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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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Systemic oestrogen signalling is mediated by EGFR and Notch. (A) Representative Western blot showing expression of cleaved (active) Notch1 (N1-ICD) following culture ± 1 nM 17β-estradiol ± 10 μM GSI. (Bi) Representative Western blot showing expression of Notch ligands in sorted MCF7 cells (left) and, where available, metastatic cells (right). (Bii) Densitometric analysis of three independent repeats of MCF7 sorting and of a single experiment for primary cells. Comparisons between population 1 (CSC enriched) and other populations are displayed. (C and D) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gamma secretase inhibitor (GSI) alone and in combination with gefitinib. Fold change is normalised to control, untreated cells represented as line. (E) Representative image of protein levels of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 10 μM GSI. 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 4: Systemic oestrogen signalling is mediated by EGFR and Notch. (A) Representative Western blot showing expression of cleaved (active) Notch1 (N1-ICD) following culture ± 1 nM 17β-estradiol ± 10 μM GSI. (Bi) Representative Western blot showing expression of Notch ligands in sorted MCF7 cells (left) and, where available, metastatic cells (right). (Bii) Densitometric analysis of three independent repeats of MCF7 sorting and of a single experiment for primary cells. Comparisons between population 1 (CSC enriched) and other populations are displayed. (C and D) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gamma secretase inhibitor (GSI) alone and in combination with gefitinib. Fold change is normalised to control, untreated cells represented as line. (E) Representative image of protein levels of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 10 μM GSI. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.

Mentions: Next, we investigated Notch signalling as another likely paracrine pathway since it has been reported [21] and we confirm here that Notch1 signalling activity increases following exposure to oestrogen (Figure 4A). Ligands of the Notch pathway are expressed at significantly lower levels in the CSC enriched sub-populations (Figure 4B) similar to the EGF ligand amphiregulin (Figure 3D) suggesting signalling may be initiated by ligands derived from the non-CSC sub-population. We predicted that Notch1 receptor signalling has a role in the oestrogenic response of breast CSCs and tested this using a gamma secretase inhibitor (GSI) known to specifically target Notch1 signalling [18]. GSI significantly reduced the oestrogenic effect on MFC in primary cells and MCF7 cells (Figure 4C) but the MFC number remained significantly increased compared to control MS cultures. However, GSI used in combination with gefitinib caused MFC number to fall below that seen in control conditions (Figure 4D). GSI had no effect on ERK phosphorylation, (Figure 4E) suggesting that the Notch and EGFR signalling pathways have distinct roles in the mediation of local signalling. We conclude from these data that both EGF and Notch1 receptor signalling are paracrine mediators of the effects of systemic oestrogen on breast CSC activity.


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)

Systemic oestrogen signalling is mediated by EGFR and Notch. (A) Representative Western blot showing expression of cleaved (active) Notch1 (N1-ICD) following culture ± 1 nM 17β-estradiol ± 10 μM GSI. (Bi) Representative Western blot showing expression of Notch ligands in sorted MCF7 cells (left) and, where available, metastatic cells (right). (Bii) Densitometric analysis of three independent repeats of MCF7 sorting and of a single experiment for primary cells. Comparisons between population 1 (CSC enriched) and other populations are displayed. (C and D) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gamma secretase inhibitor (GSI) alone and in combination with gefitinib. Fold change is normalised to control, untreated cells represented as line. (E) Representative image of protein levels of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 10 μM GSI. 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

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

Figure 4: Systemic oestrogen signalling is mediated by EGFR and Notch. (A) Representative Western blot showing expression of cleaved (active) Notch1 (N1-ICD) following culture ± 1 nM 17β-estradiol ± 10 μM GSI. (Bi) Representative Western blot showing expression of Notch ligands in sorted MCF7 cells (left) and, where available, metastatic cells (right). (Bii) Densitometric analysis of three independent repeats of MCF7 sorting and of a single experiment for primary cells. Comparisons between population 1 (CSC enriched) and other populations are displayed. (C and D) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gamma secretase inhibitor (GSI) alone and in combination with gefitinib. Fold change is normalised to control, untreated cells represented as line. (E) Representative image of protein levels of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 10 μM GSI. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.
Mentions: Next, we investigated Notch signalling as another likely paracrine pathway since it has been reported [21] and we confirm here that Notch1 signalling activity increases following exposure to oestrogen (Figure 4A). Ligands of the Notch pathway are expressed at significantly lower levels in the CSC enriched sub-populations (Figure 4B) similar to the EGF ligand amphiregulin (Figure 3D) suggesting signalling may be initiated by ligands derived from the non-CSC sub-population. We predicted that Notch1 receptor signalling has a role in the oestrogenic response of breast CSCs and tested this using a gamma secretase inhibitor (GSI) known to specifically target Notch1 signalling [18]. GSI significantly reduced the oestrogenic effect on MFC in primary cells and MCF7 cells (Figure 4C) but the MFC number remained significantly increased compared to control MS cultures. However, GSI used in combination with gefitinib caused MFC number to fall below that seen in control conditions (Figure 4D). GSI had no effect on ERK phosphorylation, (Figure 4E) suggesting that the Notch and EGFR signalling pathways have distinct roles in the mediation of local signalling. We conclude from these data that both EGF and Notch1 receptor signalling are paracrine mediators of the effects of systemic oestrogen on breast CSC activity.

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