Breast Cancer DOI 10.1007/s12282-014-0580-9
ORIGINAL ARTICLE
Antitumor and anticancer stem cell activities of eribulin mesylate and antiestrogens in breast cancer cells Junichi Kurebayashi • Naoki Kanomata • Tetsumasa Yamashita • Toshiro Shimo • Takuya Moriya
Received: 10 November 2014 / Accepted: 23 December 2014 Ó The Japanese Breast Cancer Society 2014
Abstract Background Eribulin mesylate (eribulin), a non-taxane microtubule dynamic inhibitor, has been widely used in the treatment of patients with advanced or metastatic breast cancer. The combined antitumor and anticancer stem cell (CSC) activities of eribulin with endocrine therapeutic agents have not yet been examined in breast cancer cells. We herein investigated the combined effects of eribulin and antiestrogens. Methods A panel of eight breast cancer cell lines, including five estrogen receptor (ER)-positive and three ER-negative cell lines, was used. These cells were treated with eribulin and/or the antiestrogen, 4-hydroxytamoxifen or fulvestrant. Their growth inhibitory activities and effects on cell cycle progression, apoptosis, and the CSC population were investigated. CSCs were detected using the CD44/CD24/EpCAM, Aldefluor, and mammosphere assays. Results The 50 % growth inhibitory concentrations of eribulin were 0.38–2.64 nM for the eight cell lines tested. Eribulin exhibited significant antitumor activity under estrogen-supplemented conditions in ER-positive breast cancer cells. The combined antitumor activity of eribulin with an antiestrogen was evaluated using the combination index. The combination index was 0.43–1.46 for ERpositive cell lines. The additive antitumor effect of eribulin
J. Kurebayashi (&) T. Yamashita T. Shimo Department of Breast and Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan e-mail: [email protected] N. Kanomata T. Moriya Department of Pathology 2, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
with 4-OHT was only significant in MCF-7 cells. Eribulin induced the accumulation of G2/M and apoptosis, while antiestrogens induced the retardation of G1–S cell cycle and apoptosis, respectively. Estrogen markedly increased the proportion of CSCs, whereas antiestrogens inhibited increases in ER-positive cell lines. Moreover, eribulin decreased the proportion of CSCs in either ER-positive or ER-negative cell lines. The combined treatment of eribulin with an antiestrogen did not additively decrease the proportion of CSCs in ER-positive cell lines. Discussion The results of the present study demonstrated that eribulin had potent antitumor effects on estrogenstimulated ER-positive breast cancer cells and the combined treatment of eribulin with an antiestrogen resulted in a weakly additive antitumor effect. We herein suggested for the first time that eribulin exhibited anti-CSC effects on either ER-positive or ER-negative breast cancer cells. Keywords Eribulin Tamoxifen Fulvestrant Cancer stem cells
Introduction Eribulin mesylate (eribulin), a non-taxane microtubule dynamic inhibitor, has been approved for the treatment of advanced breast cancer in the United States, Japan, and EU countries. Two different clinical phase 3 trials reported that eribulin significantly prolonged the overall survival (OS) of advanced breast cancer patients [1, 2]. Although retrospective subgroup analyses on these trials suggested that eribulin had more potent antitumor effects on patients with estrogen receptor (ER)-negative than ER-positive breast cancer, eribulin displayed marked antitumor effects in patients with ER-positive breast cancer [1, 2].
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A limited number of clinical trials indicated that the concomitant treatment of a cytotoxic agent with endocrine therapy may lead to modest increases in clinical response rates, but not in progression-free survival, and clearly increased the incidence of adverse effects such as thromboembolism in patients with advanced ER-positive breast cancer [3–5]. However, our previous experimental studies showed that the concomitant treatment of 5-fluorouracil (FU), but not paclitaxel and doxorubicin with an antiestrogen additively enhanced their antitumor activities in ERpositive breast cancer cells [6, 7]. Furthermore, some clinical trials have suggested the existence of a synergistic interaction between a 5-FU derivative and tamoxifen (TAM) in a postoperative adjuvant setting for patients with ER-positive early breast cancer [8–10]. These findings prompted us to investigate the interaction between eribulin and the antiestrogen, TAM or steroidal antiestrogen fulvestrant in terms of antitumor activity in ER-positive breast cancer cells. Emerging preclinical and clinical evidence has revealed that the cancer stem cell (CSC) population plays important roles in progression, invasion, metastasis, and resistance to cytotoxic agents and radiation therapy in certain malignancies [11]. Previous studies reported that some cytotoxic agents such as docetaxel and doxorubicin exhibited antitumor activities, but increased the CSC proportion in the surviving tumor cells [12, 13]. The anti-CSC activity of eribulin has not yet been investigated in preclinical and clinical studies. In addition, preclinical studies recently indicated that estrogen increased the proportion of CSCs while antiestrogens inhibited this increase [14, 15]. These findings also prompted us to explore the anti-CSC activities of eribulin and antiestrogens in association with their antitumor effects in the present study.
Materials and methods Compounds Eribulin was kindly provided by Eisai Co., Ltd (Tsukuba, Japan). 17b-estradiol (E2), an active metabolite of TAM, 4-hydroxytamoxifen (OHT), and the steroidal antiestrogen, fulvestrant were obtained from Sigma-Aldrich (St. Louis, MO, USA). Breast cancer cell lines and cell culture KPL-1, KPL-3C, and KPL-4 breast cancer cell lines were established in our laboratory. Their biological characteristics were described elsewhere [16–18]. MCF-7, T-47D and MDA-MB-231 cell lines were kindly provided by the late Dr. Robert B. Dickson (Lombardi Cancer Research Center,
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Washington DC, USA). BT-474 and MDA-MB-157 cell lines were obtained from the American Type Cell Collection (Rockville, MD, USA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (D-MEM, Sigma Co, St. Louis, MI, USA) supplemented with 10 % fetal bovine serum (FBS). We and others previously demonstrated that MCF-7 and T-47D cells expressed a high level of ER-a, a low level of progesterone receptor (PgR), and no detectable level of human epidermal growth factor receptor (HER) 2. KPL-1 and KPL-3C cells have been shown to express a low level of ER-a and no detectable level of either PgR or HER2 [19]. MDA-MB-231 and MDA-MB-157 cells were found to express no detectable level of ER-a or HER2. Both cell lines are also known to express high levels of mesenchymal markers such as vimentin and have been classified as the basal B subtype [20]. BT-474 cells have been shown to express high levels of both ER-a and HER2 [21]. Antitumor activity To investigate the effects of eribulin and/or antiestrogens on cell growth, breast cancer cells (1–5 9 104 cells per well) were seeded on 24-well plates (SB Medical, Tokyo, Japan) and grown in D-MEM supplemented with 10 % FBS at 37 °C in a 5 % CO2 