Thesis, FRANZEN Rachelle

Abstract

In 2022, colorectal cancer (CRC) was the third most diagnosed and the second deadliest cancer type worldwide. Radiotherapy (RT) is a major therapeutic modality for this type of cancer, yet radioresistance remains a critical barrier to treatment efficacy, contributing to tumour recurrence and poor patient outcomes. RT induces tumour cell death by DNA damage and through ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation. However, the metabolic mechanisms enabling tumour cells to evade ferroptosis and survive RT remain poorly understood. Preliminary data identify cholesterol biosynthesis (CB) as a key metabolic adaptation driving ferroptosis resistance and radioresistance in CRC cells, suggesting that this pathway may represent a therapeutic vulnerability that could be targeted to sensitize cancer cells to RT. To investigate whether CB contributes to ferroptosis resistance and its mechanistic role in radioresistance, radioresistant CRC cell lines (SW480 and HCT116) were generated in our laboratory and compared to their wild-type counterparts. Experiments included clonogenic assays with increasing doses of RT, cell death assays, and flow cytometry analysis using C11-BODIPY and FerroOrange to measure lipid peroxidation and intracellular ferrous iron content, respectively. Combination treatments were performed using ferroptosis inducers (RSL3, erastin) and the CB inhibitor TASIN-30. Molecular and cellular analyses (western blot, RT-qPCR) were also used to assess the expression and functional roles of CB enzymes and ferroptosis regulators. Importantly, 7-dehydrocholesterol (7-DHC), a CB intermediate, was identified as a key metabolite contributing to ferroptosis resistance. Pharmacological targeting of CB restored ferroptosis sensitivity and enhanced RT efficacy, identifying cholesterol metabolism as a novel and actionable metabolic vulnerability in radioresistant CRC cells.