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. 2012 Dec;9(4):248-53.
doi: 10.7497/j.issn.2095-3941.2012.04.005.

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation, Invasion, and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells

Yu-Qing Wang  1 , Fei Zhang, Ran Tian, Wei Ji, Yan Zhou, Xiu-Mei Sun, Yuan Liu, Zhi-Yong Wang, Rui-Fang Niu
Affiliations

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation, Invasion, and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells

Yu-Qing Wang et al. Cancer Biol Med. 2012 Dec.

Abstract

Objective: To investigate the role of tyrosine 23 (Tyr23) phosphorylation of Annexin A2 (Anxa2) in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells.

Methods: A panel of lentivirus plasmids expressing Anxa2-wide type (Ana2-WT), Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation.

Results: The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells (P<0.05). Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D.

Conclusions: Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus.

Keywords: Annexin A2; Stat3 transcription factor; mutation; phosphorylation; tyrosine.

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Conflict of interest statement

No potential conflicts of interest are disclosed.

Figures

Figure 1
Figure 1
Results of the testing sequence for the three lentivirus plasmids. Lentivirus-based plasmids pCDH-EGFP-Anxa2WT, pCDH-EGFP-Anxa2Y23A, and pCDH-EGFP-Anxa2Y23D were established successfully.
Figure 2
Figure 2
Anxa2-WT and Anxa2-Y23D enhanced the proliferation of SK-BR-3 cells. A: Western blot analysis of GFP in SK-BR-3, control, Anxa2-WT, Anxa2-Y23A and Anxa2-Y23D cells. B: Immunofluorescence study of Anxa2-WT. Anxa2 was evidently overexpressed in SK-BR-3-Anxa2 cells. C: Proliferation ability was markedly enhanced in Anxa2-WT and Anxa2-Y23D cells. Triplicate experiments were done for B and C. The statistical significance was assessed by one-way ANOVA.
Figure 3
Figure 3
Anxa2-WT and Anxa2-Y23D increased the migration and invasion of SK-BR-3 cells in vitro. A and C: Migration assay revealed that the numbers of Anxa2-WT and Anxa2-Y23D cells attached to the bottom of the membrane were notably higher than those of the control cells. B and D: Invasion assay showed the same trend as the migration assay. Each column and bar shows the mean ± SD. Triplicate experiments were done for A and B. The statistical significance was assessed by one-way ANOVA. * P<0.05 vs. control.
Figure 4
Figure 4
Co-immunoprecipitation of Anxa2 and Stat3 in SK-BR-3 cells. Immunoprecipitation (IP) and immunoblotting (IB). A and B: The SK-BR-3 cell lysate was immunoprecipitated with the indicated antibodies and immunoblotted with the corresponding antibodies.
Figure 5
Figure 5
Anxa2-Y23D increased the expression of phosphorylation Stat3 in nucleus. A: Phosphorylation Anxa2 expression increased as the exposure time was lengthened, whereas the expression of other proteins did not show any significant changes. B: Western blot analysis of the expression of Stat3 and phosphorylation Stat3, Anxa2 and Phosphorylation Anxa2 in SK-BR-3, Anxa2-WT, Anxa2-Y23A and Anxa2-Y23D cells, with no significant differences. C: Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells. Western blot analysis showed that the expression of phosphorylation Stat3 was notably enhanced in Anxa2-Y23D cells.
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