atmosphere for 2 days. After washing with phosphate-buffered saline (PBS, Nissui Co., Tokyo, Japan), the cells were treated with an estrogendeprived medium [phenol red-free RPMI1640 (Life Technologies, Carlsbad, California, USA) supplemented with 10 % dextran-coated charcoal-treated FBS (SigmaAldrich)] plus 1 nM E2 and the indicated concentrations of eribulin and/or antiestrogens for 3 days. After the treatments, the cells were harvested and counted with a Coulter counter (Coulter Electronics, Harpenden, UK). Reproducibility was confirmed in at least two separate experiments. To evaluate the antitumor effects of combined treatments, a combination index based on the 50 % inhibitory concentration (IC50) was calculated according to the following formula: combination index = IC50 with the combined treatment/IC50 with the single treatment. A combination index \0.5 was considered evidence of an additive interaction [6, 7]. Cell cycle and apoptosis assays To investigate the effects of agents on cell cycle progression, harvested cells were stained with propidium iodide using the CycleTest Plus DNA Reagent kit (Becton– Dickinson, San Jose, CA, USA). Apoptotic cells were stained with an Annexin-V-FLUOS staining kit (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s recommendations. Flow cytometry was
Breast Cancer
performed with a FACSCalibur flow cytometer (Becton– Dickinson), and the DNA histogram was analyzed using a CELLQuest version 6.0 (Becton–Dickinson). Reproducibility was confirmed in at least two separate experiments. CSC analysis by the CD44/CD24/EpCAM assay To analyze cell surface markers, harvested cells were treated with three fluorescence-labeled antibodies: a PEconjugated anti-CD24 antibody (clone G44-26, Becton– Dickinson), FITC-conjugated anti-CD44 antibody (clone ML5, Becton–Dickinson), and PerCP–Cy5.5 conjugated anti-EpCAM antibody (clone EBA-1, Becton–Dickinson). Flow cytometry was performed with a FACSCalibur flow cytometer (Becton–Dickinson), and analyzed using CELLQuest Software version 6.0 (Becton–Dickinson). Cells that were CD44-high, CD24-negative or low, and EpCAM-positive were recognized as CSCs [18]. CSC analysis by the Aldefluor assay The ALDEFLUOR kit (StemCell Technologies, Durham, NC, USA) was used to isolate the cell population exhibiting high aldehyde dehydrogenase (ALDH) activity. Harvested cells were suspended in the Aldefluor assay buffer containing ALDH substrate (BODIPYTM-aminoacetaldehyde, 1 lmol/l per 1 9 106 cells) and incubated for 30 min at 37 °C. As a negative control, cells were treated with 50 mmol/l diethylaminobenzaldehyde, a specific ALDH inhibitor [19]. CSC analysis by the mammosphere assay Breast cancer cells (0.3–1.5 9 105 cells per well) were seeded on 35-mm dishes (SB Medical) and grown in D-MEM supplemented with 10 % FBS at 37 °C in a 5 % CO2 atmosphere for 2 days. After washing with PBS, cells were treated with estrogen-deprived medium supplemented with 1 nM E2 and the indicated concentrations of eribulin and/or antiestrogens for 3 days. These cells were then dispersed, and single-cell suspensions (5 9 103 cells/well) were incubated in MammoCultTM basal medium (Stem Cell Technologies Co., Tokyo, Japan) supplemented with 10 % MammoCultTM proliferation supplements (Stem Cell Technologies Co.) plus heparin (Stem Cell Technologies Co.) and hydrocortisone (Stem Cell Technologies Co.) in non-adhesive 6-well plates (CORNING Co., NY, USA) for 7 days. Mammosphere colonies more than 60 lm in size were counted with an Olympus phase-contrast microscope [22]. Statistical analysis All values are expressed as the mean ± SE. An ANOVA analysis with StatView computer software (ATMS Co.,
Tokyo, Japan) was used to compare differences between two groups. A two-sided P value less than 0.05 was considered significant.
Results Growth inhibitory activity of eribulin All cell lines were treated with estrogen-deprived medium supplemented with or without 1 nM E2 plus 0–10 nM eribulin for 3 days. Although eribulin did not inhibit the growth of ER-positive and HER2-negative breast cancer cell lines (MCF-7, T-47D, KPL-1 and KPL-3C) under the estrogen-deprived condition, it did inhibit the growth of the ER-positive and HER2-positive BT-474 cell line under the estrogen-deprived condition. In contrast, eribulin dose-dependently inhibited the growth of the ERpositive and HER2-negative breast cancer cell lines as well as the ER-positive and HER2-positive cell line under the E2-supplemented condition (Fig. 1). The 50 % growth inhibitory concentrations (IC50) of the ER-positive cell lines under E2-supplemented conditions are shown in the Table 1. Eribulin dose-dependently inhibited the growth of ERnegative and HER2-positive KPL-4 cells and ER-negative and HER2-negative MDA-MB-231 and MDA-MB-257 cells irrespective of the E2 supplementation (Fig. 1). The IC50 of the ER-negative cell lines under the E2-supplemented condition are shown in the Table 1. Combined antitumor activity of eribulin with antiestrogens To investigate the possible synergistic antitumor activity of eribulin and the antiestrogen, 4-OHT or fulvestrant, all cell lines were treated with estrogen-deprived medium supplemented with 1 nM E2 plus the indicated concentration of 4-OHT or fulvestrant and 0–5 nM eribulin for 3 days. The indicated concentrations of the antiestrogens were selected according to the experimental results obtained with cells treated with the estrogen-deprived medium supplemented with 1 nM E2 plus various concentrations of antiestrogens (data not shown). The combined treatment of 4-OHT or fulvestrant with eribulin showed a modest antitumor effect on the ERpositive and HER2-negative cell lines (Fig. 2). The additive antitumor interaction (the combination index was less than 0.5) between 4-OHT and eribulin was only significant in MCF-7 cells. The other combinations did not show a significant additive interaction in the other cell lines (Table 2).
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Cell growth (% control)
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Effects of eribulin and/or antiestrogens on cell cycle progression and apoptosis To explore the action mechanisms responsible for the antitumor activities of eribulin and the antiestrogens, their effects on cell cycle progression and apoptosis were
0.1
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Fig. 1 Growth inhibitory curves of eribulin with (filled circle) or without (open circle) E2 supplementation in MCF-7 cells (a), T-47D cells (b), KPL1 cells (c), KPL-3C cells (d), BT-474 cells (e), KPL-4 cells (f), MDA-MB-231 cells (g), and MDA-MB-157 cells (h). Cells were treated with the indicated concentrations of eribulin for 3 days. The values are means ± SEs
Cell growth (% control)
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investigated. The selected cell lines were treated with estrogen-deprived medium supplemented with 1 nM E2 plus the indicated concentrations of 4-OHT or fulvestrant and eribulin for 2 days. The treatment with eribulin resulted in the accumulation of G2/M and induction of apoptosis in either ER-positive
Breast Cancer Table 1 The status of ER and HER2 and IC50s of eribulin under E2-supplemented conditions in eight breast cancer cells lines tested MCF-7
T-47D
KPL-1
KPL-3C
BT-474
KPL-4
MDA-MB-231
MDA-MB-157
ER
?
?
?
?
?
-
-
-
HER2
-
-
-
-
?
?
