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Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signalling.

Harrison H, Simões BM, Rogerson L, Howell SJ, Landberg G, Clarke RB - Breast Cancer Res. (2013)

Bottom Line: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression.This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA.We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.

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ABSTRACT

Introduction: Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity.

Methods: Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects.

Results: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.

Conclusions: These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.

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Knock-down or inhibition of ER signalling blocks the effect of 17β-estradiol. (A) Representative Western blot showing ER knock-down with siRNA. (B) Cells were exposed to oestrogen and the effect of ER siRNA was assessed with mammosphere culture. Cells were cultured for 48 hours in the presence of 1 nM 17β-estradiol ± tamoxifen before CSC assays were performed. (C) Mammosphere formation and (D) holoclone formation were assessed. Fold change is normalised to control, untreated cells represented as line. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.
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Figure 2: Knock-down or inhibition of ER signalling blocks the effect of 17β-estradiol. (A) Representative Western blot showing ER knock-down with siRNA. (B) Cells were exposed to oestrogen and the effect of ER siRNA was assessed with mammosphere culture. Cells were cultured for 48 hours in the presence of 1 nM 17β-estradiol ± tamoxifen before CSC assays were performed. (C) Mammosphere formation and (D) holoclone formation were assessed. Fold change is normalised to control, untreated cells represented as line. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.

Mentions: To verify that this effect was due to activation of oestrogen signalling we first knocked down ER gene expression using siRNA (Figure 2A). In cells cultured in the presence of ER siRNA and 17β-estradiol, no significant increase in MFC is seen, establishing ER as the major player in the oestrogenic response (Figure 2B).


Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signalling.

Harrison H, Simões BM, Rogerson L, Howell SJ, Landberg G, Clarke RB - Breast Cancer Res. (2013)

Knock-down or inhibition of ER signalling blocks the effect of 17β-estradiol. (A) Representative Western blot showing ER knock-down with siRNA. (B) Cells were exposed to oestrogen and the effect of ER siRNA was assessed with mammosphere culture. Cells were cultured for 48 hours in the presence of 1 nM 17β-estradiol ± tamoxifen before CSC assays were performed. (C) Mammosphere formation and (D) holoclone formation were assessed. Fold change is normalised to control, untreated cells represented as line. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672803&req=5

Figure 2: Knock-down or inhibition of ER signalling blocks the effect of 17β-estradiol. (A) Representative Western blot showing ER knock-down with siRNA. (B) Cells were exposed to oestrogen and the effect of ER siRNA was assessed with mammosphere culture. Cells were cultured for 48 hours in the presence of 1 nM 17β-estradiol ± tamoxifen before CSC assays were performed. (C) Mammosphere formation and (D) holoclone formation were assessed. Fold change is normalised to control, untreated cells represented as line. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.
Mentions: To verify that this effect was due to activation of oestrogen signalling we first knocked down ER gene expression using siRNA (Figure 2A). In cells cultured in the presence of ER siRNA and 17β-estradiol, no significant increase in MFC is seen, establishing ER as the major player in the oestrogenic response (Figure 2B).

Bottom Line: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression.This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA.We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity.

Methods: Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects.

Results: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.

Conclusions: These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.

Show MeSH
Related in: MedlinePlus