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. 2010 Jan 15;70(2):709-18.
doi: 10.1158/0008-5472.CAN-09-1681. Epub 2010 Jan 12.

Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor

Hannah Harrison  1 , Gillian Farnie, Sacha J Howell, Rebecca E Rock, Spyros Stylianou, Keith R Brennan, Nigel J Bundred, Robert B Clarke
Affiliations

Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor

Hannah Harrison et al. Cancer Res. .

Abstract

Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA(+)/CD44(+)/CD24(low). Using these breast cancer stem cell populations, we compared the activation status of Notch receptors with the status in luminally differentiated cells, and we evaluated the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher in stem cell-enriched cell populations compared with differentiated cells, whereas Notch1 signaling activity was 4-fold lower in the stem cell-enriched cell populations. Pharmacologic or genetic inhibition of Notch1 or Notch4 reduced stem cell activity in vitro and reduced tumor formation in vivo, but Notch4 inhibition produced a more robust effect with a complete inhibition of tumor initiation observed. Our findings suggest that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence, as it is initiated by breast cancer stem cells.

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Figures

Figure 1
Figure 1
Percentage mammosphere forming units in different cell types (A). Cell viability assessed using Annexin/PI staining and analysed using the Becton Dickinson FACS Calibur after a time course of non-adherent culture (B). Mammosphere forming unit number in anoikis resistant cells, collected at 16 hours (C). Percentage tumour formation of AR and monolayer cells (D, number represents mice in group, 50% formation represented as dotted line). * P < 0.05 compared to 0 hour control and *** P <0.001, A-C data presented as mean ± SEM. AR anoikis resistant
Figure 2
Figure 2
AR and monolayer MCF7 cells were analysed for cell surface phenotypes of breast cancer cells ranging from basal like BCSCs (CD44+/CD24low) to more differentiated, luminal cells (CD44−/CD24+) using the Becton Dickinson FACS Calibur (A). MCF7, T47D, MDA-MB-231, BT474 and primary pleural effusion cells were sorted (P1 BCSC enriched ESA+/CD44+/CD24low, P2 ESA+/CD44−/CD24low, P3 ESA+/CD44+/CD24+, and P4 ESA+/CD44−/CD24+) and plated in mammosphere culture. Percentage mammosphere forming unit number was calculated at day 7 in BSCS enriched cells (P1) and BSCS depleted cells (P2-4) (B). Percentage tumour formation measured over 5 weeks in mice injected with a limiting dilution of sorted and unsorted MCF7 cells (B). Immuno-blots for full length and cleaved Notch receptors (Notch1-ICD, Notch4 and Notch4-ICD) in sorted cell populations from MCF7, MDA-MB-231 cell lines and a primary pleural effusion sample. Black arrowhead represents Notch1 extracellular truncation (Notch1-EXT), red arrowhead represents Notch1-ICD. Blue and green arrowheads represent Notch4-EXT and Notch4-ICD, respectively (C). Representative photomicrographs of breast tissue sections stained with antibodies to Notch receptors (D). Cleaved Notch1 (Notch1-ICD) expression was assessed in normal (i, red arrow shows highly positive nucleus in luminal cell, red arrowhead shows weak staining in basal cell nucleus) and invasive tumour tissue (iii). Notch4-ICD staining in normal (ii, black arrow shows inactive cytoplasmic staining, filled black arrowhead shows positive nucleus, hollow black arrowhead indicates cell negative for the stain) and invasive tumour samples (iv). Scale bar equals 100μm. ** P < 0.01 *** P<0.001, B data represented as mean ± SEM). AR anoikis resistant, ESA epithelial specific antigen, ICD Intracellular domain, N1 Notch1, N4 Notch4
Figure 3
Figure 3
ESA+/CD44+/CD24low cells assessed in vitro following Notch inhibition of MCF7 cells with gamma secretase inhibitor, 10μM DAPT or DMSO control and siRNA to Notch1 and Notch4 (A). Percentage mammosphere forming unit number was assessed in MCF7, MDA-MB-231 and BT474 cells, pleural effusion and primary IDC samples (BB3RC2, BB3RC3 and BB2RC2) after 7 days in non-adherent culture with / without DAPT (B). Cell lines were produced with Doxycycline inducible expression of Notch1-ICD. These lines, MCF7_N1-ICD and MDA-MB-231_N1-ICD, were cultured with / without DAPT either in the absence (control) or presence (activated N1-ICD) of Doxycycline (C). Immunoblot of cleaved Notch receptors (Notch1-ICD and Notch4-ICD) was carried out in MCF7, BT474 and MDA-MB-231 cells with or without DAPT treatment and in MCF7 and MDA-MB-231 with or without DBZ (D). * P=0.05, ** P<0.01 and *** P<0.001, A-C, E and H data are presented as mean ± SEM. ICD intracellular domain, N1 Notch1, N4 Notch4, PE pleural effusion, IDC invasive ductal carcinoma, DOX Doxycycline.
Figure 4
Figure 4
Immunoblot of Notch1 and Notch4 receptors to test the extent of knock-down, and the target specificity of each shRNA cell line (A, Actin as loading control). Mammosphere forming unit number in scrambled control (MCF7scr) and inducible shRNA cell lines (MCF7Notch1 and MCF7Notch4) was measured after 7 days of culture with (grey bars) and without (black bars) Doxycycline (B, * P < 0.05; Small sample t-test, data are presented as mean ± SEM). Mean tumour volumes over 28 days in vivo growth (C, * P<0.05, ** P<0.01). A putative model for Notch signalling activity in the cellular hierarchy of breast cancer (D). MFU mammosphere forming unit, DOX Doxycycline
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