Palma, Gabriella Bianca Henriques2024-02-062024-02-062024https://hdl.handle.net/10539/37522A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023Breast cancer (BC) is the most prevalent cancer in women, with 70% of BC cases being hormone responsive (estrogen receptor positive (ER+)). This ER+ BC subtype relies on estrogen for enhanced cell proliferation and survival. The main therapeutic strategy to prevent hormone responsive BC recurrence is with the use of endocrine therapy such as Tamoxifen (TAM). Despite the success in reducing mortality rates of BC patients with the use of adjuvant TAM and chemotherapy, cancer drug resistance remains a significant challenge. A major contributor to this resistance is the dysregulation of cholesterol homeostasis in these cells. BC cells have elevated intracellular cholesterol levels, which is associated with cancer progression. In our previous research, we observed that the use of a cholesterol depleting agent, Acetyl Plumbagin (AP) in combination with TAM, led to the induction of cell death via cholesterol depletion. These results therefore warranted further investigation into the molecular mechanisms in which TAM + AP are involved in. MicroRNAs (miRNAs) regulate cholesterolrelated and cancer drug resistance pathways, and the aberrant expression of these miRNAs are often associated with increased cancer proliferation and resistance. It was therefore predicted that manipulating the expression of these target miRNAs could lead to a reduction in BC related drug resistance via cholesterol depletion. Thus, we aimed at investigating the roles of miRNA128 and miRNA-223 in cholesterol-mediated TAM resistance. Three BC cell lines (MCF-7 (estrogen-dependent), MDA-MB-231 (estrogen-independent), and Long-Term Estrogen Deprived (LTED)) were treated with a combination of 1 µM TAM and 10 µM AP following transfection with a miR-128 inhibitor or a miR-223 mimic. Cell viability and cholesterol levels were assessed following treatments. In addition, gene and protein expression levels involved in cancer drug resistance and cholesterol homeostasis were also assessed. It was found that the combination treatment with altered miRNA expression led to reduced cell viability and proliferation due to a reduction in free cholesterol, cholesteryl esters, and lipid rafts in all three BC cell lines. Moreover, miR-128 inhibition lowered the expression of genes involved in cholesterol synthesis and transport (HMGCR, HMGCS1, SREBF1/2, CETP, LCAT, and LDLR), drug resistance (ABCC5 and UGCG), and cell signalling (ESR1, EGFR, and IGF1R) in MCF7 cells. Whereas overexpression of miR-223 led to decreased expression in EGFR, ESR1, ABCC5, CETP, LCAT, LDLR, HMGCR, SREBF1, and SREBF2, with increased expression in ABCG1, PTEN, and TP53 in MDA-MB-231 cells. Therefore, the current study demonstrated that miR-128 and miR-223 could be potential targets in reducing TAM resistance through the depletion of excess cholesterol.enBreast cancerDrug resistanceThesis