix proteins, and on resistance to anoikis, (iii) regulates clonogenic potential of prostate carcinoma, thereby increasing anchorage-independent growth and self-renewal, prostasphere formation, tumor onset, dissemination to bone, and growth of metastatic colonies. Our finding indicate that EphA2-overexpressing prostate carcinoma cells gain an invasive benefit from their amoeboid motility style to escape from primary tumors and then, enhancing their clonogenic potential successfully target bone and grow metastases, thereby acknowledging EphA2 as a target for antimetastatic therapy of aggressive prostate cancers. Mol Cancer Res; 9(2); 149-60. Ó2011 AACR.

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The biological significance of the almost constant presence of macrophages in the tumoral microenvironment is an issue debated by several authors. The major difficulty in understanding the role played by tumor-associated macrophages (TAMs) in tumor progression is due to the contrasting effects of TAMs found in different studies. In addition, there is a limited information on which of the many biological activities expressed by TAMs are critical in inducing stimulatory or inhibitory effect on tumor growth. The aim of our study was: (a) to explore to what extent cyclo-oxygenase-2 (COX-2) in TAMs associated with human melanoma is expressed at different stages of tumor progression; and (b) to explore whether COX-2 expression in TAMs is stimulated by melanoma cells. In order to answer this question, we determined COX-2 positive TAMs associated with cutaneous melanocytic nevi, in situ, invasive and metastatic melanoma. In addition, we investigated whether COX-2 is expressed in peritoneal thioglycollate-elicited macrophages after co-cultivation with murine B16 melanoma cells. We found that COX-2-positive TAMs, as revealed by immunohistochemical analysis, were rare in common nevi and "dysplastic nevi", but present in a high percentage in in situ and thin melanoma. COX-2-positive TAMs were also found in more advanced tumors and metastatic melanoma, although at a significantly lower percentage in these latter. The in vitro protocol revealed that COX-2 was expressed in peritoneal macrophages upon contact with B16 murine melanoma cells, but not with normal murine fibroblasts. On the whole, the results of in vivo and in vitro studies suggest that COX-2 expressed in TAMs appears to act as an effective biomarker of melanoma progression, and melanoma cells themselves might stimulate COX-2 in macrophages.

The aim of this study was to investigate whether tumor cells as well as tumor-associated macrophages (TAMs) contribute to the generation of protease activities essential to tumor cell invasiveness, such as matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9), and the urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR). We found that the enhanced invasiveness through Matrigel-coated filters of B16 murine melanoma cells stimulated with IFNc was associated with an higher expression of uPAR and MMP-9 in these cells. Moreover, treatment with anti-MMP-9 or anti-uPAR monoclonal antibodies abrogated the increase of invasiveness in IFNcstimulated melanoma cells, suggesting a cooperation of uPA system and MMP-9 in cytokine-stimulated invasiveness. Invasiveness through Matrigel was also enhanced in B16 melanoma cells exposed to a medium conditioned by TAMs, represented in our experimental model by thioglycollate-elicited macrophages co-cultivated with melanoma cells. Macrophages isolated from these co-cultures were found to express higher levels of uPAR and MMP-9 compared to macrophage cultures alone, and the pro-invasive activity of the co-culture-conditioned medium was abrogated by anti-MMP-9 monoclonal antibodies, but not anti-uPAR monoclonal antibodies. Furthermore, the enhanced uPAR and MMP-9 expression in macrophages cocultivated with tumor cells seems a rather specific phenomenon, generated through a cell-to-cell contact mechanism. On the whole, our data point to a cooperation between tumor cells and macrophages elicited by tumor cells themselves in generating key enzymes essential in the promotion of tumor invasiveness, such as uPAR and MMP-9.Keywords Murine melanoma cells Á Macrophages Á Cell invasiveness Á Matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) Á Urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR)