Silencing of P2X7 receptor reduces B16 melanoma growth both in vitro and in vivo

Malignantmelanoma is an aggressive cancer that originates from melanocytes, the pigment-producing cells of the skin. Despite its increasing incidence, the outcome for patients with advanced disease remains poor (Jemal A. et al. CA Cancer J Clin. Sep-Oct;60(5):277–300. 2010). Therefore, investigating the biological mechanism underlying melanoma progression is crucial for developing effective therapeutic intervention. B16 cell line derives from a spontaneous melanoma in C57Black/6 mice and is a well established and widely used tumor model. B16 express high level of P2X7 receptor (P2RX7), an ATP-gated ion channel known for mediating the effects of ATP in inflammation and cytokine secretion. P2X7R expression has been proposed as biomarker of malignancies (Di Virgilio F. et al. Purinergic Signal. Jun;5(2):251–6. 2009). We have recently described P2X7R oncogenic activity in different mice models of cancer, showing that in B16 melanoma derived tumors, antagonism of P2RX7 is effective in reducing both tumor growth and vessels formation (Adinolfi E. et al. Cancer Res. Jun 15;72(12):2957–69. 2012). To further extend our understanding of receptor’s supported tumoral growth inmelanoma, we investigated the effect of P2X7R silencing in B16 either in vitro and in vivo. The downregulation of P2RX7 expression through RNA interference, with two different short-harpinRNA constructs, caused a significant decrease in its activity. In vitro, P2X7 silencing led to a reduced proliferation rate, even in presence of growth factors. A similar behavior was also reflected after in vivo subcutaneous injection of B16 silenced clones in 6 weeks-old C57Black/6 male mice. P2RX7 downmodulation significantly reduced tumor growth and dimensions. These results corroborate our hypothesis that P2X7 receptor could be a good therapeutic target in melanoma. Although further studies are required to understand the molecular mechanisms underlying P2RX7 mediated melanoma transformation, a possible explanation could be related to cell differentiation. Indeed, when analyzed at a contrast microscope, B16 silenced clones showed melanocyte-like features, such as enhanced formation of cellular projection, thus suggesting an involvement of P2RX7 in dedifferentiation of pigment-producing cells of the skin