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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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EGFR protein is stabilized by ER-α36 in ER-negative breast cancer cells(a & b). Western blot and RT-PCR analysis of EGFR expression in variants of MDA-MB-231 and 436 cells. (c). Western blot analysis of EGFR expression in MG132 treated variants of MDA-MB-231 cells. All experiments were repeated at least three times, and the representative results are shown.
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Figure 4: EGFR protein is stabilized by ER-α36 in ER-negative breast cancer cells(a & b). Western blot and RT-PCR analysis of EGFR expression in variants of MDA-MB-231 and 436 cells. (c). Western blot analysis of EGFR expression in MG132 treated variants of MDA-MB-231 cells. All experiments were repeated at least three times, and the representative results are shown.

Mentions: The finding that ER-α36 down-regulation dramatically suppresses the tumorigenicity of these ER-negative breast cancer cells in the absence of estrogen was surprising since these cells also express high levels of EGFR, which would promote malignant growth in vivo. To elucidate the underlying mechanisms, Western blot analysis was performed to examine the expression levels of EGFR protein in the cells with ER-α36 expression knocked-down. Figure 4A shows that expression levels of EGFR protein were dramatically decreased in MDA-MB-231 cell lines with ER-α36 expression knocked-down compared with control cells. However, we did not observe significant change for the mRNA levels of EGFR in these cells, suggesting that the steady state levels of EGFR protein were decreased in ER-α36 knocked-down MDA-MB-231 cells (Figure 4A). A similar destabilization of EGFR protein was also observed in MDA-MB-436 cells with knocked-down levels of ER-α36 expression (Figure 4B). Western blot analysis further demonstrated that upon treatment with MG132, a proteasome inhibitor, the levels of EGFR protein in the ER-α36 knocked-down cells were restored to levels comparable with those of control cells (Figure 4C), indicating that the protein degradation of EGFR was enhanced in ER-α36 down-regulated cells. Thus, ER-a36 is involved in regulation of the steady state levels of EGFR protein.


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)

EGFR protein is stabilized by ER-α36 in ER-negative breast cancer cells(a & b). Western blot and RT-PCR analysis of EGFR expression in variants of MDA-MB-231 and 436 cells. (c). Western blot analysis of EGFR expression in MG132 treated variants of MDA-MB-231 cells. All experiments were repeated at least three times, and the representative results are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: EGFR protein is stabilized by ER-α36 in ER-negative breast cancer cells(a & b). Western blot and RT-PCR analysis of EGFR expression in variants of MDA-MB-231 and 436 cells. (c). Western blot analysis of EGFR expression in MG132 treated variants of MDA-MB-231 cells. All experiments were repeated at least three times, and the representative results are shown.
Mentions: The finding that ER-α36 down-regulation dramatically suppresses the tumorigenicity of these ER-negative breast cancer cells in the absence of estrogen was surprising since these cells also express high levels of EGFR, which would promote malignant growth in vivo. To elucidate the underlying mechanisms, Western blot analysis was performed to examine the expression levels of EGFR protein in the cells with ER-α36 expression knocked-down. Figure 4A shows that expression levels of EGFR protein were dramatically decreased in MDA-MB-231 cell lines with ER-α36 expression knocked-down compared with control cells. However, we did not observe significant change for the mRNA levels of EGFR in these cells, suggesting that the steady state levels of EGFR protein were decreased in ER-α36 knocked-down MDA-MB-231 cells (Figure 4A). A similar destabilization of EGFR protein was also observed in MDA-MB-436 cells with knocked-down levels of ER-α36 expression (Figure 4B). Western blot analysis further demonstrated that upon treatment with MG132, a proteasome inhibitor, the levels of EGFR protein in the ER-α36 knocked-down cells were restored to levels comparable with those of control cells (Figure 4C), indicating that the protein degradation of EGFR was enhanced in ER-α36 down-regulated cells. Thus, ER-a36 is involved in regulation of the steady state levels of EGFR protein.

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