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. 2008 Nov 18;99(10):1695-703.
doi: 10.1038/sj.bjc.6604745. Epub 2008 Oct 28.

Blockade of SDF-1/CXCR4 signalling inhibits pancreatic cancer progression in vitro via inactivation of canonical Wnt pathway

Z Wang  1 , Q Ma, Q Liu, H Yu, L Zhao, S Shen, J Yao
Affiliations

Blockade of SDF-1/CXCR4 signalling inhibits pancreatic cancer progression in vitro via inactivation of canonical Wnt pathway

Z Wang et al. Br J Cancer. .

Abstract

Extra-pancreatic metastasis is a difficult problem for surgical intervention in pancreatic cancer. CXC chemokine receptor 4 (CXCR4) was considered to have an important role in this process. We hypothesized it may contribute to the pancreatic cancer progression through influencing canonical Wnt pathway. The purpose of this study was to examine the functional role of CXCR4 in the progression of pancreatic cancers and explore the possible mechanism. To this end, the relation between CXCR4 and clinical characteristics was analysed. shRNA against CXCR4 was applied to disrupt the SDF-1/CXCR4 signal transduction pathways in pancreatic cancer cell lines. Our results showed that overall survival in the case of patients positive for CXCR4 expression was significantly lower than that in the case of patients negative for CXCR4 expression. Notably, in vitro studies we observed that the abrogation of CXCR4 could obviously influence the pancreatic cancer cell phenotype including cell proliferation, colony formation, cell invasion and also inhibit the TOPflash activity. In addition, Wnt target genes and mesenchymal markers such as Vimentin and Slug were also inhibited in CXCR4 knockdown cells. Collectively, these data reported here demonstrate CXCR4 could modulate the canonical Wnt pathway and perhaps be a promising therapeutic target for pancreatic cancer progression.

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Figures

Figure 1
Figure 1
The expression of CXCR4 on pancreatic cancers. (A) immunohistochemistry staining of CXCR4 on normal pancreatic tissues (a) and pancreatic cancer tissues (b–d) × 200: negative expression (a), weak expression (b), moderate expression (c), strong expression (d); (B) Kaplan–Meier curve showed that the median survival time of the CXCR4-positive and CXCR4-negative groups was 5.5 and 13.5 months; (C). Western blot analysis revealed the CXCR4 expression for 5 pancreatic cancer cells (PC-2, PC-3, Miapaca-2, Bxpc-3, Panc-1). GAPDH was used as a loading control.
Figure 2
Figure 2
Establishment of stable CXCR4 knockdown. (A) QT–PCR analysis of CXCR4 expression after the transfection of different CXCR4 shRNA expression vectors: pRNAT-M1 (target position: 301), pRNAT-M2 (target position: 1093), pRNAT-MN (negative control) and Miapaca-2, *P<0.05 compared with pRNAT-MN cells; (B) western blot with CXCR4 antibody for different transfected pancreatic cancer cells. GAPDH was detected as a loading control; (C) Western blot showed the different expressions of CXCR4, phosphorylated CXCR4 on Serine 339 (P339-CXCR4) on transfected cells. GAPDH was detected as a loading control.
Figure 3
Figure 3
The influence of CXCR4 knockdown on the cell phenotype. (A) MTT assay was analysed for pRNAT-M1, pRNAT-MN, and Miapaca-2 on days 1, 2, 3, 4, and 5; (B) Distribution of cell cycle phases was demonstrated by flow cytometric analysis for pRNAT-M1 and pRNAT-MN; (C) Soft agar assay was assessed to evaluate the cell colony formation ability. The count number of the colony was shown in the diagram; *P<0.05 compared with pRNAT-MN cells.
Figure 4
Figure 4
The influence of CXCR4 knockdown on Wnt target gene and invasion-related genes. (A) β-Catenin/Tcf transcription reporter assay. Normalised with control reporter plasmid, the relative luciferase activity was demonstrated. *P<0.05 compared with pRNAT-MN cells; (B) QT–PCR analysis to examine the CTNNB1 (β-catenin), UPA, SLUG, CD44, MYC(c-MYC), Vim (Vimentin), CCND1 (cyclin D1) gene expression by the 2−ΔΔCt method. *P<0.05 compared with pRNAT-MN cells; (C) western blot analysis to detect MMP-9 and Vimentin protein expression. GAPDH was used as a loading control.
Figure 5
Figure 5
The abrogation of CXCR4 inhibits the invasive potential ability of pancreatic cancer cells. × 200; (A) representative staining figure: pRNAT-MN invading cells without SDF-1 stimulation (a), pRNAT-MN invading cells with 100 ng ml−1 SDF-1 stimulation (b), pRNAT-M1 invading cells without SDF-1 stimulation (c), pRNAT-M1 invading cells with 100 ng ml−1 SDF-1 stimulation (d); (B) the diagram of the count analysis, *P<0.05 compared with pRNAT-MN cells.
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