Antiproliferative Mechanism of Action of the Novel Taxane Cabazitaxel As Compared with the Parent Compound Docetaxel in MCF7 Breast Cancer Cells

Published OnlineFirst June 30, 2014; DOI: 10.1158/1535-7163.MCT-14-0265

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

Antiproliferative Mechanism of Action of the Novel Taxane Cabazitaxel as Compared with the Parent Compound Docetaxel in MCF7 Breast Cancer Cells

Olga Azarenko, Gregoriy Smiyun, Jeffrey Mah, Leslie Wilson, and Mary Ann Jordan

Abstract Cabazitaxel, a novel chemotherapeutic taxane, is effective against docetaxel-resistant cells and tumors. It is approved for treatment of metastatic hormone-refractory prostate cancer in patients pretreated with docetaxel. Objective responses have been observed in many other cancers, including pretreated metastatic breast cancer. Cabazitaxel and docetaxel share a high degree of structural similarity. The basis for cabazitaxel’s efficacy is unclear, and its mechanism has not been described. We compared the effects of cabazitaxel and docetaxel on MCF7 human breast cancer cells expressing fluorescent tubulin. Both drugs inhibited cell proliferation (IC50s, cabazitaxel, 0.4 0.1 nmol/L, docetaxel, 2.5 0.5 nmol/L) and arrested cells in metaphase by inducing mitotic spindle abnormalities. Drug concentrations required for half- maximal mitotic arrest at 24 hours were similar (1.9 nmol/L cabazitaxel and 2.2 nmol/L docetaxel). Cabazitaxel suppressed microtubule dynamic instability significantly more potently than docetaxel. In particular, cabazitaxel (2 nmol/L) suppressed the microtubule shortening rate by 59% (compared with 49% for 2 nmol/L docetaxel), the growing rate by 33% (vs. 19%), and overall dynamicity by 83% (vs. 64%). Cabazitaxel was taken up into cells significantly faster than docetaxel, attaining an intracellular concentration of 25 mmol/L within 1 hour, compared with 10 hours for docetaxel. Importantly, after washing, the intracellular cabazitaxel concentration remained high, whereas the docetaxel concentration was significantly reduced. The data indicate that the potency of cabazitaxel in docetaxel-resistant tumors is due to stronger suppression of microtubule dynamics, faster drug uptake, and better intracellular retention than occurs with docetaxel. Mol Cancer Ther; 13(8); 2092–103. 2014 AACR.

Introduction acquired resistance to these drugs (5–7). Paclitaxel and The chemotherapeutic taxanes paclitaxel (Taxol) and docetaxel both have a high affinity for multidrug resistance docetaxel (Taxotere) play important, well-established roles proteins (7–9). in the treatment of a variety of malignancies, including a Cabazitaxel (Jevtana) is a novel third-generation semi- broad spectrum of solid tumors (1, 2). Paclitaxel is widely synthetic analog of docetaxel (8, 10–12). Structurally, used for the treatment of breast, ovarian, and non–small cell cabazitaxel and docetaxel are very similar with the excep- lung cancer, whereas docetaxel-containing regimens are tion of 2 methoxy side chains in cabazitaxel that substitute indicated for metastatic prostate, breast, and other cancers for hydroxyl groups in docetaxel (Fig. 1A). In preclinical (3, 4). Despite the clinical success of microtubule-targeting testing, cabazitaxel demonstrated activity in both doce- agents, toxicity (mostly neutropenia and neurotoxicity), taxel-sensitive and docetaxel-resistant cancers (13, 14). In complex formulations (use of CremophorEL), and limited addition, the terminal half-life for cabazitaxel in humans oral bioavailability restrict their clinical use in cancer ther- is 95 hours (15) as compared with 12 hours for docetaxel apy (1). Many tumor types are refractory and/or develop (16). Cabazitaxel was approved in 2010 as a second-line treatment in men with metastatic castration-resistant prostate cancer that failed docetaxel-containing regimens Authors' Affiliation: Department of Molecular, Cellular, and Developmen- (17, 18). Like paclitaxel and docetaxel, cabazitaxel stabi- tal Biology and the Neuroscience Research Institute, University of Cali- fornia Santa Barbara, Santa Barbara, California lizes microtubules against cold-induced depolymeriza- tion and promotes assembly of tubulin into microtubules Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). (12, 14). Microtubules are major components of the eukaryotic Corresponding Author: Mary Ann Jordan, Neuroscience Research Insti- tute, Life Sciences Building, Room 1117, University of California, Santa cytoskeleton. They are involved in cell division, migra- Barbara, Santa Barbara, CA 93106. Phone: 805-893-5317; Fax: 805-893- tion, signaling, and intracellular trafficking and are 8094; E-mail: [email protected] important in cancer cell proliferation and metastasis (2). doi: 10.1158/1535-7163.MCT-14-0265 Microtubules are composed of ab-tubulin heterodimers 2014 American Association for Cancer Research. that link together noncovalently during polymerization to

