AURKA suppression induces DU145 apoptosis and sensitizes DU145 to docetaxel treatment

Abstract

The palliative therapy effect by docetaxel for CRPC patients makes it urgent to improve the therapy. It was suggested that PI3K and androgen receptor-directed combination therapy may be effective for prostate cancer (PCa) patients PTEN negative. However, for those patients PTEN positive, the mechanism of anti-apoptosis survival of cancer cells is not yet well defined. Amplification of AURKA has been detected in 5% of PCa. In this work, Du145, a PTEN positive PCa cell model, was employed to investigate the role of aurora kinase a (AURKA) on cell growth. Inhibition of AURKA expression by shRNA markedly reduced prostate cancer cell viability. Furthermore, we demonstrate that AURKA inhibition induced a remarkable downregulation of AKT activity and Bax induction. Moreover, specific inhibition of the activity of AURKA, but not other aurora family members, by small molecular chemical inhibitors induced significant cell killing effects. Notably, AURKA inhibition sensitized prostate cancer cells to docetaxel treatment. Our work suggests that AURKA-directed monotherapy or combination therapy with docetaxel could be a potent treatment for PCa patients in future.

Keywords: AURKA; Prostate cancer; aurora kinases; castration-resistant prostate cancer; docetaxel; p53.

Figure 1

Specific knockdown of AURKA by shRNAs suppresses Du145 cell growth. A. Surviving fractions following treatment with the four individual shRNAs (shAURKA1-4) and the mixture shRNA (shAURKA) targeting AURKA, and shControl. Individual shAURKA 1 and shAURKA3 and the shAURKA most significantly reduced cell viability. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to shControl-infected cells (Student’s t test). Error bars represent the SEM. B. AURKA mRNA levels quantified by qPCR following infection of shRNA into Du145 cells. AURKA expression is shown relative to expression in shControl-infected cells. Individual shAURKA1 and shAURKA3 caused the most significant reduction in AURKA mRNA expression. **p < 0.01 compared to shControl-infected cells (Student’s t test). Error bars represent the SEM.

Figure 2

AURK inhibitors have significant cytotoxic effect on PCa cells. The cytotoxic effects of the AURK inhibitors were measured by MTS assay after 72 h treatment with these inhibitors, as described in the materials and methods. The viability fraction was compared to the untreated cells for each cell line. Error bars represent the SEM. A. Relative cell vability of PCa cells treated with Danusertib at the concentrations of 0.3, 1.5 and 7.5 μmol/l. B. Relative cell vability of PCa cells treated with AURKA inhibitor I at the concentrations of 0.2, 1.0 and 5.0 μmol/l. C. Relative cell vability of PCa cells treated with AMG 900 at the concentrations of 0.05, 0.25 and 1.25 μmol/l.

Figure 3

AURKA silencing induces Du145 cells apoptosis and inactives the AKT pathway. A. AURKA knockdown resulted in increasing of sub-G1 fraction. Cells were infected with shAURKA1, shAURKA3 or shControl. Cell cycle profiles were assessed by propidium iodide (PI) staining and fluorescence-activated cell scanning (FACS) from cell aliquots. B. Histogram represented the percentage of cells in each phase of the cell cycle. Significantly larger differences in sub-G1 phase and smaller differences in G1 phase after shAURKA1 or shAURKA3 infection were observed, indicating apoptosis induced by AURKA silencing. C. shAURKA1 or shAURKA3 infected-cells have significantly lower expression of p-AKT protein than shControl-infected cells. Du145 cells were infected with shAURKA1, shAURKA3 or shControl. Lysates were made 72 h following infection. Antibodies recognizing phosphoserine 473 and total AKT were used with GAPDH as a loading control. D. shAURKA1 or shAURKA3 infected-cells have significantly higher expression of p53 and Bax protein than shControl-infected cells. Du145 cells were infected with shAURKA1, shAURKA3 or shControl. Lysates were made 72 h following infection. Antibodies recognizing p53 and Bax were used with GAPDH as a loading control.

Figure 4

Synergistic enhancement of cytotoxicity between AURKA shRNA and docetaxel. Survival cells were quantitated by MTS assay after infection of shAURKA3 or shControl only or subsequent addition of 0.9 nmol/L docetaxel. These experiments were performed for three times. Doc, docetaxel.