- Research article
- Open Access
The estrogen receptor influences microtubule-associated protein tau (MAPT) expression and the selective estrogen receptor inhibitor fulvestrant downregulates MAPT and increases the sensitivity to taxane in breast cancer cells
Breast Cancer Research volume 12, Article number: R43 (2010)
Microtubule-associated protein tau (MAPT) inhibits the function of taxanes and high expression of MAPT decreases the sensitivity to taxanes. The relationship between estrogen receptor (ER) and MAPT in breast cancer is unclear. In this study, we examined the correlation of MAPT expression with the sensitivity of human breast cancer cells to taxanes, and the relationship between ER and MAPT.
The correlation between MAPT expression and sensitivity to taxanes was investigated in 12 human breast cancer cell lines. Alterations in cellular sensitivity to taxanes were evaluated after knockdown of MAPT expression. ER expression was knocked down or stimulated in MAPT- and ER-positive cell lines to examine the relationship between ER and MAPT. The cells were also treated with hormone drugs (tamoxifen and fulvestrant) and taxanes.
mRNA expression of MAPT did not correlate with sensitivity to taxanes. However, expression of MAPT protein isoforms of less than 70 kDa was correlated with a low sensitivity to taxanes. Downregulation of MAPT increased cellular sensitivity to taxanes. MAPT protein expression was increased by stimulation with 17-β-estradiol or tamoxifen, but decreased by ER downregulation and by fulvestrant, an ER inhibitor. The combination of fulvestrant with taxanes had a synergistic effect, whereas tamoxifen and taxanes had an antagonistic effect.
Expression of MAPT protein isoforms of less than 70 kDa is correlated with a low sensitivity to taxanes in breast cancer cells. ER influences MAPT expression and fulvestrant increases the sensitivity to taxanes in MAPT- and ER-positive breast cancer cells.
Taxanes are important drugs for treatment of breast cancer [1–3]. These drugs bind to tubulin and suppress spindle microtubule dynamics, which leads to cell cycle arrest in G2/M phase followed by apoptosis [4–6].
Several mechanisms of taxane resistance have been described, including overexpression of the drug efflux pump MDR-1/P-gp, HER-2 overexpression, tubulin mutation, and variable expression of tubulin isotypes and stathmin [4, 7–12]. Microtubule-associated protein-tau (MAPT), which is implicated in the pathogenesis of Alzheimer's disease, is associated with another mechanism of taxane resistance. MAPT binds to both the outer and inner surfaces of microtubules, leading to tubulin assembly and microtubule stabilization. Since taxanes also bind to the inner surface of microtubules, MAPT obstructs the function of the drug [5, 6, 13, 14]. Rouzier et al. found that low MAPT expression was associated with higher rates of a pathologic complete response to preoperative paclitaxel and 5-fluorouracil, doxorubicin, cyclophosphamide (paclitaxel/FAC) chemotherapy . This group also showed that MAPT overexpression was correlated with resistance to paclitaxel and that knockdown of MAPT with small interfering RNA (siRNA) reversed the resistance to taxanes in vitro .
MAPT has six isoforms that are spliced from a single gene. These isoforms differ by having three or four conserved repeat motifs in the microtubule-binding domain and none, one or two insertions in the N-terminal projection domain. Isoforms with four C-terminal repeats have a higher affinity for microtubules than isoforms with three such repeats [13–17]. However, the function of each isoform is unknown.
Previous experimental studies have shown that MAPT expression is increased by estrogen in vitro and in vivo [18, 19], and clinical studies have shown a positive correlation of MAPT levels with estrogen receptors (ER) expression [20, 21]. Jonna et al. found that estrogen stimulation upregulated MAPT mRNA in MCF-7 cells in microarray analysis , and the MAPT gene is considered to contain an imperfect ER response element upstream of its promoter. The ER plays a key role in the development and progression of breast cancer, but it is unknown if ER stimulation induces MAPT expression in breast cancer cells.
