Dose-dependent induction of distinct phenotypic responses to Notch pathway activation in mammary epithelial cells

Abstract

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.

Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo

“…Stimulation of acini growth in Matrigel cultures has already been observed upon Notch1 ICD overexpression in MCF10A cells (39). In MCF10ANCT cells treatment with DAPT, Wortmannin, LY294002, and DNAKT, partially reverted NCT-induced growth, suggesting that potentially other GS substrates (HER4, CD44, β1-integrin, etc.…”

Loss of the Notch effector RBPJ promotes tumorigenesis

“…We have previously shown that expression of constitutively active Notch increases the resistance of MCF10A, a breast epithelial cell line, to anoikis and others have demonstrated induction of hyperproliferative acinar structures and colonies in soft agar (Leong et al, 2007;Mazzone et al, 2010). Compatible with a role in inhibiting apoptosis, mammary-specific deletion of Rbpj did not block Notch-induced tumor formation (Raafat et al, 2009).…”

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

“…In this respect, it is attractive to speculate that the interaction between Notch and vimentin would serve as a hub to coordinate cytoskeletal and developmental signaling in the regulation of angiogenesis. Vimentin is required for efficient Jagged-Notch signaling and provides dynamic control of the strength of signal activity as cellular effects of Notch signaling are highly dose sensitive (36,53,66). Such control is important, because Jagged recycling and surface accumulation is enhanced, but Jagged surface levels are decoupled from signaling strength in vimentin-depleted cells.…”