-
-
IC50 (mean ± SE, nM)
0.52 ± 0.21
2.64 ± 0.35
1.17 ± 0.04
1.09 ± 0.92
0.48 ± 0.06
0.65 ± 0.27
0.38 ± 0.04
1.01 ± 0.16
A
120
B
120
100
Cell growth (% control)
100
80 60 40 20
80 60 40 20 0
0 0
0.5
1.0
0
5.0
Eribulin (nM) 120 100 80 60 40 20
D
120 100
0
1.0
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80 60 40 20 0
0
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120
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100
Cell growth (% control)
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120 100 80 60 40 20 0
0 0
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0
Eribulin (nM)
0.5
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Eribulin (nM)
140
H
120
120
Cell growth (% control)
G
0.5
Eribulin (nM)
Cell growth (% control)
Cell growth (% control)
C
Cell growth (% control)
Fig. 2 Growth inhibitory curves of the concomitant treatment of eribulin with antiestrogens under the E2supplemented condition in MCF-7 cells (a), T-47D cells (b), KPL-1 cells (c), KPL-3C cells (d), BT-474 cells (e), KPL4 cells (f), MDA-MB-231 cells (g), and MDA-MB-157 cells (h). Cells were treated with eribulin (0–5 nM) and the antiestrogen, 4-OHT (0.01–1 lM) or fulvestrant (0.01–0.1 lM), for 3 days. The values are means ± SEs. Closed circles, E2; open circles, E2 plus 4-OHT; and gray circles, E2 plus fulvestrant
Cell growth (% control)
Values are means ± SEs
100
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0 0
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Breast Cancer Table 2 The combination index of eribulin with 4-OHT or fulvestrant for eight breast cancer cells lines tested
MCF-7
4-OHT
Fulvestrant
0.43 ± 0.00
0.60 ± 0.01
T-47D
0.78 ± 0.25
0.62 ± 0.21
KPL-1
0.57 ± 0.36
0.92 ± 0.49
KPL-3C
0.70 ± 0.50
1.48 ± 0.69
BT-474
1.21 ± 0.29
1.46 ± 0.17
KPL-4
0.79 ± 0.05
0.84 ± 0.05
MDA-MB-231
0.75 ± 0.06
0.89 ± 0.15
Values are means ± SEs
or ER-negative cell lines (Fig. 3). The treatment with antiestrogens resulted in the retardation of the G1–S cell cycle in MCF-7 cells, but not in KPL-1 cells and MDAMB-231 cells (Fig. 3).The treatment with antiestrogens resulted in enhanced apoptosis in the ER-positive cell lines, but not in the ER-negative cell line (Fig. 3). No significant interaction was observed between eribulin and an antiestrogen in terms of cell cycle progression and apoptosis. Effects of eribulin and/or antiestrogens on the CSC proportion To explore the effects of eribulin and/or an antiestrogen on the CSC proportion, the selected cell lines were treated with estrogen-deprived medium supplemented with 1 nM E2 plus the indicated concentrations of 4-OHT or fulvestrant and eribulin for 3 days. The CSC population was detected and measured using the CD44/CD24/EpCAM assay for ER-positive cell lines. Because the basal B breast cancer cell lines, MDA-MB-231 and MDA-MB-157, strongly acquired epithelial–mesenchymal transition (EMT) and this assay could not be applied to them, we decided to use the Aldefluor assay to detect the CSC population for the basal B cell lines. The E2 treatment significantly increased the CSC proportion in ER-positive cell lines. The treatment with antiestrogens markedly reduced the E2-induced increase in the CSC proportion (Fig. 4). Eribulin decreased the E2induced increase in the CSC proportion in ER-positive cell lines. The combined treatment of eribulin with an antiestrogen did not enhance the decrease in the CSC proportion (Fig. 4). Representative plot charts of the CD44/CD24/ EpCAM assay for MCF-7 cells are shown in Fig. 4. Eribulin significantly decreased the CSC proportion, as detected by the Aldefluor assay, in the basal B cell lines (Fig. 5). Representative plot charts of the Aldefluor assay for MDA-MB-231 cells are shown in Fig. 5. To confirm the anti-CSC effects of eribulin, the mammosphere assay was applied to both ER-positive and ER-
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negative cell lines. Eribulin significantly decreased the CSC proportion and mammosphere size in all cell lines tested (Fig. 6).
Discussion Eribulin has recently been introduced into clinics and widely used for the treatment of patients with advanced breast cancer. Two pivotal clinical trials comparing eribulin versus physicians’ choices or capecitabine showed that eribulin significantly prolonged OS in patients with advanced or metastatic breast cancer [1, 2]. Two recent preclinical studies suggested that eribulin could reverse EMT back to mesenchymal–epithelial transition (MET) in triple-negative breast cancer (TNBC) cells, and also that the eribulin-induced remodeling of an abnormal tumor vasculature led to a more functional microenvironment that may reduce the aggressiveness of tumors due to the elimination of tumor hypoxia [23, 24]. These preclinical findings may provide a scientific basis for the clinical observations of prolonged OS in breast cancer patients treated with eribulin. Retrospective subgroup analyses on the two pivotal trials suggested that eribulin had a more potent antitumor effect on patients with ER-negative breast cancer than those with ER-positive breast cancer. However, eribulin also exhibited potent antitumor effects in patients with ERpositive advanced breast cancer. We speculated that the combined use of eribulin with an endocrine therapeutic agent may lead to synergistic antitumor effects in patients with ER-positive breast cancer. To clarify this hypothesis, we investigated the potential synergistic antitumor activity of the combined treatment of eribulin with the antiestrogens, 4-OHT and fulvestrant in breast cancer cells in the present study. However, eribulin and the antiestrogens did not display an additive antitumor interaction in most ERpositive cell lines, as shown in Fig. 2 and Table 2. However, a slight additive interaction was observed in ERpositive and HER2-negative cell lines, but not in the ERpositive and HER2-positive cell line and ER-negative cell lines (Fig. 2; Table 2). The results of the cell cycle and apoptosis analyses in this study (Fig. 3) suggested that the retardation of G1–S cell cycle induced by antiestrogens interfered with the G2/M-directed antitumor activity of eribulin. Further preclinical and clinical studies are needed to clarify the antitumor interaction between eribulin and antiestrogens in ER-positive breast cancers. Our previous experimental studies and others indicated that estrogen markedly increased the CSC population, whereas antiestrogens inhibited it in ER-positive breast cancer cell lines, but not in ER-negative breast cancer cell lines. These findings have been strongly supported by the
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Percentage of apoptoc cells
70
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9
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6 5 4 3 2 1 0
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Fig. 3 Effects of concomitant treatments with E2, eribulin, and/or 4-OHT on cell cycle progression and apoptosis in ER-positive MCF-7 cells (a, b), KPL-1 cells (c, d), and ER-negative MDA-MB-231 cells (e, f). Cells were treated with 1 nM E2 and the indicated concentrations of eribulin and/or 4-OHT for 2 days. Percentages of cells at each cell cycle phase and in the apoptotic fraction were analyzed as
described in the ‘‘Materials and methods’’. The values are means ± SEs. White bars, control; black bars, E2; the most light gray bars, E2 plus 0.5 or 1 nM eribulin; the second light gray bars, E2 plus 1 or 5 nM eribulin; the third light gray bars, E2 plus 0.05 or 0.1 lM 4-OHT; the fourth light gray bars, E2 plus 0.5 or 1 nM eribulin and 0.05 or 0.1 lM 4-OHT. *P \ 0.05, **P \ 0.01
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Percentage of CD44high/ CD24low or-/EpCAM+ cells
35
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Percentage of CD44high/ CD24low or-/EpCAM+ cells
A 30 25 20 15 10 5 0
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E2 E2 Eribulin (3 nM) Eribulin (0.5 nM) Eribulin (5 nM) Eribulin (1.0 nM) 4-OHT ( 0.05 䃛M) 4-OHT (0.05 䃛M)
C 0.21%
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E2
E2 + 4-OHT
E2 + 0.5 nM eribulin
E2 + 1.0 nM eribulin