Hormonal drugs play an important role in breast cancer therapy. The selective ER inhibitor, fulvestrant, inhibits estrogen signaling through the ER in two ways: by competing with estradiol binding to the ER, and by increasing the turnover of ER to decrease the ER protein level in breast cancer cells. In contrast, tamoxifen, a selective ER modulator, is an ER antagonist but often displays estrogen-like agonist activity [22–24]. Therefore, fulvestrant and tamoxifen may have different effects on MAPT expression via the ER.
Previous in vitro studies show that tamoxifen has an antagonistic effect on anti-cancer drugs [25, 26]. Several clinical studies that used tamoxifen for hormone therapy have found that it has an antagonistic effect on chemotherapy drugs when it is used concurrently with them, and that the results of the combined use of tamoxifen with chemotherapy drugs is inferior, compared with using the drugs sequentially [27–30]. The effect of combination treatment using other modern hormone therapies, such as aromatase inhibitors or fulvestrant, has not been examined thoroughly.
In this study, we examined the relationship between the MAPT expression and the sensitivity to taxanes, the effect of ER expression or modulation on MAPT expression, and the combined impact of hormones and taxanes on anti-cancer activity and taxane resistance in breast cancer cell lines.
Materials and methods
Cell culture and agents
Twelve human breast cancer cell lines were used in the study: MCF-7, MDA-MB-231, SK-BR-3 and ZR75-1 were obtained from the American Type Culture Collection (Rockville, MD, USA); YMB1-E was kindly provided by the Tohoku University Institute of Development, Aging and Cancer Cell Resource Center for Biomedical Research; and MDA-MB-134-VI, HCC38, HCC1143, HCC1569, HCC1806, HCC1937 and HCC3153 were kindly provided by Adi F. Gazdar (Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA). Cells were maintained at 37°C in 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin.
Paclitaxel, docetaxel, fulvestrant, 17-β estradiol and tamoxifen were purchased from Sigma-Aldrich. Vinorelbine and doxorubicin were obtained from Kyowa Hakkoh (Tokyo, Japan). Cells were cultured in a phenol-free medium containing 10% dextran-coated, charcoal-treated FCS (Thermo Scientific, Waltham, MA, USA) and then treated with the above agents alone or in combination.
Small interfering RNA
Expression levels of MAPT and ER alpha were knocked down by transfection of the cells with two anti-MAPT siRNAs (5'-CGG GAC TGG AAG CGA TGA CAA-3' and 5'-CCG CCA GGA GTT CGA AGT GAT-3'; Qiagen, Valencia, CA, USA) and an anti-ER alpha siRNA (5'-GAG ACT TGA ATT AAT AAG TGA-3'; Qiagen), respectively. Scrambled siRNA (AllStars Negative Control siRNA, Qiagen) was used as the control. Transfection of siRNA was performed using HiPerfect Transfection Reagent (Qiagen) according to the manufacturer's protocol.
mRNA and protein expression analysis
Total RNA was extracted from cell pellets and cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's protocol. Quantitative real-time PCR was performed using the Step One™ Real-Time PCR System (Applied Biosystems) with 18S rRNA as an internal control (Applied Biosystems). The sequences of primers and the probe were as follows: MAPT, forward primer: 5'-TAG GCA ACA TCC ATC ATA AAC CA-3'; reverse primer: 5'-TCG ACT GGA CTC TGT CCT TGA A-3'; and the FAM-TAMR probe: 5'-TGG CCA GGT GGA AG-3' (Invitrogen, Carlsbad, CA, USA). Data were analyzed using the relative standard curve method.
Samples from cultured cells were prepared for Western blot analysis, as previously described . The samples were separated on a NuPAGE Bis-Tris Gel 4 to 12% (Invitrogen) and electroblotted onto a polyvinylidene fluoride membrane. Primary antibodies for Western blotting were as follows: MAPT (T1029, United States Biological, Swampscott, MA, USA) [5, 6]; ERα (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and actin (Sigma-Aldrich). Blots were exposed to a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) with development using enhanced chemiluminescence detection (ECL Kit, Amersham Pharmacia Biotech, Chandler, AZ, USA).
