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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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Related in: MedlinePlus

Tamoxifen treatment decreases the cellular levels of Twist1 protein. A. In pQCXIH-Luc and pQCXIH-Twist1-Luc plasmids, the expression of luciferase or Twist1-Luciferase was driven by the CMV promoter. B. HeLa cells transfected with pQCXIH-Luc or pQCXIH-Twist1-Luc plasmids and with a β-galactosidase expression plasmid were treated with vehicle (ethanol) or tamoxifen (10 μM) for 24 hours. Then, the luciferase activity was measured and normalized to the β-galactosidase activity for each sample. Relative luciferase activity was calculated by (Luc activity in tamoxifen-treated cells/Luc activity in vehicle-treated cells) × 100. Data are presented as mean ± SD. ***, P < 0.0001 by t test. C. HEK293 cells with an inducible Twist1 expression system were treated with 1 μg/ml of doxycycline for 12 hours to induce stable Twist1 expression and then co-treated with 1 μg/ml of doxycycline and vehicle or 10 μM of tamoxifen for the time periods as indicated. HEK293 cells without inducible Twist1 expression served as a negative control (Lane 1). Western blot analyses were performed with Twist1 and β-actin antibodies. D. HEK293 cells with inducible Twist1 expression were treated with 1 μg/ml of doxycycline for 12 hours, and then co-treated with 1 μg/ml of doxycycline and different concentrations of tamoxifen as indicated for 24 hours before Western blotting was performed. E. Western blot analyses of Twist1 in 168FARN and 4T1 cells treated with vehicle or tamoxifen (10 μM) for different time periods as indicated. F. Western blot analyses of Twist1 in 168FARN and MDA-MB-435 cells treated with the indicated concentrations of tamoxifen for 24 hours. Results shown in panels B to F are representative results of at least three repeat assays.
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Figure 1: Tamoxifen treatment decreases the cellular levels of Twist1 protein. A. In pQCXIH-Luc and pQCXIH-Twist1-Luc plasmids, the expression of luciferase or Twist1-Luciferase was driven by the CMV promoter. B. HeLa cells transfected with pQCXIH-Luc or pQCXIH-Twist1-Luc plasmids and with a β-galactosidase expression plasmid were treated with vehicle (ethanol) or tamoxifen (10 μM) for 24 hours. Then, the luciferase activity was measured and normalized to the β-galactosidase activity for each sample. Relative luciferase activity was calculated by (Luc activity in tamoxifen-treated cells/Luc activity in vehicle-treated cells) × 100. Data are presented as mean ± SD. ***, P < 0.0001 by t test. C. HEK293 cells with an inducible Twist1 expression system were treated with 1 μg/ml of doxycycline for 12 hours to induce stable Twist1 expression and then co-treated with 1 μg/ml of doxycycline and vehicle or 10 μM of tamoxifen for the time periods as indicated. HEK293 cells without inducible Twist1 expression served as a negative control (Lane 1). Western blot analyses were performed with Twist1 and β-actin antibodies. D. HEK293 cells with inducible Twist1 expression were treated with 1 μg/ml of doxycycline for 12 hours, and then co-treated with 1 μg/ml of doxycycline and different concentrations of tamoxifen as indicated for 24 hours before Western blotting was performed. E. Western blot analyses of Twist1 in 168FARN and 4T1 cells treated with vehicle or tamoxifen (10 μM) for different time periods as indicated. F. Western blot analyses of Twist1 in 168FARN and MDA-MB-435 cells treated with the indicated concentrations of tamoxifen for 24 hours. Results shown in panels B to F are representative results of at least three repeat assays.

Mentions: To establish a high throughput screening system for identifying bioactive small molecules that reduce Twist1 protein in cancer cells, we constructed a pQCXIH-Twist1-Luc vector to express the Twist1-luciferase fusion protein and a pQCXIH-Luc vector to express only the luciferase protein as a control (Fig. 1A). The relative cellular amounts of both Twist1-Luc and luciferase proteins can be conveniently measured by their luciferase activities. We equally transfected HeLa cells in 96-well plates with either pQCXIH-Twist1-Luc or pQCXIH-Luc plasmid, followed by treatment with one of the 1280 pharmacologically active compounds in the LOPAC library. Measurements of luciferase activities in all transfected and treated cells led to the identification of several small compounds that selectively reduced the luciferase activity in cells transfected with pQCXIH-Twist1-Luc plasmid, but had no significant effects on the luciferase activity in cells transfected with the pQCXIH-Luc plasmid. These compounds include tamoxifen citrate (tamoxifen), sertraline, indatraline, CGS-12066A, methiothepin, GR 127935, parthenolide and MK-886. tamoxifen is a well-known SERM for treating ER-positive breast cancer. Sertraline, indatraline and CGS-12066A are a selective serotonin reuptake inhibitor (SSRI), a serotonin receptor agonist and a serotonin antagonist, respectively. Methiothepin and GR-127935 are 5-HT1B/1D receptor antagonists. Parthenolide is an inhibitor of serotonin release from platelets. MK-886 is a potent and specific inhibitor of leukotriene biosynthesis. In this study, tamoxifen was selected for further characterization because of its known therapeutic role in ER-positive breast cancers and the crucial role of Twist1 in driving epithelial-mesenchymal transition and metastasis in breast cancer cells 2.


