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. 2010;12(3):R43.
doi: 10.1186/bcr2598. Epub 2010 Jun 28.

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

Hirokuni Ikeda  1 , Naruto Taira, Fumikata Hara, Takeo Fujita, Hiromasa Yamamoto, Junichi Soh, Shinichi Toyooka, Tomohiro Nogami, Tadahiko Shien, Hiroyoshi Doihara, Shinichiro Miyoshi
Affiliations

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

Hirokuni Ikeda et al. Breast Cancer Res. 2010.

Abstract

Introduction: 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.

Methods: 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.

Results: 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-beta-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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
MAPT expression and drug sensitivity. A: MAPT mRNA expression in human breast cancer cell lines. PCR was used to assess MAPT mRNA levels in 12 human breast cancer cell lines. Since MAPT expression was lowest in SK-BR-3 cells, this expression level was taken as the standard for comparison with other cell lines. Cell line ZR75-1 has a naturally high MAPT expression [5,6]. Other cell lines with an mRNA expression higher than that of cell line ZR75-1 were classified as high mRNA expression cell lines. Real-time PCR revealed six cell lines in this category: MDA-MB-134-VI, ZR75-1, YMB1-E, MCF-7, HCC1143, and HCC3153. B: The correlation between MAPT protein expression and sensitivity to anti-cancer drugs. Western blot analysis shows that four cell lines (MDA-MB-134-VI, ZR75-1, YMB1-E, and MCF-7) have multiple protein bands ranging from 50 to 70 kDa. Cell lines HCC1143 and HCC3153 each show only one band at around 70 kDa, despite having a high MAPT mRNA expression. Cell lines HCC38, MDA-MB-231 and SK-BR-3 have low MAPT mRNA expression, but show one band at around 70 kDa. MTS assays were used to assess the sensitivity to paclitaxel, docetaxel, vinorelbine, and doxorubicin, and to determine the IC50 values for drug sensitivity. The upper figure shows the sensitivity to vinorelbine and doxorubicin, and the lower figure shows the sensitivity to paclitaxel and docetaxel. Four cell lines with multiple protein bands show a low sensitivity to taxanes, and five cell lines with only one band at around 70 kDa show a high sensitivity to taxanes. This trend was not observed for vinorelbine and doxorubicin. Cell lines HCC1569 and HCC1937, both having a low MAPT expression, show a low sensitivity to all four anti-cancer drugs. Cell line HCC1937 was the BRCA1-defective breast cancer cell line. The characteristics of HCC1569 are unclear.
Figure 2
Figure 2
Downregulation of MAPT expression and alteration of cellular sensitivity to taxanes. A: Downregulation of MAPT expression with siRNA. siRNA was used to knock down MAPT expression in ZR75-1 and HCC3153 cells. Cells were harvested 72 hours after transfection for Western blot analysis. B: Cell viability determined by an MTS assay after the knockdown of MAPT. Cells were seeded 24 hr after transfection on a 96-well plate at 5 × 103 cells/well and incubated for 24 hr. The cells were then cultivated for 72 hr in the presence of various concentrations of drugs. After this treatment, four independent MTS assays were performed. The data shown are the average of these four assays. In ZR75-1 cells knockdown of MAPT significantly increased sensitivity to taxanes, but did not alter sensitivity to vinorelbine or doxorubicin. In HCC3153 cells knockdown of MAPT did not alter sensitivity to taxanes. *P < 0.05, indicates a significant difference, compared with the control (unpaired Student's test). C: Analysis of the cell cycle using flow cytometry. At 24 hr after transfection, cells were exposed to low-dose paclitaxel (25 nM) for 72 hr. Afterwards, a cell cycle analysis was performed using flow cytometry. The percentage of cells in the G2/M phase was higher in cells that had been exposed to low-dose taxanes after MAPT knockdown, compared with the controls. (The data show ZR75-1 cells with paclitaxel.) D: Analysis of cell cycle and cell proliferation using immunofluorescence. After transfection, cells were seeded on six-well plates and incubated for 24 hr. After this treatment, the cells were exposed to low-dose paclitaxel (25 nM) for 24 hr. Immunofluorescence with an anti-α-tubulin antibody was then performed. Paclitaxel caused an increase in apoptotic cells from 14/100 cells (that is, the control cells) to 29/100 cells (that is, MAPT knockdown cells). There was also repressed proliferation, compared with the controls. (The arrows indicate apoptotic ZR75-1 cells.)
