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Tamoxifen inhibits ER-negative breast cancer cell invasion and metastasis by accelerating Twist1 degradation.

Ma G, He J, Yu Y, Xu Y, Yu X, Martinez J, Lonard DM, Xu J - Int. J. Biol. Sci. (2015)

Bottom Line: In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression.However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells.These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Breast and Thyroid Cancer Surgery, The First Affiliated Hospital of Xi'an Jiaotong University Medical School, Xi'an, China; ; 2. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA;

ABSTRACT
Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers.

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Tamoxifen accelerates Twist1 protein degradation. A. Q-PCR analyses of Twist1 mRNA in HEK293 cells with inducible Twist1 expression and 4T1 and 168FARN cells with endogenous Twist1 expression. These cells were treated with different concentrations of tamoxifen for 24 hours. B. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression. Cells were treated with 1 μg/ml of doxycycline for 12 hours to induce Twist1 expression, then treated with 50 μM of cycloheximide to inhibit protein synthesis and with vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. Analysis of β-actin served as a loading control. HEK293 parent cells without Twist1 expression (Lane 1) served as a negative control. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.41 hours in the vehicle-treated cells or 3.80 hours in the tamoxifen-treated cells. C. Western blot analysis of Twist1 in 168FARN cells treated with vehicle (ethanol) or 10 μM of tamoxifen and with 50 μM of cycloheximide for different time periods as indicated. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.91 hours in the vehicle-treated cells or 4.26 hours in the tamoxifen-treated cells. Results shown in panels B and C are representative results of at least three repeat assays.
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Figure 2: Tamoxifen accelerates Twist1 protein degradation. A. Q-PCR analyses of Twist1 mRNA in HEK293 cells with inducible Twist1 expression and 4T1 and 168FARN cells with endogenous Twist1 expression. These cells were treated with different concentrations of tamoxifen for 24 hours. B. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression. Cells were treated with 1 μg/ml of doxycycline for 12 hours to induce Twist1 expression, then treated with 50 μM of cycloheximide to inhibit protein synthesis and with vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. Analysis of β-actin served as a loading control. HEK293 parent cells without Twist1 expression (Lane 1) served as a negative control. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.41 hours in the vehicle-treated cells or 3.80 hours in the tamoxifen-treated cells. C. Western blot analysis of Twist1 in 168FARN cells treated with vehicle (ethanol) or 10 μM of tamoxifen and with 50 μM of cycloheximide for different time periods as indicated. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.91 hours in the vehicle-treated cells or 4.26 hours in the tamoxifen-treated cells. Results shown in panels B and C are representative results of at least three repeat assays.

Mentions: To determine whether tamoxifen could change Twist1 mRNA levels, we treated HEK293 cells with inducible Twist1 expression and 168FARN and 4T1 mammary tumor cells with endogenous Twist1 expression with different concentrations of tamoxifen for 24 hours and measured Twist1 mRNA by qPCR. Our results demonstrate that tamoxifen treatment had no effect on Twist1 mRNA levels in HEK293 and 4T1 cells and even slightly increased Twist1 mRNA levels in 168FARN cells (Fig. 2A). These results indicate that tamoxifen-induced decreases in Twist1 protein in these cells are not due to changes at its mRNA level.


Tamoxifen inhibits ER-negative breast cancer cell invasion and metastasis by accelerating Twist1 degradation.

Ma G, He J, Yu Y, Xu Y, Yu X, Martinez J, Lonard DM, Xu J - Int. J. Biol. Sci. (2015)

Tamoxifen accelerates Twist1 protein degradation. A. Q-PCR analyses of Twist1 mRNA in HEK293 cells with inducible Twist1 expression and 4T1 and 168FARN cells with endogenous Twist1 expression. These cells were treated with different concentrations of tamoxifen for 24 hours. B. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression. Cells were treated with 1 μg/ml of doxycycline for 12 hours to induce Twist1 expression, then treated with 50 μM of cycloheximide to inhibit protein synthesis and with vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. Analysis of β-actin served as a loading control. HEK293 parent cells without Twist1 expression (Lane 1) served as a negative control. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.41 hours in the vehicle-treated cells or 3.80 hours in the tamoxifen-treated cells. C. Western blot analysis of Twist1 in 168FARN cells treated with vehicle (ethanol) or 10 μM of tamoxifen and with 50 μM of cycloheximide for different time periods as indicated. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.91 hours in the vehicle-treated cells or 4.26 hours in the tamoxifen-treated cells. Results shown in panels B and C are representative results of at least three repeat assays.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400392&req=5

Figure 2: Tamoxifen accelerates Twist1 protein degradation. A. Q-PCR analyses of Twist1 mRNA in HEK293 cells with inducible Twist1 expression and 4T1 and 168FARN cells with endogenous Twist1 expression. These cells were treated with different concentrations of tamoxifen for 24 hours. B. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression. Cells were treated with 1 μg/ml of doxycycline for 12 hours to induce Twist1 expression, then treated with 50 μM of cycloheximide to inhibit protein synthesis and with vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. Analysis of β-actin served as a loading control. HEK293 parent cells without Twist1 expression (Lane 1) served as a negative control. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.41 hours in the vehicle-treated cells or 3.80 hours in the tamoxifen-treated cells. C. Western blot analysis of Twist1 in 168FARN cells treated with vehicle (ethanol) or 10 μM of tamoxifen and with 50 μM of cycloheximide for different time periods as indicated. Band intensities were measured by densitometry and presented in the right panel. The half-life time of Twist1 protein is 4.91 hours in the vehicle-treated cells or 4.26 hours in the tamoxifen-treated cells. Results shown in panels B and C are representative results of at least three repeat assays.
Mentions: To determine whether tamoxifen could change Twist1 mRNA levels, we treated HEK293 cells with inducible Twist1 expression and 168FARN and 4T1 mammary tumor cells with endogenous Twist1 expression with different concentrations of tamoxifen for 24 hours and measured Twist1 mRNA by qPCR. Our results demonstrate that tamoxifen treatment had no effect on Twist1 mRNA levels in HEK293 and 4T1 cells and even slightly increased Twist1 mRNA levels in 168FARN cells (Fig. 2A). These results indicate that tamoxifen-induced decreases in Twist1 protein in these cells are not due to changes at its mRNA level.

Bottom Line: In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression.However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells.These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Breast and Thyroid Cancer Surgery, The First Affiliated Hospital of Xi'an Jiaotong University Medical School, Xi'an, China; ; 2. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA;

ABSTRACT
Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers.

Show MeSH
Related in: MedlinePlus