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Figure 1. Concentration-dependent effects of cabazitaxel and docetaxel (A) on inhibition of proliferation (B), induction of cell death (C), mitotic arrest (D), and G2–M arrest (E and F) in MCF7 cells. A, the chemical structures of docetaxel (Taxotere) and cabazitaxel (Jevtana). In cabazitaxel methoxy side chains substitute for hydroxyl groups in docetaxel (highlighted in red). B, cells were incubated with cabazitaxel (-&-) or docetaxel (-^-) for 72 hours, and inhibition of proliferation was measured using a sulforhodamine B assay. Cabazitaxel and docetaxel inhibited cell proliferation with IC50 values of 0.4 0.1 (SEM) nmol/L and 2.5 0.5 (SEM) nmol/L, respectively. C, cells were incubated with vehicle control (x), cabazitaxel for 24 (-&-), 48 (-*-), and 72 (-~-) hours, and docetaxel for 24 (-&-), 48 (-*-), and 72 (-~-) hours and the population of combined apoptotic/dead cells was determined by flow cytometry. Controls at 24, 48, and 72 hours displayed similar levels of apoptosis (1.5%, -x-). D, cells were incubated with cabazitaxel (-&-) and docetaxel (-^-) for 24 hours, and the percentage of mitotic cells was determined using immunofluorescence microscopy. Cabazitaxel and docetaxel induced mitotic arrest with IC50's of 1.9 and 2.2 nmol/L, respectively. Cells were incubated with cabazitaxel (CBZ) for 24 (-&-), 48 (-*-), and 72 (-~-) hours (E), and with docetaxel (DOC) for 24 (-&-), 48 (-*-), and 72 (-~-) hours (F), and the population of G2–M cells was determined by flow cytometry. Control (CTR) cells in G2–Mat24(^), 48 (^), and 72 hours gray diamond. Results are means and SEM of at least 3 independent experiments. , P < 0.01; , P < 0.001 by the Student t test.

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form 25-nm-diameter hollow cylindrical filaments (19). nificantly more effective suppression of microtubule Microtubules are highly dynamic structures that alternate dynamics, rapid intracellular uptake, and prolonged between periods of growth and shortening through the drug retention play significant roles in its efficacy in addition or loss of tubulin subunits at microtubule ends docetaxel-resistant tumors. (20). This nonequilibrium behavior is termed "dynamic instability" and occurs both in vitro with microtubules reassembled from purified tubulin and in cells. Dynamic Materials and Methods microtubules are especially important during mitosis Materials when they are required for the capture and complex Chemicals and materials were from Sigma-Aldrich movements of chromosomes, including chromosome unless otherwise noted. Cabazitaxel and docetaxel were alignment during metaphase and separation during ana- provided by Sanofi-Aventis (France) and stored as ali- phase (21, 22). Dynamic spindle microtubules are crucial quots of 10 mmol/L stock solutions in DMSO at 20C. to successful cell division, making cells highly susceptible Stock solutions were diluted in DMSO to 1 mmol/L and to drugs that suppress microtubule dynamic instability at further diluted in cell culture medium. low concentrations (23, 24). The taxanes, including paclitaxel and docetaxel, are Cell culture microtubule-stabilizing antitumor drugs that enhance MCF7 human breast adenocarcinoma cells were cul- microtubule polymerization at high concentrations (25, tured in DMEM with 10% fetal bovine serum (Atlanta 26). All taxanes bind to the same or to an overlapping Biologicals, Inc.), 1% final concentration of nonessential taxoid-binding site on b-tubulin, located on the inner amino acids, 100 units/mL penicillin, and 100 mg/mL surface of the microtubule (27, 28). Taxanes and other streptomycin at 37 C, 5.5% CO2. Shortly after purchase microtubule-targeting drugs inhibit cancer cell prolif- (ATCC, 2007), the cells were transfected with an enhanced eration by binding to microtubules and suppressing green fluorescent protein EGFP-a-tubulin vector (Clon- microtubule dynamics during the particularly vulner- tech Laboratories; ref. 34). The EGFP-a-tubulin-expres- able mitotic stage of the cell cycle without significantly sing MCF7 cells were indistinguishable from unmodified altering the mass or organization of microtubules (26). MCF7 cells except for their fluorescent microtubules and Stabilization of mitotic spindle microtubule dynamics their doubling time of 35 hours, which was 20% slower leads to mitotic arrest, inhibition of cell proliferation, than unmodified MCF7 cells. They continue to express and cell death (23, 24). EGFP-a-tubulin, were used for all experiments, were free The basic mechanisms of action of cabazitaxel on of mycoplasma, and are referred to hereafter as MCF7 microtubules have not been elucidated. It is especially cells. important to understand the differences in the mechanisms that may be responsible for cabazitaxel’s efficacy Cell proliferation in docetaxel-refractory cancers (17). We used EGFP- Proliferation was measured by a modified sulforhoda- a-tubulin-expressing MCF7 human breast cancer cells mine B assay (32). MCF7 cells were seeded in medium to compare the concentration- and time-dependent (5,000 cells/200 mL) in 96-well plates and incubated 24 effects of docetaxel and cabazitaxel on cell proliferation, hours. Fresh medium with or without the drugs was microtubule arrangement and dynamic instability, mito- added, and incubation continued for 72 hours. Cells were sis, apoptosis, and drug uptake and retention. We have fixed, stained, and the optical density determined (490 previously used EGFP-a-tubulin-expressing MCF7 cells nm; Victor3V Wallac 1420 Spectrophotometer, Perkin- extensively for determination of drug effects on micro- Elmer). Triplicates of each condition were tested in each tubule dynamic instability (29–32) because they are flat experiment. Results are mean and SEM of at least 4 enough to enable tracing the length changes of individ- experiments. The inhibitory concentrations that inhibited ual microtubules. In addition, both docetaxel and caba- cell proliferation by 50% were calculated using Prism 4.0 zitaxel are effective in breast cancer singly or in combi- software (GraphPad Software, Inc). nation (10, 33). In comparing cabazitaxel and docetaxel, we found that Cell viability and apoptosis/cell death both drugs inhibit cell proliferation and induce mitotic Cells were seeded (6 104 cells/2 mL) in 6-well plates arrest in association with suppression of microtubule (24 hours), and incubated with drug for 24, 48, and 72 dynamic instability. However, cabazitaxel suppresses hours. For viability, all cells were harvested and stained microtubule dynamic instability significantly more with ViaCount DNA binding dyes (5 minutes; EMD potently than docetaxel and induces a more sustained Millipore) and analyzed by flow cytometry (EasyCyte G2–M arrest over 72 hours. The rate of cabazitaxel’s flow cytometer; Guava Technologies, Inc.) to distin- uptake into cells is significantly faster than that of doc- guish live and apoptotic/dead cells. For cell-cycle anal- etaxel, and the intracellular concentration of cabazitaxel ysis, floating and adherent cells were collected, permea- does not change significantly upon washing the cells, bilized with ice-cold 70% ethanol, washed with PBS, whereas docetaxel levels are significantly reduced. and stained with cell-cycle reagent (EMD Millipore). Together, these findings suggest that cabazitaxel’s sig- The DNA content of 5,000 cells was measured by flow

