Mechanisms of mechanotransduction in pancreatic fibroblasts

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all major cancers, and is characterised by an intense desmoplastic reaction. The upregulation and remodelling of extracellular matrix (ECM) by activated fibroblasts results in an extremely rigid tumour environment that contributes to tumorigenic processes such as cell proliferation and invasion. Cells mediate attachment to the ECM through integrin adhesion complexes (IACs). IACs sense external and internal mechanical cues, such as matrix rigidity and actomyosin contractility, to govern cell behaviour through a variety of IAC-associated molecules, collectively known as the adhesome. However, precisely how IACs regulate cell behaviour in response to force is not fully understood. To address this, a proximity-dependent labelling method, BioID, was used to study force- regulated changes in adhesome interaction networks in a disease-relevant cell line. A protocol was developed and optimised for the detection of proximity interactions in mouse pancreatic fibroblasts, and using 16 IAC bait proteins, label-free quantitative proteomics was performed to define a proximity-dependent adhesome under standard tissue culture conditions. Stringent analysis revealed a network of 147 enriched proteins with 360 proximity interactions. Many known IAC components were identified, together with candidates that may have underappreciated roles in adhesion. Unbiased analysis revealed 5 clusters of IAC BioID baits representing groups of proximal proteins that link to common groups of prey, which may represent distinct functional modules within IACs. To examine force-dependent interactions in the proximity-dependent adhesome, actomyosin contractility was disrupted through myosin II inhibition with blebbistatin, resulting in a loss of actin stress fibres and an enrichment of nascent adhesions. The effects of substrate rigidity on the proximity-dependent adhesome were also examined, with cells seeded on stiff substrates reflecting the cancerous pancreatic environment exhibiting increased proliferation and larger IACs compared to those grown on soft substrates that reflect the normal pancreas. A number of actomyosin-dependent and substrate-dependent interactions were identified using BioID, providing some insight into mechanoregulatory mechanisms within the adhesome. Alongside multiple established mechanoresponsive components, several potential new candidates were identified that may be involved in mechanotransduction by mouse pancreatic fibroblasts. Future experiments will probe these mechanisms further. This study presents the first empirically defined view of proximal interactions in the adhesome on a global level, and has demonstrated that BioID is a valuable tool that complements existing methods to study the effects of force on IACs. It is hoped that an increased understanding of how adhesome networks are regulated may provide insights into the relationship between the ECM and pancreatic cancer.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Humphries (Supervisor) & Stephen Taylor (Supervisor)