Impact of Nitric Oxide Inhibitors on Triple Negative Breast Cancer
Ciara E O'Neill (1), Sugunapriyadharshini Sundararaman (1,2) and Sharon A Glynn (1)
(1) Discipline of Pathology, Lambe Institute for Translational Research, University of Galway, (2) CÚRAM, SFI Research Centre for Medical Devices, University of Galway.
Introduction
Nitric Oxide (NO) is a gaseous signalling molecule most commonly associated with the cardiovascular system. It is produced endogenously by nitric oxide synthase enzymes and high levels of inducible nitric oxide synthase (iNOS) expression have been linked with poor outcomes in ER-negative breast cancer. NO signalling is mediated through cGMP and s-nitrosation of proteins.
EGFR & HER2 are drivers of tumour proliferation and metastatic potential, and several targeted therapies have been developed. S-nitrosation of EGFR is linked with the activation of oncogenic signalling, and therefore NO has been proposed as an alternative activator of the HER family.
This study utilises an iNOS overexpression system to investigate the impact of NO signalling in Triple Negative Breast Cancer (TNBC), and the ability of NOS inhibitors to modulate this.
Material and Methods
iNOS was overexpressed in HCC1806 and MDA-MB-231 cells, using a Lenti-X GFP-tagged constitutive expression system. iNOS expression and functionality was confirmed by PCR, western blot, and Greiss assay respectively. The effect of iNOS expression on proliferation was measured by MTS assay and the impact on the phosphorylation status of EGFR was assessed by western blot. The ability of NOS and cGMP inhibitors to reverse these effects was then assessed.
Results
iNOS mRNA and protein expression were significantly increased in GFP-iNOS transduced HCC1806s and MDA-MB-231s compared to the corresponding GFP-Control cells. NO production by iNOS was specific to GFP-iNOS cells and successfully inhibited by the NOS inhibitors L-NMMA and 1400W but not by the cGMP inhibitor ODQ. iNOS expression also resulted in altered proliferation, and phosphorylation of EGFR.
Conclusion
NO is known to induce EGFR s-nitrosation and it has been proposed as an alternative activator of the HER family. Further research is ongoing into the impact of NO on HER2 signalling. As iNOS can be inhibited therapeutically it represents a new therapeutic strategy for the treatment of ER-negative breast cancer. Gaining further understanding of its mechanism of action and the effects of NOS inhibitors in combination with current therapeutics is key.