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ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.

Deng H, Zhang XT, Wang ML, Zheng HY, Liu LJ, Wang ZY - PLoS ONE (2014)

Bottom Line: We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression.Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo.We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, Nebraska, United States of America ; Jiangda Pathology Center, Jianghan University, Wuhan, Hubei, P. R. China.

ABSTRACT
The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3β pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

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ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.(A). Western blot analyses of ER-α36 expression in different MC7 and T47D cell variants; control cells (MCF7/V and T47D/V: cells transfected with the empty expression vector); cells with forced expression of ER-α36 (MCF7/36 and T47D/36: cells transfected with a ER-α36 expression vector); and ER-α36 expression knocked-down cells (MCF7/Si36 and T47D/Si36. (B). ER-α36-mediated estrogen signaling increases the population of the CD44−/CD24+ cells. The monolayer (parental, P) and tumorspheres (T) of MCF7 and T47D variants were treated with vehicle (ethanol) or 0.1 nM of E2β for five days. The population of CD44−/CD24+ cells in these cells were analyzed after staining with fluorochrome-conjugated antibodies. The columns represent the means of three experiments; bars, SE. *, P<0.05 for vehicle treated cells vs cells treated with E2β. (C). ER-α36-mediated estrogen signaling positively regulates the size and number of tumorspheres from MCF7 and T47D cells. Representative tumorspheres from MCF7 and T47D cell variants treated with vehicle or 0.1 nM E2β for seven days. Scale bar = 100 µm. (D). The numbers of tumorspheres and cells from dissociated tumorspheres of different cell variants were determined. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated with E2β.
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pone-0088034-g002: ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.(A). Western blot analyses of ER-α36 expression in different MC7 and T47D cell variants; control cells (MCF7/V and T47D/V: cells transfected with the empty expression vector); cells with forced expression of ER-α36 (MCF7/36 and T47D/36: cells transfected with a ER-α36 expression vector); and ER-α36 expression knocked-down cells (MCF7/Si36 and T47D/Si36. (B). ER-α36-mediated estrogen signaling increases the population of the CD44−/CD24+ cells. The monolayer (parental, P) and tumorspheres (T) of MCF7 and T47D variants were treated with vehicle (ethanol) or 0.1 nM of E2β for five days. The population of CD44−/CD24+ cells in these cells were analyzed after staining with fluorochrome-conjugated antibodies. The columns represent the means of three experiments; bars, SE. *, P<0.05 for vehicle treated cells vs cells treated with E2β. (C). ER-α36-mediated estrogen signaling positively regulates the size and number of tumorspheres from MCF7 and T47D cells. Representative tumorspheres from MCF7 and T47D cell variants treated with vehicle or 0.1 nM E2β for seven days. Scale bar = 100 µm. (D). The numbers of tumorspheres and cells from dissociated tumorspheres of different cell variants were determined. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated with E2β.

Mentions: We then examined ER-α36 function in the stem/progenitor cells derived from ER-positive breast cancer cells. MCF7 and T47D cells transfected with the empty expression vector (MCF7/V and T47D/V), MCF7 and T47D cells with knocked-down levels of ER-α36 expression (MCF7/Si36 and T47D/Si36), and MCF7/36 and T47D/36 cells with high levels of recombinant ER-α36 expression were used (Figure 2A). The CD44+/CD24− cell populations in parental MCF7 and T47D cells as well as different variants treated with or without E2β for five days were assessed. We found that in the MCF7 and T47D cells that express high levels of ER-α36, MCF7/36 and T47D/36, the populations of CD44+/CD24− cells were significantly increased compared to the control cells transfected with the empty expression vector, suggesting that ER-α36 is involved in positive regulation of ER-positive breast cancer stem/progenitor cells (Figure 2B). Estrogen treatment further increased the populations of CD44+/CD24− stem-like cells in MCF7/36 and T47D/36 cells (Figure 2B). We also examined the CD44+/CD24− cell populations in the tumorspheres formed by these cells treated with or without E2β. We found that in the tumorspheres formed by MCF736 and T47D/36 cells, the populations of CD44+/CD24− cells were dramatically increased compared to the control MCF7/V and T47D/V cells, which was further increased by estrogen treatment (Figure 2B). However, we found that the cells with knocked-down levels of ER-α36 expression, MCF7/Si36 and T47D/Si36, exhibited decreased populations of the CD44+/CD24− cell and weakly responded to estrogen treatment (Figure 2B).


ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.