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cytometry and analyzed with ModFit LT software (Ver- was replaced with fresh media containing 50 nmol/L ity Software House). Results are mean SEM, 3 [14C] cabazitaxel or 50 nmol/L [14C] docetaxel (final spe- independent experiments. cific activity: 83–86 mCi/mmol/L; Sanofi-Aventis Deutschland GmbH, Germany) or unlabeled drug. Media Mitotic arrest was removed 30 minutes to 20 hours after drug addition, þ MCF7 cells were seeded as described above for cell vials rinsed twice with 1 mL PBS, and Ready Protein viability and incubated with drug for 24 hours; both (Beckman Coulter, Inc.) added for scintillation counting floating and attached cells were collected and fixed in (LS 6500 Multi-Purpose Scintillation Counter; Beckman 3.7% formaldehyde (30 minutes), followed by cold meth- Coulter, Inc.). Intracellular drug concentration ¼ (moles of anol (10 minutes; ref. 29). Fixed cells were mounted on intracellular drug bound/mean cell volume) (the num- glass slides with antifade agent Prolong Gold-DAPI (Life ber of cells per vial). Mean cell volume (4.57 10 12 L) is Technologies, Inc.) to visualize DNA and examined by the mean diameter of cells rounded by trypsinization (n ¼ fluorescence microscopy (Nikon Eclipse E800, 40 objec- 30). After 20 hours drug, some cell vials were washed (2 tive). Mitotic cells were rounded cells with condensed washes fresh media, 30 minutes/wash) and intracellular chromosomes and no nuclear envelope. The percentage of drug concentration was determined 1 to 20 hours after mitotic cells was determined by counting 500 cells for wash. All time points were measured in duplicate. Results each condition. Results are mean SEM, 3 experiments. are means SEM of 3 experiments.

Immunofluorescence microscopy Cells were seeded as described above for cell viability Results on poly-L-lysine-treated coverslips, incubated with drug Cabazitaxel and docetaxel inhibit MCF7 cell 24 hours, fixed with 3.7% formaldehyde followed by cold proliferation and arrest cells in mitosis methanol (29) and stained with mouse monoclonal The effects of cabazitaxel and docetaxel on prolifera- a-tubulin antibody (1:1,000 DM1A) and FITC-conjugated tion of MCF7 cells were determined after 72 hours incu- goat anti-mouse antibody (1:1,000, Cappel MP Biochem- bation (Fig. 1B). Both taxanes inhibited proliferation with icals). Centrosomes were stained with rabbit polyclonal cabazitaxel exhibiting greater potency than docetaxel anti-pericentrin antibody (1:500, AB4448; Abcam) and (IC50’s SEM, 0.4 0.1 nmol/L and 2.5 0.5 nmol/ rhodamine-conjugated goat anti-rabbit antibody (1:500, L, respectively, P < 0.05). Minimal inhibition (9%) Cappel). Cells were mounted as above and imaged using a occurred at 1 pmol/L cabazitaxel or docetaxel, and spectral confocal Olympus Fluoview1000 microscope approximately 80% inhibition occurred at 1,000 nmol/ (FLV1000S, 60 oil, N.A. 1.4 objective; Olympus). L for each drug (Fig. 1B). The numbers of viable, and apoptotic/dead MCF7 cells Live cell analysis of microtubule dynamic instability induced by cabazitaxel and docetaxel (0.1–100 nmol/L) at Cells were seeded on coverslips in 6-well plates (3 24, 48, and 72 hours of incubation were determined by flow 104 cells/mL, 2 mL/well) for 24 hours, and then medi- cytometry. Both cabazitaxel (dashed lines, open symbols) um was replaced with fresh medium containing drug. and docetaxel (solid lines, filled symbols) induced time- Time-lapse images (38 frames/4-second intervals/1 and concentration-dependent apoptosis (Fig. 1C). At 24 hour at 37 1C) were taken with a 100 objective on hours (lowest curves), cabazitaxel (open squares) and doc- a Nikon Eclipse E800 microscope equipped with a Cool- etaxel (filled squares) induced similar numbers of apopto- SNAP HQ2 camera (Roper Scientific GmbH), Meta- tic/dead cells at all concentrations tested. For example, Morph 4.6 software (Molecular Devices; ref. 35). cabazitaxel and docetaxel (100 nmol/L) induced 3.5% and Changes in length of individual microtubules were 4% apoptotic/dead cells, respectively, compared with 1.5% graphed versus time as "life history" plots. Microtubule in controls (Fig. 1C). With increased time of incubation, dynamics parameters were analyzed with IGOR Pro 6.0 cabazitaxel induced significantly more cell death than (32). Changes 0.5 mm were growth or shortening events, docetaxel at all concentrations tested. For example, at 72 <0.5 mm were pause (attenuation). Time-based catastrophe hours, 100 nmol/L cabazitaxel induced 37% apoptotic/ frequency is the number of catastrophes (transitions from dead cells as compared with 27% for 100 nmol/L docetaxel growth or pause to shortening) divided by total time spent (P < 0.05; Fig. 1C). growing and paused. Rescue frequency is the number of To examine whether the antiproliferative effects were transitions from shortening to growth or pause divided by associated with mitotic arrest, we counted mitotic cells total time spent shortening. Dynamicity is total length at 24 hours incubation with a range of drug concentra- grown and shortened divided by total duration of imaging tions. Only 2.3% of control cells were mitotic (Fig. 1D). a microtubule. Results are mean SEM, 3 experiments. Half-maximal mitotic arrest occurred at 1.9 nmol/L cabazitaxel (squares) and 2.2 nmol/L docetaxel (dia- Uptake of [14C]-cabazitaxel and [14C]-docetaxel monds). At high drug concentrations (10–1,000 nmol/ To determine intracellular drug concentration (36), L), the number of mitotically-arrested cells at 24 hours MCF7 cells were seeded into poly-L-lysine-treated scin- was also similar for both drugs, reaching 60% to 65% tillation vials (1 105 cells, 2 mL). After 24 hours, media (Fig. 1D).

