Author ORCID Identifier
Date Available
5-9-2026
Year of Publication
2024
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Medicine
Department/School/Program
Toxicology and Cancer Biology
Advisor
Dr. Qiou Wei
Abstract
Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Despite the use of chemotherapeutic agents, CRCs remains to be difficult to cure at least partially due to their intrinsic and/or acquired resistance to cell death inducers. Ferroptosis is a form of regulated cell death that caused by the accumulation of lipid peroxides. Studies have shown that dysregulation of ferroptotic pathways is associated with the development of resistance to various cancer treatment. Glutathione peroxidase 4 (GPX4) is a known target for inducing ferroptosis due to its critical role in detoxifying lipid hydroperoxides, and ferroptosis suppressor protein (FSP1) also play a role in this process. However, more and more evidence suggest that additional factors may involve ferroptosis resistance of cancer cells. The aim of this study is to identify and understand how cellular factors may contribute to resistance to ferroptosis in CRC cells. Ultimately, our goal is to target these factors to eliminate ferroptosis resistance in cancer cells.
Firstly, we studied the contribution and mechanism of the peroxiredoxin (PRX) family of proteins in the resistance of CRC cells to ferroptosis. PRXs play a pivotal role as peroxidase to react with hydrogen peroxide and other reactive oxygen species to maintain cellular redox homeostasis. Due to their involvement in disulfide bond exchanging, they are also essential to mediate oxidative signaling in both physiological and pathological conditions. However, the contribution of PRXs to ferroptosis resistance of cancer cells has not been studied. Individual member of the 2-Cys containing PRXs, including PRX1, PRX2, PRX3, and PRX4, was depleted in CRC cell lines using stable shRNA knockdown technique. We evaluated the consequences of PRX depletion on cell viability in response to the treatment of erastin, a known chemical inducer of cell death primarily through ferroptosis. To determine ferroptotic cell death, we measured the levels of lipid peroxidation using the molecular probe BODIPY 581/591 C11, and various inhibitors to distinguish ferroptosis from other modes of cell death. Overexpression was used to validate the results observed in PRX4-depleted cells. We found that the depletion of PRX1, PRX2, or PRX4, but not PRX3, sensitizes CRC cells to erastin-induced cell death. However, only the depletion of PRX4 sensitizes CRC cells to cell death specifically through ferroptosis, characterized by the accumulation of lipid peroxidation and a complete rescue by specific inhibitors of ferroptosis. Moreover, RNA sequencing and gene set enrichment analysis (GSEA) were used to identify genes and pathways that are dysregulated in PRX4-depleted cells. We identified and validated that the elevation of arachidonic acid (AA) metabolism pathway in PRX4-depleted cells leads to the upregulation of lipoxygenases (ALOXs) including ALOX15, which plays a role in sensitizing these cells to ferroptosis. These findings highlight the crucial role of PRX4 in protecting colorectal cancer from ferroptosis, suggesting that PRX4 can be used as a potential target for ferroptosis-based therapies.
Secondly, to further understand the molecular basis of acquired resistance to ferroptosis, we established ferroptosis-resistant CRC cell lines through a long-term, chronical exposure of these cells to increasing doses of erastin. The established, ferroptosis resistant cells were then subjected to gene expression profiling by RNA-seq and GSEA. We identified and validated that the activation of NF-κB and AP-1 pathways contributes to their resistance by upregulating cellular antioxidant enzymes, including heme oxygenase 1 (HMOX1), NAD(P)H quinone oxidoreductase 1 (NQO1), and ferritin heavy chain 1 (FTH1). In addition, we found that increased cancer stem cell population of the resistant cells may also contribute to their resistance to ferroptosis. Furthermore, a high throughput screening of the FDA-approved drug library was performed to identify drugs that can kill resistant cells. We found that parthenolide, an NF-κB inhibitor, kills ferroptosis resistant cells through the downregulation of NQO1, a critical enzyme that is significantly upregulated in resistant cells. Taken together, our study demonstrates that NF-κB activation plays an essential role in the development of ferroptosis resistance and targeting this pathway can eliminate ferroptosis resistance in CRC cells.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2024.101
Recommended Citation
Alshahrani, Aziza, "UNDERSTANDING THE MECHANISM OF FERROPTOSIS SUSCEPTIBILITY VARIATION IN COLORECTAL CANCER" (2024). Theses and Dissertations--Toxicology and Cancer Biology. 54.
https://uknowledge.uky.edu/toxicology_etds/54
