Investigating breast cancer progression in the context of deregulated cholesterol metabolism and determining a role for estrogen receptor beta

Abstract

All breast cancer subtypes, except triple negative breast cancer (TNBC), possess at least one of the targetable receptors: estrogen receptor α (ERα), progesterone receptor (PR), and/or human epidermal growth factor receptor 2 (HER2). Having none of these biomarkers, TNBC patients face poor prognoses, especially those suffering from obesity. Obesity is associated with upregulated IGF signalling and hypercholesterolemia. Deregulated cholesterol metabolism is a hallmark of cancer. 27-OHC is the most abundant cholesterol metabolite. Notably, 30%-60% of TNBC express estrogen receptor β (ERβ) to which 27-OHC binds preferentially. This project investigates whether ERβ and de-regulated cholesterol metabolism, interlinked by a positive feedback loop with EGFR and IGF1 R signalling, drive TNBC progression. Western blotting and qPCR were used to assess the abundance of ERβ proteins and mRNA in normal and cancerous breast cells, and the impact of estradiol (E2), 27-OHC, and IGF-I on MDA-MB 231, MCF-7 and BT-20 cells. Subsequently, the effect of ERβ silencing by siRNA, and ERβ inhibition by 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a] pyrimidin-3-yl] phenol (PHTPP) on ERβ, EGFR, and IGF1 R levels was assessed. Tritiated thymidine incorporation assay and quantitative phase imaging (HoloMonitor) were used to investigate changes in cell proliferation upon simvastatin treatment. Additionally, HoloMonitor was used to investigate the impact of simvastatin on motility, and cell fate. This project found that MDA-MB 231 and BT-20 cells have high levels of ERβ protein and mRNA. Upon ERβ stimulation by E2, EGFR, and IGF1 R levels increase. In line with that ERβ silencing results in a dose-dependent decrease in EGFR and IGF1 R proteins in TNBC cells. Simvastatin significantly decreases proliferation in ERα+ERβ- MCF-7 cells and in ERβ+ERα- TNBC cells, being cytotoxic at 8µM doses. Due to unsuccessful ERβ silencing/inhibition and data inconsistencies stemming from the use of phenol-containing cell culture media, the data does not support the hypothesis where ERβ and deregulated cholesterol metabolism drive TNBC progression.

Date of Award	3 Oct 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Claire M Perks (Supervisor) & Kalina Biernacka (Supervisor)