Understanding how Extracellular Matrix Stiffness Drives Mammary Epithelial Cell Transformation

Abstract

The aim of this thesis is to understand how increased mammographic density, a modifiable breast cancer risk factor, predisposes an individual to breast cancer. It has previously been demonstrated that tissue from high-density breast is mechanically stiffer than that from low-density breast. To investigate this, I have evaluated the effects of extracellular matrix (ECM) stiffness, finding that the 3D alginate Matrigel gel (AMG) system was the most appropriate culture model for my aims. With this model, I have shown that increased environmental stiffness is associated with increased DNA damage in mammary epithelial cells (MECs). I have also shown that inhibition of apoptosis, using apoptotic protease activating factor 1 (APAF1) knockout line, is associated with an increase in DNA damage when MECs are cultured in soft AMGs, whereas inhibition of proliferation reduces DNA damage when MECs are cultured in stiff AMGs. To identify signalling pathways that are involved in stiffness-induced DNA damage, I performed RNASeq experiments on cells cultured in or on ECM of different stiffness. These data suggest that the stiff microenvironment downregulated several isoforms of aldehyde dehydrogenase, which resulted in an increase in reactive aldehydes observed in MEC in stiff AMGs. Stably expressing an aldehyde dehydrogenase (ALDH) 3B2 in cells grown in stiff environment reduced the levels of DNA damage and transformation to those seen in soft ECM. In this introduction, I will discuss the risk factors associated with breast cancer, with a focus on mammographic density, how cells sense matrix stiffness, and how DNA damage occurs and is repaired, with a concluding summary of the project aims.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Gilmore (Supervisor) & Keith Brennan (Supervisor)