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Fig. 4 Effects of E2, eribulin, and/or antiestrogens on the CSC population detected by the CD44/CD24/EpCAM assay in ER-positive MCF-7 (a) and T-47D (b) breast cancer cells. Cells were treated with 1 nM E2 plus the indicated concentrations of eribulin and/or 4-OHT for 3 days. The values are means ± SEs. White bars, control; black bars, E2; the most light gray bars, E2 plus the lower concentration of
eribulin; the second light gray bars, E2 plus the higher concentration of eribulin; the third light bars, E2 plus 4-OHT; the fourth light gray bars, E2 plus the lower concentration of eribulin and 4-OHT.*P \ 0.05, **P \ 0.01. Representative plot charts for MCF7 cells are shown (c). The left upper portions in the figure were defined as the CSC populations
results of this study (Fig. 4) [14, 15, 18]. The action mechanisms responsible for regulating the CSC population in ER-positive breast cancer such as possible paracrine mediators induced by estrogen are being examined in detail in our laboratory. Eribulin not only inhibited cell growth, but also reduced the CSC proportion in both ER-positive and ER-negative
breast cancer cell lines. A previous study reported that a possible CSC population expressing a high level of CD44, low level of CD24, and high level of EpCAM in breast cancer cells more efficiently produced xenograft tumors in an SCID mouse model [25]. In the present study, eribulin significantly decreased cell growth and the proportion of the CSC population in ER-positive breast cancer cell lines
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Percentage of ALDH1-posive cells
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C 2.42%
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1.02% Eribulin (0.5 nM) Eribulin (1.0 nM)
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4-OHT (0.1 䃛M)
Percentage of ALDH1-posive cells
B 1.2 1
**
.8 .6 .4 .2 0
E2 Eribulin (1 nM) Eribulin (5 nM) 4- OHT (0.1 䃛M)
Fig. 5 Effects of E2, eribulin, and/or antiestrogens on the CSC proportion detected by the Aldefluor assay in ER-negative MDA-MB231 (a) and MDA-MB-157 (b) breast cancer cells. Cells were treated with 1 nM E2 plus the indicated concentrations of eribulin and/or 4-OHT for 3 days. The values are means ± SEs. White bars, control; black bars, E2; the most light gray bars, E2 plus the lower
concentration of eribulin; the second light gray bars, E2 plus the higher concentration of eribulin; the third light bars, E2 plus 4-OHT; the fourth light gray bars, E2 plus the lower concentration of eribulin and 4-OHT.*P \ 0.05, **P \ 0.01. Representative plot charts for MDA-MB-231 cells are shown (c). The R3 portions in the figure were defined as the CSC populations
(Fig. 4). This is the first study to suggest the anticancer stem cell activity of eribulin in ER-positive breast cancer cells. In our CD44/CD24/EpCAM assay, TNBC cell lines such as MDA-MB-231 and MDA-MB-157 consisted of an almost 100 % CSC population. This may have been because of their high level of EMT. Our previous and present studies as well as those by others indicated that this CD44/CD24/EpCAM assay was not suitable for investigating the CSC population in TNBC cell lines [19, 26, 27]. Therefore, we used the Aldefluor assay, in which ALDH activity was recognized as a marker of the CSC population, to detect the CSC population in TNBC cell lines [28]. Eribulin significantly decreased the cell growth and
proportion of the CSC population detected by the Aldefluor assay in TNBC cell lines (Fig. 5). This is the first study to demonstrate the significant anti-CSC activity of eribulin in TNBC cells. Another CSC assay, the mammosphere assay, was also used to clarify the anti-CSC effects of eribulin. As expected, eribulin significantly decreased the proportion of the CSC population and reduced the size of mammospheres in either ER-positive or ER-negative cell lines (Fig. 6). The findings of a recent preclinical study suggested that eribulin reversed EMT back to MET in TNBC cells both in vitro and in vivo [22]. After the treatment with eribulin, the surviving cells showed an increase in both the mRNA and protein expression levels of epithelial markers, such as
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**
B Number of mammosphere/5,000 cells
Number of mammosphere/5,000 cells
A 250 225 200 175 150 125 100 75 50 25 0
** 200 180 160 140 120 100 80 60 40 20 0
E2
E2
Eribulin (1 nM)
Eribulin (1 nM)
C
Control
Eribulin-treated
D
Control Fig. 6 Effects of E2, eribulin, and/or antiestrogens on the CSC proportion detected by the mammosphere assay in ER-positive MCF7 (a) and ER-negative MDA-MB-231 (b) breast cancer cells. Cells were treated with 1 nM E2 ± 1 nM eribulin for 3 days. The values are means ± SEs. White bars, control; black bars, E2; and gray bars,
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Eribulin-treated E2 plus eribulin.*P \ 0.05, **P \ 0.01. Representative microphotographs of mammospheres for MCF-7 cells (c) and MDA-MB-231 cells (d) with or without the eribulin treatment are shown. The scale bar indicates 500 lm in size
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E-cadherin, and a decrease in those of mesenchymal markers, such as vimentin. A previous study indicated that CSCs undergo EMT more than non-CSCs [29]. We speculated that eribulin may decrease the proportion of CSCs undergoing EMT and increase that of non-CSCs undergoing MET. This may result in the reversal of EMT back to MET in the surviving cells after the treatment with eribulin. Eribulin may preferentially kill the CSC population or inhibit the self-renewal of CSCs in breast cancer cells. These hypothetical issues should be investigated further in future studies. In conclusion, our preclinical study showed that eribulin exhibited potent antitumor activity in breast cancer cell lines of various subtypes. The concomitant treatment of eribulin with the antiestrogen, 4-OHT or fulvestrant, did not lead to a synergistic antitumor interaction in any ERpositive breast cancer cell lines, but did cause a modestly additive antitumor interaction in some ER-positive breast cancer cell lines. Eribulin significantly reduced the proportion of the CSC population in the surviving cells. Further preclinical and clinical studies are needed to elucidate these phenomena. Acknowledgments Esai Co. kindly provided us with eribulin for this study. We thank Dr. Yasumitsu Nishimura and Dr. Takemi Otsuki of the Department of Hygiene, Kawasaki Medical School for their technical advice. We also thank Mrs. Kaoru Tsuboi and Ms. Megumi Ogo for their technical assistance. This work was supported by Research Project Grants from Kawasaki Medical School (25-12 and 26-5) and by Research Grants from Eisai Co. and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (23591911 and 26461964). Conflict of interest Junichi Kurebayashi received a research grant from Esai Co. The other authors declare that they have no conflict of interest.
References 1. Cortes J, O’Shaughnessy J, Loesch D, Blum JL, Vahdat LT, Petrakova K, et al. EMBRACE (Eisai Metastatic Breast Cancer Study Assessing Physician’s Choice Versus E7389) investigators: Eribulin monotherapy versus treatment of physician’s choice in patients with metastatic breast cancer (EMBRACE): a phase 3 open-label randomised study. Lancet. 2011;377(9769):914–23. 2. Kaufman PA, Awada A, Twelves C, Yelle L, Perez E, Wanders J, et al. A phase III, open-label, randomized, multicenter study of eribulin mesylate versus capecitabine in patients with locally advanced or metastatic breast cancer previously treated with anthracyclines and taxanes. 2012 San Antonio Breast Cancer Symposium. Abstract S6-6. 3. Bezwoda WR, Derman D, De Moor NG, Lange M, Levin J. Treatment of metastatic breast cancer in estrogen receptor positive patients. A randomized trial comparing tamoxifen alone versus tamoxifen plus CMF. Cancer. 1982;50:2747–50. 4. Viladiu P, Alonso MC, Avella A, Beltra´n M, Borra´s J, Ojeda B, Bosch FX. Chemotherapy versus chemotherapy plus hormonotherapy in postmenopausal advanced breast cancer patients. A randomized trial. Cancer. 1985;56:2745–50.
