Limits...
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.

Show MeSH

Related in: MedlinePlus

Tamoxifen treatment decreases cell invasiveness of 4T1 mammary tumor cells. 4T1 cells pre-treated with vehicle (ethanol) or 10 μM tamoxifen for 24 hours were seeded in the upper chambers of the transwell invasion assay system (2.5 × 104 cells/chamber). The upper chamber contains serum-free medium with vehicle (ethanol) or 10 μM tamoxifen, while the lower chamber contains medium with 1% serum and vehicle (ethanol) or 10 μM tamoxifen. After culturing for 24 hours, invaded cells on the lower side of the membrane were stained with DAPI, and imaged (Panel A) and counted (Panel B) under a fluorescence microscope. **, P < 0.01 by two-tailed Student's t test, n = 5 for each.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4400392&req=5

Figure 4: Tamoxifen treatment decreases cell invasiveness of 4T1 mammary tumor cells. 4T1 cells pre-treated with vehicle (ethanol) or 10 μM tamoxifen for 24 hours were seeded in the upper chambers of the transwell invasion assay system (2.5 × 104 cells/chamber). The upper chamber contains serum-free medium with vehicle (ethanol) or 10 μM tamoxifen, while the lower chamber contains medium with 1% serum and vehicle (ethanol) or 10 μM tamoxifen. After culturing for 24 hours, invaded cells on the lower side of the membrane were stained with DAPI, and imaged (Panel A) and counted (Panel B) under a fluorescence microscope. **, P < 0.01 by two-tailed Student's t test, n = 5 for each.

Mentions: Twist1 is known to increase breast cancer cell invasiveness 2, 7, 8. Therefore, we treated 4T1 mammary tumor cells with endogenous Twist1 expression with tamoxifen and measured the impact of tamoxifen treatment on cell invasion. We found that tamoxifen treatment significantly reduced the number of 4T1 cells that could invade through a layer of Matrigel in a transwell cell invasion assay system (Fig. 4A and B). These results suggest that tamoxifen-induced Twist1 degradation can partially inhibit EMT and invasiveness of breast cancer cells.


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 cell invasiveness of 4T1 mammary tumor cells. 4T1 cells pre-treated with vehicle (ethanol) or 10 μM tamoxifen for 24 hours were seeded in the upper chambers of the transwell invasion assay system (2.5 × 104 cells/chamber). The upper chamber contains serum-free medium with vehicle (ethanol) or 10 μM tamoxifen, while the lower chamber contains medium with 1% serum and vehicle (ethanol) or 10 μM tamoxifen. After culturing for 24 hours, invaded cells on the lower side of the membrane were stained with DAPI, and imaged (Panel A) and counted (Panel B) under a fluorescence microscope. **, P < 0.01 by two-tailed Student's t test, n = 5 for each.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4400392&req=5

Figure 4: Tamoxifen treatment decreases cell invasiveness of 4T1 mammary tumor cells. 4T1 cells pre-treated with vehicle (ethanol) or 10 μM tamoxifen for 24 hours were seeded in the upper chambers of the transwell invasion assay system (2.5 × 104 cells/chamber). The upper chamber contains serum-free medium with vehicle (ethanol) or 10 μM tamoxifen, while the lower chamber contains medium with 1% serum and vehicle (ethanol) or 10 μM tamoxifen. After culturing for 24 hours, invaded cells on the lower side of the membrane were stained with DAPI, and imaged (Panel A) and counted (Panel B) under a fluorescence microscope. **, P < 0.01 by two-tailed Student's t test, n = 5 for each.
Mentions: Twist1 is known to increase breast cancer cell invasiveness 2, 7, 8. Therefore, we treated 4T1 mammary tumor cells with endogenous Twist1 expression with tamoxifen and measured the impact of tamoxifen treatment on cell invasion. We found that tamoxifen treatment significantly reduced the number of 4T1 cells that could invade through a layer of Matrigel in a transwell cell invasion assay system (Fig. 4A and B). These results suggest that tamoxifen-induced Twist1 degradation can partially inhibit EMT and invasiveness of breast cancer cells.

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