The potential role of the oxidoreductase , NQO2 in breast cancer

Abstract

NRH: quinone oxidoreductase 2 (NQO2) is a cytosolic flavin dependent enzyme that is ubiquitously expressed in human tumours. The physiological role of NQO2 in breast cancer is still not fully characterised. NQO2 was shown to play a role in stabilising the tumour suppressor gene, p53. NQO2 has been shown to act as a detoxifying enzyme and catalyse the reduction of electrophilic estrogen quinones. The enzymatic activity of NQO2 on estrogen derived quinones and its crosstalk with p53 has led to the hypothesis that NQO2 might be a candidate susceptible gene that could have a potential role in breast cancer. The potential role of NQO2 in two different types of breast cancer cell lines, MCF-7 and MDA-MB-231 was investigated in this study. MCF-7 cells are estrogen positive (ER+) and carry wild type p53; whereas MDA-MB-231 cells are estrogen negative (ER-) and harbour mutant p53.We have investigated whether NQO2 plays a different role in both cell lines. Initially, the expression and the catalytic activity of NQO2 in MDA-MB-231 and MCF-7 cells were assessed. It was found that NQO2 is highly expressed and active in MDA-MB- 231; whereas NQO2 protein levels and enzymatic activity were not detected in MCF-7 cells. Therefore, the NQO2 biological activity was studied by generating a stable cell line which induced a ShRNA targeting NQO2 under the inducible control of doxycycline in MDA-MB-231, and by using lenti-viral vector system to overexpress NQO2 in MCF-7 cells. The consequences of genetic modification of NQO2 on different cellular characteristics were examined in the two cell lines. The findings indicate that NQO2 modulated p53 expression in both cell lines. NQO2 downregulation in MDA-MB-231 reduced the mutant p53 expression and its overexpression in MCF-7 cells upregulated the wild type p53 expression. The modulation of p53 in MDA-MB 231 and MCF-7 cells was accompanied by reduced cell growth, G1 cell cycle arrest, and induction of caspase dependent apoptosis. The apoptosis observed in MCF-7 cells was also p53 dependent. The intracellular ROS levels were also assessed to indicate whether the apoptosis observed was ROS induced. It was found that the genetic silencing of NQO2 in MDA-MB-231 increased the ROS levels; whereas no effect on ROS levels was observed in NQO2 overexpressing MCF-7cells. The effect of NQO2 gene modification on doxorubicin sensitivity was examined in MDA-MB-231 and MCF-7 cells. The downregulation of NQO2 in MDA-MB-231 cells significantly enhanced the cytotoxiciy of doxorubicin; whereas no effect on doxorubicin cytotoxicity was observed in NQO2 overexpressing MCF-7 cells.A series of potent inhibitors of NQO2 have been identified in this study. These inhibitors were shown to inhibit NQO2 at nano molar concentrations, with the most potent one exhibiting NQO2 inhibition at IC50 of 15nM. These inhibitors were shown to be cytotoxic in MDA-MB-231 and MCF-7 cells. The effect of these inhibitors on DNA binding ability as an adverse effect was assessed by measuring the DNA thermal temperature. None of the novel inhibitors demonstrated any change in the thermal temperature of DNA, indicating that they do not have DNA intercalating ability.In summary, the findings of this study indicate that the role of NQO2 in breast cancer is cell type dependent. It is a novel therapeutic target and a tumour sensitizer to doxorubicin in estrogen negative breast cancers which express mutant p53. However, NQO2 is a protective enzyme in estrogen positive breast cancers which express wild type p53.

Date of Award	31 Dec 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Stratford (Supervisor) & Costas Demonacos (Supervisor)