Figure 3
Figure 3
The relationship between ER and MAPT expression. A: Downregulation of ER expression with siRNA. siRNA was used to knock down ER expression in MCF-7 and ZR75-1 cells. Cells were harvested 72 hours after transfection for Western blot analysis. The MAPT protein expression decreased after ER knockdown in both cells lines. B: Treatment with 17-β estradiol and tamoxifen. Cells were seeded in serum-free medium and incubated. After incubation for 24 hr, cells were cultivated in a medium containing 17-β estradiol alone, tamoxifen alone, or various combinations of these two agents for 72 hr. They were then harvested for Western blot analysis. In MCF7 cells, MAPT expression was increased by 17-β estradiol. In the absence of 17-β estradiol, tamoxifen increased MAPT expression. This effect was highest at concentrations of 500 nM and 1 μM, and decreased at higher concentrations. With 17-β estradiol, tamoxifen at low concentrations decreased MAPT expression, but a high stimulatory effect was found at 500 nM. This effect decreased at high concentrations. In ZR75-1 cells, these changes were observed for protein isoforms of less than 70 kDa. MCF-7 cells were exposed to 1.0 nM 17-β estradiol and ZR75-1 cells to 10 nM 17-β estradiol. E2: 17-β estradiol; Tam: tamoxifen. C: Stimulation with 17-β estradiol and fulvestrant. Cells were seeded in a serum-free medium and incubated. After a 24-hr incubation, cells were cultivated in a medium containing 17-β estradiol alone, fulvestrant alone, or various combinations of the two agents for 72 hr. They were then harvested for Western blot analysis. Fulvestrant decreased ER and MAPT protein expression in both cell lines. These changes were more noticeable for protein isoforms of less than 70 kDa in ZR75-1 cells. MCF-7 cells were exposed to 1.0 nM 17-β estradiol and ZR75-1 cells to 10 nM 17-β estradiol. E2, 17-β estradiol; Ful, fulvestrant.
Figure 4
Figure 4
Combination treatment with hormone drugs and taxanes. A: Effect of combination treatment with hormone drugs and taxanes using a constant ratio design. The combination effect was evaluated using the Combination Index (C.I). Combination treatment with fulvestrant and taxanes showed a synergistic effect. Combination treatment with tamoxifen and taxanes had an antagonistic effect at low doses, but this effect became additive in both cell lines as the tamoxifen dose increased. (Data for MCF-7 cells are shown.) C.I, Combination Index; Fa, fraction affected. B: Effect of combination treatment with hormone drugs and taxanes using a non-constant ratio design. Cells were cultivated with 1 μM paclitaxel alone, with tamoxifen at various concentrations, with fulvestrant alone at various concentrations, and with combinations of these agents. The combination effect was evaluated using the Combination Index (C.I). In combination treatment with tamoxifen and paclitaxel, an antagonistic effect was observed at low concentrations, while an additive effect was found at high concentrations. The strongest antagonistic effect was observed at 500 nM. Combinations of fulvestrant and taxanes gave a synergistic effect. (Data are shown for MCF-7 cells with paclitaxel.) Pacli, paclitaxel; Tam, tamoxifen; Ful, fulvestrant; C.I, Combination Index. C: Evaluation of combination treatment by flow cytometry. Cells were cultivated with 50 nM paclitaxel alone, 100 nM tamoxifen alone, 75 nM fulvestrant alone, or with combinations of these agents. Combination treatment with fulvestrant and paclitaxel increased the percentage of cells in G2/M phase, compared with paclitaxel alone or combination treatment with paclitaxel and tamoxifen. (Data are shown for MCF-7 cells with paclitaxel.) D: Evaluation of combination treatment using immunofluorescence. Cells were cultivated with 50 nM paclitaxel alone, 100 nM tamoxifen alone, 75 nM fulvestrant alone, or combinations of these agents. 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 combination treatment with tamoxifen. Apoptotic cells occurred in 11/100 cells with paclitaxel treatment alone; in 12/100 cells in combination treatment using tamoxifen; and in 25/100 cells in combination treatment using fulvestrant. (Arrows indicate apoptotic MCF-7 cells.)
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