Effects of agents on cells
A cell viability assay was performed as previously described , in which IC50 values were determined for the anti-proliferative activity of each drug. Experiments were performed independently four times and the data shown are the average of the four assays. The combination effect of two agents was evaluated using the Combination Index (C.I), which was calculated using Calcusyn software (Biosoft, Cambridge, UK). The definition of C.I is as follows: C.I = (D)1/(Dx)1 + (D)2/(Dx)2 + (D)1(D)2/(Dx)1(Dx)2, where (Dx)1 is the dose of Drug 1 alone required to produce an X% effect; (D)1 is the dose of Drug 1 required to produce the same X% effect in combination with Drug 2; (Dx)2 is the dose of Drug 2 alone required to produce an X% effect; and (D)2 is the dose of Drug 2 required to produce the same X% effect in combination with Drug 1. C.I < 1, 1 and > 1 indicates a synergistic effect, an additive effect, and an antagonistic effect, respectively. Cell cycle effects were examined by flow cytometry as previously described [32, 33].
Cells were fixed in 4% paraformaldehyde, washed with cold PBS, and incubated in PBS containing 0.1% Triton X-100. After permeabilization, the cells were incubated in blocking buffer (PBS containing 0.1% Tween-20 and 3% BSA) containing antibodies against α-tubulin (Sigma-Aldrich). After washing with 0.1% Tween PBS, the cells were incubated in blocking buffer containing an anti-mouse AlexaFluor 488-conjugated secondary antibody (green) (Invitrogen). After washing again with 0.1% Tween PBS, the cells were incubated in PBS containing DAPI (blue) (Invitrogen). Immunofluorescence microscopy was performed using Biozero (Keyence, Osaka, Japan).
MAPT expression and drug sensitivity
MAPT mRNA expression was assessed using quantitative real-time PCR and MAPT protein expression was assessed using Western blot analysis in 12 human breast cancer cell lines (Figure 1A, B). Six cell lines showed high MAPT mRNA expression. Four of the six cell lines showed multiple protein bands of 50 to 70 kDa, and the other two cell lines showed only one band at around 70 kDa. Three cell lines with low MAPT mRNA expression also showed MAPT protein expression at around 70 kDa.
To analyze the correlation between MAPT expression and drug sensitivity, MTS assays were performed to determine the IC50 values for the sensitivity of the 12 cell lines to paclitaxel, docetaxel, vinorelbine, and doxorubicin. Four of the six cell lines that showed high MAPT mRNA expression had a low sensitivity to taxanes, while the remaining two cell lines showed a high sensitivity. The five cell lines expressing only the MAPT protein isoform at around 70 kDa had a high sensitivity to taxanes, but all four cell lines with MAPT protein isoforms of less than 70 kDa showed a low sensitivity to taxanes. This trend was not observed for vinorelbine and doxorubicin. Cell line HCC1937, which has a low MAPT expression, showed a low sensitivity to all four anti-cancer drugs. HCC1937 is the BRCA1-defective breast cancer cell line and has a low sensitivity to paclitaxel and doxorubicin . Cell line HCC1569, which has a low MAPT expression, showed a low sensitivity to all four anti-cancer drugs, but its character is unclear. Our results suggest that MAPT mRNA expression is insufficient as a predictor of taxane sensitivity; that the MAPT protein isoform at around 70 kDa does not cause a low sensitivity to taxanes; and that MAPT protein isoforms of less than 70 kDa are most correlated with a low sensitivity to taxanes.
