Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas

Abstract

Background: The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas.

Methods: The expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry.

Results: Quantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 +/- 0.74 (p < 0.01) and 6.25 +/- 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 +/- 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 +/- 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 +/- 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size.

Conclusions: We demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression.

Figure 1

Messenger RNA levels of the uPAS components in 14 human seminomas analyzed by quantitative RT-PCR. Fold of variations for uPAS components mRNAs in seminoma tissues have been calculated considering equal to 1 the mean value of uPAS component/β-Actin ratios found in 6 normal testicular tissues. The bars reported in the figure represent the median values. *p < 0.01.

Figure 2

Western blot analysis of uPA (A) and uPAR (C) and zymographic analysis (B) of 3 normal matched seminoma cancer tissues. Fifty μg of the different tissue protein extracts were loaded in each lane and subjected to western blot as described in the Methods section, using specific monoclonal antibodies against uPA (panel A), uPAR (panel C) and β-actin as protein loading control. In panel B, is reported the zymographic analysis of the uPA activity in protein tissues extracts of 3 normal matched seminoma cancer tissues. The conditioned medium of human breast carcinoma cell line MDA-MB-231 has been used as positive control for urokinase and tissue type PA activity, as described in the Materials and Methods section. Data reported represents one out of three similar experiments.

Figure 3

Immunohistochemistry analysis of uPA and uPAR expression in human testicular germ cell tumor. Tissue sections from 10 cases of seminomas were incubated with antibodies against human uPA or uPAR and processed as described in the Methods section. A) A representative normal testis stained with uPA antibody. B) A representative normal testis stained with uPAR antibody. C) A representative seminoma stained with uPA antibody. D) A representative seminoma stained with uPAR antibody. E) Negative control for seminoma tissue obtained omitting the first antibody. Tissue sections were counterstained with Mayer hematoxylin. Scale bar = 30 μm.

Figure 4

Correlation analysis between tumor size or patient's age and uPA or uPAR mRNA variations. Folds of mRNA variations in seminomas versus normal tissues were calculated as described in Materials and Methods section. The correlation analysis between uPA or uPAR mRNA variations and tumor size or patient's age were performed by the Spearman correlation test.