The neurotrophic factor GDNF has multiple functions that promote cell survival, proliferation and migration in different cell types. The experimental over-expression of GDNF in mouse testis leads to infertility and promotes seminomatous germ cell tumors in older animals, which suggests that although the underlying mechanisms are unknown, deregulation of the GDNF pathway may be implicated in germ cell carcinogenesis. To explore the involvement of the GDNF pathway in the onset and progression of testicular germ cell tumors, we firstly analyzed the expression pattern of GFRA1 and Ret, the two major co-receptors for GDNF, in seminoma samples. We report that GFRA1 was expressed more extensively in carcinoma in situ (CIS) cells and intratubular invasive seminoma compared with normal testis.
Functional analysis of the GDNF biological activity was performed on TCam-2 human seminoma cell line. RT-PCR and immunohistochemical analyses demonstrate that TCam-2 cells express both GFRA1 and Ret mRNA, but only GFRA1 was detected at the protein level. It is well known that GDNF plays a central role in spermatogonial stem cell self-renewal and proliferation (Hoffman MC et al., 2008). Therefore, in order to evaluate if GDNF could act as a mitogenic factor in TCam-2 cells, we performed proliferation assays and cell cycle analyses. Interestingly we observed that GDNF doesn’t induce increase in total cell number or S-phase entry in TCam-2 cell line.
Several evidences suggested that GDNF is able to induce cell migration and invasion in several normal and tumor cell types (Okada Y et al., 1999; Veit C et al., 2004; Su CM et al., 2009; Song H et al., 2006; Paratcha G et al., 2006; Tang MJ et al., 1998; Young HM et al., 2001). Therefore we have hypothesized that GDNF can act as a chemoattractant also in seminoma cells. On this purpose we stimulated directional TCam-2 cell migration and invasion in the presence of GDNF gradients and we investigated the downstream pathways responsible for the GDNF-induced invasive behaviour. We demonstrated that GDNF is able to induce migration, possibly through the Src and MEK pathways. It is documented that tumor cell invasion can occur through two modalities: a proteolytic mesenchymal-like or non-proteolytic amoeboid-like modality (Sahai E and Marshall CJ, 2003; Wolf K et al., 2003). GDNF is able to induce TCam-2 seminoma cell invasion in a mesenchymal-like metalloprotease-dependent manner.
In conclusion, GFRA1 over-expression in CIS and seminoma cells, along with the functional analyses in TCam-2 cells, suggests an involvement of the GDNF pathway in the progression of testicular germ cell cancer
