Iwakun, Oluwasegun
2021,
Investigating the role of NEIL3 in colorectal cancer resistance to oxaliplatin
, PhD thesis, University of Salford.
Abstract
Colorectal cancer (CRC) is the third most common type of cancer and accounts for 8 % of cancer related deaths worldwide. While the likelihood of colorectal carcinogenesis increases with age, genetics and lifestyle are major predisposing factors. Treatment can be curative and promises a 39 – 65% survival rate depending on the stage at cancer diagnosis. Current treatment options include standalone or a combination of surgery, radiotherapy, chemotherapy, and targeted therapy. An essential part in the treatment of advanced stage CRC employs an oxaliplatin-based chemotherapy regimen, which has shown a response rate of 50 – 60%. Nevertheless, the majority of CRC patients experience relapse after surgery and develop resistance to oxaliplatin thereby limiting its efficacy and usage at tolerable doses. Like other platinum compounds such as cisplatin, the molecular target of oxaliplatin is DNA, where it produces DNA intra- and inter-strand crosslinks that prevent transcription and DNA replication to cause cell death. Although numerous mechanisms of tumour resistance to oxaliplatin have been reported, normal healthy cells also rely on the majority of these mechanisms for survival and genome stability. Recently, NEIL3, a cell-cycle dependent and tissue-specific DNA glycosylase has been implicated in the repair of psoralen- and AP (apurinic) site-induced interstrand crosslinks (ICLs), thus prompting these studies into the possible contribution of NEIL3 to oxaliplatin resistance in CRC treatment via its ability to unhook ICLs. Previous studies have also reported the overexpression of NEIL3 in CRC tissues and associated it with the poor prognosis in a range of cancer types. Therefore, this current research project was conducted to identify a relationship between cellular levels of NEIL3 in a panel of cancer cell lines and their sensitivity/resistance to oxaliplatin. The findings confirmed that there is no significant difference in basal NEIL3 levels between oxaliplatin-sensitive and oxaliplatin-resistant CRC cells. However, a concentration-dependent upregulation of NEIL3 was observed in the oxaliplatin-sensitive HCT116 cell line (wildtype p53) following short-term exposure to different concentrations of oxaliplatin. Surprisingly, NEIL3 levels were not elevated in the same cell line following the development of resistance to oxaliplatin after chronic continuous exposure to incremental concentrations of oxaliplatin. Finally, an attempt to knockout NEIL3 from the genome of HCT116 cells using CRISPR failed, though RNAi-mediated silencing of NEIL3 mRNA confirmed that functional downregulation of NEIL3 contributed a 13.2-fold increase in sensitivity of U2OS osteosarcoma cells to oxaliplatin. Collectively, these results suggest that a strategy to inhibit or downregulate NEIL3 should improve the clinical outcome of oxaliplatin-based chemotherapy for CRC patients.
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