A Novel Mechanism of Epigenetic Regulation by Nitric Oxide: Inhibition of DNA Demethylation in Cancer

The expression of inducible nitric oxide synthase (iNOS) predicts poor outcomes in triple-negative breast cancer (TNBC) patients, yet molecular mechanisms to explain this association are lacking. Epigenetics, and particularly DNA methylation, contributes significantly to the pathology of cancers including TNBC. DNA methylation (5mC) at CpG sites in the human genome is a function of the concerted activities of DNA methyltransferases (DNMT) which catalyze methylation and ten-eleven translocation (TET) dioxygenases which demethylate cytosine residues. AlkB Homolog 2 (ALKBH2) is a DNA damage response demethylase that repairs 1-methyladenine and 3-methylcytosine on alkylated DNA. Both these DNA demethylases are in the family of Fe(II)/2-OG-dependent dioxygenases which contains other enzymes that have been shown to be inhibited by nitric oxide (NO). Here, we report that the signaling molecule NO is a potent, dose-dependent, and reversible inhibitor of TET and ALKBH2 enzyme activity. Electron paramagnetic resonance (EPR) spectroscopy revealed that NO directly binds to ferrous iron in the TET catalytic pocket, forming a dinitrosyliron complex (DNIC). Genome-wide oxidative bisulfite sequencing and ELISA showed that NO causes global increases in 5mC while global 5-hydroxymethylcytosine (5hmC) levels remain unchanged. These effects were confirmed in cell-line derived and patient-derived mouse xenografts treated with a NOS inhibitor. The enrichment or absence of these adducts at gene-regulatory loci correlated to the expression of tumor-associated genes, and western blot verified that these changes are not due to altered TET or ALKBH2 protein levels or methyltransferase expression. We also observed for the first time that NO affects chromatin conformation by significantly increasing chromatin packing scaling. In summary, we have discovered a completely novel epigenetic signaling mechanism of NO by demonstrating for the first time how NO can regulate DNA methylation to control the expression of tumor-permissive genes. These studies provide a causal link between tumor NO production and more aggressive cancers and reveal potential new therapeutic pathways to target.