The role of β-catenin in the initiation and metastasis of TA2 mice spontaneous breast cancer

Abstract

Purpose: Tientsin Albino 2 (TA2) mice have a high incidence of spontaneous breast cancer. Tumor initiation is related to mouse mammary tumor virus (MMTV) infection. MMTV is hormonally regulated and may promote tumor formation via Wnt/β-catenin signaling pathway. This study attempts to clarify the relationship between β-catenin expression and the initiation and metastasis of spontaneous breast cancer in TA2 mice. Materials and Methods: Pathological samples illustrating the development of spontaneous breast cancer in TA2 mice were collected and the presence of virus particles was verified in the cancer tissue by electron microscope. Expression of Wnt/β-catenin signaling-pathway-related proteins including β-catenin, Wnt 5a, GSK-3β, and cyclin D1 were detected. MA-891 cell line derived from TA2 spontaneous breast cancer was cultured and siRNA was used to inhibit the expression of β-catenin in the primary culture cell line. Cell cycle analyses and comparisons of the invasiveness and migration capability of tumor cells were performed before and after β-catenin inhibition. Downstream protein expression of β-catenin was studied by western blot, co-immunoprecipitation assay. Tumorigenesis and metastasis were compared with that of negative control, siRNA control, and siRNA β-catenin-1512. Furthermore, proteins related to the proliferation and invasion of tumor were detected by western blot. Results: β-catenin expression was found to be located in the membrane and cytoplasm in normal mammary tissue and precancerous lesions, respectively. However, in the breast cancer tissue, β-catenin expression was located in the nuclei. After transfection with siRNA-1512, the cells showed decreased proliferation, invasiveness and migration capability, tumorigenicity, and metastasis, and the expression of the proteins related to tumor proliferation and metastasis such as c-myc, Cyclin D1, MMP-9, and VEGF were down-regulated. Conclusion: These results confirmed that the expression and location of β-catenin were associated with the initiation and metastasis of spontaneous breast cancer in TA2 mice.

Keywords: Breast cancer.; MA-891; Tientsin Albino 2; β-catenin.

Figure 1

A. Morphological changes in different breast lesions in TA2 mice. a) Normal breast mammary gland tissue from TA2 mice without SBC (×100, black arrowheads). b) Ductal hyperplasia of breast mammary gland tissue from TA2 mice with SBC (×200, black arrowheads). c) Atypical ductal hyperplasia of breast mammary gland tissue from TA2 mice with SBC (×200, black arrowheads). d) Spontaneous intraductal carcinoma of TA2 mice (×100, black arrowheads). e) Spontaneous intraductal carcinoma of TA2 mice, the black arrow points at the intact basement membrane (×200, black arrowheads). f) Spontaneous invasive breast carcinoma of TA2 mice (×100, black arrowheads). g) The metastatic foci of lung in TA2 mice with SBC (×200, black arrowheads). h) The metastatic foci of liver in TA2 mice with SBC (×100, black arrowheads). i) This electron micrograph of SBC from TA2 mice showing the virus particles (×3000, black arrowheads). B. Expression of Wnt/β-catenin signaling pathway proteins. a) β-catenin expression in normal mammary tissue and the membranes of mammary gland epithelium were positive for β-catenin IHC staining (IHC, ×200, black arrowheads). b) β-catenin expression in precancerous lesion in TA2 mice and the cytoplasm of mammary gland epithelium were positive for β-catenin IHC staining (IHC, ×200). c) β-catenin expression of SBC tissue in TA2 mice and the nuclei of mammary gland epithelium were positive for β-catenin IHC staining (IHC, ×200). d) Wnt 5a expression was located in the cytoplasm of SBC in TA2 mice (IHC, ×200). e) GSK-3β expression was located in the cytoplasm of SBC in TA2 mice (IHC, ×200). f) Cyclin D1 expression was located in the nuclei of SBC in TA2 mice (IHC, ×200).

Figure 2

Different expression level of β-catenin regulated the cell cycle progression in MA-891 cells. A. Effects of the negative control, siRNA control and siRNA β-catenin-1512 treatments on the cell cycle of MA891 were analyzed by FCM. B. The ratios of MA-891 cells G1, S and G2 stages of cell division were analyzed by FCM. C. The proliferation of MA-891 cells in different groups was assessed by MTT. Proliferative potential of MA-891 cells with the negative control, siRNA control and siRNA β-catenin-1512 treatments at 72 h after plating was determined using a modified MTT assay with 5×10⁴/mL cells. MA-891 cells treated with siRNA β-catenin-1512 exhibit a decrease in growth in comparison with the negative control and siRNA blank control.

Figure 3

Low-expression of β-catenin inhibited the migration and invasion capability of MA-89 cell. A. Histogram shows the number of invading MA-891 cells with the negative control, siRNA control and siRNA β-catenin-1512 treatments. B. Results of Transwell experiments with MA-891 showing that siRNA β-catenin-1512 inhibits cell invasiveness. a) Negative control, b) siRNA control, c) siRNA β-catenin-1512. C. Histogram shows the number of migrating MA-891 cells with the negative control, siRNA control and siRNA β-catenin-1512 treatments. D. The number of migrations of MA-891 cells treated with siRNA β-catenin-1512 was lower than that found in the other two groups. a) Negative control, b) siRNA control, c) siRNA β-catenin-1512. E. The expression levels of C-myc, CyclinD1, MMP-9, and VEGF as determined by western blot analysis. F. Interaction between β-catenin and TCF-4 in MA-891 cells using Co-IP.

Figure 4

β-catenin activation associated with tumor growth and metastasis in cancer cells derived from TA2 SBC. A. Tumor growth curve in TA2 mice injected with MA-891 cells with negative control, siRNA control, and siRNA β-catenin-1512. The volumes of xenografts were calculated and presented as mean ± SD. B. All the mice were sacrificed and the xenografts were removed and photographed on the 19th day after injection. C. The average tumor weight, presented as mean ± SD. D. The gross images of hepatic and lung metastases in TA2 mice injected with MA-891 cells with different treatments. E. Hepatic metastases were counted after H&E staining. F. Lung metastases counted after H&E staining. G. The expression levels of β-catenin, C-myc, CyclinD1 MMP-9, and VEGF in TA2 xenografts derived from MA-891 injection with different treatments after western blot analysis.