Identification of genetic markers that predict cancer sensitivity to the anticancer drugs 5-FU and Irinotecan

• Main Author: Jordan, Tamara
• Format: Dissertation
• Language: English
• Published: Prifysgol Bangor University 2015

The MRN complex (consisting of Mre11 Rad50 and Nbs1), and CtIP, functions to repair DNA double strand breaks. They are also known to have a role in the removal of topoisomerases from DNA, and can therefore provide resistance to topoisomerase inhibitors, such as camptothecin, which are used as anti-cancer drugs and function by increasing the half-life of topoisomerase-DNA cleavage complexes leading to the persistence of DNA breaks which can lead to cell death. Therefore it has been hypothesised that patient mutations in the MRE11, RAD50, NBS1and CtIP genes can confer an increased sensitivity to topoisomerase inhibitors. A recent study, the NWCOG-2 trial, involving the treatment of colorectal carcinomas with irinotecan (a topoisomerase inhibitor) and capecitabine (which is metabolised to form the nucleoside analogue 5-FU) showed a pathological complete response in 22% of patients and a three year survival of 88%, it is unknown which genetic factors influence the response this method of treatment. The aforementioned genes are known to be mutated at high frequency in colorectal cancers associated with microsatellite instability. In this study, mutations in these genes which have been previously identified in tumours have been recreated in Schizosaccharomyces pombe, using the Cre-lox system, and tested for sensitivity to camptothecin and MMS. The results show that many of these mutants exhibit a severe sensitivity to these drugs. The nbs1-MSI mutant, which encodes a mutant protein lacking the C-terminal Mre11 and Tel1 ATM binding domains appeared to show an increased sensitivity to MMS, with only a slight increase in sensitivity to camptothecin. This mutant is a separation of function mutant and shows that the Mre11 and Tel1 ATMbinding domains of Nbs1 may not be essential for topoisomerase removal, but are required for downstream repair of MMS induced lesions. Sequencing of 25 NWCOG-2 patient tumour samples initially identified the presence of 30 somatic mutations in. However, identification of all but one of these mutations, the CtIP-poly(A)9 1 bp deletion of patient R48, was irreproducible and were not found in subsequent resequencing reactions. Fluorescent fragment analysis of the MRE11-poly(T)11 tract revealed the presence of a single base-pair deletion in one patient (patient R12). Fluorescent fragment analysis of CtIP-poly(A)9 tract confirmed the presence of a single base-pair deletion in patient R48 and identified patient R51 to possible harbour the mutation also. Bothe patients R12 and R48 responded well to the treatment regimen of irinotecan, capecitabine, radiation and excision; however it cannot be confirmed that this response was due to the presence of the mutations. S. pombe mre11Δ, rad50Δ, nbs1Δ and ctp1Δ strains were also tested for sensitivity to 5-FU, these strains showed no increase in sensitivity to 5-FU compared to the wild-type, suggesting that these genes may not confer sensitivity to 5-FU in tumours. A screen of the Bioneer genome wide deletion library was carried out to identify other genes for which mutations could potentially confer increased sensitivity or resistance to 5-FU. This identified a total of 181 mutations which confer increased 5-FU sensitivity, and 316 which conferred an increased resistance. Many of the genes found to sensitise to 5-FU when deleted function in chromatin remodelling and centromere function, suggesting a possible role for the centromere in 5-FU sensitivity. Many of the genes found to confer resistance to 5-FU function in the processing of uridine in tRNA; this suggests that the processing of incorporated fluorouridine in tRNA may be a significant contributing factor to 5-FU cytotoxicity in S. pombe.