Date of Award

2016

Document Type

Open Access Dissertation

Department

Biological Sciences

Sub-Department

College of Arts and Sciences

First Advisor

Franklin G. Berger

Second Advisor

Maria Marjorette O. Peña

Abstract

Nuclear factor-erythroid-2-related factor 2 (NRF2), a member of the cap ‘n’ collar family of bZIP transcription factors, confers protection against oxidative and electrophilic stress. NRF2 is of great interest in cancer research, due to its role in response to chemotherapy. The class of drugs targeting thymidylate synthase (TYMS) has been useful in the treatment of colorectal cancer, among other cancers. It has long been known that inhibition of TYMS leads to depletion of thymidine levels and the onset of programmed cell death, deriving from the enzyme’s function as the sole de novo source of thymidine for DNA replication and repair. Exposing cells to TYMS inhibitors such as fluoropyrimidine antimetabolites (5-fluorouracil, or FUra; 5’-fluoro-2’-deoxyuridine, or FdUrd), as well as anti-folate analogs (raltitrexed, or RTX), induce intracellular concentrations of reactive oxygen species, which are a primary cause of drug-mediated toxicity. This prompted our focus on assessing the impact of NRF2 on cellular response to TYMS inhibitors. Using human colon tumor-derived cell line HCT116, we have shown by gene expression profiling that drug exposure induces expression of a number of genes that are regulated by NRF2. Quantitative PCR assays of several colon tumor cell lines verified that FUra, FdUrd, and RTX induce transcription of several genes known to be NRF2-targets, including AKR1B10, ALDH3A1, HSPB8, HMOX1, and SERPINE1, among others. Such induction mirrors that in response to the classical NRF2 activator tert-butylhydroquinone (tBHQ). NRF2 protein concentrations in the nucleus are increased by FdUrd treatment, though not to the same extent as with tBHQ. Reporter gene constructs were used to show that both FdUrd and tBHQ induce transcription mediated by the NRF2-binding antioxidant response element (ARE). Furthermore, chromatin-immunoprecipitation experiments revealed that TYMS inhibitors promote occupancy by NRF2 of the ARE regions of several genes; again, tBHQ had a much greater effect. Additionally, chromatin-immunoprecipitation experiments indicated that TYMS inhibitors do not alter the acetylation of histones near the promoter regions. We observed no correlation between the activity of the target gene, and the acetylation of histones. We also showed that the PI3K/AKT pathway does not affect the stability of NRF2 in HCT116 cells. Finally, we observed that increases in the apoptotic index following exposure to TYMS inhibitors were greater in cells in which the transcription factor was subjected to siRNA-mediated “knockdown” or CRISPR/Cas9-mediated “knockout”, indicating that reduced NRF2 expression sensitizes cells to TYMS inhibitors. Overall, we conclude that TYMS inhibitors activate NRF2 and its downstream target genes, thereby constraining drug response. Reducing such activation of NRF2 or its consequences may be an effective strategy to sensitizing tumor cells to chemotherapy.

Rights

© 2016, Sarah Ashley Clinton

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