Downregulation of MAPT expression and alteration of cellular sensitivity to taxanes
To examine the role of MAPT protein in taxane resistance, siRNA was used to knock down MAPT expression in MAPT-positive cell lines as confirmed by Western blot analysis. ZR75-1, MCF-7 and HCC3153 cells were used in our study. The level of siRNA knockdown was approximately 65%. MDA-MB-134-VI, YMB1-E and HCC1143 cells could not be knocked down MAPT expression successfully. After MAPT knockdown, the cells were cultured with various concentrations of drugs and MTS assays were performed. ZR75-1, MCF-7 cells has MAPT protein isoforms of less than 70 kDa, all isoforms were knocked down by siRNA, and knockdown of MAPT increased the sensitivity to taxanes in both cell lines, but did not alter the sensitivity to vinorelbine and doxorubicin. In contrast, in HCC3153 cells, in which only the MAPT isoform at around 70 kDa is expressed, siRNA knockdown of this isoform did not change the sensitivity to taxanes (Figure 2A, B). Based on these results, we suggest that MAPT protein isoforms of less than 70 kDa largely determine the sensitivity to taxanes.
Taxanes bind to tubulin and suppress spindle microtubule dynamics, which leads to arrest of the cell cycle in G2/M phase. We hypothesized that MAPT expression, and especially MAPT protein isoforms of less than 70 kDa, might block this activity of the drug. Using flow cytometry and immunofluorescence, changes in the cell cycle and cell proliferation were analyzed after knockdown of MAPT and exposure of the cells to low doses of taxanes. The percentage of cells in G2/M phase was higher in those exposed to low doses of taxanes after MAPT knockdown than in controls (Figure 2C). An increased percentage of sub-G1 phase cells was also noted for cells exposed to taxanes after MAPT knockdown (data not shown). Immunofluorescence with an anti-α-tubulin antibody (Figure 2D) showed that taxanes increased the amount of apoptotic cells and repressed cell proliferation after MAPT knockdown, in comparison with controls.
The relationship between ER and MAPT expression
Three of the 12 cell lines were ER-positive and all three had expression of MAPT protein isoforms of less than 70 kDa (Figure 1A). To examine the relationship between ER and MAPT expression, siRNA was used to knock down ER expression in the MAPT- and ER-positive cell lines (MCF-7 and ZR75-1), as confirmed by Western blot analysis (The level of siRNA knockdown was approximately 85%). MAPT protein expression decreased after ER knockdown in both cells lines (Figure 3A). These results indicate that the MAPT level is influenced by ER in breast cancer cell lines. To examine this relationship in more detail, cells were stimulated with 17-β estradiol and treated with tamoxifen and fulvestrant. The cells were seeded in a serum-free medium, incubated for 24 hr, and then cultivated in a medium containing 17-β estradiol alone; tamoxifen alone; fulvestrant alone; or various combinations of these agents. Subsequent Western blot analysis was performed. In MCF7 cells, MAPT expression was increased by 17-β estradiol. In the absence of 17-β estradiol, exposure to tamoxifen increased MAPT expression. This effect was strongest at low concentrations of 500 nM to 1 μM. With 17-β estradiol, MAPT expression was decreased by tamoxifen at low concentrations, but increased by tamoxifen at 500 nM. The effect of increased MAPT expression was reduced at higher concentrations of tamoxifen. In ZR75-1 cells, these changes were observed for protein isoforms of less than 70 kDa (Figure 3B). Exposure to fulvestrant decreased ER and MAPT expression regardless of the drug concentration. In ZR75-1 cells, similar changes were observed for MAPT protein isoforms of less than 70 kDa (Figure 3C). These findings suggest that the ER modulates MAPT expression, especially for MAPT isoforms of less than 70 kDa. Tamoxifen stimulates the ER in the presence of 17-β estradiol, and significantly increases MAPT expression at low concentration, whereas fulvestrant decreases ER and MAPT expression at all concentrations.
Combination treatment with hormone drugs and taxanes
Based on the above results, we hypothesized that a combination of hormone drugs and taxanes would increase anti-cancer activity and the sensitivity to taxanes. This hypothesis was tested in the MAPT- and ER-positive cell lines (MCF-7 and ZR75-1), with evaluation of the combination treatment using the Combination Index. The molar ratios for combinations of paclitaxel, docetaxel, tamoxifen and fulvestrant were determined based on the individual IC50 values (Table 1). This study was performed using a constant ratio design. The combination treatment with tamoxifen and taxanes had an antagonistic effect at a low dose, but this effect became additive in both cell lines as the dose of tamoxifen increased. In contrast, the combination of fulvestrant with taxanes showed a synergistic effect (Figure 4A, Table 2).
