Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma

Abstract

Background: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours.

Methods: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines.

Results: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments.

Conclusion: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

Figure 1

Gene expression profiling results obtained by using the hg-u133 plus 2.0 Affymetrix microarrays and analysed with the R2 web application (http://r2.amc.nl). (A) Box plot graphical presentation of the AURK-A and AURK-B expression levels in 21 human osteosarcoma clinical samples in comparison with human normal muscles and other normal tissues. Numbers inside parenthesis indicate the number of analysed samples. Asterisks indicate statistically significant differences by ANOVA test (P<0.01). (B) Event-free and (C) overall survival probability of the 21 high-grade OS patients stratified according to the median expression level of AURK-A or AURK-B. The group of high expressors (HIGH) included patients showing kinase expression levels equal or higher than the cut-off value.

Figure 2

Growth inhibition (GI) induced by AURK-A (left) or AURK-B (right) silencing in the three human OS cell lines U-2OS, Saos-2, IOR/OS18. CTR, control, non-treated cells; SCR, cells transfected with scrambled siRNA.

Figure 3

Reversal of cross-resistance against Aurora kinase-targeting drugs in U-2OS/DX580 and Saos-2/DX580 cell lines. Columns represent the fold increase in VX-680 and ZM447439 sensitivity of cells transfected with scrambled oligonucleotide (SCR), with ABCB1 silencing siRNAs or in the presence of two different doses of the ABCB1 inhibitor CBT-1 (CBT-1 0.5 μM and CBT-1 1 μM). The fold increase in drug sensitivity was calculated by dividing the VX-680 and ZM447439 IC50 values of controls (cell treated with VX-680 or ZM447439 only) by the IC50 values of silenced- or CBT-1-treated cells.

Figure 4

Cell cycle perturbations induced by treatment with the IC50 doses of VX-680 and ZM447439 in drug-sensitive (A) and drug-resistant (B) human OS cell lines. Graphs were derived from cell cycle analyses performed after 48 h of drug treatment. The intensity of propidium iodide fluorescence (representative for the DNA content) is plotted on x axis. The intensity of the incorporated BrdU fluorescence (representative for the DNA synthesis) is plotted on y axis. The different cell populations were identified according to the scheme shown in the plot on the top left of panel A. CTR, control non-treated cells cultured in drug-free and DMSO-free medium; DMSO, control non-treated cells cultured in the presence of DMSO concentrations corresponding to those of drug-treated samples; VX-680 or ZM447439, cells treated with IC50 dosage of each Aurora kinase inhibitor drug.

Figure 5

Western blot analyses for Caspase 2, Caspase 3, and PARP-1 in the four drug-sensitive human osteosarcoma cell lines after treatment with VX-680 and ZM447439. Lines with U-2OS+CDDP5 μg and Saos-2+CDDP10 μg were used as positive controls to detect Caspase 3 and PARP-1 cleavage induced by the treatment for 48 h with CDDP 5 μg ml⁻¹ or CDDP 10 μg ml⁻¹, respectively. CTR, control non-treated cells cultured in drug-free and DMSO-free medium; DMSO, control non-treated cells cultured in the presence of DMSO concentrations corresponding to those of drug-treated samples; VX-680 or ZM447439, cells treated with IC50 or two-fold IC50 (VX-680 × 2 or ZM447439 × 2) dosage of each Aurora kinase inhibitor drug.