Nedd4L modulates the transcription of metalloproteinase-1 and -13 genes to increase the invasive activity of gallbladder cancer

Abstract

The aim of this study was to examine whether Nedd4L (neural precursor cell expressed, developmentally down-regulated 4-like) participated in gallbladder carcinogenesis. We first immunohistochemically examined the expression of Nedd4L in various gallbladder tissue specimens. Weak immunoreactivity to Nedd4L-specific antibody was observed in normal or dysplastic epithelial cells. Cancer cells in non-invasive regions exhibited little immunoreactivity, whereas strong immunostaining was found in cytoplasm of many invasive cancers, especially at cancer invasive front with desmoplastic reaction. Notably, siRNA-mediated silencing of the Nedd4L gene significantly decreased the Matrigel-invasion activity and collagen invasion activity of cultured gallbladder cancer cells, without affecting the cell growth. The subtractive mRNA hybridization followed by RT-PCR and immunoblotting revealed that down-regulation of Nedd4L significantly decreased the expression of collagenases, matrix metalloproteinase (MMP)-1 and -13, in gallbladder cancer cells. Finally, immunohistochemical staining showed that many Nedd4L-expressing invasive gallbladder cancer cells co-expressed MMP-1 and MMP-13. These results indicated that over-expression of Nedd4L might lead to gallbladder cancer invasion by regulating the transcription of the MMP-1 and MMP-13 genes.

Figure 1

Representative immunohistochemical staining of various gallbladder tissues. Weak Nedd4L immunoreactivity was observed in non-cancerous epithelium (a) dysplastic epithelial cells (b) or non-invasive foci of gallbladder cancer (c, d). In contrast, invasive gallbladder cancer cells were focally immunostained with anti-Nedd4L antibody (e), but not with control preadsorbed antibody, as described in Materials and methods (f).

Figure 2

The Western blot demonstrating the specificity of the Nedd4L antibody used in this study. Expression vector, harbouring the entire coding region of human Nedd4L cDNA, or empty vector alone, was transfected into 293FT cells using the DOTAP reagent. After 48 h, the cells were harvested and used for Western blotting (lane 1, 3: empty vector alone; lane 2, 4: expression vector containing Nedd4L cDNA.) Note the Nedd4L band, which was detected with anti-Nedd4L (lane 4) and anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA) antibodies (lane 2).

Figure 3

siRNA-silencing of Nedd4L decreased gallbladder cancer invasion without affecting cell growth. (a) Nedd4L was significantly downregulated with use of specific siRNA for Nedd4L (lane 2), but not control siRNA (lane 1) in TGBC1TKB cells (left: RT-PCR, right: Western blotting). (b) There was no significant difference in cell growth between Nedd4L-specific siRNA-treated and control TGBC1TKB cells (c) and (d): In contrast, Nedd4L-negative TGBC1TKB cells exhibited much weaker invasive activity than control cells (P < 0.01) on Matrigel (c) and collagen-gel invasion assay (d). Bars represent standard errors of the mean.

Figure 4

Correlation of expression of Nedd4L, MMP-1 and MMP-13 in human tissues. Representative immunohistochemical staining of non-invasive gallbladder cancer foci (a: HE staining) with anti-MMP-1 antibody (b). Note the staining in infiltrating stromal cells, including macrophage, whereas non-invasive gallbladder cancer cells did not exhibit staining. Invasive gallbladder cancer cells (c: HE staining) were stained with anti-Nedd4L (d), anti-MMP-1 (e), and anti-MMP-13 antibodies (f). Note that Nedd4L-expressing cancer cells expressed both MMP-1 and MMP-13.