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 treatment decreases the cellular levels of Twist1 protein. A. In pQCXIH-Luc and pQCXIH-Twist1-Luc plasmids, the expression of luciferase or Twist1-Luciferase was driven by the CMV promoter. B. HeLa cells transfected with pQCXIH-Luc or pQCXIH-Twist1-Luc plasmids and with a β-galactosidase expression plasmid were treated with vehicle (ethanol) or tamoxifen (10 μM) for 24 hours. Then, the luciferase activity was measured and normalized to the β-galactosidase activity for each sample. Relative luciferase activity was calculated by (Luc activity in tamoxifen-treated cells/Luc activity in vehicle-treated cells) × 100. Data are presented as mean ± SD. ***, P < 0.0001 by t test. C. HEK293 cells with an inducible Twist1 expression system were treated with 1 μg/ml of doxycycline for 12 hours to induce stable Twist1 expression and then co-treated with 1 μg/ml of doxycycline and vehicle or 10 μM of tamoxifen for the time periods as indicated. HEK293 cells without inducible Twist1 expression served as a negative control (Lane 1). Western blot analyses were performed with Twist1 and β-actin antibodies. D. HEK293 cells with inducible Twist1 expression were treated with 1 μg/ml of doxycycline for 12 hours, and then co-treated with 1 μg/ml of doxycycline and different concentrations of tamoxifen as indicated for 24 hours before Western blotting was performed. E. Western blot analyses of Twist1 in 168FARN and 4T1 cells treated with vehicle or tamoxifen (10 μM) for different time periods as indicated. F. Western blot analyses of Twist1 in 168FARN and MDA-MB-435 cells treated with the indicated concentrations of tamoxifen for 24 hours. Results shown in panels B to F are representative results of at least three repeat assays.
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Related In: Results  -  Collection

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Figure 1: Tamoxifen treatment decreases the cellular levels of Twist1 protein. A. In pQCXIH-Luc and pQCXIH-Twist1-Luc plasmids, the expression of luciferase or Twist1-Luciferase was driven by the CMV promoter. B. HeLa cells transfected with pQCXIH-Luc or pQCXIH-Twist1-Luc plasmids and with a β-galactosidase expression plasmid were treated with vehicle (ethanol) or tamoxifen (10 μM) for 24 hours. Then, the luciferase activity was measured and normalized to the β-galactosidase activity for each sample. Relative luciferase activity was calculated by (Luc activity in tamoxifen-treated cells/Luc activity in vehicle-treated cells) × 100. Data are presented as mean ± SD. ***, P < 0.0001 by t test. C. HEK293 cells with an inducible Twist1 expression system were treated with 1 μg/ml of doxycycline for 12 hours to induce stable Twist1 expression and then co-treated with 1 μg/ml of doxycycline and vehicle or 10 μM of tamoxifen for the time periods as indicated. HEK293 cells without inducible Twist1 expression served as a negative control (Lane 1). Western blot analyses were performed with Twist1 and β-actin antibodies. D. HEK293 cells with inducible Twist1 expression were treated with 1 μg/ml of doxycycline for 12 hours, and then co-treated with 1 μg/ml of doxycycline and different concentrations of tamoxifen as indicated for 24 hours before Western blotting was performed. E. Western blot analyses of Twist1 in 168FARN and 4T1 cells treated with vehicle or tamoxifen (10 μM) for different time periods as indicated. F. Western blot analyses of Twist1 in 168FARN and MDA-MB-435 cells treated with the indicated concentrations of tamoxifen for 24 hours. Results shown in panels B to F are representative results of at least three repeat assays.
Mentions: To establish a high throughput screening system for identifying bioactive small molecules that reduce Twist1 protein in cancer cells, we constructed a pQCXIH-Twist1-Luc vector to express the Twist1-luciferase fusion protein and a pQCXIH-Luc vector to express only the luciferase protein as a control (Fig. 1A). The relative cellular amounts of both Twist1-Luc and luciferase proteins can be conveniently measured by their luciferase activities. We equally transfected HeLa cells in 96-well plates with either pQCXIH-Twist1-Luc or pQCXIH-Luc plasmid, followed by treatment with one of the 1280 pharmacologically active compounds in the LOPAC library. Measurements of luciferase activities in all transfected and treated cells led to the identification of several small compounds that selectively reduced the luciferase activity in cells transfected with pQCXIH-Twist1-Luc plasmid, but had no significant effects on the luciferase activity in cells transfected with the pQCXIH-Luc plasmid. These compounds include tamoxifen citrate (tamoxifen), sertraline, indatraline, CGS-12066A, methiothepin, GR 127935, parthenolide and MK-886. tamoxifen is a well-known SERM for treating ER-positive breast cancer. Sertraline, indatraline and CGS-12066A are a selective serotonin reuptake inhibitor (SSRI), a serotonin receptor agonist and a serotonin antagonist, respectively. Methiothepin and GR-127935 are 5-HT1B/1D receptor antagonists. Parthenolide is an inhibitor of serotonin release from platelets. MK-886 is a potent and specific inhibitor of leukotriene biosynthesis. In this study, tamoxifen was selected for further characterization because of its known therapeutic role in ER-positive breast cancers and the crucial role of Twist1 in driving epithelial-mesenchymal transition and metastasis in breast cancer cells 2.

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