5. Pritchard KI, Paterson AH, Paul NA, Zee B, Fine S, Pater J. Increased thromboembolic complications with concurrent tamoxifen and chemotherapy in a randomized trial of adjuvant therapy for women with breast cancer. National Cancer Institute of Canada Clinical Trials Group Breast Cancer Site Group. J Clin Oncol. 1996;14:2731–7. 6. Kurebayashi J, Nukatsuka M, Nagase H, Nomura T, Hirono M, Yamamoto Y, et al. Additive antitumor effect of concurrent treatment of 4-hydroxy tamoxifen with 5-fluorouracil but not with doxorubicin in estrogen receptor-positive breast cancer cells. Cancer Chemother Pharmacol. 2007;59:515–25. 7. Kurebayashi J, Nukatsuka M, Sonoo H, Uchida J, Kiniwa M. Preclinical rationale for combined use of endocrine therapy and 5-fluorouracil but neither doxorubicin nor paclitaxel in the treatment of endocrine-responsive breast cancer. Cancer Chemother Pharmacol. 2010;65:219–25. 8. Sugimachi K, Maehara Y, Akazawa K, Nomura Y, Eida K, Ogawa M, et al. Postoperative chemo-endocrine treatment with mitomycin C, tamoxifen, and UFT is effective for patients with premenopausal estrogen receptor-positive stage II breast cancer. Nishinihon Cooperative Study Group of Adjuvant Therapy for Breast Cancer. Breast Cancer Res Treat. 1999;56:113–24. 9. Noguchi S, Koyama H, Uchino J, Abe R, Miura S, Sugimachi K, et al. Postoperative adjuvant therapy with tamoxifen, tegafur plus uracil, or both in women with node-negative breast cancer: a pooled analysis of six randomized controlled trials. J Clin Oncol. 2005;23:2172–84. 10. Ohashi Y, Watanabe T, Sano M, Koyama H, Inaji H, Suzuki T. Efficacy of oral tegafur–uracil (UFT) as adjuvant therapy as compared with classical cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) in early breast cancer: a pooled analysis of two randomized controlled trials (N.SAS-BC 01 trial and CUBC trial). Breast Cancer Res Treat. 2010;119:633–41. 11. Liu S, Wicha MS. Targeting breast cancer stem cells. J Clin Oncol. 2010;28:4006–12. 12. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008;100:672–9. 13. Tanei T, Morimoto K, Shimazu K, Kim SJ, Tanji Y, Taguchi T, et al. Association of breast cancer stem cells identified by aldehyde dehydrogenase 1 expression with resistance to sequential Paclitaxel and epirubicin-based chemotherapy for breast cancers. Clin Cancer Res. 2009;15:4234–41. 14. Fillmore CM, Gupta PB, Rudnick JA, Caballero S, Keller PJ, Lander ES, Kuperwasser C. Estrogen expands breast cancer stemlike cells through paracrine FGF/Tbx3 signaling. Proc Natl Acad Sci USA. 2010;107:21737–2142. 15. Deng H, Zhang XT, Wang ML, Zheng HY, Liu LJ, Wang ZY. ER-a36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells. PLoS One. 2014;9:e88034. 16. Kurebayashi J, Kurosumi M, Sonoo H. A new human breast cancer cell line, KPL-1 secretes tumour-associated antigens and grows rapidly in female athymic nude mice. Br J Cancer. 1995;71:845–53. 17. Kurebayashi J, Kurosumi M, Sonoo H. A new human breast cancer cell line, KPL-3C, secretes parathyroid hormone-related protein and produces tumours associated with microcalcifications in nude mice. Br J Cancer. 1996;74:200–7. 18. Kurebayashi J, Otsuki T, Tang CK, Kurosumi M, Yamamoto S, Tanaka K, et al. Isolation and characterization of a new human breast cancer cell line, KPL-4, expressing the Erb B family receptors and interleukin-6. Br J Cancer. 1999;79:707–17. 19. Yamashita T, Kurebayashi J, Kanomata N, Shimo T, Moriya T, Sonoo H. Effects of antiestrogens on regulation of cancer stem cell population in breast cancer cells. Breast. 2013;22:S33–4.
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ORIGINAL ARTICLE
Antitumor and anticancer stem cell activities of eribulin mesylate and antiestrogens in breast cancer cells Junichi Kurebayashi • Naoki Kanomata • Tetsumasa Yamashita • Toshiro Shimo • Takuya Moriya
Received: 10 November 2014 / Accepted: 23 December 2014 Ó The Japanese Breast Cancer Society 2014
Abstract Background Eribulin mesylate (eribulin), a non-taxane microtubule dynamic inhibitor, has been widely used in the treatment of patients with advanced or metastatic breast cancer. The combined antitumor and anticancer stem cell (CSC) activities of eribulin with endocrine therapeutic agents have not yet been examined in breast cancer cells. We herein investigated the combined effects of eribulin and antiestrogens. Methods A panel of eight breast cancer cell lines, including five estrogen receptor (ER)-positive and three ER-negative cell lines, was used. These cells were treated with eribulin and/or the antiestrogen, 4-hydroxytamoxifen or fulvestrant. Their growth inhibitory activities and effects on cell cycle progression, apoptosis, and the CSC population were investigated. CSCs were detected using the CD44/CD24/EpCAM, Aldefluor, and mammosphere assays. Results The 50 % growth inhibitory concentrations of eribulin were 0.38–2.64 nM for the eight cell lines tested. Eribulin exhibited significant antitumor activity under estrogen-supplemented conditions in ER-positive breast cancer cells. The combined antitumor activity of eribulin with an antiestrogen was evaluated using the combination index. The combination index was 0.43–1.46 for ERpositive cell lines. The additive antitumor effect of eribulin
J. Kurebayashi (&) T. Yamashita T. Shimo Department of Breast and Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan e-mail: [email protected] N. Kanomata T. Moriya Department of Pathology 2, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
with 4-OHT was only significant in MCF-7 cells. Eribulin induced the accumulation of G2/M and apoptosis, while antiestrogens induced the retardation of G1–S cell cycle and apoptosis, respectively. Estrogen markedly increased the proportion of CSCs, whereas antiestrogens inhibited increases in ER-positive cell lines. Moreover, eribulin decreased the proportion of CSCs in either ER-positive or ER-negative cell lines. The combined treatment of eribulin with an antiestrogen did not additively decrease the proportion of CSCs in ER-positive cell lines. Discussion The results of the present study demonstrated that eribulin had potent antitumor effects on estrogenstimulated ER-positive breast cancer cells and the combined treatment of eribulin with an antiestrogen resulted in a weakly additive antitumor effect. We herein suggested for the first time that eribulin exhibited anti-CSC effects on either ER-positive or ER-negative breast cancer cells. Keywords Eribulin Tamoxifen Fulvestrant Cancer stem cells
Introduction Eribulin mesylate (eribulin), a non-taxane microtubule dynamic inhibitor, has been approved for the treatment of advanced breast cancer in the United States, Japan, and EU countries. Two different clinical phase 3 trials reported that eribulin significantly prolonged the overall survival (OS) of advanced breast cancer patients [1, 2]. Although retrospective subgroup analyses on these trials suggested that eribulin had more potent antitumor effects on patients with estrogen receptor (ER)-negative than ER-positive breast cancer, eribulin displayed marked antitumor effects in patients with ER-positive breast cancer [1, 2].
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A limited number of clinical trials indicated that the concomitant treatment of a cytotoxic agent with endocrine therapy may lead to modest increases in clinical response rates, but not in progression-free survival, and clearly increased the incidence of adverse effects such as thromboembolism in patients with advanced ER-positive breast cancer [3–5]. However, our previous experimental studies showed that the concomitant treatment of 5-fluorouracil (FU), but not paclitaxel and doxorubicin with an antiestrogen additively enhanced their antitumor activities in ERpositive breast cancer cells [6, 7]. Furthermore, some clinical trials have suggested the existence of a synergistic interaction between a 5-FU derivative and tamoxifen (TAM) in a postoperative adjuvant setting for patients with ER-positive early breast cancer [8–10]. These findings prompted us to investigate the interaction between eribulin and the antiestrogen, TAM or steroidal antiestrogen fulvestrant in terms of antitumor activity in ER-positive breast cancer cells. Emerging preclinical and clinical evidence has revealed that the cancer stem cell (CSC) population plays important roles in progression, invasion, metastasis, and resistance to cytotoxic agents and radiation therapy in certain malignancies [11]. Previous studies reported that some cytotoxic agents such as docetaxel and doxorubicin exhibited antitumor activities, but increased the CSC proportion in the surviving tumor cells [12, 13]. The anti-CSC activity of eribulin has not yet been investigated in preclinical and clinical studies. In addition, preclinical studies recently indicated that estrogen increased the proportion of CSCs while antiestrogens inhibited this increase [14, 15]. These findings also prompted us to explore the anti-CSC activities of eribulin and antiestrogens in association with their antitumor effects in the present study.