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We also determined the drug effects on G2–M arrest. Cabazitaxel and docetaxel induced similar changes in G2–M arrest measures the number of cells in G2,inM, microtubule organization and also cells that have slipped out of mitosis without MCF7 cells were incubated with a range of drug con- undergoing cytokinesis or division. After 24 hours, both centrations for 24 hours, fixed, and stained for microtu- drugs induced similar G2–M levels but their effects bules, centrosomes, and DNA and examined by immu- diverged after longer drug incubation. By flow cytometry nofluorescence microscopy. In control interphase cells cells in G2–M increased from 25% in controls to 84% to 88% microtubules (stained green) were fine filaments through- at 10 to 100 nmol/L of either drug at 24 hours (Fig. 1E and out the cell including the cell periphery, with a pair of F). Interestingly, with cabazitaxel the high degree of G2–M centrosomes (red) next to the nucleus (Fig. 2A). At caba- arrest was sustained with longer incubation times, but zitaxel and docetaxel concentrations that induced half- docetaxel did not maintain the same high level of G2–M maximal mitotic arrest (2 nmol/L), most interphase cells arrest at 48 and 72 hours. For example, in the presence of 10 were similar to controls, although a few were slightly and 100 nmol/L docetaxel, the number of cells in G2–M rounded (Fig. 2B and C). At 10 nmol/L of either drug decreased from 84% at 24 hours to 61% at 72 hours. (approximately 5 IC50), microtubules were bundled in

Figure 2. Effects of cabazitaxel and docetaxel on microtubule organization in interphase and mitotic MCF7 cells. Interphase control cell (A), and cells incubated with 2 nmol/L (1 mitotic IC50) cabazitaxel (B) and docetaxel (C) for 24 hours have intact microtubules with similar arrangements. At 100 nmol/L (50 mitotic IC50, 24 hours) cabazitaxel (D) and docetaxel (E), microtubules were signiﬁcantly bundled in the cell center, and showed a patchy distribution in the cell periphery. The very small cell-like body on the right in D may be an apoptotic body. Multinucleated cells were also observed (100 nmol/L cabazitaxel and docetaxel). In control mitotic cells (F), spindles were normal with well separated centrosomes and with chromosomes congressed to a compact metaphase plate. The majority of cells arrested in mitosis at 1 nmol/L cabazitaxel (G), 1 nmol/L docetaxel (H), 2 nmol/L cabazitaxel (I), and 2 nmol/L docetaxel (J) displayed some metaphase abnormalities, including uncongressed chromosomes (arrowheads), poorly deﬁned bipolar spindles or monopolar spindles (100 nmol/L cabazitaxel, K; and 100 nmol/L docetaxel, L), an increased number of astral microtubules, and poorly separated centrosomes. Arrows, multipolar mitotic spindles. Microtubules, green; centrosomes, red; DNA, blue. Scale bars, 5 mm.

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the interphase cell center and were shorter, more curved, centrations that induced little or no detectable change in and fragmented at the cell periphery than in controls (not the interphase microtubule network (1 nmol/L, 0.5 IC50 shown). At 100 nmol/L cabazitaxel or docetaxel, most for mitotic arrest for both drugs; Fig. 2G and H). A few cells were rounded, and fragmented or curved microtu- cells had normal bipolar spindles with compact meta- bules at the cell periphery increased, as did prominent phase plates of chromosomes (not shown). Most cells had microtubules bundles in the cell center (Fig. 2D and E). abnormal spindles (37), with more and longer astral Centrosome pairs were localized in the cell periphery. microtubules than in controls, with from few to many Both drugs induced some giant multinucleated cells and uncongressed chromosomes remaining at the spindle apoptotic ﬁgures (Fig. 2D and E). poles rather than forming a well-deﬁned metaphase plate Drug effects on mitotic spindle organization at 24 hours (Fig. 2G and H, arrowheads). At 1 IC50 (2 nmol/L), both are shown in Fig. 2F–L. Control mitotic cells had well- drugs induced bipolar spindle abnormalities (Fig. 2I and separated centrosomes and well-formed bipolar spindles J), and multipolar spindles were common (Fig. 2I and J, with chromosomes in a compact central metaphase plate arrows). At 1 to 2 nmol/L of both drugs, a few mitotically (Fig. 2F) and duplicated centrosomes at opposite spindle arrested cells had abnormal monoastral ball-shaped spin- poles. Both drugs induced spindle abnormalities at con- dles, with little, if any, centrosome separation. At 5 to

B Length ( m m)

Figure 3. Dynamic instability of microtubules in live interphase MCF7 cells incubated with cabazitaxel or docetaxel. A, time-lapse images of individual microtubules in the lamellar periphery of cells were recorded by video epiﬂuorescence microscopy: control (top row), cell incubated with 2 nmol/L cabazitaxel (middle row), and cell incubated with 2 nmol/L docetaxel (bottom row), at 24 hours. Microtubule ends (arrows) were tracked over time to produce life history plots (changes in length of individual microtubules over time) shown in B. Mitotic IC50 for both drugs was approximately 2 nmol/L.

