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. 2011 Mar;91(3):426-38.
doi: 10.1038/labinvest.2010.201. Epub 2011 Jan 31.

Slug enhances invasion ability of pancreatic cancer cells through upregulation of matrix metalloproteinase-9 and actin cytoskeleton remodeling

Kejun Zhang  1 , Dong Chen, Xuelong Jiao, Shaoyan Zhang, Xiangping Liu, Jingyu Cao, Liqun Wu, Dongsheng Wang
Affiliations

Slug enhances invasion ability of pancreatic cancer cells through upregulation of matrix metalloproteinase-9 and actin cytoskeleton remodeling

Kejun Zhang et al. Lab Invest. 2011 Mar.

Retraction in

Abstract

Slug, a member of the Snail family of transcription factors, has a crucial role in the regulation of epithelial-mesenchymal transition (EMT) by suppressing several epithelial markers and adhesion molecules, including E-cadherin. A recent study demonstrated that no relationship exists between Slug and E-cadherin in pancreatic cancer. Another study showed that in malignant mesothelioma effusions Slug was associated with matrix metalloproteinase (MMP) expression, but that there was no association with E-cadherin. F-ascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. In this study, we investigated Slug, E-cadherin, and MMP-9 expression using immunohistochemistry in 60 patients with pancreatic cancer and their correlation with carcinoma invasion and metastasis. Additionally, we observed the effects of Slug on invasion and metastasis in the pancreatic cancer cell line PANC-1. Alterations in Slug, MMP-9, and E-cadherin were determined by RT-PCR, western blot, and immunohistochemistry. Alterations in MMP-9 and F-actin cytoskeleton were determined by immunofluorescence staining, flow cytometry (FCM), or gelatin zymography. Slug, E-cadherin, and MMP-9 expression in pancreatic cancer was significantly associated with lymph node metastases and we found a significant correlation between Slug and MMP-9 expression; however, no significant correlation was observed between Slug and E-cadherin expression. Slug transfection significantly increased invasion and metastasis in PANC-1 cells and orthotopic tumor of mouse in vivo, and significantly upregulated and activated MMP-9; however, there was no effect on E-cadherin expression. Slug promoted the formation of lamelliopodia or filopodia in PANC-1 cells. The intracellular F-actin and MMP-9 was increased and relocated to the front of the extending pseudopodia from the perinuclear pool in Slug-transfected PANC-1 cells. These results suggest that Slug promotes migration and invasion of PANC-1 cells, which may correlate with the reorganization of MMP-9 and remodeling of the F-actin cytoskeleton, but not with E-cadherin expression.

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Figures

Figure 1
Figure 1
Expression of Slug, MMP-9, and E-cadherin in human pancreatic adenocarcinomas as determined by immunohistochemistry. Staining of Slug was found in the cytoplasm as well as in the nucleus of tumor cells (a, b). Staining of MMP-9 was found in the cytoplasm of tumor cells (c, d). E-cadherin expression was identified in the cell membrane and, to a lesser degree, in the cytoplasm (e–f). (a, c, e) Magnification, × 100. (b, d, f) Magnification, × 400.
Figure 2
Figure 2
Effect of Slug expression on in vitro MMP-9 production. (a) Immunohistology analysis of levels of Slug protein in Slug-transfected clones and vector-transfected clones. (b) Western blot analysis of levels of Slug, E-cadherin, and MMP-9 protein in Slug-transfected clones and vector-transfected clones using monoclonal antibodies directed against MMP-9 and Slug. (c) RT-PCR analysis of levels of MMP-9 mRNA and E-cadherin mRNA in Slug-transfected clones and vector-transfected clones. The levels were normalized to the control GAPDH and results are expressed as the mean of three different experiments (*P<0.05; **P<0.01). (d) Q-PCR analysis of levels of MMP-9 mRNA and E-cadherin mRNA in Slug-transfected clones and vector-transfected clones. (e) ELISA assay of supernatant medium in Slug-transfected clones and vector-transfected clones. (f) Analysis by zymography of gelatinolytic activities (92 kd, pro-MMP-9) in conditioned media of Slug-transfected clones and vector-transfected clones. An MT-1-RT-PCR was performed on corresponding cells to demonstrate involvement in MMP-9 activation. The ratio of active MMP-9 (Mr 82,000) to the total MMP-9 (Mr 92,000 +82,000), as determined by densitometry, is shown under each lane. (g) Western blot analysis of levels of MMP-9 in conditioned media of Slug-transfected clones and vector-transfected clones treated (+) or not (−) with BB94. (h) RT-PCR analysis of levels of MMP-9 in conditioned media of Slug-transfected clones and vector-transfected clones treated (+) or not (−) with BB94.
Figure 3
Figure 3
The formation of pseudopodia in PANC-1 cells mediated by Slug. (a, b) The morphology of non-treated PANC-1 cells. (c, d) The morphology of Slug-transfected clones showed obvious filopodia (Rhodamine-labeled phalloidin staining, × 400).
Figure 4
Figure 4
F-actin polymerization of PANC-1 cells mediated by Slug. (a) FCM analysis for F-actin in PANC-1 cells (untreated cells defined 1). (b, c) F-actin staining of PANC-1 cells or mock-transfected cells using confocal laser scan microscopy. The F-actin is outside PANC-1 cells. (d) F-actin staining of Slug-transfected clones using confocal laser scan microscopy. Hyperchromic F-actin outside the cells redistributed to the direction (Rhodamine-labeled phalloidin staining × 400). *P<0.05; **P>0.05.
Figure 5
Figure 5
Staining of MMP-9 and F-actin in control and Slug-transfected clones, respectively. (a–c) Staining of MMP-9 and F-actin in non-treated PANC-1 cells. (d–f) Staining of MMP-9 and F-actin in Slug-transfected PANC-1 cells. (a, d) dFITC-labeled immunofluorescent staining, (b, e) Rhodamine-labeled phalloidin, (c, f) double-labeled immunofluorescent staining × 200.
Figure 6
Figure 6
In vitro invasion activities. The number of PANC-1 cells (a), Slug-transfected cells (b), and Slug-transfected PANC-1 treated with BB94 (c) on the bottom surface of transwell containing 8-μm pores were counted as invasion activities 24 h after incubation. Data represent mean and s.d. of three independent experiments (n=6/group in each experiment) (d).
Figure 7
Figure 7
Expression of Slug, MMP-9, and E-cadherin in the in vivo xenograft model (SP × 200). Immunohistochemistry with monoclonal antibodies directed against human Slug, MMP-9, and E-cadherin was performed to compare the Slug, MMP-9, and E-cadherin expression profile of Slug- and vector-transfected clones in the in vivo human xenograft model. Bars, 21 μm. (a) Slug is weakly expressed in mock-transfected group, (b) Slug is intensely expressed in Slug-transfected group, (c) MMP-9 is weakly expressed in mock-transfected group, (d) MMP-9 is intensely expressed in Slug-transfected group, (e) E-cadherin is moderately expressed in mock-transfected group, and (f) MMP-9 is moderately expressed in Slug-transfected group.
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