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. 2012 Feb 15;83(4):452-61.
doi: 10.1016/j.bcp.2011.11.005. Epub 2011 Nov 15.

Kinome-wide siRNA screening identifies molecular targets mediating the sensitivity of pancreatic cancer cells to Aurora kinase inhibitors

Lifang Xie  1 , Michelle Kassner, Ruben M Munoz, Qiang Q Que, Jeff Kiefer, Yu Zhao, Spyro Mousses, Hongwei H Yin, Daniel D Von Hoff, Haiyong Han
Affiliations

Kinome-wide siRNA screening identifies molecular targets mediating the sensitivity of pancreatic cancer cells to Aurora kinase inhibitors

Lifang Xie et al. Biochem Pharmacol. .

Abstract

Aurora kinases are a family of mitotic kinases that play important roles in the tumorigenesis of a variety of cancers including pancreatic cancer. A number of Aurora kinase inhibitors (AKIs) are currently being tested in preclinical and clinical settings as anti-cancer therapies. However, the antitumor activity of AKIs in clinical trials has been modest. In order to improve the antitumor activity of AKIs in pancreatic cancer, we utilized a kinome focused RNAi screen to identify genes that, when silenced, would sensitize pancreatic cancer cells to AKI treatment. A total of 17 kinase genes were identified and confirmed as positive hits. One of the hits was the platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), which has been shown to be overexpressed in pancreatic cancer cells and tumor tissues. Imatinib, a PDGFR inhibitor, significantly enhanced the anti-proliferative effect of ZM447439, an Aurora B specific inhibitor, and PHA-739358, a pan-Aurora kinase inhibitor. Further studies showed that imatinib augmented the induction of G2/M cell cycle arrest and apoptosis by PHA-739358. These findings indicate that PDGFRA is a potential mediator of AKI sensitivity in pancreatic cancer cells.

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Figures

Figure 1
Figure 1. Inhibition of pancreatic cancer cell growth by AKIs
AsPC-1, BxPC-3, CFPAC-1, Mia PaCa-2, PANC-1, and SU86.86 pancreatic cancer cells were treated with a serial dilution of VX-680 (A), AKI-1 (B), or MP235 (C). Cell viability was measured at 96 hours after drug treatment by CellTiter Glo® Assay.
Figure 2
Figure 2. Imatinib sensitizes pancreatic cancer cells to the treatment of ZM447439 and PHA-739358
AsPC-1, BxPC-3, and SU86.86 cells were treated with a serial dilution of ZM447439 (A~C) or PHA-739358 (D~F) in combination with different fixed concentrations of imatinib. Each of the drug dose response curves was normalized to the percentage cell survival of imatinib only treatment at the indicated concentration based on the Bliss independence drug interaction model. ZM: ZM447439; PHA: PHA-739358
Figure 3
Figure 3. Increased apoptotic cell death induced by the combination treatment of PHA-739358 and imatinib
AsPC-1 cells were treated with PHA-739358 (PHA) (3μM), imatinib (15 μM), or combination of PHA and imatinib for 72 hours and then subjected to evaluation of cell morphology changes by microscope (A), Caspase activity levels by the Caspase 3/7 Glo® assay (B), and Bcl2 and Bcl-xL expression levels by Western blotting (C). Etopside (100μM) was used as a positive control for the Caspase 3/7 assay in B. The intensities of the Western blotting bands (Top panel in C) were quantified using the ImageJ software (63) and each band was normalized with their corresponding β-Actin loading controls (Bottom panel in C). * indicates significant differences between the two treatments (P<0.001).
Figure 3
Figure 3. Increased apoptotic cell death induced by the combination treatment of PHA-739358 and imatinib
AsPC-1 cells were treated with PHA-739358 (PHA) (3μM), imatinib (15 μM), or combination of PHA and imatinib for 72 hours and then subjected to evaluation of cell morphology changes by microscope (A), Caspase activity levels by the Caspase 3/7 Glo® assay (B), and Bcl2 and Bcl-xL expression levels by Western blotting (C). Etopside (100μM) was used as a positive control for the Caspase 3/7 assay in B. The intensities of the Western blotting bands (Top panel in C) were quantified using the ImageJ software (63) and each band was normalized with their corresponding β-Actin loading controls (Bottom panel in C). * indicates significant differences between the two treatments (P<0.001).
Figure 3
Figure 3. Increased apoptotic cell death induced by the combination treatment of PHA-739358 and imatinib
AsPC-1 cells were treated with PHA-739358 (PHA) (3μM), imatinib (15 μM), or combination of PHA and imatinib for 72 hours and then subjected to evaluation of cell morphology changes by microscope (A), Caspase activity levels by the Caspase 3/7 Glo® assay (B), and Bcl2 and Bcl-xL expression levels by Western blotting (C). Etopside (100μM) was used as a positive control for the Caspase 3/7 assay in B. The intensities of the Western blotting bands (Top panel in C) were quantified using the ImageJ software (63) and each band was normalized with their corresponding β-Actin loading controls (Bottom panel in C). * indicates significant differences between the two treatments (P<0.001).
Figure 4
Figure 4. Combination of PHA-739358 and imatinib inhibits the phosphorylation of PI3K but not ERK
AsPC-1 cells were treated with PHA-739358 (PHA) (3μM), imatinib (15 μM), or the combination of PHA and imatinib. Cells were harvested 72 hours after drug treatment and 20 μg of whole cell lysates were used in Western blotting detection of the proteins (Top panel). The intensities of the bands were quantified using the ImageJ software and normalized with their corresponding β-Actin loading controls (Bottom panel)
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