c-Src is involved in the c-MET-mediated migrating and invading behavior of NT2D1 Embryonal Carcinoma cells.

The proto-oncogene c-Met is a tyrosine kinase receptor that binds, as unique ligand, the Hepatocyte Growth Factor (HGF). c-Met/HGF system is highly involved in the regulation of the embryonic development and, in the adult, of tissue homeostasis. It is well known that the deregulation of c-Met activation is responsible of the onset and progression of different human cancer types [Koeppen H. et al., J. Pathol. 2014, 232: 210-218]. Notably, literature data of the last decades demonstrated that c-Met/HGF system is important for testicular physiology [Ricci G. and Catizone A., Frontiers in Endocrinology, 2014, 5:art.38]. More recently, our group revealed that c-Met is expressed in both seminoma and non seminoma Testicular germ cell tumors (TGCTs), also demonstrating that NT2D1 cells, a non seminoma cell line with molecular features of Embryonal Carcinoma, respond to HGF activating their proliferative, migrating and invasive behavior [Corano Scheri K. et al. Oncotarget submitted]. In the present work, first of all, we provide definitive evidence that the HGF-dependent migrating and invading behavior of NT2D1 cells are mediated by c-Met receptor, using a c-Met inhibitor, called PF04217903, that counteracts specifically c-Met phosphorilation. This drug is not toxic for NT2D1 cells, but abrogates the HGF-dependent migration and invasiveness of NT2D1 cells, demonstrating that these responses are c-Met-mediated. Activated c-Met recruits several adaptor proteins such as PI3K/AKT, STAT3, MAPK, Src etc. that are responsible of the different signaling pathways triggered by c-Met [Baldanzi G. and Graziani A., Biomedicines, 2015, 3:1-31]. In particular, Src adaptor is an oncogene with tyrosine kinase activity, that appears up-regulated in tumor progression and metastasis formation [Guarino M., J. of Cell. Physiology, 2010, 223:14-26]. In the present work we investigated the relevance of Src activation in the c-Met-mediated migrating and invading behavior of NT2D1 carcinoma cells. To this aim, we performed western blot analyses in order to evaluate Src protein both at baseline condition and after HGF administration. Notably, we observed an increase of Src protein, 8 hours after HGF administration compared with control samples. This finding suggests that Src is likely recruited during activation of the HGF-dependent and c-Met-activated pathways. To deeper investigate the role of Src in the HGF-dependent migration and invasion of NT2D1 cells, we performed different migration and invasion experiments using the Src inhibitor-1, with or without HGF. Results demonstrated that both migration and invasion, triggered by HGF administration, are reverted by the co-administration of Src inhibitor-1, suggesting that Src plays an important role in the stimulation of HGF-dependent NT2D1 cell aggressive behavior. However, unexpectedly, the administration of Src inhibitor-1 alone stimulates cell invasion, and the response is similar to that obtained using the HGF alone. This result indicates that Src is still required by opposite pathways in NT2D1 cells, promoting or alternatively inhibiting invasiveness, depending on the presence of HGF to clarify the complex role of Src activation in Embryonal Carcinoma onset and progression.