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. 2018 May 25;9(40):25842-25859.
doi: 10.18632/oncotarget.25386.

Synthetic Lethality in CCNE1-amplified High Grade Serous Ovarian Cancer Through Combined Inhibition of Polo-like Kinase 1 and Microtubule Dynamics

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Free PMC article

Synthetic Lethality in CCNE1-amplified High Grade Serous Ovarian Cancer Through Combined Inhibition of Polo-like Kinase 1 and Microtubule Dynamics

Sabrina Noack et al. Oncotarget. .
Free PMC article

Abstract

The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.

Keywords: cell cycle; ovarian cancer; paclitaxel; protein kinases; sensitization.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. BI6727 treatment sensitizes ovarian cancer cells to paclitaxel
(A) OVCAR-3 cells were treated with increasing concentrations of BI6727 or (B) of paclitaxel (Pac). Cell viability was measured over 4 d using the Cell Titer-Blue® Cell Viability Assay. (C) (Left panel) The G2/M fraction was determined over 4 d post-treatment using flow cytometry. Measurements were statistically significant by two-tailed Student's t-test (*P ≤ 0.05; **P ≤ 0.01). Each bar graph represents the mean value ± SEM (n=3). (Upper right panel) Endogenous levels of PLK1, Cyclin B1, phospho-Histone H3 and phospho-Aurora B were determined by immunoblotting. β-Actin served as loading control. (Lower right panel) The mitotic index was determined by measuring pH3(Ser10) levels. (D-G) OVCAR-3 cells were treated with either 20 nM BI6727 or increasing paclitaxel concentrations or both for 4 d. The cell viability was determined using the Cell Titer-Blue® Cell Viability Assay. Measurements were statistically significant by two-tailed Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Each measurement represents the mean value ± SEM (n=3).
Figure 2
Figure 2. The combinatorial treatment of BI6727 and paclitaxel induces pronounced levels of apoptosis
(A) OVCAR-3 cells were treated with 20 nM BI6727, increasing paclitaxel concentrations or both. Apoptosis was analyzed 24 h post-treatment by western blotting for Caspase-3, full-length and cleaved PARP. β-Actin served as loading control. (B) Caspase-3/7-activity in whole cell lysates of 20 nM BI6727, paclitaxel or both treated OVCAR-3 cells was measured 24 h and 48 h post-treatment using the Caspase-Glo® 3/7 Assay. Each bar graph represents the mean value ± SEM (n=2). Apoptosis was validated (C) by measuring the sub G0/G1 fractions or (D) by PE Annexin V staining. Measurements were statistically significant by two-tailed Student's t-test (*P ≤ 0.05; **P ≤ 0.01). Each bar graph represents the mean value ± SEM (n=3).
Figure 3
Figure 3. Analysis of apoptotic signaling in ovarian cancer cells
(A) (upper panel) Whole cell lysates of OVCAR-3 cells treated with BI6727 or 3.5 nM paclitaxel or both were analyzed evaluating marker proteins for mitochondrial-mediated apoptosis. Endogenous levels of PARP, cleaved PARP, Caspase-3, cleaved-Caspase-3, BCL-XL pS62, BCL-XL, pBCL-2, BAK, BAX, EndoG, Securin, PLK1, pHistone H3 and ß-Actin were determined by immunoblotting. Caspase-3/7-activity in whole cell lysates of 20 nM BI6727, paclitaxel or both treated OVCAR-3 cells was measured 48 h post-treatment using the Caspase-Glo® 3/7 Assay. (B) Evaluation of the mitochondrial membrane potential. The data are presented as means ± SD. Statistical analysis among treatment groups was performed. ** (P< 0.01), *** (P< 0.001). (C) Endogenous levels of FBW7, MCL-1, Cyclin E, Caspase-3 and ß-Actin were determined by immunoblotting using specific antibodies.
Figure 4
Figure 4. Combinatorial treatment of OVCAR-3 cells with 20 nM BI6727 and 3
5 nM paclitaxel displays long lasting inhibition of cell growth. (A) Experimental set up of long-term analyzes. (B) Coomassie stained regrown colonies of OVCAR-3 cells treated with 20 nM BI6727, 3.5 nM paclitaxel or both for 24 h. (C) The number of colonies was determined. Numbers were statistically significant by two-tailed Student's t-test (*P≤ 0.05; **P≤ 0.01). Each bar graph represents the mean value ± SEM (n=3). (D) OVCAR-3 cells were grown as 3-D culture over 14 d and treated with 20 nM BI6727, 2.5 nM paclitaxel or both for 5 d. Cells were stained using the LIVE/DEAD viability/cytotoxicity kit and ratios of viable/dead cells were calculated. Measurements were statistically significant by two-tailed Student's t-test (*P≤ 0.05; **P≤ 0.01). Each bar graph represents the mean value ± SEM (n=3).
Figure 5
Figure 5. BI6727 treatment sensitizes patient-derived HGSOC cells to paclitaxel
(A) Primary tumor cells were treated with increasing BI6727 concentrations and the cell cycle distribution was analyzed by flow cytometry. (B) Primary tumor cells were either treated with increasing paclitaxel concentrations or both (increasing concentrations of paclitaxel and 100 nM BI6727). The cell viability was determined for 72 h or (C) over a period of 6 d using the Cell Titer-Blue Cell® Viability Assay. (D) 3-D cultures grown out of primary tumor cells were treated with either 5 nM paclitaxel, 50 nM BI6727 or both for 7 d. Cells were stained and fluorescence intensities of dead cells were determined. Measurements were statistically significant by two-tailed Student's t-test (*P≤ 0.05; **P≤ 0.01). Each bar graph represents the mean value ± SEM (n=3).
Figure 6
Figure 6. The combinatorial treatment of paclitaxel and BI6727 reduces neurotoxicity
(A) Experimental set up of neurotoxicity analysis using PC-12 cells. (B) PC-12 cells were differentiated into neurons using β-NGF. The outgrowth of neurites was analyzed 24 h and 48 h post-treatment and cells displaying elongations were counted. Numbers were statistically significant by two-tailed Student's t-test (*P≤ 0.05). Each bar graph represents the mean value ± SEM (n=3). (C) The lengths of neurites were measured 24 h/48 h post-treatment and depicted as Scatter plots. Measurements were statistically significant by Student's t-test (*P≤ 0.05; **P≤ 0.01; ***P≤ 0.001).

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