Deng H, Zhang XT, Wang ML, Zheng HY, Liu LJ, Wang ZY - PLoS ONE (2014)

ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.(A). Western blot analyses of ER-α36 expression in different MC7 and T47D cell variants; control cells (MCF7/V and T47D/V: cells transfected with the empty expression vector); cells with forced expression of ER-α36 (MCF7/36 and T47D/36: cells transfected with a ER-α36 expression vector); and ER-α36 expression knocked-down cells (MCF7/Si36 and T47D/Si36. (B). ER-α36-mediated estrogen signaling increases the population of the CD44−/CD24+ cells. The monolayer (parental, P) and tumorspheres (T) of MCF7 and T47D variants were treated with vehicle (ethanol) or 0.1 nM of E2β for five days. The population of CD44−/CD24+ cells in these cells were analyzed after staining with fluorochrome-conjugated antibodies. The columns represent the means of three experiments; bars, SE. *, P<0.05 for vehicle treated cells vs cells treated with E2β. (C). ER-α36-mediated estrogen signaling positively regulates the size and number of tumorspheres from MCF7 and T47D cells. Representative tumorspheres from MCF7 and T47D cell variants treated with vehicle or 0.1 nM E2β for seven days. Scale bar = 100 µm. (D). The numbers of tumorspheres and cells from dissociated tumorspheres of different cell variants were determined. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated with E2β.
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Related In: Results  -  Collection

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pone-0088034-g002: ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells.(A). Western blot analyses of ER-α36 expression in different MC7 and T47D cell variants; control cells (MCF7/V and T47D/V: cells transfected with the empty expression vector); cells with forced expression of ER-α36 (MCF7/36 and T47D/36: cells transfected with a ER-α36 expression vector); and ER-α36 expression knocked-down cells (MCF7/Si36 and T47D/Si36. (B). ER-α36-mediated estrogen signaling increases the population of the CD44−/CD24+ cells. The monolayer (parental, P) and tumorspheres (T) of MCF7 and T47D variants were treated with vehicle (ethanol) or 0.1 nM of E2β for five days. The population of CD44−/CD24+ cells in these cells were analyzed after staining with fluorochrome-conjugated antibodies. The columns represent the means of three experiments; bars, SE. *, P<0.05 for vehicle treated cells vs cells treated with E2β. (C). ER-α36-mediated estrogen signaling positively regulates the size and number of tumorspheres from MCF7 and T47D cells. Representative tumorspheres from MCF7 and T47D cell variants treated with vehicle or 0.1 nM E2β for seven days. Scale bar = 100 µm. (D). The numbers of tumorspheres and cells from dissociated tumorspheres of different cell variants were determined. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated with E2β.
Mentions: We then examined ER-α36 function in the stem/progenitor cells derived from ER-positive breast cancer cells. MCF7 and T47D cells transfected with the empty expression vector (MCF7/V and T47D/V), MCF7 and T47D cells with knocked-down levels of ER-α36 expression (MCF7/Si36 and T47D/Si36), and MCF7/36 and T47D/36 cells with high levels of recombinant ER-α36 expression were used (Figure 2A). The CD44+/CD24− cell populations in parental MCF7 and T47D cells as well as different variants treated with or without E2β for five days were assessed. We found that in the MCF7 and T47D cells that express high levels of ER-α36, MCF7/36 and T47D/36, the populations of CD44+/CD24− cells were significantly increased compared to the control cells transfected with the empty expression vector, suggesting that ER-α36 is involved in positive regulation of ER-positive breast cancer stem/progenitor cells (Figure 2B). Estrogen treatment further increased the populations of CD44+/CD24− stem-like cells in MCF7/36 and T47D/36 cells (Figure 2B). We also examined the CD44+/CD24− cell populations in the tumorspheres formed by these cells treated with or without E2β. We found that in the tumorspheres formed by MCF736 and T47D/36 cells, the populations of CD44+/CD24− cells were dramatically increased compared to the control MCF7/V and T47D/V cells, which was further increased by estrogen treatment (Figure 2B). However, we found that the cells with knocked-down levels of ER-α36 expression, MCF7/Si36 and T47D/Si36, exhibited decreased populations of the CD44+/CD24− cell and weakly responded to estrogen treatment (Figure 2B).

Bottom Line: We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression.Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo.We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, Nebraska, United States of America ; Jiangda Pathology Center, Jianghan University, Wuhan, Hubei, P. R. China.

ABSTRACT
The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3β pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

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Related in: MedlinePlus