Since the effect of tamoxifen on MAPT expression differs depending on its concentration, the effect of concomitant use with taxanes may also differ depending on tamoxifen concentration. Thus, we performed combination treatment with tamoxifen at various concentrations, with the taxane concentration maintained at a certain level, and evaluated the results using the Combination Index (Figure 4B). This study was performed using a non-constant ratio design. The combination treatment with tamoxifen and taxanes gave an antagonistic effect at low concentrations, but an additive effect at high concentrations. The strongest antagonistic effect was found at 500 nM, and the differences in concomitant treatment effects caused by the tamoxifen concentration were similar to those for MAPT expression. Combination treatment with fulvestrant and taxanes gave a synergistic effect regardless of the fulvestrant concentration.
The effects of a combination of hormone drugs and taxanes on the cell cycle and cell proliferation were evaluated with flow cytometry and immunofluorescence. Treatment with tamoxifen alone or fulvestrant alone increased the percentage of cells in G1 phase and taxanes increased the percentage of cells in G2/M phase, compared with controls. The combination treatment with tamoxifen and taxanes increased the percentage of cells in G1 phase and decreased the percentage in G2/M phase slightly, compared with the respective values with each taxane alone. The combination of fulvestrant and taxanes did not alter the percentage of cells in G1 phase, despite the presence of fulvestrant, but the number of cells in G2/M phase increased compared with each taxane alone (Figure 4C). We also found that the percentage of sub-G1 phase cells increased after the combination treatment with fulvestrant and taxanes, compared to treatment with each taxane alone or the combination of tamoxifen with taxanes (data not shown).
Immunofluorescence showed that an increase in apoptotic cells and repression of cell proliferation occurred in the combination treatment with fulvestrant and paclitaxel, compared with paclitaxel alone. These changes did not occur in the combination treatment with tamoxifen (Figure 4D). These results indicate that fulvestrant supports taxane drug function and has a synergistic effect with taxanes.
In this study, we obtained several new findings on MAPT expression and the effects of taxanes. First, the sensitivity to taxanes is influenced by MAPT protein isoforms of less than 70 kDa. MAPT has six isoforms that give bands representing molecules ranging in size from 50 to 70 kDa, and MAPT protein isoforms have a significant impact on taxane sensitivity since they have different affinities for microtubules and different antagonistic effects on taxane [13–16]. Rouzier et al. provided the first report of the correlation of MAPT expression with the remission rate in subjects who received perioperative chemotherapy with a regimen including paclitaxel, and identified MAPT as a predictor of sensitivity to taxanes . However, subsequent studies did not support the utility of MAPT as a predictor of the effect of taxanes [20, 21, 35, 36]. Recently, Pusztai et al. performed a large-scale phase III clinical trial to compare doxorubicin and cyclophosphamide (AC) and AC followed by four courses of paclitaxel as adjuvant chemotherapy after surgery for breast cancer . The prognosis of patients with MAPT expression was better than that of patients with no MAPT expression, although the utility of MAPT as a predictor of the taxane effect was not shown . In clinical studies, RT-PCR and immunostaining are used for analysis of MAPT expression. The results of our in vitro study indicated that expression of MAPT protein isoforms less than 70 KDa had the most influence on sensitivity to taxanes. A discrepancy between MAPT mRNA expression and protein expression has been found previously , and thus analysis of MAPT mRNA expression may not be appropriate for examining the utility of MAPT as a predictor of taxane sensitivity. Furthermore, the status of the expression of different MAPT protein isoforms is important in determining sensitivity to taxanes, but immunohistochemistry cannot be used to evaluate each isoform. MAPT isoform expression in breast cancer tissues must be examined in detail to determine the exact correlation between MAPT expression and response to taxanes.
