Fig 1.
TNBC cell lines have diverse sensitivity to Plk1 inhibition and chemotherapy.
TNBC cell lines: SUM149, SUM159, SUM229, SUM1315 and DU4475 cells, and the non-tumorigenic human breast epithelial cell line MCF10A. TNBC cell lines were treated for 72 hours in triplicates. Three replicate data were performed for each concentration and the best dose–response model (r conformity closest to 1) was selected and graphed. After a 72 hours exposure to drug, cell number was determined by harvesting and counting nuclei with a Beckman Coulter Z1 Particle Counter. IC50, IC25, and r conformity were determined using CompuSyn software. (A) Sensitivity to GSK461364, Plk1 inhibitor, in TNBC cell lines; IC50, IC25, and r conformity were reported for GSK461364. (B) TNBC cell lines sensitivity with respective IC50 and r conformity to docetaxel. (C) TNBC cell lines sensitivity with respective IC50 and r conformity to cisplatin.
Fig 2.
Plk1 inhibition synergizes with docetaxel and cisplatin in SUM149 and SUM159.
(A), Combination index (CI) curves for the 6 cell lines treated with GSK461364 plus docetaxel or cisplatin. TNBC cell lines: SUM149, SUM159, SUM229, SUM1315 and DU4475 cells, and the non-tumorigenic human breast epithelial cell line MCF10A. TNBC cell lines were treated with the Plk1 inhibitor GSK461364 (g) at the IC25 dose plus each chemotherapy drug (d, docetaxel; c, cisplatin) at the IC50 concentration. (B), CI and DRI values for each combination of GSK461364 plus chemotherapy. CI and the dose-reduction index (DRI) values were determined using CompuSyn software. CI <1 indicates a synergism. DRI >1 indicates a favorable dose reduction.
Fig 3.
Onvansertib synergizes with paclitaxel in SUM149 and SUM159.
SUM149 and SUM159 cells were treated for 72 hours in triplicates with onvansertib and paclitaxel (A). After a 72 hours exposure to drug, cell number was determined by harvesting and counting nuclei with a Beckman Coulter Z1 Particle Counter. IC50 was determined using CompuSyn software. SUM149 and SUM159 were treated with the Plk1 inhibitor onvansertib (o) at the IC50 dose plus paclitaxel (p) at the IC50 concentration (B). Combination Index (CI) values for each combination of onvansertib plus paclitaxel were determined using CompuSyn software. CI <1 indicates a synergism.
Fig 4.
Plk1 inhibition suppresses the clonogenic potential of SUM149 and SUM159.
(A) TNBC cell lines (SUM149, SUM159, SUM229, SUM1315 and DU4475 cells, and the non-tumorigenic human breast epithelial cell line MCF10A) were incubated with GSK461364, docetaxel, and cisplatin at the indicated concentrations for 24 hours; the medium was then changed to remove the drug, and colony formation was measured 7–14 days later. (B) EC50 was defined as 50% colony inhibition. Single agents EC50 were reported for each TNBC cell line and drug. According to the synergistic combination of docetaxel and cisplatin with GSK461364 we have observed in SUM149 and SUM159 cells, we focused on these two cell lines in the next experiments. (C), Colony forming assay photographs for single agents GSK461364, docetaxel, and cisplatin, with the respective combinations, in SUM149 and SUM159. (D) Histograms show colony formation rates, normalized to DMSO colony number for each cell line, for single drug and respective combinations; GSK461364 plus docetaxel interacted synergistically to decrease the clonogenic potential of SUM149 and SUM159 (p < 0.001 and p < 0.001, respectively; Interaction Test, p = 0.001* and p = 0.03+, respectively).
Fig 5.
GSK461364 plus docetaxel reduce tumorsphere formation of SUM149 and SUM159.
(A, histograms and B, pictures) For the primary tumorsphere assay, we analyzed the tumorsphere formation in presence or absence of docetaxel, GSK461364, and the combination at the IC50 concentrations identified in the proliferation assay; SUM149 and SUM159 cells were plated at a density of 20,000 cells per well in ultra-low attachment 6 well plates and after 24 hours treated with DMSO (control), docetaxel IC50, GSK461364 IC50, or both in combination. Formed spheres were counted using the Celigo Imaging Cytometer (Nexcelom, USA). GSK461364 plus docetaxel decreased primary tumorsphere formation of SUM149 and SUM159 (Interaction Test, p = 0.01* and p < 0.001+, respectively). (C, histograms and D, pictures) Dissociated tumorspheres (N = 500) were plated in ultra-low attachment 96 well plates (Costar®, USA) in serum-free medium for an additional seven days in the absence of drug. Secondary passage tumorspheres were then counted as before. GSK461364 plus docetaxel decreased primary tumorsphere formation of SUM149 and SUM159 (Interaction Test, p < 0.001+ and p < 0.001+, respectively). Poisson regression model with drug interaction was used to assess statistical differences.
Table 1.
Statistical analysis for secondary tumorsphere formation assay.
Fig 6.
Cell cycle immunoblotting, apoptosis and DNA content analysis.
For immunoblotting, apoptosis and DNA content analysis, cells were synchronized with double thymidine block. Cells were released for 2 hours in fresh media and treated with GSK461364 (G), GSK461364 + docetaxel (G+D) at the IC50 concentrations, and DMSO (control). (A) Immunoblotting of Plk1, FoxM1, Cyclin B1, CDK1, Phosphorus-histone H3 (Ser10), Cleaved PARP (89KDa), and β-actin were repeated at 8, 24, and 48 hours. For DNA content analysis, SUM149 (B) and SUM159 (C) cells were collected at 8, 24, and 48 hours and stained with propidium iodide. Data were acquired on BD Fortessa X-20 Analytic Flow Cytometer and analyzed with FlowJo. DNA copy content data showed in B and C were summarize in the histograms in D.
Fig 7.
In vivo efficacy of onvansertib alone or in combination with paclitaxel against SUM159 xenografts.
SUM159 cells were implanted in the mammary fat pad of NOD-scid-IL2 receptor gamma null female mice, and treatments began 14–21 days later when tumors were well established (tumor volume ≥ 40 mm3). Onvansertib was given by oral gavage (PO) on two consecutive days every week; paclitaxel was given intraperitoneally (IP) once per week; controls received PO vehicle on two consecutive days every week and IP vehicle once per week. Tumor volume was assessed twice per week and mice treated for 3 weeks. Mean of tumor volume with standard errors of the mean were plotted.