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A positive feedback loop of ER-α36/EGFR promotes malignant growth of ER-negative breast cancer cells.

Zhang XT, Kang LG, Ding L, Vranic S, Gatalica Z, Wang ZY - Oncogene (2010)

Bottom Line: Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36 mediated nongenomic estrogen signaling and is highly expressed in ER-negative breast cancer cells.In this study, we found that ER-α36 was highly expressed in 10/12 cases of triple-negative breast cancer.We investigated the role of mitogenic estrogen signaling mediated by ER-α36 in malignant growth of triple-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of ER-α36 and found that these cells strongly responded to mitogenic estrogen signaling both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology & Immunology, Creighton University Medical School, Omaha, NE, USA.

ABSTRACT
It is prevailingly thought that estrogen signaling is not involved in development of estrogen receptor (ER)-negative breast cancer. However, there is evidence indicating that ovariectomy prevents the development of both ER-positive and -negative breast cancer, suggesting that estrogen signaling is involved in the development of ER-negative breast cancer. Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36 mediated nongenomic estrogen signaling and is highly expressed in ER-negative breast cancer cells. In this study, we found that ER-α36 was highly expressed in 10/12 cases of triple-negative breast cancer. We investigated the role of mitogenic estrogen signaling mediated by ER-α36 in malignant growth of triple-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of ER-α36 and found that these cells strongly responded to mitogenic estrogen signaling both in vitro and in vivo. Knockdown of ER-α36 expression in these cells using the small hairpin RNA method diminished their responsiveness to estrogen. ER-α36 physically interacted with the EGFR/Src/Shc complex and mediated estrogen-induced phosphorylation of epidermal growth factor receptor (EGFR) and Src. EGFR signaling activated ER-α36 transcription through an AP1 site in the ER-α36 promoter, and ER-α36 expression was able to stabilize EGFR protein. Our results, thus demonstrated that ER-α36 mediates nongenomic estrogen signaling through the EGFR/Src/ERK signaling pathway in ER-negative breast cancer cells and suggested that a subset of ER-negative breast tumors that expresses ER-α36, retains responsiveness to mitogenic estrogen signaling.

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Non-Genomic Estrogen Signaling Stimulates Proliferation of ER-negative Breast Cancer Cells(a). The expression of ER-α variants and EGFR in MCF7, MDA-MB-231 and MDA-MB-436 breast cancer cells. (b). The dose and time dependent pattern of E2β-stimulated phosphorylation of the MAPK/ERK1/2 in MDA-MB-231 and MDA-MB-436 cells. Starved cells were treated with indicated doses of E2β or 0.1 nM of E2β for indicated time periods. Western blot analysis was performed to assess induction of ERK1/2 phosphorylation. The columns represent the means of three experiments; bars, SE. *, P<0.05 for control cells vs cells treated under different conditions. The representative results are shown. (c). The dose dependent induction c-myc and cyclin D1 by E2β in MDA-MB-231 and MDA-MB-436 cells. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated different concentrations of E2β. The representative results are shown. (d). The effects of E2β on the proliferation rate of MDA-MB-231 and MDA-MB-436 cells. Cells maintained for three days in phenol red-free DMEM plus 2.5% dextran-charcoal-stripped fetal calf serum were treated with indicated concentrations of E2β or ethanol vehicle as a control. The cell numbers were determined using an automatic cell counter after 12 days. Five dishes were used for each concentration and experiments were repeated more than four times. The mean cell numbers ± SE are shown.
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Figure 1: Non-Genomic Estrogen Signaling Stimulates Proliferation of ER-negative Breast Cancer Cells(a). The expression of ER-α variants and EGFR in MCF7, MDA-MB-231 and MDA-MB-436 breast cancer cells. (b). The dose and time dependent pattern of E2β-stimulated phosphorylation of the MAPK/ERK1/2 in MDA-MB-231 and MDA-MB-436 cells. Starved cells were treated with indicated doses of E2β or 0.1 nM of E2β for indicated time periods. Western blot analysis was performed to assess induction of ERK1/2 phosphorylation. The columns represent the means of three experiments; bars, SE. *, P<0.05 for control cells vs cells treated under different conditions. The representative results are shown. (c). The dose dependent induction c-myc and cyclin D1 by E2β in MDA-MB-231 and MDA-MB-436 cells. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated different concentrations of E2β. The representative results are shown. (d). The effects of E2β on the proliferation rate of MDA-MB-231 and MDA-MB-436 cells. Cells maintained for three days in phenol red-free DMEM plus 2.5% dextran-charcoal-stripped fetal calf serum were treated with indicated concentrations of E2β or ethanol vehicle as a control. The cell numbers were determined using an automatic cell counter after 12 days. Five dishes were used for each concentration and experiments were repeated more than four times. The mean cell numbers ± SE are shown.

Mentions: To determine if established triple-negative breast cancer cells that express ER-α36 retain non-genomic estrogen signaling, we used breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which are triple-negative. Western blot analysis showed that both ER-α36 and EGFR are highly expressed in these breast cancer cells while ER-positive MCF7 cells expressed high levels of ER-α66 but lower levels of ER-α36 and EGFR (Figure 1A).


A positive feedback loop of ER-α36/EGFR promotes malignant growth of ER-negative breast cancer cells.

