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.
Highlights
It is estimated by the American Cancer Society that breast cancer will account for 30% of new cancer cases and 15% of cancerrelated deaths in women in 2021 (Siegel et al, 2021)
Regression analysis revealed that the expression of 3,889 genes were significantly associated with the N-acetyltransferase 1 (NAT1) N-acetylation activity of the cell lines; 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 (Figure 2)
As NAT1 is known to N-acetylate serotonin (Steinberg et al, 1969; Backlund et al, 2017), albeit at very low levels in comparison to aralkylamine N-acetyltransferase (AANAT), we find the negative association between 5-hydroxytryptamine receptor 1F (HTR1F) and NAT1 N-acetylation activity interesting
Summary
It is estimated by the American Cancer Society that breast cancer will account for 30% of new cancer cases and 15% of cancerrelated deaths in women in 2021 (Siegel et al, 2021). When breast cancer is detected at the localized stage the 5-year survival rate is 99% when detected at the distant stage the 5-year survival rate is a disappointing 28% (Siegel et al, 2018). These statistics highlight the need for a better understanding of breast cancer risk and progression so that interventions can be provided at the early stage when breast cancer is the most treatable and to identify novel molecular targets for treatment. The role of NAT1 in breast cancer, until recently, was thought to center on NAT1’s ability to metabolize/activate carcinogens, it has been shown that rats with higher Nat expression (orthologous to human NAT1) had greater mammary tumor susceptibility, independent of carcinogen metabolism (Stepp et al, 2017)
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