AbstractCancer is one of the foremost causes of death worldwide with about 14.1 million new incidences and 8.2 cancer related deaths occurring globally. NF-E2 p45-related factor 2 (Nrf2), a cap-‘n’-collar basic leucine zipper (CNC-bZIP) transcription factor, prevents carcinogenesis through expression of genes that ensure the excretion, enzymatic modification, and repair of oxidative damage in cells containing the antioxidant response element (ARE) in their promoter region. Beyond providing cytoprotection against oxidative stress and xenobiotics, Nrf2 pays a role in maintaining basic physiological processes such as energy metabolism and cell cycle regulation. Whilst Nrf2 plays a pivotal role in preventing degenerative and inflammatory disease, upregulation of Nrf2 promotes tumourigenesis in cancerous cells. Therefore, understanding the mechanisms controlling Nrf2 activity is important in translational medicine.
Nrf2 is regulated by proteasomal degradation by Kelch-like ECH-associated protein 1 (Keap1) an E3 ubiquitin ligase substrate adaptor protein that recruits of cullin-3 (Cul3) to Nrf2 via its Neh2 domain. Nrf2 is also negatively regulated by phosphorylation by glycogen synthase kinase-3 (GSK-3) causing β-transducin repeat-containing protein (β-TrCP) to ubiquitinate Nrf2 by Skp1-Cul1-F-box (SCF) ubiquitin ligase through the Neh6 domain of Nrf2. Several research groups have shown that induction of ARE-driven genes can be regulated by phosphoinositide 3- kinase- protein kinase B (PI3K-Akt/PKB) signalling pathway. The ability of tert-butylhydroquinone (tBHQ), 1-[2-cyano-3,12-dioxooleana-1,9(11)-diene-28-oyl]imidazole (CDDO-Im), diethyl maleate (DEM), curcumin, carnosol, ferulic acid and sulforaphane (SFN) to activate Nrf2-target genes in a Keap1-dependent or Keap1-independent manner was tested. It was discovered that all compounds, except for SFN, activate Nrf2-target genes in a Keap1-independent manner, inhibiting GSK-3 and functioning through the Neh6 domain of Nrf2. Analysis of the involvement of PI3K-Akt/PKB pathway in Nrf2 activation revealed that regulation of Nrf2 through the PI3K-Akt/PKB pathway is independent of Keap1 but dependent on GSK-3. Also, it was shown that tBHQ, DEM, CDDO-Im, curcumin, ferulic acid directly decreased phosphatase and tensin homolog (PTEN) activity, thereby preventing formation of the phosphodegron in the Neh6 domain of Nrf2.
With increased Nrf2 levels reported in various cancers including lung cancer, leading to the progression of these cancers, Nrf2 can be seen as a double-edged sword. Loss-of-function somatic mutations in KEAP1 as well as somatic mutation in NFE2L2 has been reported in several human cancers playing a role in the development of such cancer. Using short hairpin RNA (shRNA) and the CRISPR/Cas9 system to generate stable Nrf2 knockdown A549 and H460 cells, the second part of this thesis investigated biochemical and physiological changes that occur, when the Nrf2 is genetically downregulated, and further on to determine what mechanism(s) is responsible for decreased cell proliferation in tumours. The findings obtained confirm that downregulation of Nrf2 from the human non-small lung adenocarcinoma epithelial cell line A549 and H460, in which Nrf2 is upregulated though somatic mutations in KEAP1, results in decreased cell proliferation. Analysis of the genes involved in NADPH generation and pentose phosphate pathway (PPP) show that decrease in Nrf2 caused a decrease in the expression of genes involved in PPP. Although knockdown of Nrf2 resulted in a decrease in cell proliferation, it was shown that this decrease was not as a result of cell death. Nrf2 is able to control cell proliferation by induction of metabolic reprogramming geared towards favoring anabolic pathways and influencing the PPP as well as provide energy source required for cell proliferation.
|Date of Award
|John Hayes (Supervisor) & Albena Dinkova-Kostova (Supervisor)