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. 2013 Mar 8;15(2):R21.
doi: 10.1186/bcr3396.

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

Hannah Harrison, Bruno M Simões, Lynsey Rogerson, Sacha J Howell, Göran Landberg, Robert B Clarke

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

Hannah Harrison et al. Breast Cancer Res. .

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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Figures

Figure 1
Figure 1
Oestrogenic effects on cancer stem cells. Cells were cultured for 48 hours in a monolayer in the presence of 1 nM 17β-estradiol or vehicle control and CSC assays were performed in ER positive cells (primary, MCF7, T47D and BT474) and ER negative cells (231). (A) Mammosphere formation, (B) holoclone formation and (C) expression of ESA+ CD44+ CD24low were measured. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
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.
Figure 3
Figure 3
Breast cancer stem cells have low expression of ER. Cells were sorted based on surface markers to enrich for CSC. Representative Western blot showing ER expression in sorted primary metastatic cells (A) and MCF7 cells (B). Anoikis resistant (AR) cells from primary metastatic and cell lines (MCF7 and T47D) were collected for protein and RNA. (C) Representative Western blot showing expression of ER in total versus AR cells, (D) mRNA expression level of ER (ESR1) and responsive genes in AR cells compared to total population. (E) Representative Western blot of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 1 nM 17β-estradiol ± 1 μM tamoxifen or gefitinib. (F) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gefitinib. 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.
Figure 4
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.
Figure 5
Figure 5
In vivo assessment of oestrogenic effect on breast cancer stem cells. (A) Growth curves for xenografts produced from 1,000 cells pre-treated ± 17β-estradiol, ± inhibitors. *P = 0.01 (B) In vivo tumour formation in each group represented as mice positive for growth/mice tested displayed for each cell number tested. Tumour initiating cell frequency (95% CI) estimates calculated from limiting dilution analysis.
Figure 6
Figure 6
Schematic representation of signals involved in paracrine mediation of systemic oestrogen signalling. The signal sending cell (white) responds to oestrogen (E2) and initiates EGFR, FGFR and Notch ligand production. A signalling cascade is initiated within the signal receiving cell (grey) which includes, but may not be limited to ERK, Tbx3 and Pea3 signalling. This drives increased CSC activity. This signal mediation can be blocked by tamoxifen, gefitinib and gamma secretase inhibitors (GSI).
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