The role of restraint stress facilitated by glucocorticoids in triple negative breast cancer bone metastasis

Abstract

Stress hormone signalling is increasingly recognised in breast cancer (BC) progression and metastasis, particularly in triple negative BC (TNBC). However, the role of glucocorticoid (GC) signalling and GC receptor (GR) modulation in TNBC metastasis to bone remains unclear. This thesis investigates the effects of restraint stress, GC treatment, and GR modulation on TNBC migration in vitro and bone metastasis.
Initial investigations of mouse tibiae from collaborative 4T1 orthotopic studies demonstrated little evidence of bone metastases, with no effect of restraint stress or dexamethasone treatment on tumour burden in bone. This prompted the use of bone-homing (BH) TNBC subclones to increase bone metastasis frequency.
Immunofluorescence demonstrated that MDA-BH, 4T1-BH and E0771-BH subclones expressed GR, with nuclear translocation indicating activation. Parental and BH human and murine cells exposed to physiological (500nM) and supraphysiological (1000nM) GC doses demonstrated no significant differences in migration after 24 hours. Migration significantly increased following 7-day GC pre-exposure in 4T1-BH cells. Screening of genes associated with skeletal disease demonstrated >2-fold alteration of 23 genes, including upregulation of those associated with the RANK-RANKL-OPG pathway. However, validation did not demonstrate significant differences.
Given the limited evidence for consistent GC-mediated effects on tumour cell behaviour in vitro, subsequent investigations examined stress-mediated effects on tumour burden in bone using intracardiac administration of E0771-BH cells with restraint stress and GR modulation. After 4 days of restraint stress, serum corticosterone levels did not significantly increase, although mean levels increased by 8.9%, 4.0% and 15.01% across the three restraint stress groups relative to baseline, with no significant effect of GR modulation.
Restraint stress did not significantly alter tumour burden when compared to tumour-bearing controls, however, it significantly reduced trabecular bone volume/tissue volume ratio by 40%, an effect not observed in stress-only controls. Addition of the GR modulator did not significantly modify these effects.
Tibiae from tumour-bearing stressed mice demonstrated significantly increased IL-1β-positive area compared with stress-only controls and tumour-bearing controls, with increases of 76% and 85%, respectively. GR modulation did not significantly reduce IL-1β-positive area, with a 35% reduction relative to stressed tumour-bearing mice.
Osteoclast number remained unchanged with restraint stress, although GR modulation significantly increased osteoclast number by 74% relative to tumour-bearing mice. Osteoblast number significantly decreased by in stressed tumour-bearing mice by 70% when compared with stress-only controls, but not tumour-bearing control mice. Addition of GR modulation did not mitigate this effect.
This thesis provides a novel contribution to elucidating the role of stress in breast-to-bone metastasis, with a focus on GCs and the therapeutic potential of GR modulation. This work identifies context-dependent GC effects but does not demonstrate consistent GC-mediated regulation of TNBC bone-seeking cell migration. The data did not provide robust evidence for effects on tumour burden in bone. Furthermore, GR modulation did not mitigate stress-associated effects within

Date of Award	Sept 2026
Original language	English
Awarding Institution	University of Brighton
Supervisor	Melanie Flint (Supervisor), Katherine Staines (Supervisor) & Isabel R Orriss (Supervisor)