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Human Arylamine N-Acetyltransferase 1 (NAT1) Knockout in MDA-MB-231 Breast Cancer Cell Lines Leads to Transcription of NAT2.

ABSTRACT: Many cancers, including breast cancer, have shown differential expression of human arylamine N-acetyltransferase 1 (NAT1). The exact effect this differential expression has on disease risk and progression remains unclear. While NAT1 is classically defined as a xenobiotic metabolizing enzyme, other functions and roles in endogenous metabolism have recently been described providing additional impetus for investigating the effects of varying levels of NAT1 on global gene expression. Our objective is to further evaluate the role of NAT1 in breast cancer by determining the effect of NAT1 overexpression, knockdown, and knockout on global gene expression in MDA-MB-231 cell lines. RNA-seq was utilized to interrogate differential gene expression (genes correlated with NAT1 activity) across three biological replicates of previously constructed and characterized MDA-MB-231 breast cancer cell lines expressing parental (Scrambled), increased (Up), decreased (Down, CRISPR 2-12), or knockout (CRISPR 2-19, CRISPR 5-50) levels of NAT1. 3,889 genes were significantly associated with the NAT1 N-acetylation activity of the cell lines (adjusted p ≤ 0.05); of those 3,889 genes, 1,756 were positively associated with NAT1 N-acetylation activity and 2,133 were negatively associated with NAT1 N-acetylation activity. An enrichment of genes involved in cell adhesion was observed. Additionally, human arylamine N-acetyltransferase 2 (NAT2) transcripts were observed in the complete NAT1 knockout cell lines (CRISPR 2-19 and CRISPR 5-50). This study provides further evidence that NAT1 functions as more than just a drug metabolizing enzyme given the observation that differences in NAT1 activity have significant impacts on global gene expression. Additionally, our data suggests the knockout of NAT1 results in transcription of its isozyme NAT2.

SUBMITTER: Carlisle SM

PROVIDER: S-EPMC8762260 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.

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Human Arylamine N-Acetyltransferase 1 (NAT1) Knockout in MDA-MB-231 Breast Cancer Cell Lines Leads to Transcription of NAT2.

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