Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: C-Met-activated signalling pathways promote malignant behaviour of NT2D1 non-seminoma cell line

Biological issues – c-MET/HGF axis acts as key regulator of different physiological processes during pre-and post-natal development of many organs and tissues, including the male gonad. However, the deregulation of this system is a well-known feature of malignant phenotype in several cancers and for this reason it has been considered a potential target for therapeutic purpose. Little is known about c-MET/HGF in Testicular Germ Cell Tumours (TGCTs): in the last decade our group studied c-MET/HGF expression and role on three TGCTs derived cell lines (TCam-2, NCCIT, NT2D1) and in histological samples of all major variants of Type II TGCTs. We found NT2D1 non-seminoma cells as the highest expressing cellular line, with respect to the seminoma ones. In accordance with this observation, NT2D1 presented the greatest response to HGF administration. Even in tissue biopsies, non-seminoma lesions showed a higher c-MET membranous staining than seminoma ones; in addition, HGF immunoreactivity is more evident in embryonal carcinoma (non-seminoma) biopsies indicating a link between testicular niche and tumor type. We also studied several adaptors of c-MET-activated pathways, demonstrating firstly a key role of SRC in the HGF-dependent malignant behavior of NT2D1 cells. It is fair to highlight that even PI3K/AKT and the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathways are effective adaptors activated by HGF, and that they appear upregulated in several cancer lesions. Aim – For this reason, in this PhD thesis, we studied the effects induced by HGF treatment on NT2D1 cells, focusing on the role of both PI3K/AKT and MAPK/ERK pathways in the NT2D1 biological responses. Results – To this aim we evaluated phospho- and total AKT and ERK1/2 proteins, both in basal condition and after HGF administration. Moreover, using a pharmacological approach (that is the use of the selective PI3K inhibitor, LY294002, and MEK1/2 inhibitor U0126), we demonstrated that ERK pathway has a role in basal NT2D1 cell proliferation, whereas PI3K/AKT pathway does not. Considering the importance of the micro-environment for tumors growth, and the overexpression of HGF in embryonal carcinoma lesions, we also evaluated the effects of HGF-treatment in NT2D1 cells cultured with or without U0126 or LY294002. We analyzed cell proliferation, collective migration and chemotaxis, as well as morphological modifications. Moreover, we started to study the impact of HGF administration on NT2D1 cell secretome. The administration of LY294002 together with HGF, reverts the HGF-dependent activation of cell proliferation and chemotaxis and blocks the collective migration of NT2D1 cells at levels even below the basal culture condition. Conversely, the administration of U0126 together with HGF, reverts the HGF-dependent activation of cell proliferation, but surprisingly does not exert the same effect on cell collective migration. Intriguingly, the use of U0126 alone significantly promotes the acquisition of an «invading phenotype», and collective migration of NT2D1 cells. Trying to explain this observation we found that the inhibition of ERK activation, mediated by U0126 used alone, resulted in the compensatory activation of the AKT-mediated pathway. This observation, in our opinion, can at least in part explain the results obtained and let us hypothesize that an EMT of NT2D1 cells occurred after U0126 and HGF administration even when used as single drugs. However, the study of EMT markers gave variable results, at the moment, and needs to be further analyzed. Conclusion– All together these observations, indicate that the inhibition of single onco-adaptor protein could exert paradoxical effects. These data highlight how the molecular features of each oncological lesion should be investigated in depth before setting-up the therapy and how microenvironmental cues could modify the response to oncological therapies. This stimulates further investigations of testicular secretome that could allow predicting responders and not-responders to TGCTs personalized target therapies.