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Table 1. Effects of cabazitaxel and docetaxel on microtubule dynamic instability in living MCF7 breast cancer cells

Control Cabazitaxel, nmol/L (24 hours) Docetaxel, nmol/L (24 hours) Parameter 0 2 % Change 2 % Change Growth rate (mm/min) 8.1 0.4 5.4 0.2a 33 6.6 0.3b 19 Shortening rate (mm/min) 16.2 1.1 6.7 0.3c 59 8.2 0.7a 49 Growth length (mm) 2.0 0.2 0.58 0.03a 71 0.78 0.1a 61 Shortening length (mm) 1.8 0.2 0.55 0.04a 72 0.70 0.1a 64 Attenuation duration (min) 0.14 0.01 0.33 0.04c þ136 0.20 0.03b þ43 Time growing (%) 54 13 37 Time shortening (%) 20 10 25 Time attenuated (%) 26 77 38 Catastrophe frequency (#/min) 2.4 0.1 1.5 0.1a 38 2.3 0.2 7 Rescue frequency (#/min) 9.1 0.4 12.6 0.4a þ37 11.6 0.6b þ27 Dynamicity (mm/min) 8.9 0.7 1.5 0.2c 83 3.2 0.5c 64

NOTE: Results are mean values SEM, and the corresponding percentage of change from control. Values were analyzed for statistical significance and differ significantly from controls at: aP < 0.01, bP < 0.05, and cP < 0.001 by the Student t test. Dynamicity per microtubule is the length grown and shortened divided by the total life span of the microtubule. At least 25 microtubules were analyzed for each condition. Values for percentage of time growing, shortening, and in the attenuated state were calculated for the entire population of microtubules and, thus, the confidence intervals for these parameters were not calculated.

10 IC50 (10–100 nmol/L) both drugs induced predom- Both taxanes significantly suppressed microtubule inantly ball-shaped spindles (Fig. 2K and L). dynamic instability parameters at concentrations 2 nmol/L (Table 1, Figs. 3A and B, 4, see also Supplemen- Both drugs suppress dynamic instability of tary Videos S2 and S3). Microtubules grew and shortened microtubules in living MCF7 cells at concentrations more slowly than in control cells, and suppression that arrest mitosis increased as the concentration of either drug was The effects of cabazitaxel and docetaxel on dynamic increased from 1 nmol/L (0.5 IC50) to 6 nmol/L (3 instability at plus ends of individual microtubules in the IC50; Fig. 4). thin peripheral regions of living interphase MCF7 cells Importantly, cabazitaxel (squares) inhibited the growth were examined after 24 hours of drug incubation with 1, 2, (Fig. 4A and C) and shortening (Fig. 4B and D) parameters and 6 nmol/L (concentrations that induced increasing significantly more potently than docetaxel (diamonds). degrees of mitotic arrest; Fig. 1D). Figure 3A shows For example, at 2 nmol/L, cabazitaxel and docetaxel representative frames from time-lapse series displaying suppressed the mean microtubule growth rate by 33% length changes of individual microtubules over time in a and 19%, respectively, and the mean shortening rate by control cell (top row) and cells incubated for 24 hours with 59% and 49%, respectively (Fig. 4A and B). The differences 2 nmol/L cabazitaxel (middle row) and 2 nmol/L doc- in suppression of growth and shortening rates and lengths etaxel (bottom row). Length changes were measured, and were not significant at 1 nmol/L of either drug, but were "life history plots" (Fig. 3B) of changes in length over time significantly different (between P < 0.05 and P < 0.001) at were used to determine dynamic instability parameters higher drug concentrations (2–6 nmol/L). Cabazitaxel (Table 1). In control cells, most microtubules were dynam- significantly more potently suppressed the catastrophe ic, undergoing frequent transitions between relatively frequency than docetaxel at all concentrations tested (P < slow growth and rapid shortening (Supplementary Video 0.001; Fig. 4E). At 6 nmol/L, cabazitaxel significantly S1). Plus ends of microtubules grew at 8.1 0.4 mm/min enhanced rescue frequency as compared with 6 nmol/L and shortened at 16.2 1.1 mm/min (Table 1). They docetaxel (P < 0.05; Fig. 4F and Table 1). Overall dynami- underwent catastrophes (transitions to shortening) at a city was suppressed significantly more strongly (P < 0.01 frequency of 2.4 0.12/microtubule/min and rescues and P < 0.001) by cabazitaxel than by docetaxel at all (transitions to growth or attenuation from shortening) at concentrations tested (Fig. 4G and Table 1). For example, a frequency of 9.1 0.4/microtubule/min. Microtubules at 6 nmol/L, cabazitaxel reduced dynamicity by 89% in controls spent 54% of time growing, 20% of time compared with a 76% reduction with docetaxel (Fig. shortening, and 26% of time in an attenuated or paused 4G, P < 0.001). Strikingly, 2 nmol/L cabazitaxel caused state. Their overall dynamicity (the total rate of exchange the microtubules to pause significantly more (a 136% of tubulin at microtubule ends expressed as length per increase in attenuation, compared with only 38% increase time) was 8.9 0.7 mm/min (Table 1). for 2 nmol/L docetaxel (Table 1 and Fig. 4H, P < 0.001).

