The Role of Notch Signalling in Human Breast Carcinoma-Associated Fibroblasts

Abstract

Tumour-associated stroma is composed of a unique extracellular matrix with a number of growth factors, cytokines, matrix metalloproteases and a non-epithelial mesenchymal cell population. Carcinoma-associated fibroblasts (CAFs) are one of the most abundant mesenchymal cell types found in the tumour-associated stroma. These cells are considered hallmarks of activated fibroblasts with their presence in large numbers being linked to higher-grade malignancy and poor patient prognosis. CAFs have come under considerable examination over the last decade due to their roles in various aspects of tumour growth, invasion and metastasis. Hence elucidation of cellular pathways responsible for the tumour-promoting ability of these cells is an ultimate investigative aim.Previously, we developed a mouse xenograft model in which human breast CAFs were experimentally generated (exp-CAFs). Given the prominent roles of Notch signalling in tissue remodelling, including fibrosis and vasculogenesis, it was hypothesised that activation of this signalling may give rise to exp-CAFs and maintain the activated state of these cells. The work herein demonstrates the aberrantly activated Notch signalling in exp-CAFs. More specifically, activation of Notch signalling in exp-CAFs is mainly stimulated by high levels of the Notch ligandJagged1 and the receptor Notch3. Jagged1-induced Notch3-mediated signalling activity is responsible for the myofibroblastic active state of exp-CAFs. Loss-of-function studies, in which various Notch components were silenced using either shRNAs or a γ-secretase inhibitor DAPT, demonstrated a functional role of Notch signalling activity for the maintenance of activated state of exp-CAFs. Additionally, human immortalised mammary fibroblasts forced to express human Jagged1, Dll4 and NICD3 cDNAs exhibited activation of Notch signalling with such activity leading to transdifferentiation into a CAF-like state. The same activity was also achieved when mammary fibroblasts were stimulated by immobilised recombinant Jagged1 and Dll4 ligands. Importantly, Jagged1-induced Notch3-mediated signalling was also required for regulation of the activation of Smad2-dependent TGF-β signalling and SDF-1 mRNA expression in exp-CAFs. Moreover, forced activation of Notch3-mediated signalling was found to be sufficient to regulate TGF-β mRNA expression in mammary fibroblasts. Finally, IHC analysis of Jagged1, Dll4 and Notch3 using human primary breast cancer TMAs showed an association between Jagged1, Dll4 and Notch3 in CAFs and cancer cells in addition to some of the clino-pathologicalparameters. Collectively, these observations strongly suggest the participation of Jagged1-induced Notch3-mediatated activation of Notch signalling in mediating the myofibroblastic activated state and the activation of Smad2-dependent TGF-β pathway in exp-CAFs. Given the tumour-promoting ability of CAF, targeting the Notch activity in these cells may potentially be regarded as an attractive therapeutic approach to be considered.

Date of Award	1 Aug 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Akira Orimo (Supervisor) & Goran Landberg (Supervisor)