ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells - PubMed (original) (raw)
ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells
Hao Deng et al. PLoS One. 2014.
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Estrogen expands the population of ER-positive breast cancer stem/progenitor cells.
ER-positive breast cancer MCF7 and T47D cells were used. The tumorsphere formation assay and flow cytometry analysis of the CD44− and CD24+ cells were used to assess the population of ER-positive breast cancer stem/progenitor cells. (A). Estrogen treatment increases the population of the CD44−/CD24+ cells in MCF7 and T47D cells. The monolayer (parental) and tumorspheres of MCF7 and T47D cells 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 representative results are shown on the upper panel. Lower panel: the columns represent the means of three experiments; bars, SE. *, P<0.05 for vehicle treated cells vs cells treated with E2β. (B). Estrogen treatment increases the size of tumorspheres from MCF7 and T47D cells. A representative tumorsphere from MCF7 and T47D cells treated with vehicle or 0.1 nM E2β for seven days. (C). Estrogen treatment increases the number of tumorspheres and cells from dissociated tumorspheres derived from MCF7 and T47D cells. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated with E2β.
Figure 2. 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.
Figure 3. ER-α36-mediated estrogen signaling stimulates the self-renewal of ER-positive breast cancer stem cells.
Long-term expansion of MCF7 (A.C) and T47D (B, D) variant cells in the presence of vehicle (ethanol) or 0.1 nM of E2β. The cells from tumorspheres were passed once a week for four generations. The numbers of tumorspheres and cells from dissociated tumorspheres were determined. The numbers of tumorspheres and cells from tumorspheres from the control cells transfected with the empty expression vector and treated with vehicle were arbitrarily set as 1. Three dishes were used for each group and the experiments were repeated three times. The columns represent the means of three experiments; bars, SE.
Figure 4. ER-α36-mediated estrogen signaling enhances the tumor-seeding efficiency of ER-positive breast cancer stem/progenitor cells.
Different variants of MCF7 and T47D cells at limited dilutions were implanted in the mammary fatpad of the ovariectomized female mice supplemented with estrogen or placebo pellets. The tumor-seeding efficiency was examined by measurement of tumor weight. The data represent the mean ± SE observed in six mice in each group.
Figure 5. 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.
Figure 6. The PI3K/AKT/GSK3β/β-catenin signaling pathway is involved in ER-α36-mediated mitogenic estrogen signaling of ER-positive breast cancer stem/progenitor cells.
(A). Western blot analysis of the cell lysates from tumorspheres derived from MCF7 and T47D cells treated with ethanol (V); 0.1 nM of E2β; the AKT inhibitor IV (10 µM), IAkt; and E2β+IAkt, using indicated antibodies. (B). The effects of different inhibitors of the PI3K/AKT/GSK3β pathway on estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. Tumorspheres of MCF7 and T47D cells were treated with vehicle, E2β alone or E2β together with the PI3K inhibitor LY294002 (10 µM), the GSK-3β inhibitor IX (10 µM), the AKT inhibitor IV (10 µM). After seven days, cell numbers from dissociated tumorspheres were determined. The columns represent the means of three experiments; bars, SE.
Figure 7. 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.
References
- Oliveira LR, Jeffrey SS, Ribeiro-Silva A (2010) Stem cells in human breast cancer. Histol Histopathol 25: 371–385. - PubMed
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