CRISPR/Cas9 knockout of human arylamine N-acetyltransferase 1 in MDA-MB-231 breast cancer cells suggests a role in cellular metabolism

Samantha M Carlisle,Mark A Doll,Kyung U Hong,Patrick J Trainor,David W Hein

doi:10.1038/s41598-020-66863-4

Abstract

Human arylamine N-acetyltransferase 1 (NAT1), present in all tissues, is classically described as a phase-II xenobiotic metabolizing enzyme but can also catalyze the hydrolysis of acetyl-Coenzyme A (acetyl-CoA) in the absence of an arylamine substrate using folate as a cofactor. NAT1 activity varies inter-individually and has been shown to be overexpressed in estrogen receptor-positive (ER+) breast cancers. NAT1 has also been implicated in breast cancer progression however the exact role of NAT1 remains unknown. The objective of this study was to evaluate the effect of varying levels of NAT1 N-acetylation activity in MDA-MB-231 breast cancer cells on global cellular metabolism and to probe for unknown endogenous NAT1 substrates. Global, untargeted metabolomics was conducted via ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on MDA-MB-231 breast cancer cell lines constructed with siRNA and CRISPR/Cas9 technologies to vary only in NAT1 N-acetylation activity. Many metabolites were differentially abundant in NAT1-modified cell lines compared to the Scrambled parental cell line. N-acetylasparagine and N-acetylputrescine abundances were strongly positively correlated (r = 0.986 and r = 0.944, respectively) with NAT1 N-acetylation activity whereas saccharopine abundance was strongly inversely correlated (r = −0.876). Two of the most striking observations were a reduction in de novo pyrimidine biosynthesis and defective β-oxidation of fatty acids in the absence of NAT1. We have shown that NAT1 expression differentially affects cellular metabolism dependent on the level of expression. Our results support the hypothesis that NAT1 is not just a xenobiotic metabolizing enzyme and may have a role in endogenous cellular metabolism.

Highlights

Breast cancer is a heterogeneous disease with many underlying genetic transformations that lead to a diseased state
To focus on metabolome differences associated with varying levels of N-acetyltransferase 1 (NAT1) in breast cancer, we have focused our interpretation of this study to metabolites that agreed between the two CRISPR NAT1 KO cell lines (Table 2)
Our results support the hypothesis that NAT1 is not just a xenobiotic metabolizing enzyme and may have a role in endogenous cellular metabolism

Summary

Introduction

Breast cancer is a heterogeneous disease with many underlying genetic transformations that lead to a diseased state. A previous study investigating the polar metabolome of the same cells has revealed differences in a number of metabolites, including amino acids and palmitoleic acid[13]. Given these data, we hypothesize differences in NAT1 activity may be contributing to downstream effects on metabolic pathways that utilize acetyl-CoA such as the TCA cycle and fatty acid synthesis and degradation. As metabolic reprogramming is one of the hallmarks of cancer proposed by Hanahan and Weinberg[14] and acetyl-CoA plays a central role in metabolism[15] we have utilized untargeted metabolomics to investigate how different levels of NAT1 affect cellular metabolism in MDA-MB-231 triple negative breast cancer cells. To build upon our previous metabolomics work[13], we have expanded the scope of metabolites measured from only the polar metabolome to the global metabolome and expanded the samples to include complete NAT1 knockout (KO) cell lines as well as a cell line that has only one copy of NAT1 knocked-out (expresses half of parental NAT1 activity)

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Conclusion