Zhang XT, Kang LG, Ding L, Vranic S, Gatalica Z, Wang ZY - Oncogene (2010)

Non-Genomic Estrogen Signaling Stimulates Proliferation of ER-negative Breast Cancer Cells(a). The expression of ER-α variants and EGFR in MCF7, MDA-MB-231 and MDA-MB-436 breast cancer cells. (b). The dose and time dependent pattern of E2β-stimulated phosphorylation of the MAPK/ERK1/2 in MDA-MB-231 and MDA-MB-436 cells. Starved cells were treated with indicated doses of E2β or 0.1 nM of E2β for indicated time periods. Western blot analysis was performed to assess induction of ERK1/2 phosphorylation. The columns represent the means of three experiments; bars, SE. *, P<0.05 for control cells vs cells treated under different conditions. The representative results are shown. (c). The dose dependent induction c-myc and cyclin D1 by E2β in MDA-MB-231 and MDA-MB-436 cells. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated different concentrations of E2β. The representative results are shown. (d). The effects of E2β on the proliferation rate of MDA-MB-231 and MDA-MB-436 cells. Cells maintained for three days in phenol red-free DMEM plus 2.5% dextran-charcoal-stripped fetal calf serum were treated with indicated concentrations of E2β or ethanol vehicle as a control. The cell numbers were determined using an automatic cell counter after 12 days. Five dishes were used for each concentration and experiments were repeated more than four times. The mean cell numbers ± SE are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3020987&req=5

Figure 1: Non-Genomic Estrogen Signaling Stimulates Proliferation of ER-negative Breast Cancer Cells(a). The expression of ER-α variants and EGFR in MCF7, MDA-MB-231 and MDA-MB-436 breast cancer cells. (b). The dose and time dependent pattern of E2β-stimulated phosphorylation of the MAPK/ERK1/2 in MDA-MB-231 and MDA-MB-436 cells. Starved cells were treated with indicated doses of E2β or 0.1 nM of E2β for indicated time periods. Western blot analysis was performed to assess induction of ERK1/2 phosphorylation. The columns represent the means of three experiments; bars, SE. *, P<0.05 for control cells vs cells treated under different conditions. The representative results are shown. (c). The dose dependent induction c-myc and cyclin D1 by E2β in MDA-MB-231 and MDA-MB-436 cells. The columns represent the means of three experiments; bars, SE. *, P<0.05 for cells treated with vehicle vs cells treated different concentrations of E2β. The representative results are shown. (d). The effects of E2β on the proliferation rate of MDA-MB-231 and MDA-MB-436 cells. Cells maintained for three days in phenol red-free DMEM plus 2.5% dextran-charcoal-stripped fetal calf serum were treated with indicated concentrations of E2β or ethanol vehicle as a control. The cell numbers were determined using an automatic cell counter after 12 days. Five dishes were used for each concentration and experiments were repeated more than four times. The mean cell numbers ± SE are shown.
Mentions: To determine if established triple-negative breast cancer cells that express ER-α36 retain non-genomic estrogen signaling, we used breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which are triple-negative. Western blot analysis showed that both ER-α36 and EGFR are highly expressed in these breast cancer cells while ER-positive MCF7 cells expressed high levels of ER-α66 but lower levels of ER-α36 and EGFR (Figure 1A).

Bottom Line: Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36 mediated nongenomic estrogen signaling and is highly expressed in ER-negative breast cancer cells.In this study, we found that ER-α36 was highly expressed in 10/12 cases of triple-negative breast cancer.We investigated the role of mitogenic estrogen signaling mediated by ER-α36 in malignant growth of triple-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of ER-α36 and found that these cells strongly responded to mitogenic estrogen signaling both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology & Immunology, Creighton University Medical School, Omaha, NE, USA.

ABSTRACT
It is prevailingly thought that estrogen signaling is not involved in development of estrogen receptor (ER)-negative breast cancer. However, there is evidence indicating that ovariectomy prevents the development of both ER-positive and -negative breast cancer, suggesting that estrogen signaling is involved in the development of ER-negative breast cancer. Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36 mediated nongenomic estrogen signaling and is highly expressed in ER-negative breast cancer cells. In this study, we found that ER-α36 was highly expressed in 10/12 cases of triple-negative breast cancer. We investigated the role of mitogenic estrogen signaling mediated by ER-α36 in malignant growth of triple-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of ER-α36 and found that these cells strongly responded to mitogenic estrogen signaling both in vitro and in vivo. Knockdown of ER-α36 expression in these cells using the small hairpin RNA method diminished their responsiveness to estrogen. ER-α36 physically interacted with the EGFR/Src/Shc complex and mediated estrogen-induced phosphorylation of epidermal growth factor receptor (EGFR) and Src. EGFR signaling activated ER-α36 transcription through an AP1 site in the ER-α36 promoter, and ER-α36 expression was able to stabilize EGFR protein. Our results, thus demonstrated that ER-α36 mediates nongenomic estrogen signaling through the EGFR/Src/ERK signaling pathway in ER-negative breast cancer cells and suggested that a subset of ER-negative breast tumors that expresses ER-α36, retains responsiveness to mitogenic estrogen signaling.

Show MeSH
Related in: MedlinePlus