Investigating mechanotransduction through adhesion complexes in mammary epithelial cells

Abstract

Cells in vivo are exposed to a range of mechanical stimuli within their tissue microenvironment. Mechanotransduction is the process by which cells sense, interpret, and adapt to these stimuli. This occurs through the conversion of mechanical cues into intracellular biochemical signals which elicit gene expression changes that regulate cell behaviour. Key routes through which mechanotransduction can occur include integrin- and cadherin-based adhesion complexes, which anchor the actin cytoskeleton to the extracellular matrix (ECM) and neighbouring cells respectively. These complexes act as bidirectional signalling hubs, allowing the transduction of force between a cell and its ECM, and between cells within a tissue. As mechanotransduction is fundamental to cell identity and behaviour, changes to the mechanical environment of a cell, such as an increase in ECM stiffness, can result in the development of pathologies such as cancer and fibrosis. Increased stiffness of breast tissue has been reported in breast cancer and has been shown to contribute to disease progression. Less is known about how variations in the stiffness of normal breast tissue may predispose to pathologies. Women with a high mammographic density are at greater risk of developing breast cancer, however, the mechanisms by which this risk is conferred are poorly understood. At the molecular and cellular level, high mammographic density has been shown to manifest as a difference in the ECM stiffness of the stromal tissue surrounding mammary epithelial cells (MECs). As such, we and others have hypothesised that increased ECM stiffness and altered mechanotransduction may play a role in the increased risk of breast cancer initiation in women with a high mammographic density. We therefore sought to identify mechanotransduction mechanisms in MECs which may drive transformation in stiff ECM. The results presented in this thesis elucidate a novel, RhoA-mediated mechanotransduction pathway which drives transformation of mammary epithelial cells in stiff ECM through alterations to aldehyde metabolism. Following this, we present an adaptation of the BioID proximity labelling technique to screen for mechanosensitive differences in the composition of integrin-containing adhesion complexes and adherens junctions in mammary epithelial cells. Using this technique, we demonstrate differences in integrin-containing adhesion complexes in mammary epithelial cells compared to those of cell types widely studied in the literature. Our BioID studies culminate in the characterisation of a mechanosensitive, proximity-dependent adhesome in MECs. Taken together, this thesis provides insight into mechanotransduction mechanisms in MECs. This dataset now provides a list of candidate proteins, which may be involved in driving intracellular signalling, cytoskeletal rearrangements, and gene expression changes downstream of adhesion complexes. Further study can now focus on identifying potential roles for these proteins in the transformation of MECs in a stiff ECM.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Gilmore (Supervisor) & Keith Brennan (Supervisor)