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Primary ductal carcinoma in situmammosphere formation: importance of the epidermal growth factor and Notch receptor signalling pathways
Breast Cancer Researchvolume 10, Article number: P94 (2008)
The cancer stem cell hypothesis suggests that targeting stem-like cells in cancer will improve current therapeutic strategies. In vitro culture of mammospheres (MS), colonies that are analogous to neurospheres, has been used to study factors affecting the self-renewal and growth of ductal carcinoma in situ (DCIS) in 29 cases. The MS culture system demonstrates a small subset of DCIS cells with self-renewal clonogenic capacity showing 1.5 ± 0.1% MS forming efficiency (MFE), which is greater than normal breast MFE, 0.5 ± 0.1% (P < 0.0001). DCIS MS demonstrated an increased growth rate compared with normal, yielding MS > 60 μm within 3 days rather than 7 days. The MFE was greater in high (1.6 ± 0.1%) compared with low (1.1 ± 0.1%, P = 0.012) histological grade DCIS, suggesting a link between the number of MS-initiating cells and recurrence rates.
Since normal breast MS formation was known to depend on epidermal growth factor (EGF) and Notch receptor signalling, we investigated these pathways in DCIS MS. Only high-grade DCIS MFE was decreased in the presence of the EGF receptor inhibitor, Gefitinib, when no EGF was present in the media (1.36 ± 0.16% to 0.56 ± 0.2183, P = 0.0017). This suggests high-grade DCIS secrete growth factors that signal via the EGF receptor. Notch was aberrantly activated in DCIS compared with normal breast, demonstrated by increased levels of activated Notch intracellular domain (NICD) and downstream targets Notch 4 and Hes-1. A γ-secretase inhibitor, DAPT, which inhibits the activating cleavage of Notch receptors, reduced DCIS MFE from 0.88 ± 0.07% to 0.51 ± 0.08% (P = 0.0005). A Notch 4 receptor neutralising antibody reduced DCIS MFE, 0.97 ± 0.1% to 0.2 ± 0.05%, resulting in no MS formation in two out of six cases (P < 0.0001). Furthermore, presence of NICD by immunohistochemistry predicted recurrence in patients with 5 years' follow up after surgery (n = 50, P = 0.012).
These data indicate that Notch and EGF receptor signalling pathways are important in DCIS MS formation, and therapeutic inhibition of the Notch signalling may increase recurrence-free survival after surgery.
Funded by Breast Cancer Campaign (grant# MAY2005:21).