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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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The expression and genomic function of ER-α66 are down-regulated in ER-positive breast cancer stem/progenitor cells.(A). Western blot analysis of the expression of different proteins in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (B). Western blot analysis of ER-α66 expression in monolayer (parental) and tumorspheres of the MCF7 and T47D cells treated with or without the proteasome inhibitor MG132 (100 nM) for 12 hours. (C). Indirect Immunofluorescent staining for ER-α36 and ER-α66 in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (D). The monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells were transfected with the ERE luciferase report plasmid (2 µg). Twenty-four hours later, 0.1 nM of E2β was added and incubated for indicated time periods. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Two replicates were used in each experiment. Columns: means of four independent experiments; bars, SE.
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pone-0088034-g007: The expression and genomic function of ER-α66 are down-regulated in ER-positive breast cancer stem/progenitor cells.(A). Western blot analysis of the expression of different proteins in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (B). Western blot analysis of ER-α66 expression in monolayer (parental) and tumorspheres of the MCF7 and T47D cells treated with or without the proteasome inhibitor MG132 (100 nM) for 12 hours. (C). Indirect Immunofluorescent staining for ER-α36 and ER-α66 in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (D). The monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells were transfected with the ERE luciferase report plasmid (2 µg). Twenty-four hours later, 0.1 nM of E2β was added and incubated for indicated time periods. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Two replicates were used in each experiment. Columns: means of four independent experiments; bars, SE.

Mentions: To assess the expression levels of ER-α66 and ER-α36 in ER-positive breast cancer stem/progenitor cells, we performed Western blot analysis with cell lysates from tumorspheres. We found that the expression levels of ER-α36 protein were dramatically increased in tumorspheres from MCF7 and T47D cells while ER-α66 expression was down-regulated compared to parental cells (Figure 7A). In addition, we also found that the expression levels of ALDH1 and the basal levels of the AKT and GSK3β phosphorylation were markedly increased in tumorspheres (Figure 7A). The expression levels of growth receptors EGFR and HER2 were also increased in tumorspheres (Figure 7A). When the tumorspheres derived from MCF7 and T47D cells were treated with MG132, a proteasome inhibitor, the steady state level of ER-α66 protein was dramatically increased in both parental cells and tumorshphere cells (Figure 7B), suggesting that degradation of ER-α66 protein by the proteasome system is involved in regulation of the steady state levels of ER-α66, which was enhanced in ER-positive breast cancer stem/progenitor cells. We then examined the expression patterns of ER-α66 and 36 in parental and tumorsphere cells using the indirect immunofluorescence staining. We found that ER-α36 is expressed at the plasma membrane and in the cytoplasm of both parental and tumorsphere cells (Figure 7C). ER-α66, however, exhibited a predominant nuclear staining in the parental MCF7 and T47D cells while a weak cytoplasm staining was also observed in T47D cells. In tumorsphere cells, ER-α66 was mainly expressed in the cytoplasm (Figure 7C), indicating a great portion of ER-α66 protein was redistributed to the cytoplasms of ER-positive tumorsphere cells. When the parental MCF7 and T47D cells, and their tumorsphere cells were transfected with a ERE containing luciferase reporter plasmid and treated with or without estrogen, we found that estrogen-induced transcription activities of ER-α66 were dramatically reduced in tumorsphere cells compared to parental cells (Figure 7D), indicating the genomic estrogen signaling mediated by ER-α66 is attenuated in ER-positive breast cancer stem/progenitor cells.


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)

The expression and genomic function of ER-α66 are down-regulated in ER-positive breast cancer stem/progenitor cells.(A). Western blot analysis of the expression of different proteins in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (B). Western blot analysis of ER-α66 expression in monolayer (parental) and tumorspheres of the MCF7 and T47D cells treated with or without the proteasome inhibitor MG132 (100 nM) for 12 hours. (C). Indirect Immunofluorescent staining for ER-α36 and ER-α66 in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (D). The monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells were transfected with the ERE luciferase report plasmid (2 µg). Twenty-four hours later, 0.1 nM of E2β was added and incubated for indicated time periods. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Two replicates were used in each experiment. Columns: means of four independent experiments; bars, SE.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928099&req=5

pone-0088034-g007: The expression and genomic function of ER-α66 are down-regulated in ER-positive breast cancer stem/progenitor cells.(A). Western blot analysis of the expression of different proteins in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (B). Western blot analysis of ER-α66 expression in monolayer (parental) and tumorspheres of the MCF7 and T47D cells treated with or without the proteasome inhibitor MG132 (100 nM) for 12 hours. (C). Indirect Immunofluorescent staining for ER-α36 and ER-α66 in the monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells. (D). The monolayer cells (parental) and tumorspheres of the MCF7 and T47D cells were transfected with the ERE luciferase report plasmid (2 µg). Twenty-four hours later, 0.1 nM of E2β was added and incubated for indicated time periods. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Two replicates were used in each experiment. Columns: means of four independent experiments; bars, SE.
Mentions: To assess the expression levels of ER-α66 and ER-α36 in ER-positive breast cancer stem/progenitor cells, we performed Western blot analysis with cell lysates from tumorspheres. We found that the expression levels of ER-α36 protein were dramatically increased in tumorspheres from MCF7 and T47D cells while ER-α66 expression was down-regulated compared to parental cells (Figure 7A). In addition, we also found that the expression levels of ALDH1 and the basal levels of the AKT and GSK3β phosphorylation were markedly increased in tumorspheres (Figure 7A). The expression levels of growth receptors EGFR and HER2 were also increased in tumorspheres (Figure 7A). When the tumorspheres derived from MCF7 and T47D cells were treated with MG132, a proteasome inhibitor, the steady state level of ER-α66 protein was dramatically increased in both parental cells and tumorshphere cells (Figure 7B), suggesting that degradation of ER-α66 protein by the proteasome system is involved in regulation of the steady state levels of ER-α66, which was enhanced in ER-positive breast cancer stem/progenitor cells. We then examined the expression patterns of ER-α66 and 36 in parental and tumorsphere cells using the indirect immunofluorescence staining. We found that ER-α36 is expressed at the plasma membrane and in the cytoplasm of both parental and tumorsphere cells (Figure 7C). ER-α66, however, exhibited a predominant nuclear staining in the parental MCF7 and T47D cells while a weak cytoplasm staining was also observed in T47D cells. In tumorsphere cells, ER-α66 was mainly expressed in the cytoplasm (Figure 7C), indicating a great portion of ER-α66 protein was redistributed to the cytoplasms of ER-positive tumorsphere cells. When the parental MCF7 and T47D cells, and their tumorsphere cells were transfected with a ERE containing luciferase reporter plasmid and treated with or without estrogen, we found that estrogen-induced transcription activities of ER-α66 were dramatically reduced in tumorsphere cells compared to parental cells (Figure 7D), indicating the genomic estrogen signaling mediated by ER-α66 is attenuated in ER-positive breast cancer stem/progenitor cells.

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.

Show MeSH
Related in: MedlinePlus