Author: Sandra George
George, Sandra, 2025 Intra-tumoral drug metabolism: evaluating the impact of ugt overexpression on anticancer drug efficacy in breast cancer., Flinders University, College of Medicine and Public Health
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Breast cancer is one of the most prevalent cancers in which the treatment efficacy is reduced due to drug resistance. Estrogen receptor-positive breast cancer is commonly treated with tamoxifen; however, up to 40% of patients develop resistance to the drug. Here, it was examined whether overexpression of a specific member of the drug metabolising enzyme family, Uridine 5'-Diphospho-Glucuronosyltransferase (UGT) changes the sensitivity of MCF7 breast cancer cells to 4-hydroxytamoxifen (4-OH-TAM), which is the active form of the drug. Two paradigms were established, the first used short-term (transient) overexpression of UGT2B15 and the second used long-term (stable) overexpression of UGT2B15. MCF7 cells that were transiently transfected with UGT2B15 or control plasmids were treated with varying doses of 4-OH-TAM, and cell proliferation was measured via the crystal violet method. Cells over-expressing UGT2B15 showed resistance to 4-OH-TAM induced growth inhibition, with the effect statistically significant at low doses of the drug. Analysis of estrogen-stimulated PS2 gene expression in MCF7 cells revealed that pretreatment of 4-OH-TAM reduced E2-mediated PS2 induction in control cells and UGT2B15 overexpressing cells alike. These differences were not statistically significant, likely due to high variance between the replicate experiments. To study the effects of UGT2B15 on long-term 4-OH-TAM response, cells with stably integrated UGT2B15 or control plasmids were modified by integration of green and red fluorescent protein reporter plasmids. The overexpression of UGT2B15 was confirmed by immunoblot, and expression of fluorescent proteins was confirmed by microscopy. These ‘double-stable’ cell lines were used to establish a competitive growth assay. A pilot study showed that microscopy could be used to quantify the relative growth of labelled cells; however, preliminary data were not sufficient to conclude whether UGT2B15 provided a long-term drug-specific growth/survival advantage. Collectively, these results suggest that while UGT2B15 has the potential to impart resistance to low doses of 4-OH-TAM in vitro, further studies are required to determine the dose-dependence of this effect, and whether it translates to more natural tumour-like models that include cell-type heterogeneity. These findings are representative of the multidimensionality of tamoxifen resistance mechanisms and lend further stimulus for investigation into the clinical importance of UGT2B15 in cancer therapy.
Keywords: Breast cancer, tamoxifen resistance, MCF7 cells, drug resistance, drug metabolism, glucuronidation, UGT enzymes, UGT2B15, intratumoral UGT expression, cell growth analysis, gene expression analysis, Transient overexpression, Stable overexpression, Competitive growth assay, breast cancer therapy
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Dr. Robyn Meech