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Figure 4. Concentration-dependent effects of cabazitaxel (-&-) or docetaxel (-^-) on dynamic instability parameters of microtubules in MCF7 cells at 24 hours compared with controls. A, growth rates; B, shortening rates; C, growing lengths; D, shortening lengths; E, catastrophe frequency; F, rescue frequency; G, dynamicity; EF and H, attenuation (pause) duration. Results are the mean SEM of three independent experiments. , P < 0.05; , P < 0.01; , P < 0.001 by the Student t test. ns, not signiﬁcant.

G H

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tions, except for a slight but signiﬁcantly greater mitotic arrestbycabazitaxelat24hours atlowdrugconcentrations (0.3and1nmol/L,P<0.001;Fig.1D).Inaddition,theeffects of the 2 drugs on G2–M cell-cycle arrest, apoptosis, and cell deathweresimilarat 24hours(Fig.1C,Eand F);bothdrugs induced approximately 85% arrest in G2–M at 24 hours (Fig.1EandF)butfewcells(<3%)enteredapoptosisordied (Fig.1D).Themoststrikingdifferencebetweenthedrugsat

Drug ( m mol/L) 24 hours incubation was that cabazitaxel suppressed all parametersof microtubuledynamic instabilitysigniﬁcant- ly more strongly than docetaxel (Fig. 4). We note that a 24- hour drug incubation is not optimum for discerning cell- Time (h) cycle–dependent differences between the drugs because the doubling time of the EGFP-a-tubulin MCF7 cells we used is 35 hours,and manycells would not havetransited a Figure 5. Time course of the uptake of [14C [-cabazitaxel and [14C]- complete cell cycle. docetaxel into MCF7 cells. Cells were seeded into scintillation vials for 24 At 48 to 72 hours, all cabazitaxel’s effects were more hours, then media was replaced with fresh media containing 50 nmol/L potent than those of docetaxel. Cabazitaxel inhibited cell [14C]-cabazitaxel (-&-) or 50 nmol/L [14C]-docetaxel (-^-) or unlabeled proliferation more strongly at 72 hours (Fig. 1B; IC50’s, 0.4 drug (for determination of cell number). Media was removed at various times during drug incubation for scintillation counting. To determine the 0.1 nmol/L cabazitaxel and 2.5 0.5 nmol/L docetaxel). rate of drug loss after the washing (20 hours, arrow), vials were rinsed G2–M cell-cycle arrest by 10 to 100 nmol/L cabazitaxel twice with fresh media (1 hour) and incubated with drug-free media for an was maintained at the 24-hour level of 85%, whereas at the additional 30 hours. , P < 0.02 by the Student t test. ns, not signiﬁcant. same docetaxel concentrations, G2–M arrest decreased

14 14 and only 55% to 60% of cells were in G2–M (Fig. 1E and Cellular uptake of [ C]-cabazitaxel and [ C]- F). In addition, at 48 to 72 hours, cabazitaxel induced docetaxel greater apoptosis/cell death than docetaxel (Fig. 1C). For 14 & The uptake of 50 nmol/L [ C]-cabazitaxel (- -) and 50 example, at 100 nmol/L prolonged G –M arrest induced 14 } 2 nmol/L [ C]-docetaxel (- -) in MCF7 cells over time was by cabazitaxel resulted in 37% apoptotic/dead cells, com- determined as described (36). Docetaxel was taken up pared with only 26% at 100 nmol/L docetaxel (Fig. 1C). relatively slowly, attaining an intracellular concentration The reduction in G2–M arrest and apoptosis by doce- of 25 mmol/L after 10 hours, and remaining at approxi- taxel observed at 48 to 72 hours (Fig. 1E) may result from } mately that level for the next 20 hours (Fig. 5, - -). downstream effects of its less potent suppression of Cabazitaxel was taken up significantly more rapidly than microtubule dynamic instability at 24 hours (Fig. 4) docetaxel, attaining an intracellular concentration of 25 and/or from a possible reduction in intracellular doce- mmol/L after only 1 hour, and was retained in cells for as taxel concentration at 48 and 72 hours. It is conceivable & long as 20 hours (Fig. 5, - -). Cabazitaxel’s uptake was 10 that long-term incubation with docetaxel induces an times faster than docetaxel’s, but its accumulation extent increase in expression of multidrug resistance proteins, was similar to and only slightly greater than docetaxel’s which in turn might lead to loss of drug from the cells, (540- and 500-fold, respectively). even in the absence of any washout. Either or both of these We determined the retention of cabazitaxel and doc- phenomena (docetaxel’s lesser suppression of dynamic etaxel after washing the cells (Fig. 5, arrow). Drug-con- instability (Fig. 4) and a possible reduction in intracellular taining media was removed at 20 hours, followed by 2 docetaxel levels as a result of multidrug resistance protein washes and incubation of cells in drug-free medium for expression) may contribute to the reduced effects of doc- an additional 30 hours. Washing reduced the amount of etaxel on G2–M arrest and apoptosis that were observed at intracellular cabazitaxel by 15%, from 27 to 23 mmol/L, 48 and 72 hours as well as for the 6-fold difference in which was not statistically significant. In contrast, intra- inhibition of proliferation measured at 72 hours (Fig. 1B). cellular docetaxel was significantly reduced by 48% (P < t 0.02, Student test), from 25 to 13 mmol/L (Fig. 5). Cabazitaxel suppresses microtubule dynamic instability more potently than docetaxel Discussion Both drugs suppressed all parameters of microtubule To elucidate the mechanism of action of cabazitaxel, we dynamic instability at low concentrations (1–6 nmol/L, comparedtheactionsofcabazitaxelanddocetaxelinMCF7 24-hour incubation) in concentration-dependent man- cells and found that, in most respects, cabazitaxel was as ners, leading to highly dynamically stabilized microtu- potent as or more potent than docetaxel. At early incuba- bules. Cabazitaxel’s suppression of the growth rate, the tion times (24 hours), most effects of cabazitaxel were catastrophe frequency and dynamicity, and its enhance- similar to those of docetaxel. The 2 drugs similarly affected ment of attenuation duration were all significantly greater interphase and mitotic morphology (Fig. 2), and their than docetaxel’s at the >99.9% level of significance (P < effects on mitotic arrest were similar at most concentra- 0.001). For example, 2 nmol/L cabazitaxel suppressed the

