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. 2005 Feb 1;65(3):967-71.

Silencing of CXCR4 blocks breast cancer metastasis

Zhongxing Liang  1 , Younghyoun Yoon, John Votaw, Mark M Goodman, Larry Williams, Hyunsuk Shim
Affiliations

Silencing of CXCR4 blocks breast cancer metastasis

Zhongxing Liang et al. Cancer Res. .

Abstract

RNA interference technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. For the first time in cancer research, we show that direct injection of a pool of naked small interfering RNA (siRNA) duplexes can prevent tumorigenesis in an animal model, suggesting a novel preventive and therapeutic strategy for cancer management. As a model system, we used siRNA duplexes of CXCR4 to block breast cancer metastasis. Here, we show that blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis.

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Figures

Figure 1
Figure 1
CXCR4 expression levels of MDA-MB-231 cells at 48 hours posttransfection of CXCR4 siRNA-transfected MDA-MB-231 cells were stained with immunofluorescence by using biotinylated CXCR4 antagonistic peptide and streptavidin-phycoerythrin. Red, CXCR4 phycoerythrin staining; blue, counterstaining of nuclei. B, RT-PCR analysis of CXCR4 of the siRNA-transfected MDA-MB-231 cells showed that siRNA1+2 effectively blocked the expression of CXCR4 mRNA. Western blot results of the siRNA-transfected MDA-MB-231 cells by using anti-CXCR4 antibody Ab-2 (1:1,000) showed that siRNA1+2 blocked the expression of CXCR4 protein almost completely. β-Actin (Sigma, 1:2,500) was used as a loading control. Cont, control.
Figure 2
Figure 2
Invasion of MDA-MB-231 cells transfected with CXCR4 siRNAs. A, H&E staining showing the invasion of MDA-MB-231 cells transfected with control siRNA (Cont siRNA), siRNA1, siRNA2, or siRNA1+2 in a Matrigel invasion assay. B, invasiveness of MDA-MB-231 cells transfected with siRNA1+2, siRNA1, and siRNA2 relative to the control are 16% (P < 0.0003), 39% (P < 0.0014), and 51% (P < 0.0026), respectively. Average of three experiments.
Figure 3
Figure 3
Effect of CXCR4 siRNAs on inhibition of breast cancer metastasis in vivo. A, representative photographs of lungs and their H&E stainings (original magnification ×100) of each group from two independent experiments. B, average of real-time RT-PCR of hHPRT from siRNA-treated groups relative to that of control group. 1, group 1; 2, group 2; 3, group 3; 4, group 4; 5, group 5; 6, group 6 in Table 1. The average expression levels of hHPRT in groups 2, 3, 4, 5, and 6 were 12.5% (P < 0.0040), 21.5% (P < 0.0064), 47% (P < 0.0447), 25.3% (P < 0.0063), and 42.1% (P < 0.031), respectively, compared with that of group 1. C, percentage of human CXCR4 average expression level of each treated group is relative to that of the control group. The average expression levels of human CXCR4 in groups 2, 3, 4, 5, and 6 were 8.1% (P < 0.0006), 22.9% (P < 0.0022), 40.1% (P < 0.0157), 23.8% (P < 0.0074), and 59.1% (P < 0.152), respectively, compared with that of control group.
Figure 4
Figure 4
Effect of CXCR4 siRNAs on inhibition of breast cancer metastasis in vivo confirmed by FDG-PET. Representative images of FDG-PET of group 1 (control siRNA) and group 2 (siRNA1+2). A, maximum-intensity projection of six representative mice from group 1 (left, three mice) and group 2 (right, three mice). B, coronal sectional images from the lung area from the same animals in A. C, transaxial sectional images from the lung area from the same animals in A.
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