Materials and methods Compounds Eribulin was kindly provided by Eisai Co., Ltd (Tsukuba, Japan). 17b-estradiol (E2), an active metabolite of TAM, 4-hydroxytamoxifen (OHT), and the steroidal antiestrogen, fulvestrant were obtained from Sigma-Aldrich (St. Louis, MO, USA). Breast cancer cell lines and cell culture KPL-1, KPL-3C, and KPL-4 breast cancer cell lines were established in our laboratory. Their biological characteristics were described elsewhere [16–18]. MCF-7, T-47D and MDA-MB-231 cell lines were kindly provided by the late Dr. Robert B. Dickson (Lombardi Cancer Research Center,
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Washington DC, USA). BT-474 and MDA-MB-157 cell lines were obtained from the American Type Cell Collection (Rockville, MD, USA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (D-MEM, Sigma Co, St. Louis, MI, USA) supplemented with 10 % fetal bovine serum (FBS). We and others previously demonstrated that MCF-7 and T-47D cells expressed a high level of ER-a, a low level of progesterone receptor (PgR), and no detectable level of human epidermal growth factor receptor (HER) 2. KPL-1 and KPL-3C cells have been shown to express a low level of ER-a and no detectable level of either PgR or HER2 [19]. MDA-MB-231 and MDA-MB-157 cells were found to express no detectable level of ER-a or HER2. Both cell lines are also known to express high levels of mesenchymal markers such as vimentin and have been classified as the basal B subtype [20]. BT-474 cells have been shown to express high levels of both ER-a and HER2 [21]. Antitumor activity To investigate the effects of eribulin and/or antiestrogens on cell growth, breast cancer cells (1–5 9 104 cells per well) were seeded on 24-well plates (SB Medical, Tokyo, Japan) and grown in D-MEM supplemented with 10 % FBS at 37 °C in a 5 % CO2 atmosphere for 2 days. After washing with phosphate-buffered saline (PBS, Nissui Co., Tokyo, Japan), the cells were treated with an estrogendeprived medium [phenol red-free RPMI1640 (Life Technologies, Carlsbad, California, USA) supplemented with 10 % dextran-coated charcoal-treated FBS (SigmaAldrich)] plus 1 nM E2 and the indicated concentrations of eribulin and/or antiestrogens for 3 days. After the treatments, the cells were harvested and counted with a Coulter counter (Coulter Electronics, Harpenden, UK). Reproducibility was confirmed in at least two separate experiments. To evaluate the antitumor effects of combined treatments, a combination index based on the 50 % inhibitory concentration (IC50) was calculated according to the following formula: combination index = IC50 with the combined treatment/IC50 with the single treatment. A combination index \0.5 was considered evidence of an additive interaction [6, 7]. Cell cycle and apoptosis assays To investigate the effects of agents on cell cycle progression, harvested cells were stained with propidium iodide using the CycleTest Plus DNA Reagent kit (Becton– Dickinson, San Jose, CA, USA). Apoptotic cells were stained with an Annexin-V-FLUOS staining kit (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s recommendations. Flow cytometry was
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performed with a FACSCalibur flow cytometer (Becton– Dickinson), and the DNA histogram was analyzed using a CELLQuest version 6.0 (Becton–Dickinson). Reproducibility was confirmed in at least two separate experiments. CSC analysis by the CD44/CD24/EpCAM assay To analyze cell surface markers, harvested cells were treated with three fluorescence-labeled antibodies: a PEconjugated anti-CD24 antibody (clone G44-26, Becton– Dickinson), FITC-conjugated anti-CD44 antibody (clone ML5, Becton–Dickinson), and PerCP–Cy5.5 conjugated anti-EpCAM antibody (clone EBA-1, Becton–Dickinson). Flow cytometry was performed with a FACSCalibur flow cytometer (Becton–Dickinson), and analyzed using CELLQuest Software version 6.0 (Becton–Dickinson). Cells that were CD44-high, CD24-negative or low, and EpCAM-positive were recognized as CSCs [18]. CSC analysis by the Aldefluor assay The ALDEFLUOR kit (StemCell Technologies, Durham, NC, USA) was used to isolate the cell population exhibiting high aldehyde dehydrogenase (ALDH) activity. Harvested cells were suspended in the Aldefluor assay buffer containing ALDH substrate (BODIPYTM-aminoacetaldehyde, 1 lmol/l per 1 9 106 cells) and incubated for 30 min at 37 °C. As a negative control, cells were treated with 50 mmol/l diethylaminobenzaldehyde, a specific ALDH inhibitor [19]. CSC analysis by the mammosphere assay Breast cancer cells (0.3–1.5 9 105 cells per well) were seeded on 35-mm dishes (SB Medical) and grown in D-MEM supplemented with 10 % FBS at 37 °C in a 5 % CO2 atmosphere for 2 days. After washing with PBS, cells were treated with estrogen-deprived medium supplemented with 1 nM E2 and the indicated concentrations of eribulin and/or antiestrogens for 3 days. These cells were then dispersed, and single-cell suspensions (5 9 103 cells/well) were incubated in MammoCultTM basal medium (Stem Cell Technologies Co., Tokyo, Japan) supplemented with 10 % MammoCultTM proliferation supplements (Stem Cell Technologies Co.) plus heparin (Stem Cell Technologies Co.) and hydrocortisone (Stem Cell Technologies Co.) in non-adhesive 6-well plates (CORNING Co., NY, USA) for 7 days. Mammosphere colonies more than 60 lm in size were counted with an Olympus phase-contrast microscope [22]. Statistical analysis All values are expressed as the mean ± SE. An ANOVA analysis with StatView computer software (ATMS Co.,
Tokyo, Japan) was used to compare differences between two groups. A two-sided P value less than 0.05 was considered significant.
Results Growth inhibitory activity of eribulin All cell lines were treated with estrogen-deprived medium supplemented with or without 1 nM E2 plus 0–10 nM eribulin for 3 days. Although eribulin did not inhibit the growth of ER-positive and HER2-negative breast cancer cell lines (MCF-7, T-47D, KPL-1 and KPL-3C) under the estrogen-deprived condition, it did inhibit the growth of the ER-positive and HER2-positive BT-474 cell line under the estrogen-deprived condition. In contrast, eribulin dose-dependently inhibited the growth of the ERpositive and HER2-negative breast cancer cell lines as well as the ER-positive and HER2-positive cell line under the E2-supplemented condition (Fig. 1). The 50 % growth inhibitory concentrations (IC50) of the ER-positive cell lines under E2-supplemented conditions are shown in the Table 1. Eribulin dose-dependently inhibited the growth of ERnegative and HER2-positive KPL-4 cells and ER-negative and HER2-negative MDA-MB-231 and MDA-MB-257 cells irrespective of the E2 supplementation (Fig. 1). The IC50 of the ER-negative cell lines under the E2-supplemented condition are shown in the Table 1. Combined antitumor activity of eribulin with antiestrogens To investigate the possible synergistic antitumor activity of eribulin and the antiestrogen, 4-OHT or fulvestrant, all cell lines were treated with estrogen-deprived medium supplemented with 1 nM E2 plus the indicated concentration of 4-OHT or fulvestrant and 0–5 nM eribulin for 3 days. The indicated concentrations of the antiestrogens were selected according to the experimental results obtained with cells treated with the estrogen-deprived medium supplemented with 1 nM E2 plus various concentrations of antiestrogens (data not shown). The combined treatment of 4-OHT or fulvestrant with eribulin showed a modest antitumor effect on the ERpositive and HER2-negative cell lines (Fig. 2). The additive antitumor interaction (the combination index was less than 0.5) between 4-OHT and eribulin was only significant in MCF-7 cells. The other combinations did not show a significant additive interaction in the other cell lines (Table 2).
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Effects of eribulin and/or antiestrogens on cell cycle progression and apoptosis To explore the action mechanisms responsible for the antitumor activities of eribulin and the antiestrogens, their effects on cell cycle progression and apoptosis were
0.1
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G 120 Cell growth (% control)
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Fig. 1 Growth inhibitory curves of eribulin with (filled circle) or without (open circle) E2 supplementation in MCF-7 cells (a), T-47D cells (b), KPL1 cells (c), KPL-3C cells (d), BT-474 cells (e), KPL-4 cells (f), MDA-MB-231 cells (g), and MDA-MB-157 cells (h). Cells were treated with the indicated concentrations of eribulin for 3 days. The values are means ± SEs
Cell growth (% control)
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5.0
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investigated. The selected cell lines were treated with estrogen-deprived medium supplemented with 1 nM E2 plus the indicated concentrations of 4-OHT or fulvestrant and eribulin for 2 days. The treatment with eribulin resulted in the accumulation of G2/M and induction of apoptosis in either ER-positive
Breast Cancer Table 1 The status of ER and HER2 and IC50s of eribulin under E2-supplemented conditions in eight breast cancer cells lines tested MCF-7
T-47D
KPL-1
KPL-3C
BT-474
KPL-4
MDA-MB-231
MDA-MB-157
ER
?