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Mechanism of Action of the Novel Taxane Cabazitaxel

dynamicity by 83% in contrast with only 64% suppression uptake and loss vary with the concentration of drug added by 2 nmol/L docetaxel (Figs. 3 and 4). Statistically signif- to cells (42). One can ask if docetaxel might have been icant differences (P < 0.01 or P < 0.05) were also observed more rapidly pumped out of cells than cabazitaxel before between the 2 drugs in suppression of the growth and the dynamic instability experiments, in particular if the shortening rates and shortening lengths, with cabazitaxel intracellular drug levels were different in the dynamic again having the greatest effects. The least significant instability experiments as compared with the uptake difference between the 2 drugs was on enhancement of experiments. To examine this question, we calculated and the rescue frequency (a stabilizing effect) where the only compared the approximate intracellular drug concentra- difference was at P < 0.05, at a single concentration, but tions in the uptake and dynamic instability experiments. again cabazitaxel was a significantly more potent stabi- We know from past experiments that cells take up all or lizer than docetaxel (Table 1 and Fig. 4). nearly all of a taxane available to them in culture medium Taxanes bind along the lengths of microtubules (38), (M.A. Jordan; unpublished data). Thus, we compared the and our preliminary data indicate that cabazitaxel binds ratio of cell number to total drug in the medium to with significantly higher affinity to reassembled bovine determine whether the intracellular drug concentrations brain microtubules than does docetaxel (L. Wilson, H. were of the same magnitude in the 2 kinds of experiments. Miller, O. Azarenko, M.A. Jordan; unpublished data). The numbers of cells per volume ranged between a low of Thus, it is not surprising that cabazitaxel suppressed 1.5 104 cells/mL for dynamic instability experiments microtubule dynamic instability in cellular microtubules to a high of 5 104 cells/mL for drug uptake experiments. to a greater extent than docetaxel. The drug concentrations in dynamic instability determi- nations ranged from 1 to 6 nmol/L. The drug concentra- Differences in uptake and retention of cabazitaxel tion used in uptake experiments was 50 nmol/L. There- and docetaxel in MCF7 cells fore, the ratio of available drug to cell number in the Importantly, the rate of uptake of 50 nmol/L cabazi- dynamic instability measurements was between taxel into cells was significantly (10 times) faster than (1 nmol/L)/(1.5 104 cells/mL) and (6 nmol/L)/(1.5 that of 50 nmol/L docetaxel, attaining a plateau con- 104 cells/mL). In uptake experiments, the ratio was centration of 25 mmol/Lat1hourandamaximum (50 nmol/L)/(5 104 cells/mL). Arithmetically simpli- concentration of 27 mmol/L at 2 hours, whereas doc- fying the above ratios, the drug concentrations on a per etaxel’s attainment of essentially the same maximum cell basis were 0.66 10 16 and 4 10 16 moles per cell concentration (25 mmol/L) required 10 hours (Fig. 5). for dynamic instability measurements as compared Cabazitaxel crosses the lipid bilayer of cellular mem- with 10 10 16 moles per cell for the uptake experi- branes faster than docetaxel according to the drugs’ ments. Thus, the intracellular drug concentrations in the P calculated lipophilicity values: log CAB (octanol/ dynamic instability experiments were of the same order water) ¼ 3.3 (CAS #183133-96-2) compared with log ofmagnitudeasthoseintheuptakeexperimentsin P ¼ DOC (octanol/water) 2.45 (CAS #114977-28-5), which which the plateau concentrations stayed constant for is in agreement with our uptake observations (39–41). at least 14 hours, suggesting that our comparison of After cell washing at 20 hours, the intracellular concen- intracellular effects on dynamic instability parameters is tration of docetaxel was significantly reduced (by 48%) likely valid and that drug concentrations were main- whereas the intracellular levels of cabazitaxel did not tained over time in the dynamic instability experiments change significantly (Fig. 5, arrow). The significantly as well as they were clearly maintained in the uptake/ better retention of cabazitaxel in MCF7 cells is consistent retention experiments. with the much longer terminal g-half-life reported for Ultimately, it will be important to compare the effects of cabazitaxel elimination in humans (95 hours vs. 12 hours the 2 drugs on dynamic instability of purified microtu- for docetaxel; refs. 15 and 16). As mentioned above, bules to be absolutely sure that cabazitaxel suppresses cabazitaxel seems to bind with higher affinity to micro- microtubule dynamic instability more strongly than doc- tubules than docetaxel. It is also conceivable that doce- etaxel. These experiments are underway. From the above taxel treatment might induce increased expression of calculations, we predict that the differences between the multidrug resistance proteins that could promote drug effects of the 2 drugs on dynamic instability observed in efflux. cells (Fig. 4) will hold true with purified microtubules. Therefore, we suggest that the significantly greater inhib- Did the differential rate of uptake of cabazitaxel and itory effects of cabazitaxel on microtubule dynamic insta- docetaxel and their different susceptibilities to loss bility at 24 hours are responsible for the significantly upon washout affect the measured values of greater potency of cabazitaxel at 48 to 72 hours. The suppression of dynamic instability? greater potency of cabazitaxel may be further enhanced We chose to examine effects on microtubule dynamic by its greater retention in cells as compared with that of instability after 24 hours drug incubation specifically in docetaxel. order to examine the cells after plateau intracellular drug Overall, the results indicate that, although cabazitaxel is concentrations were attained, as evidenced in the drug structurally very similar to docetaxel and most likely uptake experiments (Fig. 5). However, rates of drug binds to microtubules at the same and/or an overlapping

