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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 treatment inhibits lung metastasis derived from the 4T1 cell xenograft tumors in mice. A. There was no significant difference in the growth of 4T1 cell xenograft tumors in the mammary fat pads of vehicle (corn oil) or tamoxifen-treated SCID mice (n = 5 for each). B. Representative images taken from the lungs of vehicle or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. *, indicate the metastasis foci on the lung surface. C. The average numbers of the metastasis foci observed in the vehicle-treated or tamoxifen-treated SCID mice (n = 5 each) carrying 4T1 xenograft mammary tumors. *, P < 0.05 by two-tailed Student's t test. D. H&E-stained lung sections prepared from vehicle- or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. M, metastasis developed in the lung tissue. E. The average value of the relative lung metastasis areas is larger in the vehicle-treated versus tamoxifen-treated SCID mice (n = 5 each) with 4T1 xenograft mammary tumors. **, P < 0.01 by two tailed Student's t test.
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Figure 5: Tamoxifen treatment inhibits lung metastasis derived from the 4T1 cell xenograft tumors in mice. A. There was no significant difference in the growth of 4T1 cell xenograft tumors in the mammary fat pads of vehicle (corn oil) or tamoxifen-treated SCID mice (n = 5 for each). B. Representative images taken from the lungs of vehicle or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. *, indicate the metastasis foci on the lung surface. C. The average numbers of the metastasis foci observed in the vehicle-treated or tamoxifen-treated SCID mice (n = 5 each) carrying 4T1 xenograft mammary tumors. *, P < 0.05 by two-tailed Student's t test. D. H&E-stained lung sections prepared from vehicle- or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. M, metastasis developed in the lung tissue. E. The average value of the relative lung metastasis areas is larger in the vehicle-treated versus tamoxifen-treated SCID mice (n = 5 each) with 4T1 xenograft mammary tumors. **, P < 0.01 by two tailed Student's t test.

Mentions: It has been previously reported that knockdown of Twist1 by expressing shRNAs in 4T1 mammary tumor cells significantly inhibits its metastasis to lung in mice 7, 8. To test whether a tamoxifen-induced decrease in Twist1 protein could reduce lung metastasis in ER-negative 4T1 mammary tumor cells, we injected 4T1 cells into the mammary fat pads of SCID mice, followed by tamoxifen or vehicle (core oil) treatment and examination of primary tumor weights and lung metastases. We found that tamoxifen treatment had no significant influence on primary tumor weight (Fig. 5A), which is consistent with previous studies showing knockdown Twist1 had no obvious effect on primary tumor growth in mice 8. However, tamoxifen treatment significantly reduced the number of metastatic foci observed on the lung surface (Fig. 5B and C) and the lung metastasis index calculated based on the metastasis area versus the lung area (Fig. 5D and E). These results indicate that tamoxifen treatment can significantly inhibit the lung metastasis derived from the xenograft tumors of the ER-negative 4T1 mammary tumor cells in the mammary gland.


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 inhibits lung metastasis derived from the 4T1 cell xenograft tumors in mice. A. There was no significant difference in the growth of 4T1 cell xenograft tumors in the mammary fat pads of vehicle (corn oil) or tamoxifen-treated SCID mice (n = 5 for each). B. Representative images taken from the lungs of vehicle or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. *, indicate the metastasis foci on the lung surface. C. The average numbers of the metastasis foci observed in the vehicle-treated or tamoxifen-treated SCID mice (n = 5 each) carrying 4T1 xenograft mammary tumors. *, P < 0.05 by two-tailed Student's t test. D. H&E-stained lung sections prepared from vehicle- or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. M, metastasis developed in the lung tissue. E. The average value of the relative lung metastasis areas is larger in the vehicle-treated versus tamoxifen-treated SCID mice (n = 5 each) with 4T1 xenograft mammary tumors. **, P < 0.01 by two tailed Student's t test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400392&req=5

Figure 5: Tamoxifen treatment inhibits lung metastasis derived from the 4T1 cell xenograft tumors in mice. A. There was no significant difference in the growth of 4T1 cell xenograft tumors in the mammary fat pads of vehicle (corn oil) or tamoxifen-treated SCID mice (n = 5 for each). B. Representative images taken from the lungs of vehicle or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. *, indicate the metastasis foci on the lung surface. C. The average numbers of the metastasis foci observed in the vehicle-treated or tamoxifen-treated SCID mice (n = 5 each) carrying 4T1 xenograft mammary tumors. *, P < 0.05 by two-tailed Student's t test. D. H&E-stained lung sections prepared from vehicle- or tamoxifen-treated SCID mice carrying 4T1 xenograft mammary tumors. M, metastasis developed in the lung tissue. E. The average value of the relative lung metastasis areas is larger in the vehicle-treated versus tamoxifen-treated SCID mice (n = 5 each) with 4T1 xenograft mammary tumors. **, P < 0.01 by two tailed Student's t test.
Mentions: It has been previously reported that knockdown of Twist1 by expressing shRNAs in 4T1 mammary tumor cells significantly inhibits its metastasis to lung in mice 7, 8. To test whether a tamoxifen-induced decrease in Twist1 protein could reduce lung metastasis in ER-negative 4T1 mammary tumor cells, we injected 4T1 cells into the mammary fat pads of SCID mice, followed by tamoxifen or vehicle (core oil) treatment and examination of primary tumor weights and lung metastases. We found that tamoxifen treatment had no significant influence on primary tumor weight (Fig. 5A), which is consistent with previous studies showing knockdown Twist1 had no obvious effect on primary tumor growth in mice 8. However, tamoxifen treatment significantly reduced the number of metastatic foci observed on the lung surface (Fig. 5B and C) and the lung metastasis index calculated based on the metastasis area versus the lung area (Fig. 5D and E). These results indicate that tamoxifen treatment can significantly inhibit the lung metastasis derived from the xenograft tumors of the ER-negative 4T1 mammary tumor cells in the mammary gland.

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