Deciphering the role of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer cell metabolism using a systems biology approach.

Abstract

Background: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can additionally hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. Additionally, multiple studies have shown the knockdown of NAT1, by both small molecule inhibition and siRNA methods, in breast cancer cells leads to decreased invasive ability and proliferation and decreased anchorage-independent colony formation. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Additionally, consequences of the hydrolysis of acetyl-CoA by NAT1 on cellular metabolism remains uninvestigated. Hypothesis and Rationale: Samples with decreased levels or knockout of NAT1 will have increased free acetyl-CoA since those cell lines have less NAT1 to hydrolyze acetyl-CoA. Conversely, samples with increased NAT1 will have decreased free acetyl-CoA since those cell lines have more NAT1 to hydrolyze acetyl-CoA. These differences in free acetyl-CoA are hypothesized to lead to alterations in cellular pathways/metabolism when compared to cells with basal NAT1 that can be measured by global bioenergetics, metabolomics, and transcriptomics experiments. Methods: This dissertation utilized a systems biology approach with four layers. The first layer consists of six constructed MDA-MB-231 cell lines whose only genetic difference (theoretically) is in NAT1. The second, third, and fourth layers are bioenergetics (Chapter 3), metabolomics (Chapter 4), and transcriptomics (Chapter 5) measurements of the constructed cell lines. Resulting data were analyzed individually and also integrated and analyzed (Chapter 6). Results: The manipulation of NAT1 in MDA-MB-231 breast cancer cells severely altered cellular metabolism as measured by mitochondrial bioenergetics, metabolomics, and transcriptomics. More differences were observed in the cell lines with decreased levels and knockout NAT1 than the cell line with increased NAT1. Conclusions: This dissertation has generated novel hypotheses about the role of NAT1 in breast cancer, and more generally cellular metabolism. Furthermore, biochemicals that are likely products of NAT1 N-acetylation, N-acetylasparagine and N-acetylputrescine, have been identified. This dissertation presents strong evidence that NAT1, whether directly or through an effect of NAT1 on acetyl-CoA levels, has an effect on acyl-CoA carnitine conjugates, lysine degradation, and mitochondrial function. While the exact mechanism by which NAT1 affects cellular metabolism or breast cancer progression has not been identified, the data presented in this dissertation add important pieces to the puzzle,…