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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-accelerated Twist1 degradation is dependent on the proteasome pathway, but is independent of estrogen and MAPK signaling pathways. A. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression (HEK293+Twist1) after cells were treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. HEK293 parent cells without Twist1 expression served as a negative control. B. Western blot analysis of Twist1 in 168FARN cells treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen. C. Western blot analysis of Twist1 in HEK293+Twist1, 4T1 and 168FARN cells treated for 24 hours with vehicle (V, ethanol), or 10 μM of 17β-estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (4OH) and tamoxifen (Tam) as indicated. D. Western blot analysis of active phospho-Erk1/2, total Erk1/2, active phospho-p38, total p38, active phospho-JNK, and total JNK in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. E. Western blot analysis of phospho-Ser68-Twist1 and total Twist1 in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. Results shown in all panels are representative results of at least three repeat assays.
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Figure 3: Tamoxifen-accelerated Twist1 degradation is dependent on the proteasome pathway, but is independent of estrogen and MAPK signaling pathways. A. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression (HEK293+Twist1) after cells were treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. HEK293 parent cells without Twist1 expression served as a negative control. B. Western blot analysis of Twist1 in 168FARN cells treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen. C. Western blot analysis of Twist1 in HEK293+Twist1, 4T1 and 168FARN cells treated for 24 hours with vehicle (V, ethanol), or 10 μM of 17β-estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (4OH) and tamoxifen (Tam) as indicated. D. Western blot analysis of active phospho-Erk1/2, total Erk1/2, active phospho-p38, total p38, active phospho-JNK, and total JNK in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. E. Western blot analysis of phospho-Ser68-Twist1 and total Twist1 in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. Results shown in all panels are representative results of at least three repeat assays.

Mentions: To elucidate the mechanism of tamoxifen-induced Twist1 degradation, we examined whether inhibition of proteasome-mediated protein degradation pathway could diminish the effect of tamoxifen on Twist1 protein stability. We treated HEK293 cells with inducible Twist1 expression and 168FARN cells with endogenous Twist1 expression with the proteasome inhibitor MG132 or with MG132 and tamoxifen, followed by analysis of Twist1 protein levels at different time points. We found that Twist1 protein was accumulated in a time-dependent manner in cells treated with MG132 alone, suggesting that Twist1 was normally degraded through the proteasome-mediated pathway. Interestingly, Twist1 protein was maintained at the same level through all the time points in cells treated with both MG132 and tamoxifen, suggesting that tamoxifen-accelerated Twist1 degradation is mostly mediated by the proteasome pathway (Fig. 3A and B). These results also suggest that tamoxifen can prevent Twist1 accumulation in proteasome-inhibited cells through other unknown pathways.


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-accelerated Twist1 degradation is dependent on the proteasome pathway, but is independent of estrogen and MAPK signaling pathways. A. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression (HEK293+Twist1) after cells were treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. HEK293 parent cells without Twist1 expression served as a negative control. B. Western blot analysis of Twist1 in 168FARN cells treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen. C. Western blot analysis of Twist1 in HEK293+Twist1, 4T1 and 168FARN cells treated for 24 hours with vehicle (V, ethanol), or 10 μM of 17β-estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (4OH) and tamoxifen (Tam) as indicated. D. Western blot analysis of active phospho-Erk1/2, total Erk1/2, active phospho-p38, total p38, active phospho-JNK, and total JNK in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. E. Western blot analysis of phospho-Ser68-Twist1 and total Twist1 in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. Results shown in all panels are representative results of at least three repeat assays.
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Figure 3: Tamoxifen-accelerated Twist1 degradation is dependent on the proteasome pathway, but is independent of estrogen and MAPK signaling pathways. A. Western blot analysis of Twist1 in HEK293 cells with inducible Twist1 expression (HEK293+Twist1) after cells were treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen for different time periods as indicated. HEK293 parent cells without Twist1 expression served as a negative control. B. Western blot analysis of Twist1 in 168FARN cells treated with 20 μM of MG132 and vehicle (ethanol) or 10 μM of tamoxifen. C. Western blot analysis of Twist1 in HEK293+Twist1, 4T1 and 168FARN cells treated for 24 hours with vehicle (V, ethanol), or 10 μM of 17β-estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (4OH) and tamoxifen (Tam) as indicated. D. Western blot analysis of active phospho-Erk1/2, total Erk1/2, active phospho-p38, total p38, active phospho-JNK, and total JNK in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. E. Western blot analysis of phospho-Ser68-Twist1 and total Twist1 in 168FARN cells treated with different concentrations of tamoxifen for 6 hours. Results shown in all panels are representative results of at least three repeat assays.
Mentions: To elucidate the mechanism of tamoxifen-induced Twist1 degradation, we examined whether inhibition of proteasome-mediated protein degradation pathway could diminish the effect of tamoxifen on Twist1 protein stability. We treated HEK293 cells with inducible Twist1 expression and 168FARN cells with endogenous Twist1 expression with the proteasome inhibitor MG132 or with MG132 and tamoxifen, followed by analysis of Twist1 protein levels at different time points. We found that Twist1 protein was accumulated in a time-dependent manner in cells treated with MG132 alone, suggesting that Twist1 was normally degraded through the proteasome-mediated pathway. Interestingly, Twist1 protein was maintained at the same level through all the time points in cells treated with both MG132 and tamoxifen, suggesting that tamoxifen-accelerated Twist1 degradation is mostly mediated by the proteasome pathway (Fig. 3A and B). These results also suggest that tamoxifen can prevent Twist1 accumulation in proteasome-inhibited cells through other unknown pathways.

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