The cell fate of proliferation is used as an end point for many studies, particularly in cancer research. Primary mammary epithelial cells (MECs) are a common model in breast cancer research and MEC proliferation is limited to a few days which make it very difficult to study longer term. I describe the establishment of a new culture system to allow the analysis of cultures of primary luminal MECs over many days. Culturing the cells in a 3D microenvironment directly following tissue extraction, isolating the 3D acini using PBS/EDTA and replating onto 2D surface to study proliferation, maintained the 3D in vivo proliferation potential of the cells. Manipulation of the cellular environment, using this novel method, can maintain the proliferative potential of primary MECs. Our data further reveals that the cellular environment has profound effects on the proliferative properties of the primary MECs and is dominant over growth factors. This novel methodology, to increase the proliferation window, will allow the use of MECs over a longer time period and was used to elucidate the mechanism by which β1 integrins regulate the proliferation in MECs.Integrins are required for cell cycle progression in epithelia, but the β-integrin subunits involved and the downstream mechanisms are not known. A CreERTM strategy was used to specifically delete the β1-integrin gene in situ from primary cultures of MECs. This resulted in a block of DNA synthesis. Despite the loss of β1-integrins, no changes in cell shape, focal adhesion integrity or the ability of the cells to migrate were observed. In β1-integrin null cells, β3-integrin was expressed de novo, it associated with core adhesome components, and maintained focal adhesion signalling as determined by the presence of phospho-FAK and phospho-paxillin. β3-integrin adhesions were unable to support Rac activation and nuclear translocation of ERK. This investigation reveals that β1-integrins are uniquely required for cell cycle progression in breast epithelia, but not for their migration. Thus, the control of epithelial cell fate is dependent on the signalling pathways that emanate from specific β-integrin mediated adhesions.Disorganised morphology, in addition to uncontrolled proliferation, is a typical feature of human breast cancer. Associated with this aberrant morphology is a deregulation of integrin adhesion receptors. We investigated the role of β1-integrin in the MCF10a breast cancer progression model; a series of increasingly transformed cell lines from a common genetic background. The cell lines of the MCF10a progression series were grown in a 3D basement membrane culture, in which they formed complex disorganised multi-acinar structures, except the control normal MCF10a MEC cell line, which formed hollow acini. Analysing β1-integrin expression using flow cytometry, showed similar surface expression levels, however confocal microscopy revealed different β1-integrin localisation in the malignant cell lines compared to the normal ones. Treating the MCF10a progression panel cell lines with AIIB2, a β1 integrin inhibitory antibody resulted in the morphology of the treated 3D colonies becoming more compact, smaller, and similar to the MCF10a normal cells. All in all, the results presented in this thesis show the critical importance of integrins in regulating proliferation in MECs, the mechanism by which integrins exert this control and the role that this may have in breast cancer tumourigenesis.
|Date of Award||1 Aug 2011|
- The University of Manchester
|Supervisor||Charles Streuli (Supervisor)|