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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 estrogen signaling induced proliferation of luminal epithelial lineage specific ER-positive breast cancer progenitor cells.(A. B). Indirect Immunofluorescent staining for CK18 (red) or CD10 (red) in variants derived from MCF7 and T47D cells treated with vehicle or E2β. DAPI (blue) was used to stain the nuclear region. (C). Indirect Immunofluorescent staining for CK18 (red) or PCNA (green) in MCF7 cells treated with vehicle or E2β. DAPI (blue) was used to indicate the cell nuclei.
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pone-0088034-g005: ER-α36-mediated estrogen signaling induced proliferation of luminal epithelial lineage specific ER-positive breast cancer progenitor cells.(A. B). Indirect Immunofluorescent staining for CK18 (red) or CD10 (red) in variants derived from MCF7 and T47D cells treated with vehicle or E2β. DAPI (blue) was used to stain the nuclear region. (C). Indirect Immunofluorescent staining for CK18 (red) or PCNA (green) in MCF7 cells treated with vehicle or E2β. DAPI (blue) was used to indicate the cell nuclei.

Mentions: Breast cancer stem cells are able to differentiate into both luminal epithelial and myoepithelial cells [3]. We investigated the differentiation lineages of the stem cells derived from different MCF7 and T47D derivatives in the presence and absence of estrogen. Single cell suspensions from tumorspheres plated on collagen-coated coverslips or intact tumorspheres in suspension culture were treated with or without E2βfor five days, and the indirect immunofluoresces assay was performed to determine the effects of estrogen on differentiation lineages of these cells using cytokeratin 18 (CK18) for luminal epithelial cells and CD10 for myoepithelial cells. We found that tumorsphere cells plated on collagen-coated coverslips were fully differentiated into either luminal epithelial or myoepithelial lineages, and estrogen treatment had less or no effect on the differentiation (Figure S1), suggesting that estrogen treatment was unable to influence differentiation induced by cell attachment. We then assessed the effects of E2β on the spontaneous differentiation occurred in tumorspheres under suspension culture. In tumorspheres formed by MCF7 cells, we found that estrogen treatment increased the population of the cells that were stained positive for CK18 but without effect on the cells positive for CD10 (Figure 5A). We also found that estrogen treatment increased more number of cells expressing CK18 in MCF7/36 cells compared with MCF7/V cells (Figure 5A) while estrogen only slightly increased CK18 positive cells in MCF7/Si36 cells. Similar results were also observed in T47D cell variants; T47D/Si36 cells failed to respond to estrogen (Figure 5B).


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 estrogen signaling induced proliferation of luminal epithelial lineage specific ER-positive breast cancer progenitor cells.(A. B). Indirect Immunofluorescent staining for CK18 (red) or CD10 (red) in variants derived from MCF7 and T47D cells treated with vehicle or E2β. DAPI (blue) was used to stain the nuclear region. (C). Indirect Immunofluorescent staining for CK18 (red) or PCNA (green) in MCF7 cells treated with vehicle or E2β. DAPI (blue) was used to indicate the cell nuclei.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928099&req=5

pone-0088034-g005: ER-α36-mediated estrogen signaling induced proliferation of luminal epithelial lineage specific ER-positive breast cancer progenitor cells.(A. B). Indirect Immunofluorescent staining for CK18 (red) or CD10 (red) in variants derived from MCF7 and T47D cells treated with vehicle or E2β. DAPI (blue) was used to stain the nuclear region. (C). Indirect Immunofluorescent staining for CK18 (red) or PCNA (green) in MCF7 cells treated with vehicle or E2β. DAPI (blue) was used to indicate the cell nuclei.
Mentions: Breast cancer stem cells are able to differentiate into both luminal epithelial and myoepithelial cells [3]. We investigated the differentiation lineages of the stem cells derived from different MCF7 and T47D derivatives in the presence and absence of estrogen. Single cell suspensions from tumorspheres plated on collagen-coated coverslips or intact tumorspheres in suspension culture were treated with or without E2βfor five days, and the indirect immunofluoresces assay was performed to determine the effects of estrogen on differentiation lineages of these cells using cytokeratin 18 (CK18) for luminal epithelial cells and CD10 for myoepithelial cells. We found that tumorsphere cells plated on collagen-coated coverslips were fully differentiated into either luminal epithelial or myoepithelial lineages, and estrogen treatment had less or no effect on the differentiation (Figure S1), suggesting that estrogen treatment was unable to influence differentiation induced by cell attachment. We then assessed the effects of E2β on the spontaneous differentiation occurred in tumorspheres under suspension culture. In tumorspheres formed by MCF7 cells, we found that estrogen treatment increased the population of the cells that were stained positive for CK18 but without effect on the cells positive for CD10 (Figure 5A). We also found that estrogen treatment increased more number of cells expressing CK18 in MCF7/36 cells compared with MCF7/V cells (Figure 5A) while estrogen only slightly increased CK18 positive cells in MCF7/Si36 cells. Similar results were also observed in T47D cell variants; T47D/Si36 cells failed to respond to estrogen (Figure 5B).

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