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. 2014 Mar 26;6(229):229ra43.
doi: 10.1126/scitranslmed.3007965.

Cytotoxicity of Paclitaxel in Breast Cancer Is Due to Chromosome Missegregation on Multipolar Spindles

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Free PMC article

Cytotoxicity of Paclitaxel in Breast Cancer Is Due to Chromosome Missegregation on Multipolar Spindles

Lauren M Zasadil et al. Sci Transl Med. .
Free PMC article

Abstract

The blockbuster chemotherapy drug paclitaxel is widely presumed to cause cell death in tumors as a consequence of mitotic arrest, as it does at concentrations routinely used in cell culture. However, we determine here that paclitaxel levels in primary breast tumors are well below those required to elicit sustained mitotic arrest. Instead, cells in these lower concentrations of drug proceed through mitosis without substantial delay and divide their chromosomes on multipolar spindles, resulting in chromosome missegregation and cell death. Consistent with these cell culture data, most mitotic cells in primary human breast cancers contain multipolar spindles after paclitaxel treatment. Contrary to the previous hypothesis, we find that mitotic arrest is dispensable for tumor regression in patients. These results demonstrate that mitotic arrest is not responsible for the efficacy of paclitaxel, which occurs because of chromosome missegregation on highly abnormal, multipolar spindles. This mechanistic insight may be used to improve selection of future antimitotic drugs and to identify a biomarker with which to select patients likely to benefit from paclitaxel.

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Paclitaxel has concentration-dependent effects
(A) Paclitaxel-treated cells exhibit mitotic spindles containing the indicated number of spindle poles as assessed by staining for microtubules (red) and DNA (teal) in MDA-MB-231 cells. (B and C) MDA-MB-231 (B) and Cal51 (C) triple negative breast cancer cell lines both show a direct relationship between number of poles per spindle and paclitaxel concentration. n ≥ 100 cells from each of 3 independent experiments. (D and E) In response to increasing concentrations of paclitaxel, mitotic index in both MDA-MB-231 (D) and Cal51 (E) cells peaks and then declines. n ≥ 1500 cells from 3 separate experiments. (F and G) Duration of mitosis in response to paclitaxel in both MDA-MB-231 (F) and Cal51 (G) cells closely mimics mitotic index. Duration of mitosis was measured as length of time from rounding to flattening of the first daughter cell in 10× phase contrast movies acquired every 10 minutes for 65 hours. n ≥ 30 cells per condition.
Figure 2
Figure 2. Mitotic arrest is not required for response to paclitaxel
(A) Schematic showing the trial design. Research biopsy and serum were obtained 20 hours after the start of the first 3 hour paclitaxel infusion. Tumor measurements were obtained by mammogram and/or ultrasound, depending on patient insurance. AC = a combination of Adriamycin and cyclophosphamide. (B through D) Mammograms (B) and ultrasounds (C, D) used to measure tumor response. Yellow lines in C and D indicate tumor measurements. (E) Waterfall plot showing change in largest tumor diameter. Response to paclitaxel is determined as a decrease of ≥30% in largest tumor diameter, according to RECIST 1.1 criteria (23). The grey line indicates this threshold. *Note that the second tumor measurement was obtained after cycle 1 of AC in patient 1, but before AC in all other patients. (F) Mitotic index before (open square) and after 20 hours of paclitaxel treatment (arrowhead; the direction indicates increase or decrease in mitotic index in response to paclitaxel). n ≥ 500 cells. (G) Patient 5 tumor stained with phosphorylated histone H3 (pH3; to mark mitotic cells), DAPI, and cytokeratin (to identify epithelial cells). Insets, enlargements of mitotic cells. Scale bar = 50 μm.
Figure 3
Figure 3. Clinically relevant concentrations of paclitaxel cause cell death
(A) Low nM concentrations of paclitaxel cause a decrease in live cell number over 120 hours in MDA-MB-231 (left) and Cal51 (right) cells. (B) Trypan blue assay showing an increase in the proportion of dead cells in both MDA-MB-231 (left) and Cal51 (right) cell lines by 120 hours of paclitaxel treatment. (C) Low nM paclitaxel inhibits the ability of cells to form colonies after 14 days of paclitaxel treatment. No colony formation was observed at concentrations above 100 nM paclitaxel in either cell line. (A–C) n ≥ 3 independent experiments. * = p < 0.05 as compared to DMSO control by Wilcoxon (A, C) or Mantel-Haenszel (B) statistical analysis. Exact p values are listed in Table S2.
Figure 4
Figure 4. Low concentrations of paclitaxel increase aneuploidy and chromosome missegregation
(A) Chromosome spread from a Cal51 cell containing 46 chromosomes. (B–C) Cal51 cells treated for 48 hours with 5, 10, or 50 nM paclitaxel show an increase in aneuploidy (B) that is predominantly near-diploid with little effect on triploidy or tetraploidy (C). n = 50 cells from each of 3 independent experiments. (D–E) Low dose paclitaxel increases abnormal mitotic events. (D) Cal51 cells expressing H2B-RFP (red) and GFP-tubulin (green) were filmed at 60× with 2-minute intervals during mitosis in DMSO (top row) or 10 nM paclitaxel (center and bottom rows). Shown are still frames from Videos S1–3. Time is shown in hours:minutes. Cells are able to divide in the presence of drug on multipolar spindles. (E) Quantitation of mitotic defects in Cal51 cells. The increase in defects is mainly due to multipolar spindles and >2–way DNA divisions. n=20–36 cells per condition. * = p < 0.05 as compared to DMSO control by Wilcoxon statistical analysis. Exact p values are listed in Table S2.
Figure 5
Figure 5. Paclitaxel causes multipolar spindles in patient tumors
(A) Immunofluorescence of patient tumors reveals additional spindle poles, indicated by NuMA staining. Not all poles contain γ-tubulin. Asterisks denote misaligned chromosomes, which occur even on bipolar spindles. Scale bar = 2.5 μm. (B) The incidence of multipolar spindles, defined as containing >2 NuMA foci, increased in every patient after paclitaxel treatment. Samples sizes for patients 1, 3, 4, 5 and 6, respectively, are 9, 9, 25, 53, and 19 mitotic cells pre-treatment and 11, 31, 29, 35 and 26 mitotic cells post-paclitaxel.

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