The second finding in the study involved clarification of the effect of the ER on the MAPT protein level in breast cancer cells. Clinical studies have suggested that MAPT expression has a positive correlation with ER expression and is influenced by ER signaling [20, 21]. In our study in ER-positive and MAPT-positive breast cancer cell lines, expression of MAPT protein isoforms of less than 70 kDa, which have a large impact on sensitivity to taxanes, was affected by ER signaling. Furthermore, treatment of MAPT- and ER-positive cells with tamoxifen or fulvestrant had different effects on MAPT expression via the ER, which suggests that these drugs can alter cellular sensitivity to taxanes. In clinical treatment for breast cancer, the advantages and disadvantages of concomitant use of chemotherapeutic drugs and endocrine therapy have long been discussed. Several clinical studies of tamoxifen as hormone therapy have found that an antagonistic effect on concurrent chemotherapeutic agents, and that the results of giving tamoxifen concurrently with these agents are inferior to that of sequential administration [27–30]. These results suggest that concomitant chemotherapy and endocrine therapy should be avoided clinically. However, the effect of combination treatment using other modern hormone therapies, such as aromatase inhibitors or fulvestrant, has not been examined thoroughly.
Our third finding supports and complements the current idea on concomitant use of chemotherapy and endocrine therapy, and indicated a new possibility for concomitant use. Tamoxifen is an ER antagonist that also has estrogen-like agonist activity. It has been used as hormone drug for long-term therapy, but its effect are complicated and incompletely understood. Our results suggested that the effect of tamoxifen on ER signaling differs depending on the dose. MAPT protein expression was increased at low concentrations of tamoxifen of 500 nM - 1 μM, but decreased at higher concentrations. Several factors associated with resistance to chemotherapy via regulation by ER signaling have been identified [4, 22, 38–40]. Tamoxifen is thought to exert an antagonistic effect in concomitant use with chemotherapeutic drugs by increasing the expression of these factors via an agonistic effect on the ER. Active metabolites of tamoxifen also have different functions compared with the parent drug [41–43], and more detailed studies are needed to determine how tamoxifen and its metabolites influence chemotherapy in vivo and in vitro.
Fulvestrant decreased ER and MAPT expression at all concentrations. An MTS assay, flow cytometry, and immunofluorescence all showed that the combination of fulvestrant and taxanes had a synergistic effect, consistent with the finding of Sui et al. that fulvestrant combined with paclitaxel was effective in breast cancer cells in vitro . Fulvestrant assists taxane function by downregulating the ER and ER-regulated factors associated with taxane resistance, and the combination of fulvestrant with taxanes increases the sensitivity of MAPT- and ER-positive breast cancer cells to taxanes.
ER-positive breast cancers clinically show a lower sensitivity to chemotherapy than do ER-negative breast cancers. This may be caused by the ER itself or by ER modulation of factors that result in resistance to chemotherapy. Our study indicates that the combination of modern hormone therapy with modern chemotherapy may become an effective therapy to ER-positive breast cancers.
Expression of MAPT protein isoforms of less than 70 kDa is correlated with a low sensitivity to taxanes in breast cancer cells. ER influences MAPT expression and the selective ER inhibitor fulvestrant downregulates MAPT expression and increases the sensitivity to taxanes in MAPT- and ER-positive breast cancer cells.
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This study was supported by a Grant-in-Aid for Scientific Research (c) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr. Adi F. Gazdar for providing several breast cancer cell lines.
The authors declare that they have no competing interests.
NT, FH, TF, TS and HD designed this study. HI wrote the manuscript with NT. HI and TN performed the experiments. HY and JS analyzed the data. ST and SM gave expert advice throughout the study.
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Ikeda, H., Taira, N., Hara, F. et al. The estrogen receptor influences microtubule-associated protein tau (MAPT) expression and the selective estrogen receptor inhibitor fulvestrant downregulates MAPT and increases the sensitivity to taxane in breast cancer cells. Breast Cancer Res 12, R43 (2010). https://doi.org/10.1186/bcr2598
- Estrogen Receptor