?
?
?
?
-
-
-
HER2
-
-
-
-
?
?
-
-
IC50 (mean ± SE, nM)
0.52 ± 0.21
2.64 ± 0.35
1.17 ± 0.04
1.09 ± 0.92
0.48 ± 0.06
0.65 ± 0.27
0.38 ± 0.04
1.01 ± 0.16
A
120
B
120
100
Cell growth (% control)
100
80 60 40 20
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Fig. 2 Growth inhibitory curves of the concomitant treatment of eribulin with antiestrogens under the E2supplemented condition in MCF-7 cells (a), T-47D cells (b), KPL-1 cells (c), KPL-3C cells (d), BT-474 cells (e), KPL4 cells (f), MDA-MB-231 cells (g), and MDA-MB-157 cells (h). Cells were treated with eribulin (0–5 nM) and the antiestrogen, 4-OHT (0.01–1 lM) or fulvestrant (0.01–0.1 lM), for 3 days. The values are means ± SEs. Closed circles, E2; open circles, E2 plus 4-OHT; and gray circles, E2 plus fulvestrant
Cell growth (% control)
Values are means ± SEs
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Breast Cancer Table 2 The combination index of eribulin with 4-OHT or fulvestrant for eight breast cancer cells lines tested
MCF-7
4-OHT
Fulvestrant
0.43 ± 0.00
0.60 ± 0.01
T-47D
0.78 ± 0.25
0.62 ± 0.21
KPL-1
0.57 ± 0.36
0.92 ± 0.49
KPL-3C
0.70 ± 0.50
1.48 ± 0.69
BT-474
1.21 ± 0.29
1.46 ± 0.17
KPL-4
0.79 ± 0.05
0.84 ± 0.05
MDA-MB-231
0.75 ± 0.06
0.89 ± 0.15
Values are means ± SEs
or ER-negative cell lines (Fig. 3). The treatment with antiestrogens resulted in the retardation of the G1–S cell cycle in MCF-7 cells, but not in KPL-1 cells and MDAMB-231 cells (Fig. 3).The treatment with antiestrogens resulted in enhanced apoptosis in the ER-positive cell lines, but not in the ER-negative cell line (Fig. 3). No significant interaction was observed between eribulin and an antiestrogen in terms of cell cycle progression and apoptosis. Effects of eribulin and/or antiestrogens on the CSC proportion To explore the effects of eribulin and/or an antiestrogen on the CSC proportion, the selected cell lines were treated with estrogen-deprived medium supplemented with 1 nM E2 plus the indicated concentrations of 4-OHT or fulvestrant and eribulin for 3 days. The CSC population was detected and measured using the CD44/CD24/EpCAM assay for ER-positive cell lines. Because the basal B breast cancer cell lines, MDA-MB-231 and MDA-MB-157, strongly acquired epithelial–mesenchymal transition (EMT) and this assay could not be applied to them, we decided to use the Aldefluor assay to detect the CSC population for the basal B cell lines. The E2 treatment significantly increased the CSC proportion in ER-positive cell lines. The treatment with antiestrogens markedly reduced the E2-induced increase in the CSC proportion (Fig. 4). Eribulin decreased the E2induced increase in the CSC proportion in ER-positive cell lines. The combined treatment of eribulin with an antiestrogen did not enhance the decrease in the CSC proportion (Fig. 4). Representative plot charts of the CD44/CD24/ EpCAM assay for MCF-7 cells are shown in Fig. 4. Eribulin significantly decreased the CSC proportion, as detected by the Aldefluor assay, in the basal B cell lines (Fig. 5). Representative plot charts of the Aldefluor assay for MDA-MB-231 cells are shown in Fig. 5. To confirm the anti-CSC effects of eribulin, the mammosphere assay was applied to both ER-positive and ER-
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negative cell lines. Eribulin significantly decreased the CSC proportion and mammosphere size in all cell lines tested (Fig. 6).
Discussion Eribulin has recently been introduced into clinics and widely used for the treatment of patients with advanced breast cancer. Two pivotal clinical trials comparing eribulin versus physicians’ choices or capecitabine showed that eribulin significantly prolonged OS in patients with advanced or metastatic breast cancer [1, 2]. Two recent preclinical studies suggested that eribulin could reverse EMT back to mesenchymal–epithelial transition (MET) in triple-negative breast cancer (TNBC) cells, and also that the eribulin-induced remodeling of an abnormal tumor vasculature led to a more functional microenvironment that may reduce the aggressiveness of tumors due to the elimination of tumor hypoxia [23, 24]. These preclinical findings may provide a scientific basis for the clinical observations of prolonged OS in breast cancer patients treated with eribulin. Retrospective subgroup analyses on the two pivotal trials suggested that eribulin had a more potent antitumor effect on patients with ER-negative breast cancer than those with ER-positive breast cancer. However, eribulin also exhibited potent antitumor effects in patients with ERpositive advanced breast cancer. We speculated that the combined use of eribulin with an endocrine therapeutic agent may lead to synergistic antitumor effects in patients with ER-positive breast cancer. To clarify this hypothesis, we investigated the potential synergistic antitumor activity of the combined treatment of eribulin with the antiestrogens, 4-OHT and fulvestrant in breast cancer cells in the present study. However, eribulin and the antiestrogens did not display an additive antitumor interaction in most ERpositive cell lines, as shown in Fig. 2 and Table 2. However, a slight additive interaction was observed in ERpositive and HER2-negative cell lines, but not in the ERpositive and HER2-positive cell line and ER-negative cell lines (Fig. 2; Table 2). The results of the cell cycle and apoptosis analyses in this study (Fig. 3) suggested that the retardation of G1–S cell cycle induced by antiestrogens interfered with the G2/M-directed antitumor activity of eribulin. Further preclinical and clinical studies are needed to clarify the antitumor interaction between eribulin and antiestrogens in ER-positive breast cancers. Our previous experimental studies and others indicated that estrogen markedly increased the CSC population, whereas antiestrogens inhibited it in ER-positive breast cancer cell lines, but not in ER-negative breast cancer cell lines. These findings have been strongly supported by the
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A
**
B
90
Percentage of apoptoc cells
70
Percentage of cells
**
9
80
60 50 40 30
*
**
20 10
8 7
**
6 5 4 3 2 1 0
0 sub G1
G1
S
G2/M E2 Eribulin (0.5 nM) Eribulin (1.0 nM) 4-OHT (0.05 M)
C
90
D Percentage of apoptoc cells
70
Percentage of cells
**
14
80
60 50 40
**
30 20 10 0 sub G1
G1
S
12
**
10 8 6 4 2 0
G2/M E2
Eribulin (1 nM) Eribulin (5 nM)
Percentage of cells
E
80
F
16
70
Percentage of apoptoc cells
4-OHT (0.1 M)
14
60 50 40 30
**
20 10
12
**
10 8 6 4 2 0
0 sub G1
G1
S
G2/M
E2 Eribulin (0.5 nM) Eribulin (1..0 nM) 4-OHT (0.1 M)
Fig. 3 Effects of concomitant treatments with E2, eribulin, and/or 4-OHT on cell cycle progression and apoptosis in ER-positive MCF-7 cells (a, b), KPL-1 cells (c, d), and ER-negative MDA-MB-231 cells (e, f). Cells were treated with 1 nM E2 and the indicated concentrations of eribulin and/or 4-OHT for 2 days. Percentages of cells at each cell cycle phase and in the apoptotic fraction were analyzed as
described in the ‘‘Materials and methods’’. The values are means ± SEs. White bars, control; black bars, E2; the most light gray bars, E2 plus 0.5 or 1 nM eribulin; the second light gray bars, E2 plus 1 or 5 nM eribulin; the third light gray bars, E2 plus 0.05 or 0.1 lM 4-OHT; the fourth light gray bars, E2 plus 0.5 or 1 nM eribulin and 0.05 or 0.1 lM 4-OHT. *P \ 0.05, **P \ 0.01
