Role of N-cadherin in proliferation, migration, and invasion of germ cell tumours

Abstract

Germ cell tumors (GCTs) are the most common malignancies in young men. Most patients with GCT can be cured with cisplatin-based combination chemotherapy, even in metastatic disease. In case of therapy resistance, prognosis is usually poor. We investigated the potential of N-cadherin inhibition as a therapeutic strategy. We analyzed the GCT cell lines NCCIT, NTERA-2, TCam-2, and the cisplatin-resistant sublines NCCIT-R and NTERA-2R. Effects of a blocking antibody or siRNA against N-cadherin on proliferation, migration, and invasion were investigated. Mouse xenografts of GCT cell lines were analyzed by immunohistochemistry for N-cadherin expression. All investigated GCT cell lines were found to express N-cadherin protein in vitro and in vivo. Downregulation of N-cadherin in vitro leads to a significant inhibition of proliferation, migration, and invasion. N-cadherin-downregulation leads to a significantly higher level of pERK. N-cadherin-inhibition resulted in significantly higher rates of apoptotic cells in caspase-3 staining. Expression of N-cadherin is preserved in cisplatin-resistant GCT cells, pointing to an important physiological role in cell survival. N-cadherin-downregulation results in a significant decrease of proliferation, migration, and invasion and stimulates apoptosis in cisplatin-naive and resistant GCT cell lines. Therefore, targeting N-cadherin may be a promising therapeutic approach, particularly in cisplatin-resistant, therapy refractory and metastatic GCT.

Keywords: GCT-cell lines; N-cadherin; cisplatin resistance; germ cell tumours.

Figure 1. N-cadherin protein is expressed in cisplatin-sensitive and resistant GCT-cell lines

N-cadherin protein expression was found in the GCT cell lines NCCIT, NTERA-2, and in TCam-2 cells A. and the two cisplatin-sensitive and –resistant cell line pairs NCCIT/-R and NTERA-2/-R B. The siRNA against CDH2 (siCDH2) efficiently reduced N-cadherin expression in all investigated GCT cell lines C+D.

Figure 2. N-cadherin expression in mouse xenografts

On immunohistochemical analysis in xenografts of NCCIT (n = 4; A + B.), NTERA-2 (n = 4; C + D.) and TCam-2 (n = 4; E + F.) N-cadherin was expressed in the cytoplasm and on the membrane of the tumor cells. The, expression of N-cadherin was higher in NTERA-2 and NCCIT, whereas the expression was lower in TCam-2 xenografts.

Figure 3. N-cadherin expression in metastasis of TGCT

Seminomas (n = 3, A + B.) and yolk sack tumors (n = 5, C + D.) strongly expressed N-cadherin. Primitive neuroectodermal tissues (n = 4, E + F.) and neuronal tissue (n = 14, G+H) within mature teratomas strongly expressed N-cadherin, also.

Figure 4. Silencing of N-cadherin significantly reduces proliferation in GCT cell lines

Proliferation of NCCIT was significantly reduced after 12 h, 24 h and 48 h. After 72 h, no significant change in proliferation was distinguishable A. Proliferation of NCCIT-R was significantly reduced after 12 h, 24 h, 48 h and 72 h B. In NTERA-2, proliferation was significantly reduced after 48 h and 72 h. After 12 h and 24 h, no significant reduction of proliferation was detectable C. In NTERA-2R, proliferation was significantly reduced after 12 h, 48 h and 72 h. After 24 h, no significant reduction of proliferation was detectable D. The proliferation of TCam-2 was significantly reduced after 12 h, 24 h, 48 h and 72 h E. (n.s. = not significant, *= p < 0.05, **= p < 0.005, ***= p < 0.0005).

Figure 5. Migration and invasion is significantly reduced after treatment with a siRNA against CDH2 for 48 hours

Migration of NCCIT A. NTERA-2 B. and TCam-2 C. NCCIT-R- D. and NTERA-2R- E. tumor cells was significantly reduced. Invasiveness of NCCIT F. NTERA-2 G. and TCam-2 H. NCCIT-R- I. and NTERA-2R- J. tumor cells was significantly reduced. (n.s. = not significant, *= p < 0.05, **= p < 0.005, ***= p < 0.0005).

Figure 6. Downregulation of N-cadherin leads to significant higher levels of pERK

The downregulation of N-cadherin leads to a significant increase of pERK in NCCIT A. NCCIT-R B. NTERA-2 C. NTERA-2R D. and TCam-2 E. cell lines (n.s. = not significant, *= p < 0.05, **= p < 0.005, ***= p < 0.0005).

Figure 7. Blocking N-cadherin causes significantly higher numbers of apoptotic cells

GCT cell lines NCCIT A. NCCIT-R B. NTERA-2 C. NTERA-2R D. and TCam-2 E. showed a significant increase in the number of apoptotic cells in immunocytochemistry analysis for Caspase-3. (n.s. = not significant, *= p < 0.05, **= p < 0.005, ***= p < 0.0005).