Vinsamlegast notið þetta auðkenni þegar þið vitnið til verksins eða tengið í það: http://hdl.handle.net/1946/10468
The breast is composed of branching epithelial ducts terminating in structures referred to as terminal duct lobular units (TDLUs) surrounded by vascular-rich stroma. Continous cell renewal and expansion during breast morphogenesis is dependent on epithelial stem cells. During branching, epithelial cells acquire partial mesenchymal-like phenotype with increased migration and loss of polarization, similar to epithelial-mesenchymal transition (EMT). In breast cancer, EMT has been linked to basal-like breast cancer, a highly vascularized and aggressive subtype rich in cancer stem cells. Signals through receptors tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR) family, have been shown to be critical in branching morphogenesis in different epithelial systems. RTKs are regulated through negative feedback by the sprouty protein family and lack of sprouty has been shown to result in abnormal and increased branching.
The aim of this thesis was to study extrinsic (endothelial) and intrinsic (Sprouty-2) regulation of breast morphogenesis and EMT. Towards that goal I cocultured normal and malignant breast epithelial cells with breast endothelial cells (BRENCs) in 3D cell culture to unravel their morphogenic potential.
I have demonstrated that BRENCs stimulate growth and morphogenesis of breast epithelial cells and in coculture with D492, a breast epithelial stem cell line, a dramtic increase in branching morphogenesis was seen. In addition to branching, BRENCs induced the formation of spindle like colonies in D492. D492M, a (mesenchymal) subline was isolated from a spindle-like colony and characterization showed irreversible EMT, as evidenced by reduced expression of keratins, a switch from E- to N-cadherin and acquisition of cancer stem cell characteristics. The EMT phenotype could be partially blocked by inhibition of hepatocyte growth factor. Furthermore, I showed that tumor cells close to vascular rich areas within basal-like breast cancers had a characteristic EMT phenotype. Finally, I showed that Sprouty-2 expression is linked to critical steps in branching morphogenesis. Sprouty-2 knock down in D492 resulted in more complex branching structures in 3D culture indicating the loss of negative feedback control of branching morphogenesis. Interestingly, using coculture with endothelial cells I demonstrated that Sprouty-2 knock down cells were more prone to undergo EMT further tightening the link between branching and EMT in breast morphogenesis.
Collectively, I have shown that endothelial cells are potent inducers of breast epithelial growth, branching morphogenesis and in the conversion of cells towards an EMT phenotype. I hypothesize that endothelial cells may be an important regulator of the stem cell niche in the human breast gland and may also play an active role in the progression of basal-like breast cancers through induction of EMT. In addition, I have shown that Spry-2 is instrumental for correct branching morphogenesis of the human breast gland and in safeguarding breast epithelial integrity.
|Valgardur Sigurdsson PhD-1.pdf