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Percentage of CD44high/ CD24low or-/EpCAM+ cells
35
**
*
**
B
**
Percentage of CD44high/ CD24low or-/EpCAM+ cells
A 30 25 20 15 10 5 0
**
80 70 60 50 40 30 20 10 0
E2 E2 Eribulin (3 nM) Eribulin (0.5 nM) Eribulin (5 nM) Eribulin (1.0 nM) 4-OHT ( 0.05 䃛M) 4-OHT (0.05 䃛M)
C 0.21%
20.54%
Control
30.0%
22.15%
7.47%
7.24%
E2
E2 + 4-OHT
E2 + 0.5 nM eribulin
E2 + 1.0 nM eribulin
E2 + 0.5 nM eribulin + 4-OHT
Fig. 4 Effects of E2, eribulin, and/or antiestrogens on the CSC population detected by the CD44/CD24/EpCAM assay in ER-positive MCF-7 (a) and T-47D (b) breast cancer cells. Cells were treated with 1 nM E2 plus the indicated concentrations of eribulin and/or 4-OHT for 3 days. The values are means ± SEs. White bars, control; black bars, E2; the most light gray bars, E2 plus the lower concentration of
eribulin; the second light gray bars, E2 plus the higher concentration of eribulin; the third light bars, E2 plus 4-OHT; the fourth light gray bars, E2 plus the lower concentration of eribulin and 4-OHT.*P \ 0.05, **P \ 0.01. Representative plot charts for MCF7 cells are shown (c). The left upper portions in the figure were defined as the CSC populations
results of this study (Fig. 4) [14, 15, 18]. The action mechanisms responsible for regulating the CSC population in ER-positive breast cancer such as possible paracrine mediators induced by estrogen are being examined in detail in our laboratory. Eribulin not only inhibited cell growth, but also reduced the CSC proportion in both ER-positive and ER-negative
breast cancer cell lines. A previous study reported that a possible CSC population expressing a high level of CD44, low level of CD24, and high level of EpCAM in breast cancer cells more efficiently produced xenograft tumors in an SCID mouse model [25]. In the present study, eribulin significantly decreased cell growth and the proportion of the CSC population in ER-positive breast cancer cell lines
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Percentage of ALDH1-posive cells
A 3
*
C 2.42%
2.5 2
E2
1.5 1 .5 0
E2
1.02% Eribulin (0.5 nM) Eribulin (1.0 nM)
E2 + 1 nM eribulin
4-OHT (0.1 䃛M)
Percentage of ALDH1-posive cells
B 1.2 1
**
.8 .6 .4 .2 0
E2 Eribulin (1 nM) Eribulin (5 nM) 4- OHT (0.1 䃛M)
Fig. 5 Effects of E2, eribulin, and/or antiestrogens on the CSC proportion detected by the Aldefluor assay in ER-negative MDA-MB231 (a) and MDA-MB-157 (b) breast cancer cells. Cells were treated with 1 nM E2 plus the indicated concentrations of eribulin and/or 4-OHT for 3 days. The values are means ± SEs. White bars, control; black bars, E2; the most light gray bars, E2 plus the lower
concentration of eribulin; the second light gray bars, E2 plus the higher concentration of eribulin; the third light bars, E2 plus 4-OHT; the fourth light gray bars, E2 plus the lower concentration of eribulin and 4-OHT.*P \ 0.05, **P \ 0.01. Representative plot charts for MDA-MB-231 cells are shown (c). The R3 portions in the figure were defined as the CSC populations
(Fig. 4). This is the first study to suggest the anticancer stem cell activity of eribulin in ER-positive breast cancer cells. In our CD44/CD24/EpCAM assay, TNBC cell lines such as MDA-MB-231 and MDA-MB-157 consisted of an almost 100 % CSC population. This may have been because of their high level of EMT. Our previous and present studies as well as those by others indicated that this CD44/CD24/EpCAM assay was not suitable for investigating the CSC population in TNBC cell lines [19, 26, 27]. Therefore, we used the Aldefluor assay, in which ALDH activity was recognized as a marker of the CSC population, to detect the CSC population in TNBC cell lines [28]. Eribulin significantly decreased the cell growth and
proportion of the CSC population detected by the Aldefluor assay in TNBC cell lines (Fig. 5). This is the first study to demonstrate the significant anti-CSC activity of eribulin in TNBC cells. Another CSC assay, the mammosphere assay, was also used to clarify the anti-CSC effects of eribulin. As expected, eribulin significantly decreased the proportion of the CSC population and reduced the size of mammospheres in either ER-positive or ER-negative cell lines (Fig. 6). The findings of a recent preclinical study suggested that eribulin reversed EMT back to MET in TNBC cells both in vitro and in vivo [22]. After the treatment with eribulin, the surviving cells showed an increase in both the mRNA and protein expression levels of epithelial markers, such as
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**
B Number of mammosphere/5,000 cells
Number of mammosphere/5,000 cells
A 250 225 200 175 150 125 100 75 50 25 0
** 200 180 160 140 120 100 80 60 40 20 0
E2
E2
Eribulin (1 nM)
Eribulin (1 nM)
C
Control
Eribulin-treated
D
Control Fig. 6 Effects of E2, eribulin, and/or antiestrogens on the CSC proportion detected by the mammosphere assay in ER-positive MCF7 (a) and ER-negative MDA-MB-231 (b) breast cancer cells. Cells were treated with 1 nM E2 ± 1 nM eribulin for 3 days. The values are means ± SEs. White bars, control; black bars, E2; and gray bars,
123
Eribulin-treated E2 plus eribulin.*P \ 0.05, **P \ 0.01. Representative microphotographs of mammospheres for MCF-7 cells (c) and MDA-MB-231 cells (d) with or without the eribulin treatment are shown. The scale bar indicates 500 lm in size
Breast Cancer
E-cadherin, and a decrease in those of mesenchymal markers, such as vimentin. A previous study indicated that CSCs undergo EMT more than non-CSCs [29]. We speculated that eribulin may decrease the proportion of CSCs undergoing EMT and increase that of non-CSCs undergoing MET. This may result in the reversal of EMT back to MET in the surviving cells after the treatment with eribulin. Eribulin may preferentially kill the CSC population or inhibit the self-renewal of CSCs in breast cancer cells. These hypothetical issues should be investigated further in future studies. In conclusion, our preclinical study showed that eribulin exhibited potent antitumor activity in breast cancer cell lines of various subtypes. The concomitant treatment of eribulin with the antiestrogen, 4-OHT or fulvestrant, did not lead to a synergistic antitumor interaction in any ERpositive breast cancer cell lines, but did cause a modestly additive antitumor interaction in some ER-positive breast cancer cell lines. Eribulin significantly reduced the proportion of the CSC population in the surviving cells. Further preclinical and clinical studies are needed to elucidate these phenomena. Acknowledgments Esai Co. kindly provided us with eribulin for this study. We thank Dr. Yasumitsu Nishimura and Dr. Takemi Otsuki of the Department of Hygiene, Kawasaki Medical School for their technical advice. We also thank Mrs. Kaoru Tsuboi and Ms. Megumi Ogo for their technical assistance. This work was supported by Research Project Grants from Kawasaki Medical School (25-12 and 26-5) and by Research Grants from Eisai Co. and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (23591911 and 26461964). Conflict of interest Junichi Kurebayashi received a research grant from Esai Co. The other authors declare that they have no conflict of interest.
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