www.aacrjournals.org Mol Cancer Ther; 13(8) August 2014 2101

Azarenko et al.

binding site, it suppresses microtubule dynamic instabil- Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): O. Azarenko, G. Smiyun, J. Mah, L. Wilson, ity significantly more strongly than docetaxel and is taken M.A. Jordan up more quickly and can be retained longer in cells, thus Writing, review, and/or revision of the manuscript: O. Azarenko, L. Wilson, M.A. Jordan producing a longer-lasting G2–M arrest followed by Administrative, technical, or material support (i.e., reporting or orga- increased apoptosis. These differences likely play a major nizing data, constructing databases): O. Azarenko, L. Wilson, M.A. Jordan role in cabazitaxel’s enhanced clinical activity over doc- Study supervision: L. Wilson, M.A. Jordan etaxel regimens. Acknowledgments The authors thank Dr. N. LaPointe for critically reading the article. Disclosure of Potential Conflicts of Interest M.A. Jordan and L. Wilson report receiving commercial research grants from Sanofi. No potential conflicts of interest were disclosed by the other Grant Support authors. This work was supported by a grant from Sanofi-Oncology, Cambridge (MA), and the NRI-MCDB Microscopy Facility at UC Santa Barbara (NIH #1 S10 OD010610-01A1). Authors' Contributions The costs of publication of this article were defrayed in part by the Conception and design: L. Wilson, M.A. Jordan payment of page charges. This article must therefore be hereby marked Development of methodology: O. Azarenko, G. Smiyun, L. Wilson, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate M.A. Jordan this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): O. Azarenko, G. Smiyun, J. Mah, L. Wilson, Received March 28, 2014; revised June 3, 2014; accepted June 9, 2014; M.A. Jordan published OnlineFirst June 30, 2014.

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32. Oroudjev E, Lopus M, Wilson L, Audette C, Provenzano C, Erickson H, 38. Parness J, Horwitz SB. Taxol binds to polymerized tubulin in vitro. et al. Maytansinoid-antibody conjugates induce mitotic arrest by J Cell Biol 1981;91:479–87. suppressing microtubule dynamic instability. Mol Cancer Ther 2010; 39. Kort A, Hillebrand MJ, Cirkel GA, Voest EE, Schinkel AH, Rosing H, 9:2700–13. et al. Quantification of cabazitaxel, its metabolite docetaxel and the 33. Alken S, Kelly CM. Benefit risk assessment and update on the use of determination of the demethylated metabolites RPR112698 and docetaxel in the management of breast cancer. Cancer Manag Res RPR123142 as docetaxel equivalents in human plasma by liquid 2013;5:357–65. chromatography-tandem mass spectrometry. J Chromatogr B Analyt 34. Bunker JM, Wilson L, Jordan MA, Feinstein SC. Modulation of micro- Technol Biomed Life Sci 2013;925:117–23. tubule dynamics by tau in living cells: implications for development and 40. Cisternino S, Bourasset F, Archimbaud Y, Semiond D, Sanderink neurodegeneration. Mol Biol Cell 2004;15:2720–8. G, Scherrmann JM. Nonlinear accumulation in the brain of the 35. Kamath K, Oroudjev E, Jordan MA. Determination of microtubule new taxoid TXD258 following saturation of P-glycoprotein at the dynamic instability in living cells. Methods Cell Biol 2010;97: blood-brain barrier in mice and rats. Br J Pharmacol 2003;138: 1–14. 1367–75. 36. Okouneva T, Azarenko O, Wilson L, Littlefield BA, Jordan MA. Inhibition 41. CAS.org. [Internet]. American Chemical Society, Chemical Abstracts of centromere dynamics by eribulin (E7389) during mitotic metaphase. Service. c1980–2014 [updated 2014 May 26; cited 2014 Apr13]. Mol Cancer Ther 2008;7:2003–11. Available from: http://www.cas.org/ 37. Jordan MA, Thrower D, Wilson L. Mechanism of inhibition of cell 42. Jordan MA, Wilson L. The use and action of drugs in analyzing mitosis. proliferation by Vinca alkaloids. Cancer Res 1991;51:2212–22. Methods Cell Biol 1999;61:267–95.

www.aacrjournals.org Mol Cancer Ther; 13(8) August 2014 2103

Antiproliferative Mechanism of Action of the Novel Taxane Cabazitaxel as Compared with the Parent Compound Docetaxel in MCF7 Breast Cancer Cells

Olga Azarenko, Gregoriy Smiyun, Jeffrey Mah, et al.

Mol Cancer Ther 2014;13:2092-2103. Published OnlineFirst June 30, 2014.

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