Relation of AURKB over-expression to low survival rate in BCRA and reversine-modulated aurora B kinase in breast cancer cell lines

Abstract

Background: New therapeutic drug for breast cancer (BRCA), especially triple negative BRCA (TNBC), is urgently needed. Even though 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) is an aurora kinase inhibitor, it also inhibits some cancer cells and human BRCA cells. However, the potential roles of reversine as a novel therapeutic agent for the treatment of BRCA remains unknown and must be further investigation. Thus, the relationship of reversine to aurora kinase in BCRA has not been reported. The relationship between AURKB and survival rate in BRCA has never been reported. Herein, we tested the roles of reversine on different BRCA cell line subtypes. We also investigated the relationship between AURKB and survival rate in BRCA as well as reversine to Aurora kinase expression in BCRA cell lines, including TNBC subtype, 4T1, MDA-MB-231, and luminal subtype MCF-7.

Methods: Cell viability and apoptosis were detected using Cell Counting Kit-8 and flow cytometry analysis, respectively. Apoptotic and tumor-related proteins were tested using Western blot analysis. Important microRNAs that regulate BRCA were analyzed using RT-PCR. UALCAN public databases were used to analyze the targeted gene profiles, and the PROGgeneV2 database was used to study the prognostic implications of genes.

Results: Reversine inhibits cell proliferation and induces cell apoptosis by modulating caspase-3 and bax/bcl-2 among the three cell lines. Data from the UALCAN public database show that BRCA tissues expressed high gene levels of AURKB, TIMP1, MMP9, and TGFB1 compared with the normal tissue. Among the over-expressed genes in BRCA, AURKB ranks 9th in TNBC, 49th in luminal subtype, and 48th in HER2 subtype. High AURKB level in BRCA is highly related to the low survival rate in patients displayed in 18 databases searched via PROGgeneV2. The protein levels of aurora B kinase (Aurora B), which is encoded by AURKB gene, are highly suppressed by reversine in the three cell lines. The tumor-related proteins TGF-β1, TIMP1, and MMP9 are partially suppressed by reversine but with different sensitivity in the three cell lines. The reversine-affected microRNAs, such as miR129-5p, miR-199a-3p, and miR-3960, in MDA-MB-231 cell line might be the research targets in TNBC regulation.

Conclusions: In BRCA, the level of AURKB are over-expressed and is related to low survival rate. Reversine contributes to anti-growth effect in BRCA cell lines, especially for TNBC, by modulating the aurora B. However, the invasiveness, metastasis, and anti-tumor effects of reversine in vivo and in vitro must be further investigated.

Keywords: AURKB; Aurora B; BRCA; Reversine; Triple-negative breast cancer cells; microRNAs.

Fig. 1

Reversine suppresses the cell proliferation and induces cell apoptosis and cell cycle arrest of human breast cancer cell lines. Time- and concentration-dependent changes of cell viability in the MDA-mB-231 (a) and MCF-7 cell lines (b) demonstrating the inhibition of reversion to human breast cancer cell lines. c Phase-contrast images of MDA-mB-231 (upper panel) and MCF-7 (low panel) in the indicated concentrations of reversine for 48 h. d Representative cell apoptosis phase images of MDA-mB-231 (upper panel) and MCF-7 (low panel) in the indicated concentrations of reversine for 48 h and the related apoptosis rate (%) in each cell lines (n = 3), compared with the control, **p < 0.01, ***p < 0.001, ^#p < 0.05, ^##p < 0.01 (one-way ANOVA), n.s., no significance. e Cell cycle analysis of MCF-7 in the indicated concentrations of reversine for 48 h and the related percent bar chart of each cell cycle stages (n = 3)

Fig. 2

Reversine suppresses the cell proliferation and induces cell apoptosis of 4T1 mouse breast cancer cell lines. a Time- and concentration-dependent changes of cell viability indicating the inhibition of reversion to mouse breast cancer cell line. b Phase-contrast images of 4T1 cell line in the indicated concentrations of reversine for 48 h. c Representative cell apoptosis phase images of the 4T1 cell line in the indicated concentrations of reversine for 48 h. d Related apoptosis rate (%) in 4T1 (n = 3), compared with the control, *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA), n.s., no significance

Fig. 3

Reversine induces apoptosis by modulating capsase-3 and bax/bcl-2 in breast cancer cell lines. The levels of apoptosis-related proteins were tested with Western blot (left panel) analysis at the indicated concentrations of reversine for 48 h in MDA-mB-231 (a), MCF7 (b) and 4T1 (c) cell lines, and the relative expression of each proteins (left panels) was listed as bar grafts (n = 2) compared with the control, *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA). d Heatmap of the three genes of CASP3, BAX, and BCL2 expressed in normal and breast cancer tumor tissues; data were acquired from UALCAN (see the materials and method for details). e Transcript expression levels of CASP, BAX, and BCL2 in BRCA based on breast cancer subclasses, indicating the over-expressed CASP3 and BAX genes and under-expressed BCL2 gene in the tumor tissues; data were acquired from UALCAN by analyzing the TCGA samples

Fig. 4

Reversine downregulates the expression of tumor-related proteins in breast cancer cell lines. The protein levels of TGF-b1, MMP9, TIMA1 m, and aurora-B were tested by Western blot analysis at the indicated concentrations of reversine for 48 h in MDA-mB-231 (a), MCF7 (b), and 4T1 (c) cell lines, and the relative expression of each proteins (inferior panels) was listed as bar grafts (n = 2) compared with the control, *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA)

Fig. 5

Overexpression of aurora B relates to lower survival rate in BRCA The heatmap of over-expressed genes in TNBC subtype (a), HER2 subtype (b), and luminal subtype (c), showing that AURKB genes ranked 19, 48, and 49 in the TNBC, HER2, and luminal subtypes. d Heatmap of the four tumor-related genes of AURKB, TGFB1, MMP9, and TIMP1 expressed in normal and breast cancer tumor tissues; data were acquired from UALCAN. e Transcript expression levels of AURKB, TGFB1, MMP9, and TIMP1 in BRCA based on breast cancer subclasses, indicating the over-expressed AURKB, TGFB1, MMP9, and TIMP1 genes in the tumor tissues; data were acquired from UALCAN by analyzing the TCGA samples. The overall survival at different AURKB expression levels in the TCGA (f), GSE3141 (g), and GSE3493_U133A database (h), demonstrating the influence of AURKB level on patient survival; data were acquired form the PROGgeneV2

Fig. 6

Reversine regulates the expression prolife of microRNAs in different breast cancer cell lines. a Three microRNAs were upregulated in the MDA-MB-231 cell line. b Six microRNAs were downregulated in the MCF-7 cell line, n = 3, values were determined relative to U6 and were presented as fold-change relative the levels in Ctrl, which was set as 1. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA)